Received: 11 November 2021 Revised: 6 February 2022 Accepted: 7 April 2022

DOI: 10.1002/cac2.12291

REVIEW

Cancer-associated fibroblasts in breast cancer: Challenges

and opportunities
Dengdi Hu1,† Zhaoqing Li2,3,† Bin Zheng1,† Xixi Lin2,3 Yuehong Pan1
Peirong Gong1 Wenying Zhuo1,2,3 Yujie Hu1 Cong Chen2,3 Lini Chen2,3
Jichun Zhou2,3 Linbo Wang2,3
1 Affiliated Cixi Hospital, Wenzhou Medical University, Ningbo, Zhejiang 315300, P. R. China
2 Affiliated
Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (Key Laboratory of Cancer Prevention and Intervention, Ministry of
Education), Hangzhou, Zhejiang 310016, P. R. China
3 Biomedical Research Center and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, Zhejiang 310016, P. R. China

Correspondence
Linbo Wang, Department of Surgical Abstract
Oncology, Affiliated Sir Run Run Shaw The tumor microenvironment is proposed to contribute substantially to the
Hospital, Zhejiang University School of
progression of cancers, including breast cancer. Cancer-associated fibroblasts

Abbreviations: α-SMA, alpha smooth muscle actin; ALDH, aldehyde dehydrogenase; ASO, antisense oligonucleotide; ATM, ataxia telangiectasia
mutated; ATRA, all-trans retinoic acid; BNIP3, BCL2 interacting protein 3; c-MET, scatter factor receptor; C9ORF135, chromosome 9 open reading
frame 135; CAF, cancer-associated fibroblast; cAMP, cyclic adenosine monophosphate; CAR, chimeric antigen receptor; Cav, caveolin; CCL, C-C motif
chemokine ligand; CCN2, connective tissue growth factor; CDC6, cell division cycle 6 homolog; CDK1, cyclin-dependent kinase 1; CDX2,
caudal-related homeobox 2; COX2, cyclooxygenase-2; CREB, cAMP response element-binding protein; CSC, cancer stem cell; CSF-2, colony
stimulating factor 2; CTHRC1, collagen triple helix repeat containing-1; CXCL, C-X-C motif chemokine ligand; CXCR, C-X-C motif chemokine
receptor; DCIS, ductal carcinoma in situ; DFS, disease-free survival; DOTA, 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid; DPP, dipeptidyl
peptidase; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; ER, estrogen receptor; ERK, extracellular signal-regulated kinase;
ET-1, endothelin-1; FAK, focal adhesion kinase; FAP, fibroblast activation protein alpha; FAPI, fibroblast activation protein alpha-specific enzyme
inhibitor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FLI1, friend leukemia virus integration 1; FOSL2, FOS-like 2; FSP1,
fibroblast-specific protein-1; FZD5, frizzled class receptor 5; GLIS1, Gli-similar 1; GLUT1, glucose transporter 1; GPER, G-protein-coupled estrogen
receptor; GPR77, G protein-coupled receptor 77; HA, hyaluronan; HDAC6, histone deacetylase 6; HGF, hepatocyte growth factor; HIF-1α,
hypoxia-inducible factor 1-alpha; HOTAIR, HOX transcript antisense RNA; HOXA5, homeobox A5; IDH3α, isocitrate dehydrogenase 3α; IFN-γ,
interferon gamma; IL, interleukin; ILC, invasive lobular carcinoma; IDC, invasive ductal carcinoma; IDH3α, isocitrate dehydrogenase 3α; IDO,
indoleamine-2,3-dioxygenase; IGFBP3, insulin growth factor-binding protein 3; LATS2, large tumor suppressor homolog 2; MAPK, mitogen-activated
protein kinase; MDSCs, myeloid-derived suppressor cells; MMP, matrix metalloproteinase; MSC, mesenchymal stem cell; mtDNA, mitochondrial
DNA; NF, normal fibroblast; NF-κB, nuclear factor kappa B subunit 1; NG2, chondroitin sulfate proteoglycan; NST, no special type; OPN, osteopontin;
P4HA3, prolyl 4-hydroxylase subunit alpha 3; OS, overall survival; PCP, planar cell polarity; PD-1, programmed cell death protein 1; PD-L1,
programmed death ligand 1; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PDPN, podoplanin; PECAM1,
platelet/endothelial cell adhesion molecule; PGE2, prostaglandin E2; PEGPH20, pegvorhyaluronidase alfa; PIK3Cδ, p110δ subunit of
phosphatidylinositol-3-OH kinase; PKA, protein kinase A; PKM2, pyruvate kinase M2; PLK1, polo-like kinase 1; PTEN, phosphatase and tensin
homolog; PTK7, protein tyrosine kinase 7; RFS, relapse-free survival; RIG-I, retinoic acid-inducible gene 1 protein; RN7SL1, RNA component of signal
recognition particle 7SL1; S100A4, S100 calcium-binding protein A4; S100A9, S100 calcium binding protein A9; scRNA-seq, single-cell RNA
sequencing; SDF-1, stromal cell-derived factor-1; SHC2, SHC-transforming protein 2; SMAD3, mothers against decapentaplegic homolog 3; SNHG3,
small nucleolar RNA host gene 3; SOCS2, suppressor of cytokine signaling 2; SOD1, superoxide dismutase 1; STAT1, signal transducer and activator of
transcription 1; STC1, stanniocalcin1; TCEAL7, transcription elongation factor A like 7; TIMP, tissue inhibitors of metalloproteinase; TGF-β,
transforming growth factor-β; TME, tumor microenvironment; TNBC, triple-negative breast cancer; TXNIP, thioredoxin-interacting protein; USP28,
ubiquitin-specific peptidase 28; VEGF, vascular endothelial growth factor; YAP1, yes associated protein 1

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium,
provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2022 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.

Cancer Communications. 2022;42:401–434. wileyonlinelibrary.com/journal/cac2 401

402 HU et al.

Medicine (Key Laboratory of Cancer

Prevention and Intervention, Ministry of (CAFs) are the most abundant components of the tumor microenvironment.
Education), Biomedical Research Center
Studies have revealed that CAFs in breast cancer originate from several types
and Key Laboratory of Biotherapy of
Zhejiang Province, No.3 Eastern of cells and promote breast cancer malignancy by secreting factors, generating
Qingchun Road, Hangzhou, Zhejiang, exosomes, releasing nutrients, reshaping the extracellular matrix, and suppress-
310016, P. R. China.
ing the function of immune cells. CAFs are also becoming therapeutic targets
Email: [email protected]
Jichun Zhou, Department of Surgical for breast cancer due to their specific distribution in tumors and their unique
Oncology, Affiliated Sir Run Run Shaw biomarkers. Agents interrupting the effect of CAFs on surrounding cells have
Hospital, Zhejiang University School of
Medicine (Key Laboratory of Cancer
been developed and applied in clinical trials. Here, we reviewed studies examin-
Prevention and Intervention, Ministry of ing the heterogeneity of CAFs in breast cancer and expression patterns of CAF
Education), Biomedical Research Center markers in different subtypes of breast cancer. We hope that summarizing CAF-
and Key Laboratory of Biotherapy of
Zhejiang Province, No.3 Eastern
related studies from a historical perspective will help to accelerate the develop-
Qingchun Road, Hangzhou, Zhejiang, ment of CAF-targeted therapeutic strategies for breast cancer.
310016, P. R. China. Email:
[email protected] KEYWORDS
† These authors contributed equally to this Cancer-associated fibroblasts, breast cancer, therapeutic target, tumor microenvironment,
work. biomarker, tumor heterogeneity

Funding information
National Natural Science Foundation of
China, Grant/Award Numbers: 81602471,
81672729, 81972453, 81972597; Natural
Science Foundation of Zhejiang Province,
Grant/Award Numbers: LY19H160055,
LY19H160059, LR22H160011; Natural
Science Foundation of Ningbo,
Grant/Award Number: 2019A610315; Cixi
Agricultural and Social Development
Science and Technology Project,
Grant/Award Number: CN2020006;
Zheng Shu Medical Elite Scholarship
Fund

1 BACKGROUND been revealed, and we reviewed related studies published

to the present.
Breast cancer is the most common malignant tumor in
the world [1–3]. The three far-reaching events affecting the
clinical prognosis of patients with breast cancer are drug 2 THE PROPERTIES OF CAFs
resistance, recurrence, and metastasis [4]. Studies have
shown that the tumor microenvironment (TME), espe- 2.1 Fibroblasts
cially cancer-associated fibroblasts (CAFs), which have
attracted attention in recent years, influences these three The discovery of fibroblasts is traced back to the observa-
significant events [5, 6]. tion of spindle-shaped cells that secrete collagen in con-
The TME comprises all cells, cytokines, and the extra- nective tissue by Virchow et al. [12] in 1858. However, the
cellular matrix (ECM) in the tumor except for tumor cells, definition of fibroblasts currently remains confusing. The
such as CAFs, vascular tissue, lymphatic tissue, nerve tis- embryonic origins of fibroblasts are the primitive mes-
sue, and factors secreted by these tissue cells [7–9]. CAFs enchyme (major) and neural crest (minor), which are also
are the main components of the TME and can reshape the embryonic origins of other mesenchymal lineages, such
ECM to exert a critical effect on the interaction between as osteoblasts, adipocytes and chondrocytes [13, 14]. Due
tumor cells and surrounding cells [10]. CAFs associated to the lack of specific markers identified in fibroblasts,
with the clinicopathological characteristics of tumors play we must consider the cell morphology, tissue position and
a major role in tumor pathogenesis [11]. The characteristics the existence of markers of leukocytes, epithelial cells, and
of CAFs and their functions in breast cancer have gradually endothelial cells when defining fibroblasts [5].
HU et al. 403

2.2 CAFs different biomarkers. Different biomarker expression pat-

terns have been identified in CAFs, and CAFs have been
Fibroblasts are activated during inflammation and fibro- divided into diverse subgroups.
sis in tumors and are thus called “cancer-associated fibrob-
lasts” [15]. Once activated, these CAFs interact with tumor
cells continuously, promoting the development of each 3.1 The heterogeneity of CAFs in
other and ultimately leading to tumor progression [16]. different molecular subtypes of breast
Moreover, the activation of certain signaling pathways sub- cancer
sequently activates nearby fibroblasts and promotes their
recruitment and proliferation [17], thus accelerating the According to the expression levels of ERα, PR and HER2,
progression of tumors, including breast cancer, through a breast cancer is divided into luminal A, luminal B, HER2-
positive feedback mechanism [18]. positive, and triple-negative subtypes, which have diverse
The most unique function of CAFs lies in their ability prognoses. Similarly, CAFs in different molecular subtypes
to synthesize and reshape the ECM. This process is called of breast cancers exhibit different expression levels of var-
the “desmoplastic reaction”, during which CAFs synthe- ious molecules and biological behaviors. In 2012, Tchou
size and secrete large amounts of type I, III, IV and V colla- et al. [33] analyzed the gene expression profiles of CAFs
gen, fibrinolytic protein, hyaluronic acid, and laminin [19, from different breast cancer subtypes and found that CAFs
20]. At the same time, they degrade the nearby ECM by from HER2-positive breast cancer were significantly differ-
secreting proteases, including matrix metalloproteinases ent from those present in triple-negative and ER-positive
(MMPs) and urokinase-type plasminogen activator [21, 22], breast cancers, especially genes related to cytoskeleton and
thus remodeling the local TME by promoting tissue hard- integrin signaling, which contribute to increased migra-
ening and stromal cell fibrosis [23]. The formed scaffold tion and an unfavorable prognosis of HER2-positive breast
structure not only prevents the entry of immune cells and cancer.
drugs (thus causing tumor immune evasion and drug resis- Although the effects of CAFs on luminal breast cancer
tance) [24] but also provides a suitable environment for the cells have been proven by many studies [34–36] and CAFs
interaction between tumor cells and cytokines, increasing in luminal breast cancer exhibit some unique characteris-
the migration, invasion and other malignant behaviors of tics [37, 38], few studies have reported the effect of estro-
cancer cells [10, 25]. Therefore, CAFs are intimately asso- gen on CAFs. Intriguingly, a transcriptionally incompe-
ciated with the progression of cancers and the prognosis of tent androgen receptor has been reported to be expressed
patients [26–28]. on prostate CAFs. Upon androgen stimulation, the recep-
tor colocalized with the scaffold protein filamin A in the
2.3 Myofibroblasts extranuclear compartment of fibroblasts, mediating their
migration and invasiveness [39]. This process was inter-
Myofibroblasts are fibroblasts that are activated under rupted by an androgen receptor-derived stapled peptide
conditions of inflammation and are characterized by the [39]. Since estrogen plays an important role in the develop-
expression of alpha smooth muscle actin (α-SMA) [23]. ment of breast cancer, studies exploring the effect of estro-
Myofibroblasts are known to remodel the ECM by pro- gen on CAFs in breast cancer are valuable.
ducing fibrogenic factors, ECM proteins, MMPs and tissue
inhibitors of metalloproteinases (TIMPs) and regulate the
functions of surrounding cells by secreting mediators dur- 3.2 The heterogeneity of CAFs in
ing the processes of wound healing and fibrosis[23, 29]. It different pathological subtypes of breast
is no wonder that myofibroblasts and CAFs have similar cancer
phenotypes, since cancers are described as wounds that do
not heal [30, 31]. As a result, CAFs are regarded as activated In 2016, Park et al. [40] compared the expression of
myofibroblasts in tumors[32]. CAF-related proteins between invasive lobular carcinoma
(ILC) and invasive ductal carcinoma (IDC). They observed
high expression levels of prolyl 4-hydroxylase, platelet-
3 HETEROGENEITY OF CAFS IN derived growth factor receptor alpha (PDGFRα), and chon-
BREAST CANCER droitin sulfate proteoglycan (NG2) in stromal cells of IDC
and overexpression of fibroblast activation protein alpha
CAFs exhibit significant heterogeneity in cancers, includ- (FAP), fibroblast-specific protein-1 (FSP1), and platelet-
ing breast cancer [5]. Due to the lack of perfect biomarkers derived growth factor receptor beta (PDGFRβ) in ILC. In
for CAFs, CAFs are assessed by detecting a combination of ILC, stromal PDGFRα positivity was related to lymph node
404 HU et al.

metastasis. Stromal podoplanin (PDPN) positivity (P = 3.4 The ratio of different subtypes of
0.041) and stromal FSP1 negativity (P = 0.041) were related CAFs in breast cancer varies as the tumor
to shorter disease-free survival (DFS). progresses
Park et al. [38] also compared the expression of CAF-
related proteins in invasive breast cancer with differ- A team conducted a series of studies to explore the CAF-
ences in stromal histology. High levels of PDPN, prolyl 4- related protein expression pattern in breast DCIS, inva-
hydroxylase, FSP1, PDGFRα, and PDGFRβ were observed sive breast cancer, and metastatic breast cancer [37, 38,
in the desmoplastic type, whereas low expression of FAP, 45]. In DCIS, the expression levels of all CAF-related pro-
PDGFα, PDGFβ, and NG2 was observed in the sclerotic teins including FSP1, PDPN, P4HA3, NG2 and PDGFRα
type. Upregulated FAP and NG2 expression and downreg- in stromal cells were higher in the HER2-positive and
ulated PDPN expression were detected in the inflamma- triple-negative breast cancer (TNBC) subtypes than in
tory type, while downregulated prolyl 4-hydroxylase and the luminal subtypes [37]. In invasive breast cancer, FAP,
FSP1 expression were detected in the normal-like type. PDGFα, PDGFβ and NG2 were downregulated in luminal
Regarding ductal carcinoma in situ (DCIS), all 7 CAF- A breast cancer, and PDPN, P4HA3 and FSP1 were down-
related proteins FSP1, PDPN, prolyl 4-hydroxylase subunit regulated in TNBC [38]. In metastatic breast cancer, stro-
alpha 3 (P4HA3), NG2, FAP, PDGFRα and PDGFRβ were mal PDPN, FSP1 and PDGFRα were significantly upreg-
upregulated in the inflammatory stromal type compared ulated in bone metastasis; stromal PDGFRβ expression
with other stromal types [37]. was significantly elevated in lung metastasis; and stromal
FSP1 and PDGFRα levels were decreased in liver metasta-
sis [45]. Based on these results, the expression pattern of
3.3 The heterogeneity of CAFs with biomarkers in CAFs is dynamic during the progression of
different origins and intratumor spatial breast cancer, and this conclusion was verified by Fried-
distributions in breast cancer man et al. [46]. They isolated CAFs from a mouse model of
breast cancer at several time points during breast tumor
Single-cell RNA sequencing (scRNA-seq) is an excellent progression and analyzed their transcriptional profiles
tool to analyze transcriptomic profile of individual cells in using single-cell sorting. The CAFs were subgrouped into
breast cancer [41], helping us understand the heterogene- PDPN-positive CAFs (pCAFs) and S100A4-positive CAFs
ity of CAFs. In 2018, Bartoschek et al. [42] isolated mes- (sCAFs). The transcriptional programs of these subpopu-
enchymal cells from a mouse model of breast cancer and lations varied as tumors progressed, converting from an
performed a scRNA-seq analysis of the transcriptomes of immunoregulatory mode to wound-healing and antigen-
768 cells. Three different subpopulations of CAFs (vascular presentation modes, which indicated that CAFs and their
CAFs, matrix CAFs and developmental CAFs) were iden- behaviors were dynamic. In addition, they found that the
tified according to the significantly differentially expressed ratio of sCAFs and pCAFs was related to the outcome of
genes. Interestingly, the authors found that these subpop- patients with breast cancer regardless of subtype and was
ulations of CAFs were spatially and functionally distinct associated with BRCA mutations in TNBC. Moreover, the
and assumed that they had different origins. Matrix CAFs dynamic ratio of subpopulations of CAFs in breast cancer
(mCAFs) were characterized by high transcriptional levels tissue was also proven by Bartoschek et al. [42].
of ECM components and ECM-related genes. Due to their
distribution in the invasive front of tumors, the authors
regarded them as related to tumor invasion. This assump- 3.5 Different subtypes of CAFs
tion was supported by another study conducted by Jung- cooperate to promote cancer progression
wirth et al. [43]. They proved that Endo180 (MRC2) was
essential for CAFs to promote breast cancer metastasis, Costa et al. [47] grouped CAFs into four subtypes accord-
and Endo180-positive CAFs substantially overlapped with ing to the expression of CD29, FAP, α-SMA, FSP1, PDGFRβ
mCAFs in the study by Bartoschek et al. [42]. and caveolin-1 (Cav-1). Among the four subtypes, S1 sub-
In 2020, Sebastian et al. [44] conducted a single-cell type CAFs (CD29Med FAPHigh FSP1Low-High α-SMAHigh
transcriptomic analysis of CAFs from BALB/c-derived 4T1 PDGFRβMed-High Cav-1Low ) recruited CD4-positive and
mammary tumors. Six CAF subpopulations were identi- CD25-positive T cells, constructing an immunosuppressive
fied, among which “myofibroblastic CAFs”, “inflamma- microenvironment. In another study, Bonneau et al. [48]
tory’ CAFs” and “MHC class II-expressing CAF” subpopu- proved that S1 subtype CAFs contributed to distant recur-
lations also existed in pancreatic cancer, and the latter two rence in early luminal breast cancer. Pelon et al. [49] iso-
subpopulations also existed in normal breast/pancreatic lated CAFs from breast cancer metastatic lymph nodes
tissue. and divided them into 4 groups based on the expression
HU et al. 405

of FAP, PDPN, α-SMA and PDGFRβ. The S1 (FAPHigh all survival (OS) and relapse-free survival (RFS) [65, 66].
CD29Med-High α-SMAHigh PDPNHigh PDGFRβHigh ) and S4 α-SMA-positive CAFs promoted tumor progression by pro-
(FAPLow-Med CD29High α-SMAHigh PDPNLow PDGFRβMed ) ducing lactate and pyruvate during metabolism, providing
subtypes were enriched in lymph nodes and associated cancer cells with nutrients [67].
with the invasion of breast cancer cells. S1 CAFs pro-
moted the migration and epithelial-mesenchymal tran-
sition (EMT) of breast cancer cells via C-X-C motif 4.2 Vimentin
chemokine ligand 12 (CXCL12) and transforming growth
factor beta (TGF-β) signaling, and the S4 subtype enhanced Vimentin, a type III intermediate filament protein, is
the invasion of cancer cells via NOTCH signaling. Thus, often used as a marker of the maintenance of the cellu-
different CAF subtypes drive breast cancer metastasis lar structure and motility during cell migration [68], and
through complementary mechanisms. it is often expressed at high levels in CAFs and asso-
In conclusion, CAFs are induced to differentiate into dif- ciated with the migration and invasion potential [69].
ferent subtypes by different stimuli. Different spatial distri- Higher vimentin expression in the stromal compartment
butions and different disease phases also result in hetero- was related to higher malignant potential of the tumor
geneity of CAFs. The “CAF subtype” is presumed to be a and predicted shorter survival of patients with colorectal
status rather than a fixed category of CAFs. cancer [70] and pancreatic ductal adenocarcinoma [71].
However, vimentin is widely expressed by NFs, cells of
mesenchymal origin (such as adipocytes and myocytes)
4 BIOMARKERS OF CAFS
and epithelial cells (including cancer cells) undergoing the
EMT [51].
Studies have revealed that CAFs differ from normal fibrob-
lasts (NFs) in biological behavior, function and the expres-
sion levels of certain proteins. These proteins (Table 1) may
4.3 FSP1
thus be used as biomarkers to distinguish CAFs from NFs.
However, due to the high heterogeneity of CAFs, these so-
FSP1, also named S100 calcium-binding protein A4
called markers are somewhat deficient in specificity and
(S100A4), is a common marker of CAFs [72, 73]. The
sensitivity [50, 51]. Some common CAF markers are briefly
biological functions of FSP1-positive CAFs are controver-
summarized below, with others concluded in Table 1 [11,
sial. On the one hand, FSP1-positive CAFs were related to
52-57].
lymphovascular invasion and the presence of tumor bud-
ding in colorectal cancer [74]. Stromal FSP1 expression
4.1 α-SMA was related to the expression of E-cadherin and Zeb1 in
tumor cells and was also associated with tumor metasta-
α-SMA is a skeletal protein expressed in cells and has sis in urothelial carcinoma [75]. On the other hand, FSP1-
been applied in many studies as a marker of activated positive fibroblasts contributed to the immune surveil-
fibroblasts. As one of the earliest discovered and most lance capacity of the body by producing collagen and
extensively used biomarkers of CAFs, α-SMA is associated engulfing carcinogens [76].
with TGF-β production and a highly contractile pheno- In breast cancer, FSP1-positive CAFs increased tumor
type [58, 59]. Previous studies have reported a role for α- metastasis by secreting vascular endothelial growth factor
SMA-positive CAFs as key regulators of cancer progres- (VEGF)-A and tenascin-C [52], and their expression was
sion, therapeutic resistance and immune suppression [15, higher in the stromal cells of ILC than in those of invasive
60-62]. However, in mouse models, the ablation of α-SMA- carcinoma of no special type (NST) [40]. A high ratio of
positive CAFs accelerated pancreatic ductal carcinoma, FSP1-positive CAFs and PDPN-positive CAFs was related
and a lower level of myofibroblasts in tumors was also asso- to prolonged RFS and OS of patients with breast cancer
ciated with reduced survival of patients with pancreatic [46]. Furthermore, FSP1 is also expressed in breast can-
ductal adenocarcinoma [63]. In addition, in patients with cer cells, and its expression in breast cancer cells was also
pancreatic ductal carcinoma and lung cancer, high α-SMA higher in ILC than in NST [40].
expression predicted a good prognosis [64]. Therefore, the
effect of α-SMA-positive CAFs on the malignant pheno-
type of tumors requires further study. 4.4 FAP
In breast cancer, the proportion of α-SMA-positive
myofibroblasts was positively correlated with the prolifer- FAP is another widely distributed biomarker of CAFs and
ation of tumor cells and negatively correlated with over- a serine protease that is involved in the remodeling of
406 HU et al.

TA B L E 1 Biomarkers of CAFs
CAF Expression level in Other cell types expressing this
marker Description CAFs marker References
Vimentin Type III intermediate Upregulated Endothelial and epithelial cells [32, 68]
filament undergoing the EMT, and
neurons
α-SMA Associated with cell Upregulated Normal fibroblasts, pericytes, [19, 359, 360]
contraction, (downregulated in smooth muscle cells and
movement, structure prostate cancer) cardiomyocytes
and integrity
FSP1 Related to cell Upregulated Normal fibroblasts, epithelial cells [40, 76, 201,
movement, collagen (downregulated in undergoing the EMT, 360, 361]
induction and tissue prostate cancer) macrophages and tumor cells
fibrosis
FAP Related to fiber Upregulated Reactive stromal fibroblasts, [77, 362, 363]
production and ECM resting mesoderm cells and
remodeling CD45-positive immune cells
Tenascin-C ECM glycoprotein that Upregulated Tumor cells [52, 53]
is related to cell
adhesion
Desmin Type III intermediate Downregulated Skin fibroblasts, pericytes and [54, 55]
filament myocytes
PDGFRα Tyrosine kinase receptor Upregulated Normal fibroblasts, pericytes, [54, 98, 100,
vascular smooth muscle cells, 364]
skeletal muscle cells,
cardiomyocytes and tumor cells
PDGFRβ Tyrosine kinase receptor Upregulated Normal fibroblasts, pericytes, [54, 98, 100,
vascular smooth muscle cells, 364]
skeletal muscle cells,
cardiomyocytes and tumor cells
Caveolin-1 Scaffold protein in the Upregulated or Adipocytes, endothelial cells, [111, 117, 265,
caveolae membrane downregulated normal fibroblasts, type I 365]
alveolar cells and tumor cells
CD10 Metalloproteinase Upregulated Bone marrow stromal cells, [11, 56]
especially pre-B lymphocytes
GPR77 Associated with Upregulated Polynuclear neutrophilic [11, 57]
complement leukocytes
activation and
pro-inflammatory
signaling pathways
Podoplanin Type-I integral Upregulated Tumor cells [125, 126, 129]
membrane
glycoprotein
Abbreviations: CAF, cancer-associated fibroblast; α-SMA, alpha smooth muscle actin; FSP1: fibroblast-specific protein-1; EMT, epithelial-mesenchymal transition;
FAP, fibroblast activation protein alpha; ECM, extracellular matrix; PDGFRα, platelet-derived growth factor receptor alpha; PDGFRβ, platelet-derived growth
factor receptor beta; GPR77, G protein-coupled receptor 77.

the ECM and fibrosis, thus accelerating tumor progression ing FAP such as gene knockout [81], small-molecule agents
[77]. FAP-positive CAFs helped build an immunosuppres- (PT630 and PT-100) [82, 83], monoclonal antibody (mAb)
sive TME through diverse mechanisms [78, 79]. A study of FAP5-DM1 [84], diphtheria toxins [85], alpha FAP-PE38
ovarian cancer revealed that CAFs expressing high levels [86] and immunotherapy targeting FAP, including DNA
of FAP were correlated with a poor prognosis for patients vaccines [87], chimeric antigen receptor (CAR)-T cells [88,
[80]. 89], and adenoviruses [90, 91], have been proven to be
FAP has been regarded as one of the most promising effective in preclinical studies [50]. Although FAP-targeted
therapeutic targets for CAFs. Therapeutic strategies target- treatment failed to show significant efficacy in clinical tri-
HU et al. 407

als [92–95], FAP remains one of the most potential thera- tumor-promoting factors released by fibroblasts, promot-
peutic targets in CAFs and requires further study. ing the proliferation and migration of tumor cells [110].
In breast cancer, FAP-positive CAFs mediated Treg Cav-1 was also essential for fibroblast-mediated microen-
activation and exerted immunosuppressive activity in a vironmental remodeling [111].
dipeptidyl peptidase (DPP) 4-dependent manner that was In breast cancer, Cav-1 expression was downregulated
related to a poor outcome [47]. Unexpectedly, a study also in CAFs [106], and its expression was positively correlated
indicated a positive relationship between abundant FAP with the patient prognosis [112]. However, dissenting
expression and longer OS and DFS of patients with IDC studies also exist. Goetz et al. [111] reported that the
[96]. expression of Cav-1 in breast cancer was negatively cor-
related with the prognosis and that Cav-1 knockout led
to a decrease in the contractility of fibroblasts. Moreover,
4.5 PDGFRα and PDGFRβ CAFs in metastatic axillary lymph nodes exhibited higher
Cav-1 expression than those in normal/reactive axillary
PDGFRα and PDGFRβ levels are increased in the stroma lymph nodes [113], implying a role for Cav-1 in breast
of many types of tumors [97], and these proteins partici- cancer metastasis. Importantly, Cav-1 is presumed to be
pate in fibroblast activation and transformation [98, 99]. associated with the “reverse Warburg effect” in CAFs,
PDGFRα/β-positive CAFs induced the migration and M2 which promotes the malignancy of cancer cells (see
polarization of macrophage, thus modulating the immune detailed content in 6.1.3) [114–116]. Notably, Cav-1 is also
microenvironment [15]. Inhibition of PDGFR signaling expressed in breast cancer cells and plays a multifaceted
transformed CAFs into resting fibroblasts and inhibited role [117].
angiogenesis and tumor growth [100, 101], suggesting that
approaches targeting PDGFR pathways may be a poten-
tially effective tumor treatment strategy. Notably, PDGFRα 4.7 PDPN
and PDGFRβ are widely expressed in fibroblasts and do
not show specific upregulation in CAF populations [51]. Studies have revealed that PDPN expression in CAFs pre-
PDGFRs are also expressed in multiple types of cancer cells dicts poor prognosis for patients with multiple types of
[98]. solid tumors, including lung cancer [118–121], cholangio-
In breast cancer, the expression of PDGFRβ in stromal carcinoma [120], breast cancer [120] and pancreatic can-
cells was positively correlated with the histopathological cer [120], and is associated with higher numbers of sin-
grade and HER2 expression, but negatively correlated with gle nucleotide variants in lung adenocarcinoma cells [122].
estrogen receptor (ER) expression [102, 103], reducing the PDPN expression in CAFs also enhanced tumor progres-
efficacy of tamoxifen [104]. The expression of PDGFRβ in sion in IDC of the pancreas [123]. On the other hand,
stromal cells was also negatively correlated with the radio- PDPN-positive CAFs suppressed the growth of small cell
therapy benefit, RFS and breast cancer-specific survival lung cancer cells [124].
[102, 103, 105]. Moreover, stromal PDGFRβ expression had In breast cancer, PDPN expression in CAFs was asso-
a better prognostic value among young and premenopausal ciated with a higher histological grade and negatively
patients with breast cancer [102, 103]. correlated with the ER status, DFS and OS [120, 125,
126]. Interestingly, Yamaguchi et al. [127] dichotomized
patients with invasive breast cancer into PDPN-positive
4.6 Caveolin-1 and PDPN-negative groups according to the existence of
PDPN-positive CAFs and reported its relationship with
Caveolins (Cavs), including Cav-1, Cav-2, and Cav-3, are magnetic resonance imaging findings. Invasive breast can-
the main structural proteins that envelop the caveolae cer with PDPN-positive CAFs tended to have a more malig-
membrane, with diameters ranging from 50 to 100 nm nant pathological status. The PDPN-positive group had
[106]. The effect of Cav-1 on the phenotypes of fibrob- a notably higher lesion-to-muscle ratio in the short-tau
lasts is controversial. On the one hand, in NIH-3T3 fibrob- inversion-recovery images. Moreover, the washout pattern
lasts, the activation of cancer genes such as H-Ras (G12V), rate was significantly higher in the PDPN-positive group in
Bcr-Abl and v-Abl caused a significant decrease in Cav- a dynamic analysis. Last, the lesions of the PDPN-positive
1 protein levels [107, 108], and the Cav-1 expression level group tended to show a circumscribed margin and a rim
was associated with a weaker ability of fibroblasts to grow enhancement. PDPN-positive CAFs are associated with
independently in agar [108]. Cav-1 knockdown in NIH-3T3 the development and metastasis of breast cancer. PDPN
fibroblasts promoted cell growth [109]. On the other hand, promoted the migration of fibroblasts and accelerated the
Cav-1 expression increased the levels of inflammatory and formation of pseudotubes by endothelial cells in breast
408 HU et al.

F I G U R E 1 Origins of cancer-associated fibroblasts in breast cancer. (A) Resident fibroblasts are transformed into α-SMA+ CAFs by
TGF-β, CXCL12, Wnt7a, miR-125b, miR-9 and chemotherapy; TGF-β and PDGF transform fibroblasts into FSP1+ CAF; miR-146a and miR-222
induce resident fibroblasts to α-SMA+ /FAP+ CAFs and FSP1+ /α-SMA+ CAFs, respectively; fibroblasts are transformed into IL-1β/IL-6/IL-8
secreting CAFs by miR-370-3p; Survivin and OPN activate fibroblasts into vimentin+ /α-SMA+ CAFs and FSP1+ /α-SMA+ /FAP+ CAFs,
respectively; when cocultured with MCF-7 breast cancer cells, normal fibroblasts are transformed into α-SMA+ /calponin+ /vimentin+ CAFs.
(B) Bone marrow-derived fibroblasts are recruited to TME as α-SMA+ /PDGFRα− CAFs. (C) Mesenchymal stem cells are transformed into
α-SMA+ CAFs by tumor cell-derived exosomes and TGF-β; marrow-derived MSCs are the origin of FSP1-positive and FAP-positive CAFs;
osteopontin transforms MSCs into α-SMA+ /vimentin+ /FSP1+ CAFs. (D) Wnt3a transforms adipocytes into FSP1+ /α-SMA− /FAP− CAFs. (E)
CAFs expressing vascular regulating genes originate from pericytes. Abbreviations: α-SMA, alpha smooth muscle actin; CAF,
cancer-associated fibroblast; TGF-β, transforming growth factor beta; CXCL12, C-X-C motif chemokine ligand 12; PDGF, platelet-derived
growth factor; FSP1, fibroblast-specific protein-1; FAP, fibroblast activation protein alpha; IL, interleukin; OPN, osteopontin; TME, tumor
microenvironment

cancer, and it was expressed at higher levels in IDC than 5.1 Local resident fibroblasts
in DCIS [128]. However, as a marker of CAFs, PDPN is also
reported to be expressed in tumor cells, including breast 5.1.1 Conversion from NFs to CAFs
cancer cells [126, 129].
The most broadly accepted hypothesis is that the major-
ity of CAFs likely originate from the activation of local
5 THE ORIGINS OF CAFS IN BREAST tissue-resident fibroblasts [5]. These NFs are the primary
CANCER generators of the ECM. They are activated following tissue
damage and participate in tissue repair, during which they
Although multiple biological markers of CAFs have been produce TGF-β and acquire a highly contractile pheno-
proposed and applied in research, none of them are spe- type with increased level of α-SMA. These activated fibrob-
cific and accepted by all researchers. The dilemma of iden- lasts are termed ‘myofibroblasts’ [5]. In 1995, Ronnov-
tifying CAFs also makes it difficult to trace their origins. Jessen et al. [131] explored the origin of myofibroblasts in
Currently, CAFs are postulated to mainly originate from breast cancer and found that fibroblasts exhibited myo-
the activation of resident fibroblasts, along with alterna- genic differentiation in a graded pattern according to the
tive origins such as adipocytes and mesenchymal stem distance to tumor cells, with the nearest fibroblasts dis-
cells (MSCs) [5, 6, 130]. In breast cancer, researchers have playing the highest myogenic differentiation. This report
obtained evidence that CAFs are derived from the origins might be one of the earliest studies exploring the origin
described below (Figure 1). of CAFs in breast cancer, and these myofibroblasts are
HU et al. 409

F I G U R E 2 Molecular mechanisms of fibroblast activation. CXCL12 and TGF-β activate fibroblasts by binding to CXCR4 and TGF-β
receptors, respectively, inducing the upregulation of α-SMA and secretion of TGF-β and CXCL12. Wnt7a also activates fibroblasts by binding
to the TGF-β receptor. TGF-β and PDGF downregulate IDH3α by binding to the TGF-β receptor and PDGFR, respectively. Downregulation of
IDH3α increased the levels of effective α-KG and HIF1α, resulting in enhanced glycolysis and attenuated OXPHOS. The binding of OPN to
CD44 and integrin αvβ3 activate fibroblasts via AKT and ERK signaling pathway, respectively, which converge to upregulate α-SMA, FSP1 and
FAP, and the activated fibroblasts secrete increased CXCL1, CXCL2, COX2, IL-6, OPN and collagen. Exosomal miR-9 increases the motility of
fibroblasts and upregulate α-SMA expression. Exosomal miR-125b downregulates TP53INP1 and TP53, activating fibroblasts characterized by
upregulated α-SMA. Exosomal miR-222 reduces LBR expression and increases α-SMA and FSP1 expression. Exosomal miR-146a
downregulates TXNIP, activating the β-catenin pathway and upregulating α-SMA and FAP expression. MiR-370-3p from exosomes
downregulates CYLD and upregulates NFκB, increasing the motility and paracrine signaling of IL-1β, IL-6 and IL-8 in fibroblasts. Exosomal
survivin increases the motility of fibroblasts and upregulates the levels of α-SMA and vimentin. Abbreviations: TGF-β, transforming growth
factor beta; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C motif chemokine receptor 4; PDGF, platelet-derived growth factor;
PDGFR, platelet-derived growth factor receptor; IDH3α, isocitrate dehydrogenase 3α; α-KG, α-ketoglutarate; HIF1α, hypoxia inducible factor 1
subunit alpha; OXPHOS, oxidative phosphorylation; OPN, osteopontin; AKT, AKT serine/threonine kinase; ERK, mitogen-activated protein
kinase 1; α-SMA, alpha smooth muscle actin; FSP1, fibroblast-specific protein-1; FAP, fibroblast activation protein alpha; COX2,
cyclooxygenase-2; IL, interleukin; OPN, osteopontin; TP53INP1, tumor protein p53 inducible nuclear protein 1; LBR, lamin B receptor; TXNIP,
thioredoxin interacting protein; CYLD, cylindromatosis; NFκB, nuclear factor kappa B subunit 1

currently termed ‘myoCAFs’ [5]. Various mechanisms by rylation and aerobic glycolysis in fibroblasts. This switch
which fibroblasts in breast cancer convert into CAFs have was activated by TGF-β and PDGF, transforming fibrob-
been proposed (Figure 2). lasts into CAFs. As shown by Sharon et al. [134], osteo-
pontin secreted by breast cancer cells bound to CD44 and
Growth factors, cytokines and other ligands αvβ3 integrin on fibroblasts and reprogrammed them into
In 2010, Kojima et al. [132] showed that autocrine TGF- a proinflammatory state. Butti et al. [135] also reported that
β and CXCL12 (also named stromal cell-derived factor- osteopontin derived from breast cancer cells differentiated
1, SDF-1) signaling converted normal mammary fibrob- NFs into myofibroblasts. They further demonstrated that
lasts into CAFs. According to Zhang et al. [133], isocitrate the binding of osteopontin to CD44 and αvβ3 activated
dehydrogenase 3α (IDH3α) was crucially responsible for Akt and extracellular signal-regulated kinase (ERK) path-
switching the metabolic mode between oxidative phospho- ways and induced the upregulation of Twist 1-dependent
410 HU et al.

genes. The activated myofibroblasts secreted CXCL12 into In conclusion, resident fibroblasts in breast cancer are
the TME, promoting the EMT of surrounding breast can- continually stimulated by diverse factors in the TME and
cer cells. Studies have also revealed a vital role for breast gradually acquire a CAF phenotype (Figure 2), promoting
cancer cell-derived Wnt7a in fibroblast activation by poten- the progression of breast cancer through different mecha-
tiating TGF-β receptor signaling rather than classical Wnt nisms (see below). Notably, NFs were also proven to par-
signaling [136]. ticipate in constructing an IL-1β-enriched microenviron-
ment by interacting with ER-positive breast cancer cells via
Exosomal miRNAs and proteins a paracrine mechanism, suggesting the potential ability of
Breast cancer cell-derived exosomes transport miRNAs to NFs in tumor-adjacent breast tissue to cause tumor recur-
NFs, transforming them into CAFs. In 2016, Baroni et al. rence [145].
[137] documented that exosomal miR-9 from breast cancer
cells induced CAF-like properties in human breast fibrob-
lasts. In 2019, Vu et al. [138] reported that miR-125b from 5.1.2 Differences between NFs and CAFs
breast cancer cells promoted the transformation of NFs to
CAFs. In the same year, Chatterjee et al. [139] reported the CAFs have more malignant phenotypes than NFs, such as
upregulation of miR-222 in CAFs compared with NFs, and increased proliferation [146–149], migration/invasion [146,
miR-222 overexpression was sufficient to induce CAF-like 147, 150], tumorigenicity [151] and chemoresistance [152].
profiles in NFs. In 2020, Yang et al. [140] proved that exo- Studies have been conducted to determine the differen-
somal miR-146a from breast cancer cells accelerated the tially expressed genes between NFs and CAFs in breast
transformation of NFs to CAFs by targeting thioredoxin- cancer. In 2010, six pairs of CAFs and NFs were iso-
interacting protein (TXNIP). In the 2021 study by Ren lated from patients with primary breast cancer by Bauer
et al. [141], breast cancer cell-derived miR-370-3p activated et al. [153], and gene expression profiles were analyzed
fibroblasts, which increased the stemness, migration and with Affymetrix Human Genome U133 Plus 2.0 arrays.
invasion of cancer cells. Twenty-one genes related to paracrine or intracellular sig-
Breast cancer cells also release protein-containing exo- naling, transcriptional regulation, ECM and cell adhe-
somes to activate fibroblasts. In 2020, Li et al. [142] reported sion/migration were upregulated in CAFs, while 10 genes
that survivin from breast cancer cells was delivered to sur- related to steroid hormone metabolism, polycyclic aro-
rounding fibroblasts via exosomes and transformed them matic hydrocarbon detoxification, transcription, migration
into CAFs by upregulating superoxide dismutase 1 (SOD1) or cell signaling were downregulated.
expression. In 2012, Zhao et al. [154] compared miRNA expression
levels in 6 pairs of NFs and CAFs from patients with breast
Unknown mechanisms cancer using miRNA microarrays. miR-221-5p, miR-31-3p,
In 2010, Martinez-Outschoorn et al. [143] reported that and miR-221-3p were upregulated in CAFs, while miR-
coculturing with MCF-7 breast cancer cells downregulated 205, miR-200b, miR-200c, miR-141, miR-101, miR-342-3p,
Cav-1, upregulated myofibroblast markers and ECM pro- let-7g and miR-26b were downregulated. Target genes of
teins, and activated TGF-β/Smad2 signaling in fibroblasts, these dysregulated miRNAs are associated with cell prolif-
which are features of the CAF phenotype. They claimed eration, differentiation, adhesion, migration, secretion and
that the autophagic/lysosomal degradation of Cav-1 was cell–cell interaction.
the key initiator of the reversion, but they did not explain In 2013, Peng et al. [147] analyzed the gene expres-
what mechanism caused the autophagic degradation. As sion profiles of human breast CAFs and paired NFs
shown by Peiris-Pagès et al. [144], chemotherapy upreg- with microarrays. A total of 809 upregulated genes and
ulated the expression of α-SMA in fibroblasts and pro- 15 downregulated genes were detected in CAFs. C-C
moted interleukin (IL)-6 secretion from fibroblasts. Addi- motif chemokine ligand (CCL) 18, CXCL12, cell divi-
tionally, chemotherapy also transformed the metabolism sion cycle 6 homolog (CDC6), cyclin-dependent kinase 1
of fibroblasts into a highly glycolytic and inactive mode, in (CDK1), friend leukemia virus integration 1 (FLI1), MMP-
which the fibroblasts produced excess lactate and released 9, platelet/endothelial cell adhesion molecule (PECAM1),
it into the microenvironment, but the mechanism remains polo-like kinase 1 (PLK1) and S100 calcium binding
unknown. In 2018, Bartoschek et al. [42] identified a sub- protein A9 (S100A9) were significantly upregulated in
group of CAFs in breast cancer with a similar marker CAFs, whereas chromosome 9 open reading frame 135
expression pattern to the dominant fibroblast population (C9ORF135) and SHC-transforming protein 2 (SHC2) were
in the normal mammary gland; thus, they proposed that downregulated. Most genes that were upregulated in
this subgroup of CAFs may originate from resident fibrob- CAFs are related to the cell cycle, adhesion and secreted
lasts domesticated by cancer cells. factors.
HU et al. 411

5.2 Bone marrow-derived fibroblasts regulating genes and located close to the vasculature. They
concluded that this subgroup of CAFs originated from a
In 2018, Raz et al. [155] reported that bone marrow-derived pool of perivascular cells.
fibroblasts converted to CAFs in breast cancer and pro-
moted tumor growth and angiogenesis by upregulating
clusterin. Bone marrow-derived CAFs did not express 6 EFFECTS AND MECHANISMS OF
PDGFRα, in contrast to resident CAFs. The recruitment CAFS ON BREAST CANCER
of bone marrow-derived CAFs decreased the percentage
of PDGFRα-positive CAFs in breast cancer tissues, and a CAFs have been reported to promote proliferation [162,
decrease in PDGFRα expression was associated with worse 163], metastasis [164–168], stemness [169–173] and treat-
prognosis. ment resistance [174] in various types of cancer. As
important members of the TME, CAFs also regulate the
metabolism of cancer cells [175–178] and suppress the func-
5.3 Mesenchymal stem cells tions of immune cells to promote cancer progression [179,
180].
In 2007, Karnoub et al. [56] injected bone marrow-derived Similarly, CAFs promote the proliferation [181], migra-
MSCs into breast cancer-bearing mice and found that they tion [49], invasion [182–184], stemness [185] and treatment
became CAFs and promoted breast cancer metastasis by resistance [186] of breast cancer cells and contribute to the
inducing paracrine signaling of the chemokine CCL5. In reconstruction of the ECM [160] and immune microenvi-
2011, Jotzu et al. [156] proved that TGF-β from breast can- ronment [187]. Here, we summarize the studies assessing
cer cells induced human adipose tissue-derived stem cells the effect of CAFs on breast cancer and the mechanisms
to differentiate into a CAF-like myofibroblastic pheno- by which CAFs function (Figure 3).
type by activating the mothers against decapentaplegic
homolog 3 (SMAD3) pathway. In 2012, Kidd et al. [157]
identified the origin of the majority of FSP1-positive and 6.1 Strengthening malignancy of cancer
FAP-positive CAFs as marrow-derived MSCs. In the same cells
year, Cho et al. [158] found that breast cancer-derived exo-
somes activated SMAD signaling and induced the conver- 6.1.1 Secreting factors
sion of adipose tissue-derived MSCs to CAFs by upregulat-
ing α-SMA, CXCL12, VEGF, CCL5 and TGF-β expression. Growth factors
In 2015, Weber et al. [159] proved that osteopontin derived TGF-β from CAFs promoted the EMT and increases
from breast cancer cells engendered MSC-CAF transfor- the expression of fibronectin, vimentin, MMP-2, MMP-
mation, as characterized by the upregulation of α-SMA, 9, SNAIL and TWIST in surrounding breast cancer cells,
vimentin, tenascin-C, FSP1 and TGF-β. increasing their motility [49, 188-190]. TGF-β also pro-
moted the drug resistance and stemness of breast cancer
cells by increasing the expression of the long non-coding
5.4 Adipocytes RNA (lncRNA) HOX transcript antisense RNA (HOTAIR),
which led to the silencing of tumor suppressor genes in
In 2012, Bochet et al. [160] found that Wnt3a secreted by breast cancer [185].
tumor cells converted adipocytes to CAFs in breast cancer Hepatocyte growth factor (HGF) secreted by CAFs
by activating the Wnt/β-catenin pathway, and these CAFs was reported to enhance breast tumorigenesis in mice
were characterized by increased expression of FSP-1 but and cancer cell colony formation in vitro [151]. CAFs
not α-SMA. stimulated with PDGF-CC released HGF, insulin growth
factor-binding protein 3 (IGFBP3) and stanniocalcin1
(STC1), which suppressed the luminal phenotype and
5.5 Pericytes maintain the triple-negative status of breast cancer
cells [191].
In 2016, Hosaka et al. [161] found that pericytes were con- Fibroblast growth factor 5 (FGF5) secreted by activated
verted into fibroblasts by activating PDGF-BB-PDGFRβ CAFs provided a supportive niche for cancer cells to
signaling, promoting thyroid cancer invasion and metas- acquire a chemoresistant cancer stem cell (CSC) pheno-
tasis. This conclusion was subsequently supported with type [192]. FGF5 also activates HER2 via the FGF recep-
evidence from a breast cancer model by Bartoschek et al. tor (FGFR)2/c-Src/HER2 axis, leading to resistance to
[42], who isolated a subgroup of CAFs expressing vascular- HER2-targeted therapies, which was overcome by FGFR
412 HU et al.

F I G U R E 3 CAFs expressing different markers function in diverse pathways. CAFs expressing different markers affect tumor cells and
the TME by secreting factors, generating exosomes, releasing nutrients, reshaping the ECM, suppressing immune cells and promoting
angiogenesis. Abbreviations: CAF, cancer-associated fibroblast; TME, tumor microenvironment; ECM, extracellular matrix
HU et al. 413

inhibitors [186]. Resistance to lapatinib induced by CAF- promote the growth of breast cancer cells by enhancing
derived FGF was also reported by Zeryantonakis et al. glycolysis [206].
[193]. CAF-derived FGF2 enhanced the growth and migra-
tion of TNBC cells by interacting with FGFR1 [194]. Other proteins
Tenascin-C secreted by CAFs was reported to reduce the
Interleukins apoptosis of breast cancer cells, thus contributing to the
CAF-derived IL-6 has been reported to enhance the inva- formation of metastatic foci [52]. CAF-derived osteopon-
sive ability of breast cancer cells and promote the transi- tin, gremlin1, and collagen triple helix repeat containing-1
tion from in situ breast cancer to invasive breast cancer (CTHRC1) have been revealed to promote migration, inva-
[195–197]. Louault et al. [34] found that IL-6 secreted from sion and EMT in breast cancer cells [207–209].
CAFs favored MCL-1 expression and apoptotic resistance Moreover, neurotrophins are the potential proteins by
in luminal cancer cells. which CAFs act on breast cancer cells. On the one hand,
IL-6 and IL-8 secreted by CD10-positive and G protein- neurotrophins and their receptors are reported to be
coupled receptor 77 (GPR77)-positive CAFs, which were expressed in breast cancer and to affect tumor cell prolifer-
enriched in chemoresistant breast cancer tissues, pro- ation [210–212], metastasis [213], treatment resistance [214,
moted the enrichment of CSCs by constructing niches [11, 215], angiogenesis [216] and CSC self-renewal [217] through
198]. various signaling pathways [218]. Neurotrophins and their
IL-32 has been found to be abundantly expressed in receptors have been regarded as potential therapeutic tar-
CAFs, and its RGD motif specifically bound to integrin gets in breast cancer [218–220]. On the other hand, neu-
β3, which was upregulated in breast cancer cells, activat- rotrophins are also released by CAFs and exert their biolog-
ing downstream intracellular p38 mitogen-activated pro- ical functions in various types of cancer [221, 222] including
tein kinase (MAPK) signaling. This signaling increased the breast cancer [223]. Thus, a reasonable assumption is that
expression of EMT markers and promoted tumor cell inva- CAFs affect the malignancy of breast cancer cells by releas-
sion [182]. ing neurotrophins, although this mechanism has rarely
been reported in breast cancer studies.
Chemokines
When exposed to neoadjuvant chemotherapy, CAFs
secreted Glu-Leu-Arg (ELR) motif-positive chemokines, 6.1.2 Generating exosomes
which acted on breast cancer cells through C-X-C motif
chemokine receptor (CXCR) 2 to enhance their invasion Exosomes are believed to play vital roles in crosstalk
[199]. CAFs in TNBC were activated by immunosuppres- between CAFs and cancer cells by transporting various
sive S100A9-positive myeloid cells, and CCL16 secreted types of substances, including DNA, RNA, proteins, and
by activated CAFs recruited monocytes, resulting in a metabolites [224].
positive feedback loop that switched the stroma to a
reactive mode [200]. In 2005, Orimo et al. [201] observed Exosomal DNA
that stromal fibroblast-secreted CXCL12 promoted breast In 2017, Sansone et al. [225] reported that CAF-derived
cancer cell growth. Boesch et al. [202] also proved that exosomes contained whole genomic mitochondrial DNA
CAF-derived CXCL12 increased the growth, invasion (mtDNA), which overcame the deficiency of oxidative
and stemness of breast cancer cells. CXCL12 released by phosphorylation in breast cancer induced by hormone
CAFs induced the downregulation of mDIA2 expression therapy, resulting in resistance to hormone therapy.
and the degradation of F-actin, resulting in increased
tumor cell motility [203]. Ahirwar et al. [204] found that Exosomal non-coding RNAs
selective knockout of CXCL12 in CAFs suppressed the Exosomal miRNAs function as essential mediators in the
growth and metastasis of breast cancer in a mouse model. communication between CAFs and breast cancer cells. In
Mechanistically, CXCL12 increased vascular permeability 2015, Shah et al. [226] revealed that exosomal miR-221 and
and the expansion of a leaky tumor vasculature, thus miR-222 derived from CAFs in ER-negative breast cancer
promoting tumor cell invasion into vessels. Based on these downregulated ER in breast cancer cells. Two years later,
findings, CXCL12 secreted by CAFs contributed to the Sansone et al. [35] also reported that miR-221 delivered
expansion of breast cancer from the primary foci. In 2017, by CAF-derived exosomes decreased ER expression and
CCL11 and CXCL14 derived from CAFs were reported to activated the ERlow /Notchhigh feedback pathway in lumi-
promote the growth, chemoresistance and metastasis of nal breast cancer, inducing resistance to endocrine ther-
breast cancer [205]. In 2020, CCL6 and CCL12 secreted by apy and the generation of CD133high CSCs. In 2020, CD63-
focal adhesion kinase (FAK)-low CAFs were reported to positive CAF-derived exosomal miR-22 was found to tar-
414 HU et al.

get ERα and phosphatase and tensin homolog (PTEN) in (ASOs); ii) editing non-coding RNA genes by techniques
breast cancer cells, conferring tamoxifen resistance [227]. such as CRISPR-Cas9; iii) replacing non-coding RNA with
The transport of miR-21, miR-378e and miR-143 in exo- RNA mimics; iv) blocking functions of non-coding RNAs
somes from CAFs to breast cancer cells promoted the EMT with small molecules [240]. In breast cancer, siRNAs and
phenotype and stemness of breast cancer cells [228]. miR- ASOs targeting non-coding RNAs have been proven to
181d-5p-containing exosomes derived from CAFs increased suppress tumor in pre-clinical studies [241–244]. Restoring
aggressiveness by targeting the caudal-related homeobox non-coding RNAs with mimics has also been applied to
2 (CDX2)/homeobox A5 (HOXA5) axis in breast cancer treatment of breast cancer [245–248]. Small molecules
[229]. Exosomal miR-3613-3p and miR-500a-5p derived attenuating the function of miRNAs in breast cancer have
from CAFs promoted the proliferation and metastasis of been developed [249, 250]. Gene knockout of cancer-
breast cancer cells by targeting suppressor of cytokine sig- promoting non-coding RNA in breast cancer cells dimin-
naling 2 (SOCS2) [230] and ubiquitin-specific peptidase ishes their malignancy [251–254]. However, few studies
28 (USP28) [231], respectively. miR-18b and miR-1-3p were have conducted gene-editing technology on non-coding
delivered from CAFs to breast cancer cells via exosomes RNA genes in CAFs to suppress breast cancer. Although
to increase the invasion and metastasis of tumor cells by these therapeutic strategies have potential value for clini-
downregulating transcription elongation factor A like 7 cal applications, multiple shortcomings exist, such as chal-
(TCEAL7) and Krüppel-like zinc-finger protein Gli-similar lenges in delivery, the short half-life of RNA molecules,
1 (GLIS1), respectively [232, 233]. activation of innate immune response and off-target
Downregulation of certain miRNAs in CAF-derived exo- effects [240].
somes also contributes to the malignancy of breast cancer.
Liu et al. [234] compared the miRNA profiles in exosomes Exosomal proteins
from NFs and CAFs and identified 14 upregulated miRNA CD81-positive exosomes secreted by CAFs were reported
and 530 downregulated miRNAs in exosomes secreted by to increase the motility of breast cancer cells by activat-
CAFs. Exosomes lacking miR-7641 promoted the stem- ing Wnt-planar cell polarity (PCP) signaling [255]. This
ness and glycolysis of breast cancer cells by upregulating result was verified by Chen et al. [256] who further proved
hypoxia-inducible factor 1-alpha (HIF-1α). that Wnt10b delivered by CD81-positive exosomes derived
Other types of non-coding RNAs derived from CAFs also from CAFs promoted breast cancer metastasis via the
promote the malignancy of breast cancer. In 2014, Boelens Wnt/β-catenin pathway. Exosomes containing metallopro-
et al. [235] reported that RNA within CAF-derived exo- teinase ADAM10 produced by CAFs activated RhoA via
somes activated signal transducer and activator of tran- the NOTCH pathway and upregulated aldehyde dehydro-
scription 1 (STAT1)-dependent antiviral signaling by stim- genase (ALDH) expression, promoting the migration of
ulating the pattern recognition receptor retinoic acid- breast cancer cells [257]. Xi et al. [258] reported that GPR64
inducible gene 1 protein (RIG-I), increasing the resistance present in hypoxic CAF-derived exosomes increased the
of breast cancer cells to treatment. Three years later, Nabet expression of MMP-9 and IL-8 in breast cancer cells,
et al. [236] further identified that CAF-derived endoge- enhancing their invasion. The phosphorylation of BCL2
nous RNA component of signal recognition particle 7SL1 interacting protein 3 (BNIP3) by oxidized ataxia telangiec-
(RN7SL1) was delivered to breast cancer cells via exosomes, tasia mutated (ATM) regulated this process.
enhancing the growth, metastasis and therapy resistance
of cancer cells by activating RIG-I. In 2020, Li et al. [237]
reported that exosomal lncRNA small nucleolar RNA host 6.1.3 Releasing nutrients
gene 3 (SNHG3) increased the expression of pyruvate
kinase M1/M2 by functioning as an miR-330-5p sponge, The “reverse Warburg effect” describes glycolysis that
switching the metabolic mode of breast cancer cells from occurs in CAFs and produces lactate and pyruvate, which
mitochondrial oxidative phosphorylation to glycolytic car- support energy generation in cancer cells [114]. It is an
boxylation. important mechanism to promote cancer progression and
These exosomal non-coding RNAs derived from CAFs has been regarded as a vital therapeutic target [115, 259].
in breast cancer are summarized in Table 2. Martinez-Outschoorn et al. [260–262], Pavlides et al.
Since exosomal non-coding RNAs contribute sub- [263, 264] and Guido et al. [265] reported that autophagy-
stantially to breast cancer progression [238], therapeutic mediated loss of Cav-1 in CAFs led to mitochondrial dys-
strategies targeting non-coding RNAs in breast cancer function, oxidative stress and aerobic glycolysis, result-
have been developed [239]. Generally, these strategies are ing in the release of essential nutrients (including lactate,
subgrouped as follows: i) degradation by small interfering ketones, glutamine and pyruvate) and chemical substrates
RNA (siRNAs) or synthetic antisense oligonucleotides (including amino acids and nucleotides), which fueled the
HU et al. 415

TA B L E 2 Exosomal non-coding RNAs derived from CAFs

Changes in cancer cell Publication

Non-coding RNAs Mechanisms functions year Reference
miR-221 and Downregulating ER Not mentioned 2015 [226]
miR-222
miR-21, miR-378e Not mentioned Enhanced the EMT 2017 [228]
and miR-143 phenotype and stemness
RN7SL1 Activating RIG-I Increased growth, 2017 [236]
metastasis and therapy
resistance
miR-221 Activating the Resistance to endocrine 2017 [35]
ERlo /Notchhi feedback therapy and the
pathway generation of CD133hi
cancer stem cells
LncRNA SNHG3 Inhibiting miR-330-5p, Increased glycolysis 2020 [237]
upregulating pyruvate carboxylation and
kinase M1/M2 proliferation
miR-22 Downregulating ERα and Resistance to tamoxifen 2020 [227]
PTEN
miR-181d-5p Downregulating Increased proliferation and 2020 [229]
CDX2/HOXA5 decreased apoptosis
miR-3613-3p Downregulating SOCS2 Increased proliferation and 2020 [230]
metastasis
miR-500a-5p Downregulating USP28 Increased proliferation and 2021 [231]
metastasis
Lack of miR-7641 Upregulating HIF-1α Increased stemness and 2021 [234]
glycolysis
miR-18b Downregulating TCEAL7 Increased invasion and 2021 [232]
metastasis
miR-1-3p Downregulating GLIS1 Increased progression and 2021 [233]
metastasis
Abbreviations: ER, estrogen receptor; EMT, epithelial-mesenchymal transition; RN7SL1, RNA component of signal recognition particle 7SL1; RIG-I, retinoic acid-
inducible gene 1 protein; SNHG3, small nucleolar RNA host gene 3; PTEN, phosphatase and tensin homolog; CDX2, caudal-related homeobox 2; HOXA5, homeobox
A5; SOCS2, suppressor of cytokine signaling 2; USP28, ubiquitin-specific peptidase 28; HIF-1α, hypoxia-inducible factor 1-alpha; TCEAL7, transcription elongation
factor A like 7; GLIS1, Gli-similar 1.

growth of nearby breast cancer cells and increased their increased glycolysis in CAFs via ATM-induced phospho-
stemness and proliferation. According to Yu et al. [266], rylation of glucose transporter 1 (GLUT1) and upregula-
tumor cells reprogrammed CAFs into an aerobic glycolysis tion of pyruvate kinase M2 (PKM2). The CAFs that had
mode by activating the estrogen/G-protein-coupled estro- undergone metabolic reprogramming released lactate into
gen receptor (GPER)/cyclic adenosine monophosphate the microenvironment, increasing the invasion of breast
(cAMP)/protein kinase A (PKA)/cAMP response element- cancer cells by activating the TGF-β1/p38 MAPK/MMP-
binding protein (CREB) signaling pathway. These CAFs 2/-9 signaling axis and increasing mitochondrial activity.
produced pyruvate and lactate and enhanced the mito- Epigenetic modification induced the dysregulated expres-
chondrial activity of breast cancer cells, conferring resis- sion of HIF-1α and metabolic enzymes such as fructose-
tance to multiple treatments. As shown in the study by 1,6-bisphosphatase (FBP1), pyruvate kinase M (PKM) and
Yan et al. [267], exosomal miR-105 from MYC-expressing lactate dehydrogenase A (LDHA) in CAFs, leading to
breast cancer cells reprogramed the metabolism of CAFs, increased lactate, pyruvate and erythrose-4P production,
and the metabolites produced by these cells in turn fueled which fueled cancer cells and promoted tumor growth [67].
the growth of nearby cancer cells. Following starvation, Chen et al. [269] reported that breast cancer cell-derived
these CAFs converted metabolic wastes such as lactic high mobility group box-1 protein (HMGB1) triggered aer-
acid and ammonium to nutrients such as lactate, acetate obic glycolysis in CAFs, which promoted metastasis of
and glutamate. Sun et al. [268] documented that hypoxia tumor cells by releasing lactate.
416 HU et al.

6.2 Reshaping the ECM and providing CAFs build a “wall” to protect breast cancer from drugs.
“mechanical pressure” Following exposure to Taxotere treatment, CAFs were acti-
vated and upregulated the expression of MMP-1 and type
As the most abundant constituent of the TME of breast IV collagen, protecting breast cancer cells from the effects
cancer, type I collagen increases the survival and aggres- of Taxotere [299]. This physical effect may also facili-
siveness of cancer cells [270]. In breast cancer, Wnt3a tate breast cancer invasion. Karagiannis et al. [300] pro-
derived from cancer cells activated the Wnt/β-catenin posed that CAFs could promote the migration of tumor
pathway in CAFs, increasing the secretion of fibronectin cells through “mechanical pressure”. More precisely, CAFs
and type I collagen [160]. Type I collagen promoted the migrated in cohorts that exerted mechanical pressure on
secretion of MMP-9 by breast cancer cells, increasing their the cancer cells, changing the tissue-tension dynamics in
migration and invasion capabilities [271, 272]. the microenvironment. Consequently, tumor cells were
Fibronectin is also an important molecule in the forced to migrate toward relatively loose tissues [300, 301].
TME, and it exerts important effects on the proliferation,
migration, EMT and angiogenesis of breast cancer cells
[273]. Fibronectin is abundant in breast cancer, and CAFs 6.3 Suppressing immune cells
continuously secrete fibronectin upon stimulation with
TGF-β and interferon gamma (IFN-γ) released by the In a mouse breast cancer model, the elimination of CAFs
tumor [274, 275]. CAFs not only increase the expression increased IL-2 and IL-7 levels and the numbers of den-
of fibronectin but also alter its arrangement and promote dritic cells and cytotoxic T cells with antitumor effects in
the migration of tumor cells [43, 276]. In breast cancer, the TME. In addition, it decreased the IL-4, IL-6, VEGF
the expression of fibronectin was associated with shorter and colony stimulating factor 2 (CSF-2) levels and num-
patient survival [277]. Fibronectin promoted the EMT bers of tumor-promoting macrophages and immunologi-
in breast cancer cells by activating the STAT3 path- cal suppressive regulatory T cells in the TME, indicating
way[278] and calpain [279]. Inhibiting the production of the vital role of CAFs in building an immunosuppressing
fibronectin reduced the aggressiveness of breast cancer microenvironment [302].
cells [280–282]. In human breast cancer tissue, CAFs recruited mono-
Studies have shown that the hardness of the tumor cytes by secreting monocyte chemotactic protein-1 (MCP-
stroma is related to tumor growth and metastasis and 1), CXCL12, CCL2 and CCL16 [200, 303]. The differen-
the prognosis of patients [283–288]. An increase in matrix tiation of monocytes toward M2-like macrophages was
hardness reduced the level of PTEN through miR-18a, induced, and these cells could exert immunosuppressive
increasing the survival, migration and invasion ability of effects via the programmed cell death protein 1 (PD-1)
breast cancer cells [289]. An increase in collagen cross- axis [304]. Additionally, exosomes containing miRNAs
linking induced by lysyl oxidase (LOX) hardened the secreted by CAFs in breast cancer promoted apoptosis and
ECM and increased the number of focal adhesions, which impaired the proliferation of T cells via the miR-92/large
increased the migration and invasion ability of breast can- tumor suppressor homolog (LATS2)/yes associated pro-
cer cells [287]. In the PyMT mouse model, loss of interstitial tein 1 (YAP1)/programmed death ligand 1 (PD-L1) pathway
LOX reduced tumor metastasis [290]. TGF-β and miR-200 [187].
induced the expression of LOX in mesenchymal cells and
tumor cells, thereby exerting a profound effect on matrix
remodeling in the TME [290, 291]. In addition, an increase 6.4 Promoting angiogenesis
in matrix stiffness affected the phenotype and biological
behavior of CAFs and may lead to increased expression 6.4.1 VEGF-dependent mechanism
of α-SMA and enhanced proliferation and migration in
response to factors such as PDGF [292, 293]. VEGF is the most important protein that promotes angio-
In breast cancer, CAFs secrete a large amount of MMPs genesis, and CAFs are the main source of VEGF in the
into the ECM of the tumor. Studies have shown that the TME [305, 306]. De Francesco et al. [307] showed that
levels of MMP-1, MMP-7, MMP-9, MMP-11, MMP-12 and hypoxia upregulated VEGF expression in CAFs and pro-
MMP-14 in the stroma are associated with tumor progres- moted angiogenesis in breast cancer via the HIF-1α/GPER
sion and a poor prognosis [294–296]. Tumor cells secreted signaling pathway. Similar results were also reported by
TGF-β and TNF-α to promote the production of MMPs Ren et al. [308]. Al-Jomah et al. [309] observed IL-6-
from CAFs, thereby further promoting their own invasion induced VEGF-A secretion from CAFs, and this process
[297, 298]. was inhibited by the IL-6 receptor inhibitor tocilizumab.
HU et al. 417

6.4.2 VEGF-independent mechanism

CAFs also promote angiogenesis in a VEGF-independent

manner. In 2005, Orimo et al. [201] observed that stro-
mal fibroblast-secreted CXCL12 promoted angiogenesis
in breast cancer. In the 2018 study by Raz et al. [155],
CAFs in breast cancer promoted tumor angiogenesis by
upregulating clusterin. FOS-like 2 (FOSL2) released by
CAFs promoted angiogenesis in breast cancer by increas-
ing the transcription of Wnt5a to activate frizzled class
receptor 5 (FZD5)/nuclear factor kappa B subunit 1 (NF-
κB)/ERK signaling in human umbilical vein endothelial
cells (HUVECs) [310]. Additionally, Sewell-Loftin et al.
[311] proved that the mechanical force provided by CAFs
in breast cancer also contributed to the formation of the
vasculature.

6.5 Uncertain mechanisms

Choi et al. [312] mimicked the blood-brain barrier with

three-dimensional in vitro models and found that CAFs F I G U R E 4 Potential therapeutic role of fibroblasts in in vitro
increased the expression of α5β1 and α5β3 integrins, scat- studies. Tumor cell-derived Hh activates fibroblasts via the
ter factor receptor (c-MET) and α2,6-siayltransferase in PTCH/SMO pathway, which is a target of the SMO inhibitors
breast cancer cells to promote their invasion. However, Vismodegib and Sonidegib. HA secreted by CAFs reshapes the ECM
the authors did not mention how the CAFs promoted the and protects tumor cells from drugs. Hyaluronidase degrades HA,
upregulation of these proteins. Jungwirth et al. [43] proved exposing tumor cells to therapeutic drugs. CAFs promote tumor
growth by secreting PIK3Cδ, which can be inhibited by CAL-101.
the vital role of CAFs expressing Endo180 in mediating
The angiotensin inhibitor losartan suppresses tumor growth by
the metastasis of breast cancer. Endo180 affects the con-
binding to ACE2 receptor. TGF-β is a critical mediator between
tractility and viability of CAFs, but the mechanism by
CAFs and tumor cells. Binding between TGF-β and TGF-βR can be
which it contributed to the metastasis of breast cancer blocked by IN-1130, Ki26894 and YR-290. The FGF inhibitors
remains unknown. According to Amornsupak et al. [313], lucitanib, erdafitinib, AZD4547 and Futibatinib block the binding of
CAFs increased the tolerance of breast cancer cells to adri- FGF and FGFR, suppressing tumor growth and overcoming
amycin by upregulating HMGB1 in cancer cells, but the resistance to fulvestrant and CDK4/6 inhibitors. Abbreviations: Hh,
mechanism underlying the upregulation of this protein is hedgehog; SMO, smoothened, frizzled class receptor; HA,
unknown. Brechbuhl et al. [36] identified CD146-negative hyaluronan; ECM, extracellular matrix; CAF, cancer-associated
CAFs in ER-positive breast cancer and found that this sub- fibroblast; ACE2, angiotensin converting enzyme 2; TGF-β,
population of CAFs downregulated the expression of ER in transforming growth factor beta; TGF-βR, transforming growth
MCF-7 cells and confers tamoxifen resistance to cells. This factor beta receptor; FGF, fibroblast growth factor; FGFR, fibroblast
growth factor receptor; CDK, cyclin dependent kinase
effect might be mediated by conditioned media, but the
authors did not explore further mechanisms. Nguyen et al.
[314] proved that CAFs could antagonize the antibody-
dependent cell-mediated cytotoxicity effect of trastuzumab
using tumor-on-chip platforms, but they did not explore
the underlying mechanism. cells in the TME [315]. CAFs have been proven by mul-
tiple studies to promote the initiation and progression
of a various tumors [52, 63, 204, 316, 317], and their
7 THERAPEUTIC APPROACHES roles in promoting breast cancer growth, metastasis and
TARGETING CAFS IN BREAST CANCER immunosuppression have also been confirmed [52, 204,
318]. Therefore, drugs targeting CAFs are a promising strat-
Antitumor studies have long focused on treating tumor egy for breast cancer treatment (Figures 4–5). Representa-
cells. In recent years, important progress has been tive drugs potentially targeting CAFs that have been inves-
achieved in research on immune cells, CAFs and other tigated in clinical trials of breast cancer are listed in Table 3.
418 HU et al.

F I G U R E 5 CAF-targeting therapeutic strategies in breast cancer. Conversion from local resident normal fibroblasts to activated CAFs is
induced by hedgehog and TGF-β. The former is inhibited by sonidegib and vismodegib, and the latter is inhibited by fresolimumab and
M7824. Activated CAFs are targeted by the FAP vaccine, FAP-CAR-T cells and HDAC6 inhibitor ACY1215. ATRA, paricalcltol and losartan
suppress the functions of CAFs, converting them into a more quiescent status. TGF-β secreted by CAFs is neutralized by fresolimumab and
M7824, and galunisertib could inhibit the TGF-β signaling by blocking TGF-β receptor I. M7824 also inhibits PD-L1 expression on tumor cells.
Balixafortide, a CXCR4 inhibitor, suppresses the function of CXCL12 secreted by CAFs. FGF/FGFR signaling is inhibited by erdafitinib,
AZD4547, futibatinib and debio1347. Hyaluronan and fibronectin produced by CAFs are involved in ECM reconstruction and are targeted by
PEGPH20 and immunization with extra domain-A of fibronectin, respectively. Abbreviations: CAF, cancer associated fibroblasts; TGF-β,
transforming growth factor beta; FAP, fibroblast activation protein alpha; CAR, chimeric antigen receptor; HDAC6, histone deacetylase 6;
CXCR4, C-X-C motif chemokine receptor 4; CXCL12, C-X-C motif chemokine ligand 12. ECM, extracellular matrix

7.1 Value of CAFs for breast cancer tracer and therapeutic target. Loktev et al. [320] developed
diagnosis, imaging and prognosis an iodinated and 1,4,7,10-tetraazacyclododecane-1,4,7,10-
prediction tetraacetic acid (DOTA)-coupled radiotracer based on a
FAP-specific enzyme inhibitor (FAPI). In vitro and in vivo
Considering the vital role of CAFs in the development of experiments were conducted to test the performance of the
breast cancer and their intratumor specificity, researchers radiotracer. Both FAPI-01 and FAPI-02 were internalized
have tried to assess their value in the early diagnosis of into FAP-expressing cells rapidly and exhibited high affin-
breast cancer and prognostic prediction for patients with ity and specificity. In a patient with metastatic breast can-
breast cancer. Giussani et al. [319] revealed higher lev- cer, FAPI-02 accumulated in the primary tumor, metastatic
els of type IX and X collagen α1 and cartilage ligament lymph nodes and remote metastases, with low uptake in
matrix in the plasma of patients with breast cancer than normal tissues. In tumors with a large stromal compart-
those in patients with benign lesions and healthy popula- ment, the radiotracer provided fast imaging with high con-
tions. In vitro experiments have shown that the expression trast. Due to relative specificity, it is a potentially valuable
of these proteins was elevated in fibroblasts after culture tool to deliver therapeutic isotopes.
with tumor cell-conditioned medium. The authors pro- Strell et al. [321] identified a PDGFRαlow /PDGFRβhigh
posed that the expression of these proteins by fibroblasts fibroblast subset as a sign of an increased risk of recur-
might be a good biomarker for distinguishing benign and rence in patient with DCIS. This subpopulation of fibrob-
malignant neoplasms. lasts was induced by contact-dependent communication
High and specific expression of FAP in tumors sug- between epithelial cells and fibroblasts via Jagged1 and
gests that it represents a promising candidate tumor Notch2, respectively.
HU et al. 419

TA B L E 3 Drugs targeting CAF-associated signaling in breast cancer investigated in clinical trials

Trial ID and current
Target Drug Class Mechanism status
Hedgehog Vismodegib Small-molecule Preventing CAF activation NCT02694224, phase II,
inhibitor recruiting
Sonidegib Small-molecule Preventing CAF activation NCT02027376, phase I,
inhibitor completed
Hyaluronic acid PEGPH20 Hyaluronidase Interfering with NCT02753595, phase II,
CAF-mediated terminated
desmoplasia
Vitamin A ATRA Metabolite of Reprogramming CAFs NCT04113863, phase I,
metabolism vitamin A unknown
Vitamin D Paricalcitol Small-molecule Reprogramming CAFs NCT00637897, phase I,
receptor agonist completed
Angiotensin Losartan Small-molecule Reducing collagen and NCT05097248, phase II,
receptor inhibitor hyaluronan levels not yet recruiting
TGF-β Fresolimumab Monoclonal Neutralizing TGF-β NCT01401062, phase II,
antibody completed
Galunisertib Small-molecule Preventing CAF activation NCT02672475, phase I,
inhibitor and interfering with active, not recruiting
CAF-mediated signaling
M7824 Anti-PD- Preventing CAF activation NCT03524170, phase I,
L1/TGF-β and immune active, not recruiting
trap fusion suppression, interfering NCT04296942, phase I,
protein with CAF-mediated completed
signaling
NCT03579472, phase I,
recruiting
FGFR Erdafitinib Small-molecule Interfering with NCT03238196, Phase I,
inhibitor CAF-mediated signaling active, not recruiting
AZD4547 Small-molecule Interfering with NCT01202591, phase I,
inhibitor CAF-mediated signaling completed
Small-molecule Interfering with NCT01791985, phase
inhibitor CAF-mediated signaling Ib/IIa, completed
Futibatinib Small-molecule Interfering with NCT04024436, phase II,
inhibitor CAF-mediated signaling recruiting
Debio1347 Small-molecule Interfering with NCT03344536, phase II,
inhibitor CAF-mediated signaling completed
CXCR4 Balixafortide Small-molecule Interfering with NCT01837095, phase I,
inhibitor CAF-mediated signaling completed
Abbreviations: CAF, cancer-associated fibroblast; PEGPH20, pegvorhyaluronidase alfa; ATRA, all-trans retinoic acid; TGF-β, transforming growth factor beta;
PD-L1, programmed cell death 1 ligand 1; FGFR, fibroblast growth factor receptor; CXCR4, C-X-C motif chemokine receptor 4.

7.2 Targeting unique and highly the drug uptake rate in the tumor by 70% [87]. FAP-CAR
expressed molecules in CAFs mouse T cells specifically eliminated FAPhigh CAFs and
suppressed tumor growth [89]. FAP is also used as a target
Currently, FAP-based vaccines are being developed to treat for drug delivery. Wang et al. [326] conjugated epirubicin
tumors by targeting CAFs [81, 322, 323]. Current research (EPI) with an FAP-specific dipeptide that delivered EPI
results show that these vaccines inhibit tumor growth, to FAP-positive tumor cells and exerted cytotoxicity. This
including breast cancer, by increasing the level of IFN- novel agent, named Z-GP-EPI, exhibited good efficiency in
γ and the number of CD8-positive T cells in tumor tis- both in vitro and in vivo experiments.
sues [87, 324, 325]. FAP vaccines also reduced the colla- Histone deacetylase 6 (HDAC6) expression is often
gen type I expression in breast cancer tissues and increased upregulated in CAFs and predicts a poor prognosis for
420 HU et al.

patients with breast cancer [327–329]. Inhibition of HDAC6 7.4 Targeting CAF-secreted proteins
with drugs decreased the tumor growth rate, prevented and -associated signaling pathways
the accumulation of bone marrow-derived monocytes
and regulatory T cells in the TME, altered the trans- Since CAFs interact with nearby tumor cells and the TME
formation of macrophages, and activated CD8-positive by secreting proteins, these proteins and the correspond-
and CD4-positive T cells. The mechanism was that ing signaling pathways are regarded as potential therapeu-
HDAC6 expressed in CAFs upregulated the expression of tic targets. Inhibitors have been developed and applied in
prostaglandin E2 (cyclooxygenase-2, COX2) by regulating clinical trials (Figures 4–5 and Table 3).
STAT3 activity [327]. Therefore, HDAC6 may be a good As an important mediator of the mutual effects of
antitumor target in breast cancer. CAFs and breast cancer cells, TGF-β is an important tar-
get expressed in CAFs. Basic researches have shown that
small-molecule inhibitors targeting TGF-β receptors sig-
7.3 Preventing CAF activation and nificantly inhibited the aggressiveness of breast cancer
reprogramming CAFs to quiescent cells [190, 337-340]. In addition, neutralizing antibodies
fibroblasts targeting TGF-β have entered clinical trials. Fresolimumab
is a monoclonal antibody that can neutralize all three iso-
Activation of resident fibroblasts is a crucial step in the forms of TGF-β. When combined with radiotherapy in
generation of CAFs and has been regarded as an important patients with breast cancer presenting distant metastases,
therapeutic target in breast cancer research (Table 3). high-dose fresolimumab resulted in longer OS than low-
Cazet et al. [192] examined a TNBC model and revealed dose fresolimumab (NCT01401062) [341]. Galunisertib, a
that Hedgehog-dependent CAF activation and ECM TGF-βRI small-molecule inhibitor, modulated T cell activ-
remodeling promote the formation of CSC niches, leading ity and inhibited tumor growth in a mouse breast can-
to docetaxel resistance. They proposed treatments target- cer model in combination with PD-L1 blockade [342]. A
ing this pathway and achieved good results in preclinical phase I study with galunisertib in patients with breast
models, which led to the initiation of phase I and phase cancer is ongoing (NCT02672475). M7824, a bifunctional
II clinical trials of the smoothened inhibitor sonidegib in fusion protein targeting TGF-β and PD-L1, exerted antitu-
combination with docetaxel. Three of 12 patients who had mor activity in pre-clinic studies [343, 344] and demon-
TNBC with distant metastasis benefited from the com- strated an acceptable safety profile and clinical benefit
bination therapy (NCT02027376). Another smoothened based on the results of a phase I study in patients with solid
inhibitor, vismodegib, blocked the growth of tamoxifen- tumors [345]. Multiple phase I studies are also ongoing in
resistant breast cancer xenografts in mice [330], and a patients with breast cancer (NCT03524170, NCT04296942
phase II study of this drug is ongoing (NCT02694224). and NCT03579472).
The angiotensin inhibitor losartan inhibited FGFs and CXCLs mediate CAF-induced stemness, pro-
angiotensin-II receptor-1 in CAFs and reduced the liferation, migration and treatment resistance in breast
expression of downstream TGF-β, connective tissue cancer cells as previously described. Thus, FGFR and
growth factor (CCN2) and endothelin-1 (ET-1) signaling, CXCR might be potential therapeutic targets. The FGFR
reducing stromal collagen and hyaluronan production in inhibitor erdafitinib has been reported to overcome resis-
tumors and increasing drug and oxygen delivery [331]. tance to fulvestrant and CDK4/6 inhibitors in MCF-7 cells
Coulson et al. [332] also proved the activity of losartan to [346]. AZD4547 was identified as a selective inhibitor of
inhibit mammary tumor development and progression in FGFR1/2/3, and suppressed the growth of tumor cells
a mouse model. A phase II clinical trial with a combina- including breast cancer cells [347, 348]. Futibatinib was
tion of camrelizumab, liposomal doxorubicin and losartan also able to inhibit the growth of breast cancer cells in
in patients with breast cancer is ongoing (NCT05097248). vitro by blocking FGFR [349]. However, breast cancer cells
All-trans retinoic acid (ATRA) has been proven to switch showed little sensitivity to the FGFR inhibitor Debio 1347
CAFs into more quiescent fibroblasts, thus suppressing reported by Nakanishi et al. [350], who attributed the
their functions [333–335]. The application of ATRA in a insensitivity to genetic alterations in other signaling path-
clinical trial of patients with breast cancer is ongoing ways in these cell lines. These FGFR inhibitors and the
(NCT04113863). CXCR4 inhibitor balixafortide are being investigated in
Paricalcitol, a vitamin D receptor agonist, was also clinical trials as treatments for breast cancer.
reported to inactivate CAFs [336]. A phase I study with As a major component of the ECM, hyaluronan (HA)
paricalcitol in patients with breast cancer is ongoing is mainly generated by CAFs and is associated with
(NCT00637897). breast cancer malignancy [351, 352]. Pegvorhyaluronidase
HU et al. 421

alfa (PEGPH20), a hyaluronidase, remodeled the TME Much effort has been made to understand the role
by degrading HA, increasing the exposure of tumor cells of CAFs in breast cancer, and great progress has been
to drugs and augmenting drug efficacy [353]. PEGPH20 achieved. However, many challenges in translating these
increased the uptake of anti-PD-L1 antibody [353] and lapa- findings from bench to bedside persist. First, since broadly
tinib [354] in animal models of breast cancer and increased accepted origins and markers of CAFs are still ill-defined,
the efficacy of these agents. In a phase I trial, PEGPH20 a precise definition of CAFs is lacking, which presents a
reduced the hyaluronan level in patients with advanced challenge to investigating CAFs. Second, do subtypes of
solid tumors. PEGPH20 was proven to increase tumor per- CAFs have different origins or form in response to different
fusion and reduce tumor metabolic activity in both ani- stimuli? Are subtypes of CAFs reversible? Third, because
mal models and clinical trials [355]. However, the addi- fibroblasts may be activated by antitumor treatment that
tion of PEGPH20 to nab-paclitaxel/gemcitabine showed no converts their phenotypes and promotes treatment resis-
benefit on OS or PFS in a randomized phase III trial of tance, which methods can develop better strategies that
patients with hyaluronan-high metastatic pancreatic ade- combine traditional antitumor agents with CAF-targeting
nocarcinoma in 2020 [356]. A phase II study of patients drugs? Fourth, as CAFs have unique characteristics,
with metastatic breast cancer was terminated due to the including marker expression patterns and secretory capa-
evolving standard of care and difficulties in enrolling par- bility, in different subtypes of breast cancer, more specific
ticipants (NCT02753595). CAF-targeting therapeutics must be chosen according to
Fibronectin is an important protein secreted by CAFs, the breast cancer subtypes. Therefore, more information
and its cancer-promoting effect has been discussed in the about the heterogeneity of CAFs in breast cancer is needed.
previous sections. Femel et al. [357] proved that immuniz- The effects of antitumor agents, such as chemotherapy,
ing mice with extra domain-A of fibronectin (EDA-FN) endocrine therapy and HER2-targeted therapy, on CAFs
significantly reduced the tumor-bearing rate and distant should be adequately tested to select better combinations
metastasis rate in mouse breast cancer models. with CAF-targeted drugs. Additionally, since a majority
Gagliano et al. [358] identified fibroblast-derived p110δ of breast cancers are estrogen-dependent, an interesting
subunit of phosphatidylinositol-3-OH kinase (PIK3Cδ) as approach is to consider the potential effect of estrogen on
a vital mediator of TNBC. Following the administration of CAFs in breast cancer. More importantly, most studies
the PIK3Cδ inhibitor CAL-101, tumor growth was reduced involve in vitro experiments to explore the properties of
in an orthotopic breast cancer xenograft model. CAFs. However, in vitro culture conditions may alter the
Novel therapeutic approaches targeting CAFs in breast phenotype of CAFs. Thus, the biological behaviors of
cancer provide us additional tools to treat the disease. An CAFs observed in these studies may be very different from
earlier and more accurate diagnosis, imaging and progno- those in vivo. For example, when CAFs are isolated from
sis might be achieved by using these approaches, and drugs cancer tissues, they are free of the TME. CAFs are not
with fewer adverse effects and more antitumor potential stimulated by surrounding cells or the ECM, which might
might be developed in the future. However, due to the wide be the origin of CAF phenotypes, as described above.
intertumor and intratumoral heterogeneity of CAFs, the When cultured in a two-dimensional model, cell-cell
effects of these therapeutic strategies on the entire popu- contact is reduced significantly, also inducing alterations
lation of patients remain to be improved. Moreover, phe- in intracellular signaling. Moreover, uncertain supple-
notypic conversion of CAFs during treatment should also ments in culture medium might affect the phenotypes
be considered. of CAFs through unknown mechanisms. Notably, the
lifespan of primary CAFs is limited, and the phenotypic
changes caused by aging may affect the repeatability of
8 CONCLUSIONS AND PERSPECTIVES experiments. Studying CAFs in animal models would
help maintain the activity of CAFs and simulate the
Currently, tumors are regarded as highly complex entities, situation in the human body; however, generating and
with diverse cells and surrounding components that affect maintaining gene-edited animals is a challenge for many
each other. As a chief component of the TME, CAFs play an laboratories. Immortalization of CAFs is an alternative,
important role in all stages of cancer from onset to growth, but the extent to which CAF traits are conserved remains
invasion and finally distant metastasis and drug resistance. unclear. Because the CAF phenotype might be lost during
This review summarizes the research on CAFs in tumors, continuous subculture, research must be completed with
especially breast cancer, and clarifies the mechanism of fresh primary CAFs and the phenotype must be repeat-
their interaction with tumor cells and the current poten- edly examined during research. The passage number and
tial strategies targeting CAFs for the treatment of breast detailed information about the culture medium should be
cancer. recorded.
422 HU et al.

Currently, based on advanced techniques such as single- ORCID

cell sequencing and novel in vitro platforms, research on Linbo Wang https://orcid.org/0000-0003-0526-1990
CAFs is advancing rapidly. CAF-targeting therapeutics are
continuously being developed, providing new weapons to REFERENCES
fight cancer. We postulate that treatment targeting CAFs 1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I,
will become an efficient strategy to improve the prognosis Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Esti-
mates of Incidence and Mortality Worldwide for 36 Cancers in
of patients with breast cancer.
185 Countries. CA Cancer J Clin. 2021;71(3):209-49.
2. Qiu H, Cao S, Xu R. Cancer incidence, mortality, and bur-
den in China: a time-trend analysis and comparison with the
DECLARATIONS United States and United Kingdom based on the global epi-
demiological data released in 2020. Cancer Commun (Lond).
2021;41(10):1037-48.
AC K N OW L E D G M E N T S 3. Lei S, Zheng R, Zhang S, Wang S, Chen R, Sun K, et al.
Illustrations were created using BioRender.com. Figure Global patterns of breast cancer incidence and mortality: A
2 and figure 4 are adapted from “Signaling Path- population-based cancer registry data analysis from 2000 to
ways Underlying EMT and EndMT in Age-related 2020. Cancer Commun (Lond). 2021;41(11):1183-94.
Macular Degeneration (AMD)” and “Interaction 4. Harbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami
between T Cell and Cancer Cell with Callout (Lay- N, Poortmans P, et al. Breast cancer. Nat Rev Dis Primers.
2019;5(1):66.
out)”, respectively, by BioRender.com. Retrieved from
5. Sahai E, Astsaturov I, Cukierman E, DeNardo DG, Egeblad
https://app.biorender.com/biorender-templates.
M, Evans RM, et al. A framework for advancing our under-
standing of cancer-associated fibroblasts. Nat Rev Cancer.
E T H I C S A P P R O VA L A N D C O N S E N T T O 2020;20(3):174-86.
PA R T I C I PA T E 6. Chen X, Song E. Turning foes to friends: targeting cancer-
Not applicable. associated fibroblasts. Nat Rev Drug Discov. 2019;18(2):99-
115.
7. Jin MZ, Jin WL. The updated landscape of tumor microenvi-
FUNDING
ronment and drug repurposing. Signal Transduct Target Ther.
This work was supported by the National Natural Sci- 2020;5(1):166.
ence Foundation of China (No. 81672729, No. 81972597, 8. Lim B, Woodward WA, Wang X, Reuben JM, Ueno NT. Inflam-
No. 81602471, and No. 81972453), Zhejiang Provincial Nat- matory breast cancer biology: the tumour microenvironment is
ural Science Foundation of China (Grant LY19H160059, key. Nat Rev Cancer. 2018;18(8):485-99.
LR22H160011, and LY19H160055), Ningbo Natural Science 9. Baghban R, Roshangar L, Jahanban-Esfahlan R, Seidi K,
Foundation (Grant No. 2019A610315), Cixi Agricultural Ebrahimi-Kalan A, Jaymand M, et al. Tumor microenviron-
ment complexity and therapeutic implications at a glance. Cell
and Social Development Science and Technology Project
Commun Signal. 2020;18(1):59.
(CN2020006) and Zheng Shu Medical Elite Scholarship
10. Gaggioli C, Hooper S, Hidalgo-Carcedo C, Grosse R, Marshall
Fund. JF, Harrington K, et al. Fibroblast-led collective invasion of car-
cinoma cells with differing roles for RhoGTPases in leading and
D A T A AVA I L A B I L I T Y S T A T E M E N T following cells. Nat Cell Biol. 2007;9(12):1392-400.
Not applicable. 11. Su S, Chen J, Yao H, Liu J, Yu S, Lao L, et al. CD10(+)GPR77(+)
Cancer-Associated Fibroblasts Promote Cancer Formation
and Chemoresistance by Sustaining Cancer Stemness. Cell.
CONFLICT OF INTEREST
2018;172(4):841-56 e16.
There are no conflicts of interest to declare. 12. Virchow R. Die Cellularpathologie in lhrer Begruendung auf
Physiologische und Pathologische Gewebelehre. 1858.
C O N S E N T F O R P U B L I C AT I O N 13. Hay ED. An overview of epithelio-mesenchymal transforma-
Not applicable. tion. Acta Anat (Basel). 1995;154(1):8-20.
14. Sharpe PT. Neural crest and tooth morphogenesis. Adv Dent
AU T H O R CO N T R I B U T I O N S Res. 2001;15:4-7.
Manuscript writing (original draft): DH, ZL, and BZ. 15. Liu T, Han C, Wang S, Fang P, Ma Z, Xu L, et al. Cancer-
associated fibroblasts: an emerging target of anti-cancer
Conceptualization/funding acquisition: DH, LW, and
immunotherapy. J Hematol Oncol. 2019;12(1):86.
JZ. 16. Ohlund D, Elyada E, Tuveson D. Fibroblast heterogeneity in
Manuscript writing, review and editing: XL, YP, YH, PG, the cancer wound. J Exp Med. 2014;211(8):1503-23.
CC, LC, and WZ. 17. Saadi A, Shannon NB, Lao-Sirieix P, O’Donovan M, Walker
Manuscript vetting and final approval: LW and JZ. E, Clemons NJ, et al. Stromal genes discriminate preinvasive
HU et al. 423

from invasive disease, predict outcome, and highlight inflam- 35. Sansone P, Berishaj M, Rajasekhar VK, Ceccarelli C, Chang
matory pathways in digestive cancers. Proc Natl Acad Sci U S Q, Strillacci A, et al. Evolution of Cancer Stem-like Cells in
A. 2010;107(5):2177-82. Endocrine-Resistant Metastatic Breast Cancers Is Mediated by
18. Ma XJ, Dahiya S, Richardson E, Erlander M, Sgroi DC. Gene Stromal Microvesicles. Cancer Res. 2017;77(8):1927-41.
expression profiling of the tumor microenvironment during 36. Brechbuhl HM, Finlay-Schultz J, Yamamoto TM, Gillen AE,
breast cancer progression. Breast Cancer Res. 2009;11(1):R7. Cittelly DM, Tan AC, et al. Fibroblast Subtypes Regulate
19. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Responsiveness of Luminal Breast Cancer to Estrogen. Clin
Myofibroblasts and mechano-regulation of connective tissue Cancer Res. 2017;23(7):1710-21.
remodelling. Nat Rev Mol Cell Biol. 2002;3(5):349-63. 37. Lee JH, Kim HM, Koo JS. Differential Expression of Cancer-
20. Rodemann HP, Muller GA. Characterization of human renal Associated Fibroblast-Related Proteins in Ductal Carcinoma in
fibroblasts in health and disease: II. In vitro growth, differenti- situ According to Molecular Subtype and Stromal Histology.
ation, and collagen synthesis of fibroblasts from kidneys with Pathobiology. 2018;85(5-6):311-21.
interstitial fibrosis. Am J Kidney Dis. 1991;17(6):684-6. 38. Park SY, Kim HM, Koo JS. Differential expression of cancer-
21. Egeblad M, Werb Z. New functions for the matrix metallopro- associated fibroblast-related proteins according to molecular
teinases in cancer progression. Nat Rev Cancer. 2002;2(3):161- subtype and stromal histology in breast cancer. Breast Cancer
74. Res Treat. 2015;149(3):727-41.
22. Dano K, Behrendt N, Hoyer-Hansen G, Johnsen M, Lund LR, 39. Di Donato M, Zamagni A, Galasso G, Di Zazzo E, Giovannelli P,
Ploug M, et al. Plasminogen activation and cancer. Thromb Barone MV, et al. The androgen receptor/filamin A complex as
Haemost. 2005;93(4):676-81. a target in prostate cancer microenvironment. Cell Death Dis.
23. Yazdani S, Bansal R, Prakash J. Drug targeting to myofibrob- 2021;12(1):127.
lasts: Implications for fibrosis and cancer. Adv Drug Deliv Rev. 40. Park CK, Jung WH, Koo JS. Expression of cancer-associated
2017;121:101-16. fibroblast-related proteins differs between invasive lobular car-
24. Cukierman E, Bassi DE. Physico-mechanical aspects of extra- cinoma and invasive ductal carcinoma. Breast Cancer Res
cellular matrix influences on tumorigenic behaviors. Semin Treat. 2016;159(1):55-69.
Cancer Biol. 2010;20(3):139-45. 41. Ding S, Chen X, Shen K. Single-cell RNA sequencing in
25. Baeriswyl V, Christofori G. The angiogenic switch in carcino- breast cancer: Understanding tumor heterogeneity and paving
genesis. Semin Cancer Biol. 2009;19(5):329-37. roads to individualized therapy. Cancer Commun (Lond).
26. Tsujino T, Seshimo I, Yamamoto H, Ngan CY, Ezumi K, 2020;40(8):329-44.
Takemasa I, et al. Stromal myofibroblasts predict disease recur- 42. Bartoschek M, Oskolkov N, Bocci M, Lovrot J, Larsson C,
rence for colorectal cancer. Clin Cancer Res. 2007;13(7):2082- Sommarin M, et al. Spatially and functionally distinct sub-
90. classes of breast cancer-associated fibroblasts revealed by single
27. Laklai H, Miroshnikova YA, Pickup MW, Collisson EA, Kim cell RNA sequencing. Nat Commun. 2018;9(1):5150.
GE, Barrett AS, et al. Genotype tunes pancreatic ductal adeno- 43. Jungwirth U, van Weverwijk A, Evans RJ, Jenkins L, Vicente
carcinoma tissue tension to induce matricellular fibrosis and D, Alexander J, et al. Impairment of a distinct cancer-associated
tumor progression. Nat Med. 2016;22(5):497-505. fibroblast population limits tumour growth and metastasis. Nat
28. Calvo F, Ege N, Grande-Garcia A, Hooper S, Jenkins RP, Commun. 2021;12(1):3516.
Chaudhry SI, et al. Mechanotransduction and YAP-dependent 44. Sebastian A, Hum NR, Martin KA, Gilmore SF, Peran I,
matrix remodelling is required for the generation and main- Byers SW, et al. Single-Cell Transcriptomic Analysis of Tumor-
tenance of cancer-associated fibroblasts. Nat Cell Biol. Derived Fibroblasts and Normal Tissue-Resident Fibroblasts
2013;15(6):637-46. Reveals Fibroblast Heterogeneity in Breast Cancer. Cancers
29. Kalluri R. The biology and function of fibroblasts in cancer. Nat (Basel). 2020;12(5).
Rev Cancer. 2016;16(9):582-98. 45. Kim HM, Jung WH, Koo JS. Expression of cancer-associated
30. Dvorak HF. Tumors: wounds that do not heal. Similarities fibroblast related proteins in metastatic breast cancer: an
between tumor stroma generation and wound healing. N Engl immunohistochemical analysis. J Transl Med. 2015;13:222.
J Med. 1986;315(26):1650-9. 46. Friedman G, Levi-Galibov O, David E, Bornstein C, Giladi A,
31. Haddow A. Molecular repair, wound healing, and carcinogene- Dadiani M, et al. Cancer-associated fibroblast compositions
sis: tumor production a possible overhealing? Adv Cancer Res. change with breast cancer progression linking the ratio of
1972;16:181-234. S100A4+ and PDPN+ CAFs to clinical outcome. Nature Can-
32. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. cer. 2020;1(7):692-708.
2006;6(5):392-401. 47. Costa A, Kieffer Y, Scholer-Dahirel A, Pelon F, Bourachot B,
33. Tchou J, Kossenkov AV, Chang L, Satija C, Herlyn M, Showe Cardon M, et al. Fibroblast Heterogeneity and Immunosup-
LC, et al. Human breast cancer associated fibroblasts exhibit pressive Environment in Human Breast Cancer. Cancer Cell.
subtype specific gene expression profiles. BMC Med Genomics. 2018;33(3):463-79 e10.
2012;5:39. 48. Bonneau C, Elies A, Kieffer Y, Bourachot B, Ladoire S, Pelon
34. Louault K, Bonneaud TL, Seveno C, Gomez-Bougie P, F, et al. A subset of activated fibroblasts is associated with dis-
Nguyen F, Gautier F, et al. Interactions between cancer- tant relapse in early luminal breast cancer. Breast Cancer Res.
associated fibroblasts and tumor cells promote MCL-1 depen- 2020;22(1):76.
dency in estrogen receptor-positive breast cancers. Oncogene. 49. Pelon F, Bourachot B, Kieffer Y, Magagna I, Mermet-Meillon
2019;38(17):3261-73. F, Bonnet I, et al. Cancer-associated fibroblast heterogeneity
424 HU et al.

in axillary lymph nodes drives metastases in breast can- 65. Surowiak P, Murawa D, Materna V, Maciejczyk A, Pudelko M,
cer through complementary mechanisms. Nat Commun. Ciesla S, et al. Occurence of stromal myofibroblasts in the inva-
2020;11(1):404. sive ductal breast cancer tissue is an unfavourable prognostic
50. Han C, Liu T, Yin R. Biomarkers for cancer-associated fibrob- factor. Anticancer Res. 2007;27(4C):2917-24.
lasts. Biomark Res. 2020;8(1):64. 66. Paulsson J, Micke P. Prognostic relevance of cancer-associated
51. Nurmik M, Ullmann P, Rodriguez F, Haan S, Letellier E. In fibroblasts in human cancer. Semin Cancer Biol. 2014;25:
search of definitions: Cancer-associated fibroblasts and their 61-8.
markers. Int J Cancer. 2020;146(4):895-905. 67. Becker LM, O’Connell JT, Vo AP, Cain MP, Tampe D, Bizarro L,
52. O’Connell JT, Sugimoto H, Cooke VG, MacDonald BA, Mehta et al. Epigenetic Reprogramming of Cancer-Associated Fibrob-
AI, LeBleu VS, et al. VEGF-A and Tenascin-C produced by lasts Deregulates Glucose Metabolism and Facilitates Progres-
S100A4+ stromal cells are important for metastatic coloniza- sion of Breast Cancer. Cell Rep. 2020;31(9):107701.
tion. Proc Natl Acad Sci U S A. 2011;108(38):16002-7. 68. Vuoriluoto K, Haugen H, Kiviluoto S, Mpindi JP, Nevo J,
53. Reardon DA, Akabani G, Coleman RE, Friedman AH, Gjerdrum C, et al. Vimentin regulates EMT induction by Slug
Friedman HS, Herndon JE, 2nd, et al. Salvage radioim- and oncogenic H-Ras and migration by governing Axl expres-
munotherapy with murine iodine-131-labeled antitenascin sion in breast cancer. Oncogene. 2011;30(12):1436-48.
monoclonal antibody 81C6 for patients with recurrent primary 69. Richardson AM, Havel LS, Koyen AE, Konen JM, Shupe
and metastatic malignant brain tumors: phase II study results. J, Wiles WGt, et al. Vimentin Is Required for Lung Ade-
J Clin Oncol. 2006;24(1):115-22. nocarcinoma Metastasis via Heterotypic Tumor Cell-Cancer-
54. Armulik A, Genove G, Betsholtz C. Pericytes: developmen- Associated Fibroblast Interactions during Collective Invasion.
tal, physiological, and pathological perspectives, problems, and Clin Cancer Res. 2018;24(2):420-32.
promises. Dev Cell. 2011;21(2):193-215. 70. Ngan CY, Yamamoto H, Seshimo I, Tsujino T, Man-i M,
55. Eyden B. The myofibroblast: phenotypic characterization as Ikeda JI, et al. Quantitative evaluation of vimentin expres-
a prerequisite to understanding its functions in translational sion in tumour stroma of colorectal cancer. Br J Cancer.
medicine. J Cell Mol Med. 2008;12(1):22-37. 2007;96(6):986-92.
56. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell 71. Maehira H, Miyake T, Iida H, Tokuda A, Mori H, Yasukawa
GW, et al. Mesenchymal stem cells within tumour stroma pro- D, et al. Vimentin Expression in Tumor Microenviron-
mote breast cancer metastasis. Nature. 2007;449(7162):557-63. ment Predicts Survival in Pancreatic Ductal Adenocarcinoma:
57. Gerard NP, Lu B, Liu P, Craig S, Fujiwara Y, Okinaga S, et al. An Heterogeneity in Fibroblast Population. Ann Surg Oncol.
anti-inflammatory function for the complement anaphylatoxin 2019;26(13):4791-804.
C5a-binding protein, C5L2. J Biol Chem. 2005;280(48):39677-80. 72. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG.
58. Lazard D, Sastre X, Frid MG, Glukhova MA, Thiery JP, Evidence that fibroblasts derive from epithelium during tissue
Koteliansky VE. Expression of smooth muscle-specific proteins fibrosis. J Clin Invest. 2002;110(3):341-50.
in myoepithelium and stromal myofibroblasts of normal and 73. Zeisberg EM, Potenta S, Xie L, Zeisberg M, Kalluri R.
malignant human breast tissue. Proc Natl Acad Sci U S A. Discovery of endothelial to mesenchymal transition as a
1993;90(3):999-1003. source for carcinoma-associated fibroblasts. Cancer Res.
59. Rockey DC, Weymouth N, Shi Z. Smooth muscle alpha actin 2007;67(21):10123-8.
(Acta2) and myofibroblast function during hepatic wound heal- 74. Choi SY, Sung R, Lee SJ, Lee TG, Kim N, Yoon SM, et al.
ing. PLoS One. 2013;8(10):e77166. Podoplanin, alpha-smooth muscle actin or S100A4 expressing
60. Huang M, Li Y, Zhang H, Nan F. Breast cancer stromal fibrob- cancer-associated fibroblasts are associated with different prog-
lasts promote the generation of CD44+CD24- cells through nosis in colorectal cancers. J Korean Med Sci. 2013;28(9):1293-
SDF-1/CXCR4 interaction. J Exp Clin Cancer Res. 2010;29:80. 301.
61. Lau EY, Lo J, Cheng BY, Ma MK, Lee JM, Ng JK, et al. Cancer- 75. Schulte J, Weidig M, Balzer P, Richter P, Franz M, Junker K,
Associated Fibroblasts Regulate Tumor-Initiating Cell Plastic- et al. Expression of the E-cadherin repressors Snail, Slug and
ity in Hepatocellular Carcinoma through c-Met/FRA1/HEY1 Zeb1 in urothelial carcinoma of the urinary bladder: relation
Signaling. Cell Rep. 2016;15(6):1175-89. to stromal fibroblast activation and invasive behaviour of carci-
62. Labernadie A, Kato T, Brugues A, Serra-Picamal X, Derzsi noma cells. Histochem Cell Biol. 2012;138(6):847-60.
S, Arwert E, et al. A mechanically active heterotypic E- 76. Zhang J, Chen L, Liu X, Kammertoens T, Blankenstein T, Qin
cadherin/N-cadherin adhesion enables fibroblasts to drive can- Z. Fibroblast-specific protein 1/S100A4-positive cells prevent
cer cell invasion. Nat Cell Biol. 2017;19(3):224-37. carcinoma through collagen production and encapsulation of
63. Ozdemir BC, Pentcheva-Hoang T, Carstens JL, Zheng X, Wu carcinogens. Cancer Res. 2013;73(9):2770-81.
CC, Simpson TR, et al. Depletion of Carcinoma-Associated 77. Huber MA, Kraut N, Park JE, Schubert RD, Rettig WJ, Peter
Fibroblasts and Fibrosis Induces Immunosuppression and RU, et al. Fibroblast activation protein: differential expres-
Accelerates Pancreas Cancer with Reduced Survival. Cancer sion and serine protease activity in reactive stromal fibroblasts
Cell. 2015;28(6):831-3. of melanocytic skin tumors. J Invest Dermatol. 2003;120(2):
64. Wang WQ, Liu L, Xu HX, Luo GP, Chen T, Wu CT, et al. 182-8.
Intratumoral alpha-SMA enhances the prognostic potency of 78. Yang X, Lin Y, Shi Y, Li B, Liu W, Yin W, et al. FAP Promotes
CD34 associated with maintenance of microvessel integrity in Immunosuppression by Cancer-Associated Fibroblasts in the
hepatocellular carcinoma and pancreatic cancer. PLoS One. Tumor Microenvironment via STAT3-CCL2 Signaling. Cancer
2013;8(8):e71189. Res. 2016;76(14):4124-35.
HU et al. 425

79. Bughda R, Dimou P, D’Souza RR, Klampatsa A. Fibroblast in patients with advanced or metastatic fibroblast activation
Activation Protein (FAP)-Targeted CAR-T Cells: Launching an protein-positive cancer. Clin Cancer Res. 2003;9(5):1639-47.
Attack on Tumor Stroma. Immunotargets Ther. 2021;10:313-23. 94. Hofheinz RD, al-Batran SE, Hartmann F, Hartung G, Jager
80. Hussain A, Voisin V, Poon S, Karamboulas C, Bui NHB, Meens D, Renner C, et al. Stromal antigen targeting by a human-
J, et al. Distinct fibroblast functional states drive clinical out- ised monoclonal antibody: an early phase II trial of sibro-
comes in ovarian cancer and are regulated by TCF21. J Exp tuzumab in patients with metastatic colorectal cancer. Onkolo-
Med. 2020;217(8). gie. 2003;26(1):44-8.
81. Kraman M, Bambrough PJ, Arnold JN, Roberts EW, Magiera 95. Narra K, Mullins SR, Lee HO, Strzemkowski-Brun B, Magalong
L, Jones JO, et al. Suppression of antitumor immunity by stro- K, Christiansen VJ, et al. Phase II trial of single agent Val-
mal cells expressing fibroblast activation protein-alpha. Sci- boroPro (Talabostat) inhibiting Fibroblast Activation Protein
ence. 2010;330(6005):827-30. in patients with metastatic colorectal cancer. Cancer Biol Ther.
82. Adams S, Miller GT, Jesson MI, Watanabe T, Jones B, Wallner 2007;6(11):1691-9.
BP. PT-100, a small molecule dipeptidyl peptidase inhibitor, 96. Ariga N, Sato E, Ohuchi N, Nagura H, Ohtani H. Stromal
has potent antitumor effects and augments antibody-mediated expression of fibroblast activation protein/seprase, a cell mem-
cytotoxicity via a novel immune mechanism. Cancer Res. brane serine proteinase and gelatinase, is associated with
2004;64(15):5471-80. longer survival in patients with invasive ductal carcinoma of
83. Santos AM, Jung J, Aziz N, Kissil JL, Pure E. Targeting breast. Int J Cancer. 2001;95(1):67-72.
fibroblast activation protein inhibits tumor stromagenesis and 97. Paulsson J, Ehnman M, Ostman A. PDGF receptors in tumor
growth in mice. J Clin Invest. 2009;119(12):3613-25. biology: prognostic and predictive potential. Future Oncol.
84. Ostermann E, Garin-Chesa P, Heider KH, Kalat M, Lamche 2014;10(9):1695-708.
H, Puri C, et al. Effective immunoconjugate therapy in can- 98. Heldin CH. Targeting the PDGF signaling pathway in tumor
cer models targeting a serine protease of tumor fibroblasts. Clin treatment. Cell Commun Signal. 2013;11:97.
Cancer Res. 2008;14(14):4584-92. 99. Aoto K, Ito K, Aoki S. Complex formation between platelet-
85. Feig C, Jones JO, Kraman M, Wells RJ, Deonarine A, Chan derived growth factor receptor beta and transforming growth
DS, et al. Targeting CXCL12 from FAP-expressing carcinoma- factor beta receptor regulates the differentiation of mesenchy-
associated fibroblasts synergizes with anti-PD-L1 immunother- mal stem cells into cancer-associated fibroblasts. Oncotarget.
apy in pancreatic cancer. Proc Natl Acad Sci U S A. 2018;9(75):34090-102.
2013;110(50):20212-7. 100. Pietras K, Pahler J, Bergers G, Hanahan D. Functions
86. Fang J, Xiao L, Joo KI, Liu Y, Zhang C, Liu S, et al. A potent of paracrine PDGF signaling in the proangiogenic tumor
immunotoxin targeting fibroblast activation protein for treat- stroma revealed by pharmacological targeting. PLoS Med.
ment of breast cancer in mice. Int J Cancer. 2016;138(4):1013-23. 2008;5(1):e19.
87. Loeffler M, Kruger JA, Niethammer AG, Reisfeld RA. Target- 101. Haubeiss S, Schmid JO, Murdter TE, Sonnenberg M, Friedel
ing tumor-associated fibroblasts improves cancer chemother- G, van der Kuip H, et al. Dasatinib reverses cancer-associated
apy by increasing intratumoral drug uptake. J Clin Invest. fibroblasts (CAFs) from primary lung carcinomas to a phe-
2006;116(7):1955-62. notype comparable to that of normal fibroblasts. Mol Cancer.
88. Kakarla S, Chow KK, Mata M, Shaffer DR, Song XT, Wu MF, 2010;9:168.
et al. Antitumor effects of chimeric receptor engineered human 102. Paulsson J, Sjoblom T, Micke P, Ponten F, Landberg G, Heldin
T cells directed to tumor stroma. Mol Ther. 2013;21(8):1611-20. CH, et al. Prognostic significance of stromal platelet-derived
89. Wang LC, Lo A, Scholler J, Sun J, Majumdar RS, Kapoor V, growth factor beta-receptor expression in human breast cancer.
et al. Targeting fibroblast activation protein in tumor stroma Am J Pathol. 2009;175(1):334-41.
with chimeric antigen receptor T cells can inhibit tumor growth 103. Strell C, Stenmark Tullberg A, Jetne Edelmann R, Akslen LA,
and augment host immunity without severe toxicity. Cancer Malmstrom P, Ferno M, et al. Prognostic and predictive impact
Immunol Res. 2014;2(2):154-66. of stroma cells defined by PDGFRb expression in early breast
90. Freedman JD, Duffy MR, Lei-Rossmann J, Muntzer A, Scott cancer: results from the randomized SweBCG91RT trial. Breast
EM, Hagel J, et al. An Oncolytic Virus Expressing a T-cell Cancer Res Treat. 2021;187(1):45-55.
Engager Simultaneously Targets Cancer and Immunosuppres- 104. Paulsson J, Ryden L, Strell C, Frings O, Tobin NP, Fornander T,
sive Stromal Cells. Cancer Res. 2018;78(24):6852-65. et al. High expression of stromal PDGFRbeta is associated with
91. de Sostoa J, Fajardo CA, Moreno R, Ramos MD, Farrera-Sal M, reduced benefit of tamoxifen in breast cancer. J Pathol Clin Res.
Alemany R. Targeting the tumor stroma with an oncolytic ade- 2017;3(1):38-43.
novirus secreting a fibroblast activation protein-targeted bispe- 105. Frings O, Augsten M, Tobin NP, Carlson J, Paulsson J, Pena
cific T-cell engager. J Immunother Cancer. 2019;7(1):19. C, et al. Prognostic significance in breast cancer of a gene
92. Welt S, Divgi CR, Scott AM, Garin-Chesa P, Finn RD, Graham signature capturing stromal PDGF signaling. Am J Pathol.
M, et al. Antibody targeting in metastatic colon cancer: a 2013;182(6):2037-47.
phase I study of monoclonal antibody F19 against a cell-surface 106. Mercier I, Casimiro MC, Wang C, Rosenberg AL, Quong J,
protein of reactive tumor stromal fibroblasts. J Clin Oncol. Minkeu A, et al. Human breast cancer-associated fibroblasts
1994;12(6):1193-203. (CAFs) show caveolin-1 downregulation and RB tumor sup-
93. Scott AM, Wiseman G, Welt S, Adjei A, Lee FT, Hopkins pressor functional inactivation: Implications for the response
W, et al. A Phase I dose-escalation study of sibrotuzumab to hormonal therapy. Cancer Biol Ther. 2008;7(8):1212-25.
426 HU et al.

107. Engelman JA, Wykoff CC, Yasuhara S, Song KS, Okamoto T, 122. Nakasone S, Mimaki S, Ichikawa T, Aokage K, Miyoshi
Lisanti MP. Recombinant expression of caveolin-1 in onco- T, Sugano M, et al. Podoplanin-positive cancer-associated
genically transformed cells abrogates anchorage-independent fibroblast recruitment within cancer stroma is associated
growth. J Biol Chem. 1997;272(26):16374-81. with a higher number of single nucleotide variants in can-
108. Koleske AJ, Baltimore D, Lisanti MP. Reduction of caveolin and cer cells in lung adenocarcinoma. J Cancer Res Clin Oncol.
caveolae in oncogenically transformed cells. Proc Natl Acad Sci 2018;144(5):893-900.
U S A. 1995;92(5):1381-5. 123. Shindo K, Aishima S, Ohuchida K, Fujiwara K, Fujino M,
109. Galbiati F, Volonte D, Engelman JA, Watanabe G, Burk R, Mizuuchi Y, et al. Podoplanin expression in cancer-associated
Pestell RG, et al. Targeted downregulation of caveolin-1 is suffi- fibroblasts enhances tumor progression of invasive ductal car-
cient to drive cell transformation and hyperactivate the p42/44 cinoma of the pancreas. Mol Cancer. 2013;12(1):168.
MAP kinase cascade. EMBO J. 1998;17(22):6633-48. 124. Takahashi A, Ishii G, Neri S, Yoshida T, Hashimoto H,
110. Shen XJ, Zhang H, Tang GS, Wang XD, Zheng R, Wang Y, et al. Suzuki S, et al. Podoplanin-expressing cancer-associated
Caveolin-1 is a modulator of fibroblast activation and a poten- fibroblasts inhibit small cell lung cancer growth. Oncotarget.
tial biomarker for gastric cancer. Int J Biol Sci. 2015;11(4):370-9. 2015;6(11):9531-41.
111. Goetz JG, Minguet S, Navarro-Lerida I, Lazcano JJ, Samaniego 125. Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek
R, Calvo E, et al. Biomechanical remodeling of the microen- T, Calik J, et al. Podoplanin expression by cancer-associated
vironment by stromal caveolin-1 favors tumor invasion and fibroblasts predicts poor outcome in invasive ductal breast car-
metastasis. Cell. 2011;146(1):148-63. cinoma. Histopathology. 2011;59(6):1249-60.
112. Simpkins SA, Hanby AM, Holliday DL, Speirs V. Clinical and 126. Schoppmann SF, Berghoff A, Dinhof C, Jakesz R, Gnant
functional significance of loss of caveolin-1 expression in breast M, Dubsky P, et al. Podoplanin-expressing cancer-associated
cancer-associated fibroblasts. J Pathol. 2012;227(4):490-8. fibroblasts are associated with poor prognosis in invasive breast
113. Scatena C, Fanelli G, Fanelli GN, Menicagli M, Aretini P, cancer. Breast Cancer Res Treat. 2012;134(1):237-44.
Ortenzi V, et al. New insights in the expression of stromal cave- 127. Yamaguchi K, Hara Y, Kitano I, Hamamoto T, Kiyomatsu K,
olin 1 in breast cancer spread to axillary lymph nodes. Sci Rep. Yamasaki F, et al. Relationship between MRI findings and inva-
2021;11(1):2755. sive breast cancer with podoplanin-positive cancer-associated
114. Pavlides S, Whitaker-Menezes D, Castello-Cros R, Flomenberg fibroblasts. Breast Cancer. 2021;28(3):572-80.
N, Witkiewicz AK, Frank PG, et al. The reverse Warburg 128. Suchanski J, Tejchman A, Zacharski M, Piotrowska A,
effect: aerobic glycolysis in cancer associated fibroblasts and Grzegrzolka J, Chodaczek G, et al. Podoplanin increases
the tumor stroma. Cell Cycle. 2009;8(23):3984-4001. the migration of human fibroblasts and affects the endothe-
115. Wilde L, Roche M, Domingo-Vidal M, Tanson K, Philp N, Curry lial cell network formation: A possible role for cancer-
J, et al. Metabolic coupling and the Reverse Warburg Effect in associated fibroblasts in breast cancer progression. PLoS One.
cancer: Implications for novel biomarker and anticancer agent 2017;12(9):e0184970.
development. Semin Oncol. 2017;44(3):198-203. 129. Chuang WY, Yeh CJ, Chao YK, Liu YH, Chang YS, Tseng CK,
116. Bonuccelli G, Whitaker-Menezes D, Castello-Cros R, Pavlides et al. Concordant podoplanin expression in cancer-associated
S, Pestell RG, Fatatis A, et al. The reverse Warburg effect: fibroblasts and tumor cells is an adverse prognostic factor in
glycolysis inhibitors prevent the tumor promoting effects of esophageal squamous cell carcinoma. Int J Clin Exp Pathol.
caveolin-1 deficient cancer associated fibroblasts. Cell Cycle. 2014;7(8):4847-56.
2010;9(10):1960-71. 130. Pan C, Liu P, Ma D, Zhang S, Ni M, Fang Q, et al. Bone mar-
117. Qian XL, Pan YH, Huang QY, Shi YB, Huang QY, Hu ZZ, et al. row mesenchymal stem cells in microenvironment transform
Caveolin-1: a multifaceted driver of breast cancer progression into cancer-associated fibroblasts to promote the progression
and its application in clinical treatment. Onco Targets Ther. of B-cell acute lymphoblastic leukemia. Biomed Pharmacother.
2019;12:1539-52. 2020;130:110610.
118. Kubouchi Y, Yurugi Y, Wakahara M, Sakabe T, Haruki T, 131. Ronnov-Jessen L, Petersen OW, Koteliansky VE, Bissell MJ.
Nosaka K, et al. Podoplanin expression in cancer-associated The origin of the myofibroblasts in breast cancer. Recapitula-
fibroblasts predicts unfavourable prognosis in patients with tion of tumor environment in culture unravels diversity and
pathological stage IA lung adenocarcinoma. Histopathology. implicates converted fibroblasts and recruited smooth muscle
2018;72(3):490-9. cells. J Clin Invest. 1995;95(2):859-73.
119. Ono S, Ishii G, Nagai K, Takuwa T, Yoshida J, Nishimura M, 132. Kojima Y, Acar A, Eaton EN, Mellody KT, Scheel C, Ben-Porath
et al. Podoplanin-positive cancer-associated fibroblasts could I, et al. Autocrine TGF-beta and stromal cell-derived factor-
have prognostic value independent of cancer cell phenotype in 1 (SDF-1) signaling drives the evolution of tumor-promoting
stage I lung squamous cell carcinoma: usefulness of combining mammary stromal myofibroblasts. Proc Natl Acad Sci U S A.
analysis of both cancer cell phenotype and cancer-associated 2010;107(46):20009-14.
fibroblast phenotype. Chest. 2013;143(4):963-70. 133. Zhang D, Wang Y, Shi Z, Liu J, Sun P, Hou X, et al. Metabolic
120. Hu G, Wang S, Xu F, Ding Q, Chen W, Zhong K, et al. Tumor- reprogramming of cancer-associated fibroblasts by IDH3alpha
Infiltrating Podoplanin+ Fibroblasts Predict Worse Outcome in downregulation. Cell Rep. 2015;10(8):1335-48.
Solid Tumors. Cell Physiol Biochem. 2018;51(3):1041-50. 134. Sharon Y, Raz Y, Cohen N, Ben-Shmuel A, Schwartz H, Geiger
121. Ito M, Ishii G, Nagai K, Maeda R, Nakano Y, Ochiai A. Prog- T, et al. Tumor-derived osteopontin reprograms normal mam-
nostic impact of cancer-associated stromal cells in patients with mary fibroblasts to promote inflammation and tumor growth
stage I lung adenocarcinoma. Chest. 2012;142(1):151-8. in breast cancer. Cancer Res. 2015;75(6):963-73.
HU et al. 427

135. Butti R, Nimma R, Kundu G, Bulbule A, Kumar TVS, 149. Yan L, Wang P, Fang W, Liang C. Cancer-associated fibroblasts-
Gunasekaran VP, et al. Tumor-derived osteopontin drives the derived exosomes-mediated transfer of LINC00355 regulates
resident fibroblast to myofibroblast differentiation through bladder cancer cell proliferation and invasion. Cell Biochem
Twist1 to promote breast cancer progression. Oncogene. Funct. 2020;38(3):257-65.
2021;40(11):2002-17. 150. Arandkar S, Furth N, Elisha Y, Nataraj NB, van der Kuip H,
136. Avgustinova A, Iravani M, Robertson D, Fearns A, Gao Q, Yarden Y, et al. Altered p53 functionality in cancer-associated
Klingbeil P, et al. Tumour cell-derived Wnt7a recruits and acti- fibroblasts contributes to their cancer-supporting features. Proc
vates fibroblasts to promote tumour aggressiveness. Nat Com- Natl Acad Sci U S A. 2018;115(25):6410-5.
mun. 2016;7:10305. 151. Tyan SW, Kuo WH, Huang CK, Pan CC, Shew JY, Chang KJ,
137. Baroni S, Romero-Cordoba S, Plantamura I, Dugo M, et al. Breast cancer cells induce cancer-associated fibroblasts to
D’Ippolito E, Cataldo A, et al. Exosome-mediated delivery of secrete hepatocyte growth factor to enhance breast tumorigen-
miR-9 induces cancer-associated fibroblast-like properties in esis. PLoS One. 2011;6(1):e15313.
human breast fibroblasts. Cell Death Dis. 2016;7(7):e2312. 152. Soon PS, Kim E, Pon CK, Gill AJ, Moore K, Spillane AJ,
138. Vu LT, Peng B, Zhang DX, Ma V, Mathey-Andrews CA, Lam et al. Breast cancer-associated fibroblasts induce epithelial-to-
CK, et al. Tumor-secreted extracellular vesicles promote the mesenchymal transition in breast cancer cells. Endocr Relat
activation of cancer-associated fibroblasts via the transfer of Cancer. 2013;20(1):1-12.
microRNA-125b. J Extracell Vesicles. 2019;8(1):1599680. 153. Bauer M, Su G, Casper C, He R, Rehrauer W, Friedl A.
139. Chatterjee A, Jana S, Chatterjee S, Wastall LM, Mandal Heterogeneity of gene expression in stromal fibroblasts of
G, Nargis N, et al. MicroRNA-222 reprogrammed cancer- human breast carcinomas and normal breast. Oncogene.
associated fibroblasts enhance growth and metastasis of breast 2010;29(12):1732-40.
cancer. Br J Cancer. 2019;121(8):679-89. 154. Zhao L, Sun Y, Hou Y, Peng Q, Wang L, Luo H, et al. MiRNA
140. Yang SS, Ma S, Dou H, Liu F, Zhang SY, Jiang C, et al. Breast expression analysis of cancer-associated fibroblasts and nor-
cancer-derived exosomes regulate cell invasion and metasta- mal fibroblasts in breast cancer. Int J Biochem Cell Biol.
sis in breast cancer via miR-146a to activate cancer associ- 2012;44(11):2051-9.
ated fibroblasts in tumor microenvironment. Exp Cell Res. 155. Raz Y, Cohen N, Shani O, Bell RE, Novitskiy SV, Abramovitz
2020;391(2):111983. L, et al. Bone marrow-derived fibroblasts are a functionally
141. Ren Z, Lv M, Yu Q, Bao J, Lou K, Li X. MicroRNA-370-3p shut- distinct stromal cell population in breast cancer. J Exp Med.
tled by breast cancer cell-derived extracellular vesicles induces 2018;215(12):3075-93.
fibroblast activation through the CYLD/Nf-kappaB axis to pro- 156. Jotzu C, Alt E, Welte G, Li J, Hennessy BT, Devarajan E, et al.
mote breast cancer progression. FASEB J. 2021;35(3):e21383. Adipose tissue derived stem cells differentiate into carcinoma-
142. Li K, Liu T, Chen J, Ni H, Li W. Survivin in breast cancer- associated fibroblast-like cells under the influence of tumor
derived exosomes activates fibroblasts by up-regulating SOD1, derived factors. Cell Oncol (Dordr). 2011;34(1):55-67.
whose feedback promotes cancer proliferation and metastasis. 157. Kidd S, Spaeth E, Watson K, Burks J, Lu H, Klopp A, et al.
J Biol Chem. 2020;295(40):13737-52. Origins of the tumor microenvironment: quantitative assess-
143. Martinez-Outschoorn UE, Pavlides S, Whitaker-Menezes D, ment of adipose-derived and bone marrow-derived stroma.
Daumer KM, Milliman JN, Chiavarina B, et al. Tumor cells PLoS One. 2012;7(2):e30563.
induce the cancer associated fibroblast phenotype via caveolin- 158. Cho JA, Park H, Lim EH, Lee KW. Exosomes from breast
1 degradation: implications for breast cancer and DCIS therapy cancer cells can convert adipose tissue-derived mesenchymal
with autophagy inhibitors. Cell Cycle. 2010;9(12):2423-33. stem cells into myofibroblast-like cells. Int J Oncol. 2012;40(1):
144. Peiris-Pages M, Sotgia F, Lisanti MP. Chemotherapy induces 130-8.
the cancer-associated fibroblast phenotype, activating 159. Weber CE, Kothari AN, Wai PY, Li NY, Driver J, Zapf MA, et al.
paracrine Hedgehog-GLI signalling in breast cancer cells. Osteopontin mediates an MZF1-TGF-beta1-dependent trans-
Oncotarget. 2015;6(13):10728-45. formation of mesenchymal stem cells into cancer-associated
145. Chatterjee S, Bhat V, Berdnikov A, Liu J, Zhang G, Buchel E, fibroblasts in breast cancer. Oncogene. 2015;34(37):4821-33.
et al. Paracrine Crosstalk between Fibroblasts and ER(+) Breast 160. Bochet L, Lehuede C, Dauvillier S, Wang YY, Dirat B, Laurent
Cancer Cells Creates an IL1beta-Enriched Niche that Promotes V, et al. Adipocyte-derived fibroblasts promote tumor progres-
Tumor Growth. iScience. 2019;19:388-401. sion and contribute to the desmoplastic reaction in breast can-
146. Shen Z, Qin X, Yan M, Li R, Chen G, Zhang J, et al. Cancer- cer. Cancer Res. 2013;73(18):5657-68.
associated fibroblasts promote cancer cell growth through a 161. Hosaka K, Yang Y, Seki T, Fischer C, Dubey O, Fredlund E,
miR-7-RASSF2-PAR-4 axis in the tumor microenvironment. et al. Pericyte-fibroblast transition promotes tumor growth and
Oncotarget. 2017;8(1):1290-303. metastasis. Proc Natl Acad Sci U S A. 2016;113(38):E5618-27.
147. Peng Q, Zhao L, Hou Y, Sun Y, Wang L, Luo H, et al. Biological 162. Jahangiri B, Khalaj-Kondori M, Asadollahi E, Sadeghizadeh
characteristics and genetic heterogeneity between carcinoma- M. Cancer-associated fibroblasts enhance cell proliferation and
associated fibroblasts and their paired normal fibroblasts in metastasis of colorectal cancer SW480 cells by provoking long
human breast cancer. PLoS One. 2013;8(4):e60321. noncoding RNA UCA1. J Cell Commun Signal. 2019;13(1):53-64.
148. Li H, Zhang J, Chen SW, Liu LL, Li L, Gao F, et al. Cancer- 163. Zhou Z, Zhou Q, Wu X, Xu S, Hu X, Tao X, et al. VCAM-
associated fibroblasts provide a suitable microenvironment for 1 secreted from cancer-associated fibroblasts enhances the
tumor development and progression in oral tongue squamous growth and invasion of lung cancer cells through AKT and
cancer. J Transl Med. 2015;13:198. MAPK signaling. Cancer Lett. 2020;473:62-73.
428 HU et al.

164. Zhuang J, Lu Q, Shen B, Huang X, Shen L, Zheng X, et al. 180. Zhang R, Qi F, Zhao F, Li G, Shao S, Zhang X, et al. Cancer-
TGFbeta1 secreted by cancer-associated fibroblasts induces associated fibroblasts enhance tumor-associated macrophages
epithelial-mesenchymal transition of bladder cancer cells enrichment and suppress NK cells function in colorectal can-
through lncRNA-ZEB2NAT. Sci Rep. 2015;5:11924. cer. Cell Death Dis. 2019;10(4):273.
165. Fessler E, Drost J, van Hooff SR, Linnekamp JF, Wang X, 181. Zellmer VR, Schnepp PM, Fracci SL, Tan X, Howe EN, Zhang
Jansen M, et al. TGFbeta signaling directs serrated adenomas to S. Tumor-induced Stromal STAT1 Accelerates Breast Can-
the mesenchymal colorectal cancer subtype. EMBO Mol Med. cer via Deregulating Tissue Homeostasis. Mol Cancer Res.
2016;8(7):745-60. 2017;15(5):585-97.
166. Heldin CH, Vanlandewijck M, Moustakas A. Regulation of 182. Wen S, Hou Y, Fu L, Xi L, Yang D, Zhao M, et al. Cancer-
EMT by TGFbeta in cancer. FEBS Lett. 2012;586(14):1959-70. associated fibroblast (CAF)-derived IL32 promotes breast can-
167. Zhang Y, Xia M, Jin K, Wang S, Wei H, Fan C, et al. Function of cer cell invasion and metastasis via integrin beta3-p38 MAPK
the c-Met receptor tyrosine kinase in carcinogenesis and asso- signalling. Cancer Lett. 2019;442:320-32.
ciated therapeutic opportunities. Mol Cancer. 2018;17(1):45. 183. Truong DD, Kratz A, Park JG, Barrientos ES, Saini H,
168. Yu B, Wu K, Wang X, Zhang J, Wang L, Jiang Y, et al. Nguyen T, et al. A Human Organotypic Microfluidic Tumor
Periostin secreted by cancer-associated fibroblasts promotes Model Permits Investigation of the Interplay between Patient-
cancer stemness in head and neck cancer by activating protein Derived Fibroblasts and Breast Cancer Cells. Cancer Res.
tyrosine kinase 7. Cell Death Dis. 2018;9(11):1082. 2019;79(12):3139-51.
169. Nakano M, Kikushige Y, Miyawaki K, Kunisaki Y, Mizuno S, 184. Hockemeyer K, Janetopoulos C, Terekhov A, Hofmeister W,
Takenaka K, et al. Dedifferentiation process driven by TGF-beta Vilgelm A, Costa L, et al. Engineered three-dimensional
signaling enhances stem cell properties in human colorectal microfluidic device for interrogating cell-cell interactions in the
cancer. Oncogene. 2019;38(6):780-93. tumor microenvironment. Biomicrofluidics. 2014;8(4):044105.
170. Jakowlew SB. Transforming growth factor-beta in cancer and 185. Ren Y, Jia HH, Xu YQ, Zhou X, Zhao XH, Wang YF, et al.
metastasis. Cancer Metastasis Rev. 2006;25(3):435-57. Paracrine and epigenetic control of CAF-induced metastasis:
171. Li Y, Wang R, Xiong S, Wang X, Zhao Z, Bai S, et al. Cancer- the role of HOTAIR stimulated by TGF-ss1 secretion. Mol Can-
associated fibroblasts promote the stemness of CD24(+) liver cer. 2018;17(1):5.
cells via paracrine signaling. J Mol Med (Berl). 2019;97(2):243- 186. Fernandez-Nogueira P, Mancino M, Fuster G, Lopez-Plana A,
55. Jauregui P, Almendro V, et al. Tumor-Associated Fibroblasts
172. Lenos KJ, Miedema DM, Lodestijn SC, Nijman LE, van Promote HER2-Targeted Therapy Resistance through FGFR2
den Bosch T, Romero Ros X, et al. Stem cell functional- Activation. Clin Cancer Res. 2020;26(6):1432-48.
ity is microenvironmentally defined during tumour expan- 187. Dou D, Ren X, Han M, Xu X, Ge X, Gu Y, et al. Cancer-
sion and therapy response in colon cancer. Nat Cell Biol. Associated Fibroblasts-Derived Exosomes Suppress Immune
2018;20(10):1193-202. Cell Function in Breast Cancer via the miR-92/PD-L1 Pathway.
173. Sung PJ, Rama N, Imbach J, Fiore S, Ducarouge B, Neves D, Front Immunol. 2020;11:2026.
et al. Cancer-Associated Fibroblasts Produce Netrin-1 to Con- 188. Yu Y, Xiao CH, Tan LD, Wang QS, Li XQ, Feng YM. Cancer-
trol Cancer Cell Plasticity. Cancer Res. 2019;79(14):3651-61. associated fibroblasts induce epithelial-mesenchymal transi-
174. Deying W, Feng G, Shumei L, Hui Z, Ming L, Hongqing W. tion of breast cancer cells through paracrine TGF-beta sig-
CAF-derived HGF promotes cell proliferation and drug resis- nalling. Br J Cancer. 2014;110(3):724-32.
tance by up-regulating the c-Met/PI3K/Akt and GRP78 sig- 189. Bellomo C, Caja L, Moustakas A. Transforming growth factor
nalling in ovarian cancer cells. Biosci Rep. 2017;37(2). beta as regulator of cancer stemness and metastasis. Br J Can-
175. Mishra R, Haldar S, Placencio V, Madhav A, Rohena-Rivera K, cer. 2016;115(7):761-9.
Agarwal P, et al. Stromal epigenetic alterations drive metabolic 190. Takai K, Le A, Weaver VM, Werb Z. Targeting the cancer-
and neuroendocrine prostate cancer reprogramming. J Clin associated fibroblasts as a treatment in triple-negative breast
Invest. 2018;128(10):4472-84. cancer. Oncotarget. 2016;7(50):82889-901.
176. Fiaschi T, Marini A, Giannoni E, Taddei ML, Gandellini P, De 191. Roswall P, Bocci M, Bartoschek M, Li H, Kristiansen G, Jansson
Donatis A, et al. Reciprocal metabolic reprogramming through S, et al. Microenvironmental control of breast cancer subtype
lactate shuttle coordinately influences tumor-stroma interplay. elicited through paracrine platelet-derived growth factor-CC
Cancer Res. 2012;72(19):5130-40. signaling. Nat Med. 2018;24(4):463-73.
177. Salem AF, Whitaker-Menezes D, Lin Z, Martinez-Outschoorn 192. Cazet AS, Hui MN, Elsworth BL, Wu SZ, Roden D, Chan CL,
UE, Tanowitz HB, Al-Zoubi MS, et al. Two-compartment tumor et al. Targeting stromal remodeling and cancer stem cell plastic-
metabolism: autophagy in the tumor microenvironment and ity overcomes chemoresistance in triple negative breast cancer.
oxidative mitochondrial metabolism (OXPHOS) in cancer cells. Nat Commun. 2018;9(1):2897.
Cell Cycle. 2012;11(13):2545-56. 193. Zervantonakis IK, Poskus MD, Scott AL, Selfors LM, Lin JR,
178. Yang L, Achreja A, Yeung TL, Mangala LS, Jiang D, Han C, et al. Dillon DA, et al. Fibroblast-tumor cell signaling limits HER2
Targeting Stromal Glutamine Synthetase in Tumors Disrupts kinase therapy response via activation of MTOR and antiapop-
Tumor Microenvironment-Regulated Cancer Cell Growth. Cell totic pathways. Proc Natl Acad Sci U S A. 2020;117(28):16500-8.
Metab. 2016;24(5):685-700. 194. Suh J, Kim DH, Lee YH, Jang JH, Surh YJ. Fibroblast growth
179. Ziani L, Chouaib S, Thiery J. Alteration of the Antitumor factor-2, derived from cancer-associated fibroblasts, stimulates
Immune Response by Cancer-Associated Fibroblasts. Front growth and progression of human breast cancer cells via FGFR1
Immunol. 2018;9:414. signaling. Mol Carcinog. 2020;59(9):1028-40.
HU et al. 429

195. Hu M, Yao J, Carroll DK, Weremowicz S, Chen H, Carrasco D, 210. Dolle L, Adriaenssens E, El Yazidi-Belkoura I, Le Bourhis X,
et al. Regulation of in situ to invasive breast carcinoma transi- Nurcombe V, Hondermarck H. Nerve growth factor recep-
tion. Cancer Cell. 2008;13(5):394-406. tors and signaling in breast cancer. Curr Cancer Drug Targets.
196. Osuala KO, Sameni M, Shah S, Aggarwal N, Simonait ML, 2004;4(6):463-70.
Franco OE, et al. Il-6 signaling between ductal carcinoma in 211. Hondermarck H. Potential role for NGF in breast cancer. Stem
situ cells and carcinoma-associated fibroblasts mediates tumor Cells. 2000;18(5):386-7.
cell growth and migration. BMC Cancer. 2015;15:584. 212. Dolle L, El Yazidi-Belkoura I, Adriaenssens E, Nurcombe
197. Studebaker AW, Storci G, Werbeck JL, Sansone P, Sasser AK, V, Hondermarck H. Nerve growth factor overexpres-
Tavolari S, et al. Fibroblasts isolated from common sites of sion and autocrine loop in breast cancer cells. Oncogene.
breast cancer metastasis enhance cancer cell growth rates and 2003;22(36):5592-601.
invasiveness in an interleukin-6-dependent manner. Cancer 213. Tsai YF, Tseng LM, Hsu CY, Yang MH, Chiu JH, Shyr
Res. 2008;68(21):9087-95. YM. Brain-derived neurotrophic factor (BDNF) -TrKB signal-
198. Huelsken J, Hanahan D. A Subset of Cancer-Associated Fibrob- ing modulates cancer-endothelial cells interaction and affects
lasts Determines Therapy Resistance. Cell. 2018;172(4):643-4. the outcomes of triple negative breast cancer. PLoS One.
199. Chan TS, Hsu CC, Pai VC, Liao WY, Huang SS, Tan KT, et al. 2017;12(6):e0178173.
Metronomic chemotherapy prevents therapy-induced stromal 214. Descamps S, Toillon RA, Adriaenssens E, Pawlowski V, Cool
activation and induction of tumor-initiating cells. J Exp Med. SM, Nurcombe V, et al. Nerve growth factor stimulates prolif-
2016;213(13):2967-88. eration and survival of human breast cancer cells through two
200. Allaoui R, Bergenfelz C, Mohlin S, Hagerling C, Salari K, Werb distinct signaling pathways. J Biol Chem. 2001;276(21):17864-70.
Z, et al. Cancer-associated fibroblast-secreted CXCL16 attracts 215. Chakravarthy R, Mnich K, Gorman AM. Nerve growth
monocytes to promote stroma activation in triple-negative factor (NGF)-mediated regulation of p75(NTR) expression
breast cancers. Nat Commun. 2016;7:13050. contributes to chemotherapeutic resistance in triple nega-
201. Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, tive breast cancer cells. Biochem Biophys Res Commun.
Delaunay T, Naeem R, et al. Stromal fibroblasts present in 2016;478(4):1541-7.
invasive human breast carcinomas promote tumor growth and 216. Romon R, Adriaenssens E, Lagadec C, Germain E,
angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. Hondermarck H, Le Bourhis X. Nerve growth factor pro-
2005;121(3):335-48. motes breast cancer angiogenesis by activating multiple
202. Boesch M, Onder L, Cheng HW, Novkovic M, Morbe U, Sopper pathways. Mol Cancer. 2010;9:157.
S, et al. Interleukin 7-expressing fibroblasts promote breast 217. Tomellini E, Touil Y, Lagadec C, Julien S, Ostyn P, Ziental-
cancer growth through sustenance of tumor cell stemness. Gelus N, et al. Nerve growth factor and proNGF simultaneously
Oncoimmunology. 2018;7(4):e1414129. promote symmetric self-renewal, quiescence, and epithelial to
203. Dvorak KM, Pettee KM, Rubinic-Minotti K, Su R, Nestor- mesenchymal transition to enlarge the breast cancer stem cell
Kalinoski A, Eisenmann KM. Carcinoma associated fibrob- compartment. Stem Cells. 2015;33(2):342-53.
lasts (CAFs) promote breast cancer motility by suppressing 218. Hondermarck H. Neurotrophins and their receptors in breast
mammalian Diaphanous-related formin-2 (mDia2). PLoS One. cancer. Cytokine Growth Factor Rev. 2012;23(6):357-65.
2018;13(3):e0195278. 219. Adriaenssens E, Vanhecke E, Saule P, Mougel A, Page A,
204. Ahirwar DK, Nasser MW, Ouseph MM, Elbaz M, Cuitino Romon R, et al. Nerve growth factor is a potential therapeutic
MC, Kladney RD, et al. Fibroblast-derived CXCL12 promotes target in breast cancer. Cancer Res. 2008;68(2):346-51.
breast cancer metastasis by facilitating tumor cell intravasa- 220. Tajbakhsh A, Mokhtari-Zaer A, Rezaee M, Afzaljavan F,
tion. Oncogene. 2018;37(32):4428-42. Rivandi M, Hassanian SM, et al. Therapeutic Potentials of
205. Liu Y, Zhang J, Sun X, Su Q, You C. Down-regulation of miR- BDNF/TrkB in Breast Cancer; Current Status and Perspectives.
29b in carcinoma associated fibroblasts promotes cell growth J Cell Biochem. 2017;118(9):2502-15.
and metastasis of breast cancer. Oncotarget. 2017;8(24):39559- 221. Jin Z, Lu Y, Wu X, Pan T, Yu Z, Hou J, et al. The cross-
70. talk between tumor cells and activated fibroblasts mediated
206. Demircioglu F, Wang J, Candido J, Costa ASH, Casado P, de by lactate/BDNF/TrkB signaling promotes acquired resistance
Luxan Delgado B, et al. Cancer associated fibroblast FAK regu- to anlotinib in human gastric cancer. Redox Biol. 2021;46:
lates malignant cell metabolism. Nat Commun. 2020;11(1):1290. 102076.
207. Xu K, Tian X, Oh SY, Movassaghi M, Naber SP, Kuperwasser 222. Jiffar T, Yilmaz T, Lee J, Miller Y, Feng L, El-Naggar A, et al.
C, et al. The fibroblast Tiam1-osteopontin pathway modu- Brain derived neutrophic factor (BDNF) coordinates lympho-
lates breast cancer invasion and metastasis. Breast Cancer Res. vascular metastasis through a fibroblast-governed paracrine
2016;18(1):14. axis in the tumor microenvironment. Cancer Cell Microenvi-
208. Ren J, Smid M, Iaria J, Salvatori DCF, van Dam H, Zhu HJ, ron. 2017;4(2).
et al. Cancer-associated fibroblast-derived Gremlin 1 promotes 223. Bloom AP, Jimenez-Andrade JM, Taylor RN, Castaneda-Corral
breast cancer progression. Breast Cancer Res. 2019;21(1):109. G, Kaczmarska MJ, Freeman KT, et al. Breast cancer-induced
209. Li H, Liu W, Zhang X, Wang Y. Cancer-associated fibroblast- bone remodeling, skeletal pain, and sprouting of sensory nerve
secreted collagen triple helix repeat containing-1 promotes fibers. J Pain. 2011;12(6):698-711.
breast cancer cell migration, invasiveness and epithelial- 224. Yang X, Li Y, Zou L, Zhu Z. Role of Exosomes in Crosstalk
mesenchymal transition by activating the Wnt/beta-catenin Between Cancer-Associated Fibroblasts and Cancer Cells.
pathway. Oncol Lett. 2021;22(6):814. Front Oncol. 2019;9:356.
430 HU et al.

225. Sansone P, Savini C, Kurelac I, Chang Q, Amato LB, Strillacci 239. Tomar D, Yadav AS, Kumar D, Bhadauriya G, Kundu GC. Non-
A, et al. Packaging and transfer of mitochondrial DNA coding RNAs as potential therapeutic targets in breast cancer.
via exosomes regulate escape from dormancy in hormonal Biochim Biophys Acta Gene Regul Mech. 2020;1863(4):194378.
therapy-resistant breast cancer. Proc Natl Acad Sci U S A. 240. Grillone K, Riillo C, Scionti F, Rocca R, Tradigo G, Guzzi PH,
2017;114(43):E9066-E75. et al. Non-coding RNAs in cancer: platforms and strategies for
226. Shah SH, Miller P, Garcia-Contreras M, Ao Z, Machlin L, Issa E, investigating the genomic “dark matter”. J Exp Clin Cancer Res.
et al. Hierarchical paracrine interaction of breast cancer asso- 2020;39(1):117.
ciated fibroblasts with cancer cells via hMAPK-microRNAs to 241. Xing Z, Lin A, Li C, Liang K, Wang S, Liu Y, et al.
drive ER-negative breast cancer phenotype. Cancer Biol Ther. lncRNA directs cooperative epigenetic regulation downstream
2015;16(11):1671-81. of chemokine signals. Cell. 2014;159(5):1110-25.
227. Gao Y, Li X, Zeng C, Liu C, Hao Q, Li W, et al. CD63(+) 242. Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong
Cancer-Associated Fibroblasts Confer Tamoxifen Resistance DJ, et al. Long non-coding RNA HOTAIR reprograms
to Breast Cancer Cells through Exosomal miR-22. Adv Sci chromatin state to promote cancer metastasis. Nature.
(Weinh). 2020;7(21):2002518. 2010;464(7291):1071-6.
228. Donnarumma E, Fiore D, Nappa M, Roscigno G, Adamo A, 243. Arun G, Diermeier S, Akerman M, Chang KC, Wilkinson
Iaboni M, et al. Cancer-associated fibroblasts release exosomal JE, Hearn S, et al. Differentiation of mammary tumors and
microRNAs that dictate an aggressive phenotype in breast can- reduction in metastasis upon Malat1 lncRNA loss. Genes Dev.
cer. Oncotarget. 2017;8(12):19592-608. 2016;30(1):34-51.
229. Wang H, Wei H, Wang J, Li L, Chen A, Li Z. MicroRNA-181d- 244. Sha S, Yuan D, Liu Y, Han B, Zhong N. Targeting long non-
5p-Containing Exosomes Derived from CAFs Promote EMT by coding RNA DANCR inhibits triple negative breast cancer pro-
Regulating CDX2/HOXA5 in Breast Cancer. Mol Ther Nucleic gression. Biol Open. 2017;6(9):1310-6.
Acids. 2020;19:654-67. 245. Mobarra N, Shafiee A, Rad SM, Tasharrofi N, Soufi-Zomorod
230. Liu Y, Yang Y, Du J, Lin D, Li F. MiR-3613-3p from carcinoma- M, Hafizi M, et al. Overexpression of microRNA-16 declines
associated fibroblasts exosomes promoted breast cancer cell cellular growth, proliferation and induces apoptosis in human
proliferation and metastasis by regulating SOCS2 expression. breast cancer cells. In Vitro Cell Dev Biol Anim. 2015;51(6):604-
IUBMB Life. 2020;72(8):1705-14. 11.
231. Chen B, Sang Y, Song X, Zhang D, Wang L, Zhao W, 246. El Helou R, Pinna G, Cabaud O, Wicinski J, Bhajun R, Guyon
et al. Exosomal miR-500a-5p derived from cancer-associated L, et al. miR-600 Acts as a Bimodal Switch that Regulates
fibroblasts promotes breast cancer cell proliferation and Breast Cancer Stem Cell Fate through WNT Signaling. Cell Rep.
metastasis through targeting USP28. Theranostics. 2021;11(8): 2017;18(9):2256-68.
3932-47. 247. Xu B, Zhang X, Wang S, Shi B. MiR-449a suppresses cell migra-
232. Yan Z, Sheng Z, Zheng Y, Feng R, Xiao Q, Shi L, et al. tion and invasion by targeting PLAGL2 in breast cancer. Pathol
Cancer-associated fibroblast-derived exosomal miR-18b pro- Res Pract. 2018;214(5):790-5.
motes breast cancer invasion and metastasis by regulating 248. Zhao J, Zou H, Han C, Ma J, Zhao J, Tang J. Circlular RNA
TCEAL7. Cell Death Dis. 2021;12(12):1120. BARD1 (Hsa_circ_0001098) overexpression in breast cancer
233. Tao S, Li H, Ma X, Ma Y, He J, Gao Y, et al. Elevat- cells with TCDD treatment could promote cell apoptosis via
ing microRNA-1-3p shuttled by cancer-associated fibroblasts- miR-3942/BARD1 axis. Cell Cycle. 2018;17(24):2731-44.
derived extracellular vesicles suppresses breast cancer progres- 249. Haga CL, Velagapudi SP, Childs-Disney JL, Strivelli J, Disney
sion and metastasis by inhibiting GLIS1. Cancer Gene Ther. MD, Phinney DG. Rapid Generation of miRNA Inhibitor Leads
2021;28(6):634-48. by Bioinformatics and Efficient High-Throughput Screening
234. Liu Y, Hua F, Zhan Y, Yang Y, Xie J, Cheng Y, et al. Carcinoma Methods. Methods Mol Biol. 2017;1517:179-98.
associated fibroblasts small extracellular vesicles with low miR- 250. Li Y, Disney MD. Precise Small Molecule Degradation of a Non-
7641 promotes breast cancer stemness and glycolysis by HIF- coding RNA Identifies Cellular Binding Sites and Modulates an
1alpha. Cell Death Discov. 2021;7(1):176. Oncogenic Phenotype. ACS Chem Biol. 2018;13(11):3065-71.
235. Boelens MC, Wu TJ, Nabet BY, Xu B, Qiu Y, Yoon T, et al. Exo- 251. Singh R, Gupta SC, Peng WX, Zhou N, Pochampally R, Atfi
some transfer from stromal to breast cancer cells regulates ther- A, et al. Regulation of alternative splicing of Bcl-x by BC200
apy resistance pathways. Cell. 2014;159(3):499-513. contributes to breast cancer pathogenesis. Cell Death Dis.
236. Nabet BY, Qiu Y, Shabason JE, Wu TJ, Yoon T, Kim BC, 2016;7(6):e2262.
et al. Exosome RNA Unshielding Couples Stromal Activa- 252. Koirala P, Huang J, Ho TT, Wu F, Ding X, Mo YY. LncRNA
tion to Pattern Recognition Receptor Signaling in Cancer. Cell. AK023948 is a positive regulator of AKT. Nat Commun.
2017;170(2):352-66 e13. 2017;8:14422.
237. Li Y, Zhao Z, Liu W, Li X. SNHG3 Functions as miRNA Sponge 253. Peng WX, Huang JG, Yang L, Gong AH, Mo YY. Linc-RoR
to Promote Breast Cancer Cells Growth Through the Metabolic promotes MAPK/ERK signaling and confers estrogen-
Reprogramming. Appl Biochem Biotechnol. 2020;191(3):1084- independent growth of breast cancer. Mol Cancer.
99. 2017;16(1):161.
238. Yi Y, Wu M, Zeng H, Hu W, Zhao C, Xiong M, et al. Tumor- 254. Hannafon BN, Cai A, Calloway CL, Xu YF, Zhang R, Fung
Derived Exosomal Non-Coding RNAs: The Emerging Mech- KM, et al. miR-23b and miR-27b are oncogenic microRNAs in
anisms and Potential Clinical Applications in Breast Cancer. breast cancer: evidence from a CRISPR/Cas9 deletion study.
Front Oncol. 2021;11:738945. BMC Cancer. 2019;19(1):642.
HU et al. 431

255. Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou through the MYC-dependent metabolic reprogramming of stro-
MR, Chiu E, et al. Exosomes mediate stromal mobilization of mal cells. Nat Cell Biol. 2018;20(5):597-609.
autocrine Wnt-PCP signaling in breast cancer cell migration. 268. Sun K, Tang S, Hou Y, Xi L, Chen Y, Yin J, et al. Oxidized ATM-
Cell. 2012;151(7):1542-56. mediated glycolysis enhancement in breast cancer-associated
256. Chen Y, Zeng C, Zhan Y, Wang H, Jiang X, Li W. Aberrant low fibroblasts contributes to tumor invasion through lactate as
expression of p85alpha in stromal fibroblasts promotes breast metabolic coupling. EBioMedicine. 2019;41:370-83.
cancer cell metastasis through exosome-mediated paracrine 269. Chen Y, Cai L, Guo X, Li Z, Liao X, Zhang X, et al. HMGB1-
Wnt10b. Oncogene. 2017;36(33):4692-705. activated fibroblasts promote breast cancer cells metastasis via
257. Shimoda M, Principe S, Jackson HW, Luga V, Fang H, RAGE/aerobic glycolysis. Neoplasma. 2021;68(1):71-8.
Molyneux SD, et al. Loss of the Timp gene family is sufficient 270. Robertson C. The extracellular matrix in breast cancer predicts
for the acquisition of the CAF-like cell state. Nat Cell Biol. prognosis through composition, splicing, and crosslinking. Exp
2014;16(9):889-901. Cell Res. 2016;343(1):73-81.
258. Xi L, Peng M, Liu S, Liu Y, Wan X, Hou Y, et al. Hypoxia- 271. Kim SH, Lee HY, Jung SP, Kim S, Lee JE, Nam SJ, et al. Role of
stimulated ATM activation regulates autophagy-associated secreted type I collagen derived from stromal cells in two breast
exosome release from cancer-associated fibroblasts to promote cancer cell lines. Oncol Lett. 2014;8(2):507-12.
cancer cell invasion. J Extracell Vesicles. 2021;10(11):e12146. 272. Liu J, Shen JX, Wu HT, Li XL, Wen XF, Du CW, et al. Colla-
259. Benny S, Mishra R, Manojkumar MK, Aneesh TP. From War- gen 1A1 (COL1A1) promotes metastasis of breast cancer and
burg effect to Reverse Warburg effect; the new horizons of anti- is a potential therapeutic target. Discov Med. 2018;25(139):
cancer therapy. Med Hypotheses. 2020;144:110216. 211-23.
260. Martinez-Outschoorn UE, Whitaker-Menezes D, Pavlides S, 273. Insua-Rodriguez J, Oskarsson T. The extracellular matrix in
Chiavarina B, Bonuccelli G, Casey T, et al. The autophagic breast cancer. Adv Drug Deliv Rev. 2016;97:41-55.
tumor stroma model of cancer or “battery-operated tumor 274. Mulsow JJ, Watson RW, Fitzpatrick JM, O’Connell PR. Trans-
growth”: A simple solution to the autophagy paradox. Cell forming growth factor-beta promotes pro-fibrotic behavior by
Cycle. 2010;9(21):4297-306. serosal fibroblasts via PKC and ERK1/2 mitogen activated pro-
261. Martinez-Outschoorn UE, Prisco M, Ertel A, Tsirigos A, Lin tein kinase cell signaling. Ann Surg. 2005;242(6):880-7, discus-
Z, Pavlides S, et al. Ketones and lactate increase cancer cell sion 7-9.
“stemness,” driving recurrence, metastasis and poor clinical 275. Czaja MJ, Weiner FR, Eghbali M, Giambrone MA, Eghbali
outcome in breast cancer: achieving personalized medicine via M, Zern MA. Differential effects of gamma-interferon on
Metabolo-Genomics. Cell Cycle. 2011;10(8):1271-86. collagen and fibronectin gene expression. J Biol Chem.
262. Martinez-Outschoorn UE, Balliet RM, Rivadeneira DB, 1987;262(27):13348-51.
Chiavarina B, Pavlides S, Wang C, et al. Oxidative stress in can- 276. Erdogan B, Ao M, White LM, Means AL, Brewer BM, Yang
cer associated fibroblasts drives tumor-stroma co-evolution: L, et al. Cancer-associated fibroblasts promote directional
A new paradigm for understanding tumor metabolism, the cancer cell migration by aligning fibronectin. J Cell Biol.
field effect and genomic instability in cancer cells. Cell Cycle. 2017;216(11):3799-816.
2010;9(16):3256-76. 277. Yao ES, Zhang H, Chen YY, Lee B, Chew K, Moore D, et al.
263. Pavlides S, Tsirigos A, Migneco G, Whitaker-Menezes D, Increased beta1 integrin is associated with decreased survival
Chiavarina B, Flomenberg N, et al. The autophagic tumor in invasive breast cancer. Cancer Res. 2007;67(2):659-64.
stroma model of cancer: Role of oxidative stress and ketone 278. Balanis N, Wendt MK, Schiemann BJ, Wang Z, Schiemann
production in fueling tumor cell metabolism. Cell Cycle. WP, Carlin CR. Epithelial to mesenchymal transition promotes
2010;9(17):3485-505. breast cancer progression via a fibronectin-dependent STAT3
264. Pavlides S, Tsirigos A, Vera I, Flomenberg N, Frank PG, signaling pathway. J Biol Chem. 2013;288(25):17954-67.
Casimiro MC, et al. Loss of stromal caveolin-1 leads to oxidative 279. Li CL, Yang D, Cao X, Wang F, Hong DY, Wang J,
stress, mimics hypoxia and drives inflammation in the tumor et al. Fibronectin induces epithelial-mesenchymal transition
microenvironment, conferring the “reverse Warburg effect”: a in human breast cancer MCF-7 cells via activation of calpain.
transcriptional informatics analysis with validation. Cell Cycle. Oncol Lett. 2017;13(5):3889-95.
2010;9(11):2201-19. 280. Hong H, Zhou T, Fang S, Jia M, Xu Z, Dai Z, et al. Pigment
265. Guido C, Whitaker-Menezes D, Capparelli C, Balliet R, epithelium-derived factor (PEDF) inhibits breast cancer metas-
Lin Z, Pestell RG, et al. Metabolic reprogramming of tasis by down-regulating fibronectin. Breast Cancer Res Treat.
cancer-associated fibroblasts by TGF-beta drives tumor 2014;148(1):61-72.
growth: connecting TGF-beta signaling with “Warburg-like” 281. He ZH, Lei Z, Zhen Y, Gong W, Huang B, Yuan Y, et al.
cancer metabolism and L-lactate production. Cell Cycle. Adeno-associated virus-mediated expression of recombinant
2012;11(16):3019-35. CBD-HepII polypeptide of human fibronectin inhibits metasta-
266. Yu T, Yang G, Hou Y, Tang X, Wu C, Wu XA, et al. Cytoplasmic sis of breast cancer. Breast Cancer Res Treat. 2014;143(1):33-45.
GPER translocation in cancer-associated fibroblasts mediates 282. Park CC, Zhang H, Pallavicini M, Gray JW, Baehner F, Park
cAMP/PKA/CREB/glycolytic axis to confer tumor cells with CJ, et al. Beta1 integrin inhibitory antibody induces apoptosis
multidrug resistance. Oncogene. 2017;36(15):2131-45. of breast cancer cells, inhibits growth, and distinguishes malig-
267. Yan W, Wu X, Zhou W, Fong MY, Cao M, Liu J, et al. Cancer- nant from normal phenotype in three dimensional cultures and
cell-secreted exosomal miR-105 promotes tumour growth in vivo. Cancer Res. 2006;66(3):1526-35.
432 HU et al.

283. Pickup MW, Mouw JK, Weaver VM. The extracellular 300. Karagiannis GS, Poutahidis T, Erdman SE, Kirsch R, Riddell
matrix modulates the hallmarks of cancer. EMBO Rep. RH, Diamandis EP. Cancer-associated fibroblasts drive the pro-
2014;15(12):1243-53. gression of metastasis through both paracrine and mechanical
284. Baker AM, Bird D, Lang G, Cox TR, Erler JT. Lysyl oxidase pressure on cancer tissue. Mol Cancer Res. 2012;10(11):1403-18.
enzymatic function increases stiffness to drive colorectal can- 301. Kumar S, Weaver VM. Mechanics, malignancy, and metasta-
cer progression through FAK. Oncogene. 2013;32(14):1863-8. sis: the force journey of a tumor cell. Cancer Metastasis Rev.
285. Provenzano PP, Cuevas C, Chang AE, Goel VK, Von Hoff DD, 2009;28(1-2):113-27.
Hingorani SR. Enzymatic targeting of the stroma ablates physi- 302. Liao D, Luo Y, Markowitz D, Xiang R, Reisfeld RA. Cancer
cal barriers to treatment of pancreatic ductal adenocarcinoma. associated fibroblasts promote tumor growth and metastasis
Cancer Cell. 2012;21(3):418-29. by modulating the tumor immune microenvironment in a 4T1
286. Provenzano PP, Eliceiri KW, Campbell JM, Inman DR, White murine breast cancer model. PLoS One. 2009;4(11):e7965.
JG, Keely PJ. Collagen reorganization at the tumor-stromal 303. Pan Y, Yu Y, Wang X, Zhang T. Tumor-Associated
interface facilitates local invasion. BMC Med. 2006;4(1):38. Macrophages in Tumor Immunity. Front Immunol.
287. Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, 2020;11:583084.
et al. Matrix crosslinking forces tumor progression by enhanc- 304. Gok Yavuz B, Gunaydin G, Gedik ME, Kosemehmetoglu K,
ing integrin signaling. Cell. 2009;139(5):891-906. Karakoc D, Ozgur F, et al. Cancer associated fibroblasts sculpt
288. Wells RG. The role of matrix stiffness in regulating cell behav- tumour microenvironment by recruiting monocytes and induc-
ior. Hepatology. 2008;47(4):1394-400. ing immunosuppressive PD-1(+) TAMs. Sci Rep. 2019;9(1):3172.
289. Mouw JK, Yui Y, Damiano L, Bainer RO, Lakins JN, Acerbi 305. Apte RS, Chen DS, Ferrara N. VEGF in Signaling and Disease:
I, et al. Tissue mechanics modulate microRNA-dependent Beyond Discovery and Development. Cell. 2019;176(6):1248-64.
PTEN expression to regulate malignant progression. Nat Med. 306. Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC, Lu N, et al.
2014;20(4):360-7. Tumor induction of VEGF promoter activity in stromal cells.
290. Pickup MW, Laklai H, Acerbi I, Owens P, Gorska AE, Chytil Cell. 1998;94(6):715-25.
A, et al. Stromally derived lysyl oxidase promotes metastasis 307. De Francesco EM, Lappano R, Santolla MF, Marsico S, Caruso
of transforming growth factor-beta-deficient mouse mammary A, Maggiolini M. HIF-1alpha/GPER signaling mediates the
carcinomas. Cancer Res. 2013;73(17):5336-46. expression of VEGF induced by hypoxia in breast cancer asso-
291. Tang X, Hou Y, Yang G, Wang X, Tang S, Du YE, et al. ciated fibroblasts (CAFs). Breast Cancer Res. 2013;15(4):R64.
Stromal miR-200s contribute to breast cancer cell invasion 308. Ren J, Guo H, Wu H, Tian T, Dong D, Zhang Y, et al. GPER
through CAF activation and ECM remodeling. Cell Death Dif- in CAFs regulates hypoxia-driven breast cancer invasion in a
fer. 2016;23(1):132-45. CTGF-dependent manner. Oncol Rep. 2015;33(4):1929-37.
292. El-Mohri H, Wu Y, Mohanty S, Ghosh G. Impact of matrix stiff- 309. Al-Jomah N, Al-Mohanna FH, Aboussekhra A. Tocilizumab
ness on fibroblast function. Mater Sci Eng C Mater Biol Appl. suppresses the pro-carcinogenic effects of breast cancer-
2017;74:146-51. associated fibroblasts through inhibition of the STAT3/AUF1
293. Asano S, Ito S, Takahashi K, Furuya K, Kondo M, Sokabe M, pathway. Carcinogenesis. 2021;42(12):1439-48.
et al. Matrix stiffness regulates migration of human lung fibrob- 310. Wan X, Guan S, Hou Y, Qin Y, Zeng H, Yang L, et al. FOSL2 pro-
lasts. Physiol Rep. 2017;5(9). motes VEGF-independent angiogenesis by transcriptionnally
294. Basset P, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM, et al. activating Wnt5a in breast cancer-associated fibroblasts. Ther-
A novel metalloproteinase gene specifically expressed in stro- anostics. 2021;11(10):4975-91.
mal cells of breast carcinomas. Nature. 1990;348(6303):699-704. 311. Sewell-Loftin MK, Bayer SVH, Crist E, Hughes T, Joison
295. Tetu B, Brisson J, Wang CS, Lapointe H, Beaudry G, Blanchette SM, Longmore GD, et al. Cancer-associated fibroblasts sup-
C, et al. The influence of MMP-14, TIMP-2 and MMP-2 port vascular growth through mechanical force. Sci Rep.
expression on breast cancer prognosis. Breast Cancer Res. 2017;7(1):12574.
2006;8(3):R28. 312. Choi YP, Lee JH, Gao MQ, Kim BG, Kang S, Kim SH, et al.
296. Radisky ES, Radisky DC. Matrix metalloproteinases as breast Cancer-associated fibroblast promote transmigration through
cancer drivers and therapeutic targets. Front Biosci (Landmark endothelial brain cells in three-dimensional in vitro models. Int
Ed). 2015;20:1144-63. J Cancer. 2014;135(9):2024-33.
297. Stuelten CH, DaCosta Byfield S, Arany PR, Karpova TS, Stetler- 313. Amornsupak K, Insawang T, Thuwajit P, P OC, Eccles SA,
Stevenson WG, Roberts AB. Breast cancer cells induce stromal Thuwajit C. Cancer-associated fibroblasts induce high mobil-
fibroblasts to express MMP-9 via secretion of TNF-alpha and ity group box 1 and contribute to resistance to doxorubicin in
TGF-beta. J Cell Sci. 2005;118(Pt 10):2143-53. breast cancer cells. BMC Cancer. 2014;14:955.
298. Saad S, Gottlieb DJ, Bradstock KF, Overall CM, Bendall LJ. 314. Nguyen M, De Ninno A, Mencattini A, Mermet-Meillon F,
Cancer cell-associated fibronectin induces release of matrix Fornabaio G, Evans SS, et al. Dissecting Effects of Anti-cancer
metalloproteinase-2 from normal fibroblasts. Cancer Res. Drugs and Cancer-Associated Fibroblasts by On-Chip Recon-
2002;62(1):283-9. stitution of Immunocompetent Tumor Microenvironments.
299. Cui Q, Wang B, Li K, Sun H, Hai T, Zhang Y, et al. Upregulating Cell Rep. 2018;25(13):3884-93 e3.
MMP-1 in carcinoma-associated fibroblasts reduces the efficacy 315. Houthuijzen JM, Jonkers J. Cancer-associated fibroblasts as
of Taxotere on breast cancer synergized by Collagen IV. Oncol key regulators of the breast cancer tumor microenvironment.
Lett. 2018;16(3):3537-44. Cancer Metastasis Rev. 2018;37(4):577-97.
HU et al. 433

316. Duyverman AM, Steller EJ, Fukumura D, Jain RK, Duda DG. tiates chemotherapy by decompressing tumour blood vessels.
Studying primary tumor-associated fibroblast involvement in Nat Commun. 2013;4:2516.
cancer metastasis in mice. Nat Protoc. 2012;7(4):756-62. 332. Coulson R, Liew SH, Connelly AA, Yee NS, Deb S, Kumar B,
317. Rhim AD, Oberstein PE, Thomas DH, Mirek ET, Palermo et al. The angiotensin receptor blocker, Losartan, inhibits mam-
CF, Sastra SA, et al. Stromal elements act to restrain, rather mary tumor development and progression to invasive carci-
than support, pancreatic ductal adenocarcinoma. Cancer Cell. noma. Oncotarget. 2017;8(12):18640-56.
2014;25(6):735-47. 333. Chronopoulos A, Robinson B, Sarper M, Cortes E,
318. Wu SZ, Roden DL, Wang C, Holliday H, Harvey K, Cazet Auernheimer V, Lachowski D, et al. ATRA mechanically
AS, et al. Stromal cell diversity associated with immune reprograms pancreatic stellate cells to suppress matrix
evasion in human triple-negative breast cancer. EMBO J. remodelling and inhibit cancer cell invasion. Nat Commun.
2020;39(19):e104063. 2016;7:12630.
319. Giussani M, Merlino G, Cappelletti V, Tagliabue E, Daidone 334. Froeling FE, Feig C, Chelala C, Dobson R, Mein CE, Tuveson
MG. Tumor-extracellular matrix interactions: Identification of DA, et al. Retinoic acid-induced pancreatic stellate cell qui-
tools associated with breast cancer progression. Semin Cancer escence reduces paracrine Wnt-beta-catenin signaling to slow
Biol. 2015;35:3-10. tumor progression. Gastroenterology. 2011;141(4):1486-97, 97 e1-
320. Loktev A, Lindner T, Mier W, Debus J, Altmann A, Jager D, 14.
et al. A Tumor-Imaging Method Targeting Cancer-Associated 335. Ene-Obong A, Clear AJ, Watt J, Wang J, Fatah R, Riches
Fibroblasts. J Nucl Med. 2018;59(9):1423-9. JC, et al. Activated pancreatic stellate cells sequester CD8+ T
321. Strell C, Paulsson J, Jin SB, Tobin NP, Mezheyeuski A, Roswall cells to reduce their infiltration of the juxtatumoral compart-
P, et al. Impact of Epithelial-Stromal Interactions on Peritu- ment of pancreatic ductal adenocarcinoma. Gastroenterology.
moral Fibroblasts in Ductal Carcinoma in Situ. J Natl Cancer 2013;145(5):1121-32.
Inst. 2019;111(9):983-95. 336. Sherman MH, Yu RT, Engle DD, Ding N, Atkins AR, Tiriac
322. Fearon DT. The carcinoma-associated fibroblast expressing H, et al. Vitamin D receptor-mediated stromal reprogramming
fibroblast activation protein and escape from immune surveil- suppresses pancreatitis and enhances pancreatic cancer ther-
lance. Cancer Immunol Res. 2014;2(3):187-93. apy. Cell. 2014;159(1):80-93.
323. Kakarla S, Song XT, Gottschalk S. Cancer-associated fibroblasts 337. Park CY, Min KN, Son JY, Park SY, Nam JS, Kim DK, et al.
as targets for immunotherapy. Immunotherapy. 2012;4(11):1129- An novel inhibitor of TGF-beta type I receptor, IN-1130, blocks
38. breast cancer lung metastasis through inhibition of epithelial-
324. Duperret EK, Trautz A, Ammons D, Perales-Puchalt A, Wise mesenchymal transition. Cancer Lett. 2014;351(1):72-80.
MC, Yan J, et al. Alteration of the Tumor Stroma Using a Con- 338. Fang Y, Chen Y, Yu L, Zheng C, Qi Y, Li Z, et al. Inhibition of
sensus DNA Vaccine Targeting Fibroblast Activation Protein breast cancer metastases by a novel inhibitor of TGFbeta recep-
(FAP) Synergizes with Antitumor Vaccine Therapy in Mice. tor 1. J Natl Cancer Inst. 2013;105(1):47-58.
Clin Cancer Res. 2018;24(5):1190-201. 339. Ehata S, Hanyu A, Fujime M, Katsuno Y, Fukunaga E, Goto K,
325. Gottschalk S, Yu F, Ji M, Kakarla S, Song XT. A vaccine that et al. Ki26894, a novel transforming growth factor-beta type I
co-targets tumor cells and cancer associated fibroblasts results receptor kinase inhibitor, inhibits in vitro invasion and in vivo
in enhanced antitumor activity by inducing antigen spreading. bone metastasis of a human breast cancer cell line. Cancer Sci.
PLoS One. 2013;8(12):e82658. 2007;98(1):127-33.
326. Wang J, Li Q, Li X, Yuan W, Huang S, Cai S, et al. A novel 340. Bandyopadhyay A, Agyin JK, Wang L, Tang Y, Lei X, Story
FAPalpha-based Z-Gly-Pro epirubicin prodrug for improving BM, et al. Inhibition of pulmonary and skeletal metastasis
tumor-targeting chemotherapy. Eur J Pharmacol. 2017;815:166- by a transforming growth factor-beta type I receptor kinase
72. inhibitor. Cancer Res. 2006;66(13):6714-21.
327. Li A, Chen P, Leng Y, Kang J. Histone deacetylase 6 regulates 341. Formenti SC, Lee P, Adams S, Goldberg JD, Li X, Xie MW, et al.
the immunosuppressive properties of cancer-associated fibrob- Focal Irradiation and Systemic TGFbeta Blockade in Metastatic
lasts in breast cancer through the STAT3-COX2-dependent Breast Cancer. Clin Cancer Res. 2018;24(11):2493-504.
pathway. Oncogene. 2018;37(45):5952-66. 342. Holmgaard RB, Schaer DA, Li Y, Castaneda SP, Murphy MY,
328. Li C, Cao L, Xu C, Liu F, Xiang G, Liu X, et al. The immuno- Xu X, et al. Targeting the TGFbeta pathway with galunis-
histochemical expression and potential prognostic value of ertib, a TGFbetaRI small molecule inhibitor, promotes anti-
HDAC6 and AR in invasive breast cancer. Hum Pathol. tumor immunity leading to durable, complete responses, as
2018;75:16-25. monotherapy and in combination with checkpoint blockade.
329. Park Y, Lee KS, Park SY, Kim JH, Kang EY, Kim SW, et al. J Immunother Cancer. 2018;6(1):47.
Potential Prognostic Value of Histone Deacetylase 6 and Acety- 343. Jochems C, Tritsch SR, Pellom ST, Su Z, Soon-Shiong P, Wong
lated Heat-Shock Protein 90 in Early-Stage Breast Cancer. J HC, et al. Analyses of functions of an anti-PD-L1/TGFbetaR2
Breast Cancer. 2015;18(3):249-55. bispecific fusion protein (M7824). Oncotarget. 2017;8(43):75217-
330. Ramaswamy B, Lu Y, Teng KY, Nuovo G, Li X, Shapiro CL, et al. 31.
Hedgehog signaling is a novel therapeutic target in tamoxifen- 344. David JM, Dominguez C, McCampbell KK, Gulley JL,
resistant breast cancer aberrantly activated by PI3K/AKT path- Schlom J, Palena C. A novel bifunctional anti-PD-L1/TGF-
way. Cancer Res. 2012;72(19):5048-59. beta Trap fusion protein (M7824) efficiently reverts mesenchy-
331. Chauhan VP, Martin JD, Liu H, Lacorre DA, Jain SR, Kozin SV, malization of human lung cancer cells. Oncoimmunology.
et al. Angiotensin inhibition enhances drug delivery and poten- 2017;6(10):e1349589.
434 HU et al.

345. Strauss J, Heery CR, Schlom J, Madan RA, Cao L, Kang 356. Van Cutsem E, Tempero MA, Sigal D, Oh DY, Fazio
Z, et al. Phase I Trial of M7824 (MSB0011359C), a Bifunc- N, Macarulla T, et al. Randomized Phase III Trial of
tional Fusion Protein Targeting PD-L1 and TGFbeta, in Pegvorhyaluronidase Alfa With Nab-Paclitaxel Plus Gemc-
Advanced Solid Tumors. Clin Cancer Res. 2018;24(6):1287- itabine for Patients With Hyaluronan-High Metastatic Pancre-
95. atic Adenocarcinoma. J Clin Oncol. 2020;38(27):3185-94.
346. Formisano L, Lu Y, Servetto A, Hanker AB, Jansen VM, 357. Femel J, Huijbers EJ, Saupe F, Cedervall J, Zhang L, Roswall
Bauer JA, et al. Aberrant FGFR signaling mediates resistance P, et al. Therapeutic vaccination against fibronectin ED-A
to CDK4/6 inhibitors in ER+ breast cancer. Nat Commun. attenuates progression of metastatic breast cancer. Oncotarget.
2019;10(1):1373. 2014;5(23):12418-27.
347. Gavine PR, Mooney L, Kilgour E, Thomas AP, Al-Kadhimi K, 358. Gagliano T, Shah K, Gargani S, Lao L, Alsaleem M, Chen J, et al.
Beck S, et al. AZD4547: an orally bioavailable, potent, and selec- PIK3Cdelta expression by fibroblasts promotes triple-negative
tive inhibitor of the fibroblast growth factor receptor tyrosine breast cancer progression. J Clin Invest. 2020;130(6):3188-204.
kinase family. Cancer Res. 2012;72(8):2045-56. 359. Yin C, Evason KJ, Asahina K, Stainier DY. Hepatic stellate cells
348. Kang J, Choi YJ, Seo BY, Jo U, Park SI, Kim YH, et al. A in liver development, regeneration, and cancer. J Clin Invest.
Selective FGFR inhibitor AZD4547 suppresses RANKL/M- 2013;123(5):1902-10.
CSF/OPG-dependent ostoclastogenesis and breast cancer 360. Ayala G, Tuxhorn JA, Wheeler TM, Frolov A, Scardino
growth in the metastatic bone microenvironment. Sci Rep. PT, Ohori M, et al. Reactive stroma as a predictor of
2019;9(1):8726. biochemical-free recurrence in prostate cancer. Clin Cancer
349. Sootome H, Fujita H, Ito K, Ochiiwa H, Fujioka Y, Ito K, et al. Res. 2003;9(13):4792-801.
Futibatinib Is a Novel Irreversible FGFR 1-4 Inhibitor That 361. Biffi G, Oni TE, Spielman B, Hao Y, Elyada E, Park Y, et al.
Shows Selective Antitumor Activity against FGFR-Deregulated IL1-Induced JAK/STAT Signaling Is Antagonized by TGFbeta
Tumors. Cancer Res. 2020;80(22):4986-97. to Shape CAF Heterogeneity in Pancreatic Ductal Adenocarci-
350. Nakanishi Y, Akiyama N, Tsukaguchi T, Fujii T, Sakata K, Sase noma. Cancer Discov. 2019;9(2):282-301.
H, et al. The fibroblast growth factor receptor genetic status 362. Wang XM, Yu DM, McCaughan GW, Gorrell MD. Fibrob-
as a potential predictor of the sensitivity to CH5183284/Debio last activation protein increases apoptosis, cell adhesion, and
1347, a novel selective FGFR inhibitor. Mol Cancer Ther. migration by the LX-2 human stellate cell line. Hepatology.
2014;13(11):2547-58. 2005;42(4):935-45.
351. Zhang G, He Y, Liu Y, Du Y, Yang C, Gao F. Reduced hyaluro- 363. Arnold JN, Magiera L, Kraman M, Fearon DT. Tumoral
nan cross-linking induces breast cancer malignancy in a CAF- immune suppression by macrophages expressing fibrob-
dependent manner. Cell Death Dis. 2021;12(6):586. last activation protein-alpha and heme oxygenase-1. Cancer
352. Auvinen P, Tammi R, Parkkinen J, Tammi M, Agren U, Immunol Res. 2014;2(2):121-6.
Johansson R, et al. Hyaluronan in peritumoral stroma and 364. Pena C, Cespedes MV, Lindh MB, Kiflemariam S, Mezheyeuski
malignant cells associates with breast cancer spreading and A, Edqvist PH, et al. STC1 expression by cancer-associated
predicts survival. Am J Pathol. 2000;156(2):529-36. fibroblasts drives metastasis of colorectal cancer. Cancer Res.
353. Clift R, Souratha J, Garrovillo SA, Zimmerman S, Blouw B. 2013;73(4):1287-97.
Remodeling the Tumor Microenvironment Sensitizes Breast 365. Cohen AW, Hnasko R, Schubert W, Lisanti MP. Role of
Tumors to Anti-Programmed Death-Ligand 1 Immunotherapy. caveolae and caveolins in health and disease. Physiol Rev.
Cancer Res. 2019;79(16):4149-59. 2004;84(4):1341-79.
354. Marusyk A, Tabassum DP, Janiszewska M, Place AE, Trinh
A, Rozhok AI, et al. Spatial Proximity to Fibroblasts Impacts
Molecular Features and Therapeutic Sensitivity of Breast How to cite this article: Hu D, Li Z, Zheng B, Lin
Cancer Cells Influencing Clinical Outcomes. Cancer Res.
X, Pan Y, Gong P, et al. Cancer-Associated
2016;76(22):6495-506.
Fibroblasts in Breast Cancer: Challenges and
355. Infante JR, Korn RL, Rosen LS, LoRusso P, Dychter SS, Zhu
J, et al. Phase 1 trials of PEGylated recombinant human Opportunities. Cancer Commun. 2022;42:401–434.
hyaluronidase PH20 in patients with advanced solid tumours. https://doi.org/10.1002/cac2.12291
Br J Cancer. 2018;118(2):153-61.