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Tumors are the main cause of death in humans. The tumor microenvironment plays an important role in regulating tumor behavior through cross-talk between tumor cells and other cell types. Cancer-associated fibroblasts (CAFs) are a main cell type in the tumor microenvironment and play a key role in tumor progression through remodeling of the extracellular matrix and secretion of growth factors. The document aims to identify CAF subtypes and their functions in the tumor microenvironment through fluorescence-activated cell sorting, single-cell RNA sequencing, and experiments using a 3D microfluidic platform to study angiogenesis and tumor migration.

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14 views2 pages

Guião

Tumors are the main cause of death in humans. The tumor microenvironment plays an important role in regulating tumor behavior through cross-talk between tumor cells and other cell types. Cancer-associated fibroblasts (CAFs) are a main cell type in the tumor microenvironment and play a key role in tumor progression through remodeling of the extracellular matrix and secretion of growth factors. The document aims to identify CAF subtypes and their functions in the tumor microenvironment through fluorescence-activated cell sorting, single-cell RNA sequencing, and experiments using a 3D microfluidic platform to study angiogenesis and tumor migration.

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© © All Rights Reserved
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Tumors are the main cause of death in humans.

Nowadays, even though the


development of new technologies, treatment, and new tools for early diagnosis, there
are many things unknown in this area. It’s well established that the tumor
microenvironment (TME) plays an important role in the regulation of the biological
behavior of tumors. The cross-talk between tumor cells and the TME provides a
mechanism and signal network for tumors to survive and progress.
The TME is a very dynamic environment composed of cellular components such: as
fibroblasts, immune cells, inflammatory cells, adipocytes, epithelial cells, endothelial
cells, mesenchymal cells, cancer-associated fibroblasts, and extracellular matrix.
CAFs are one of the main cell types in the TME and play a key role in regulating the
biological behavior of tumors. One of the major roles of CAFs is cancer invasion by
mediating the remodeling of ECM leading to secondary metastasis. Activated CAFs
secrete a high level of growth factors and inflammatory cytokines as a promoter of
tumor progression.
However, there are evidence that CAFs can have antitumorigenic effects.
At the moment, cell morphology is still the most reliable way to distinguish CAFs within
TME. We have 3 markers: α-smooth muscle actin (SMA), fibroblast-specific protein 1
(FPS-1), and fibroblast activation protein (FAP). The lack of congruency in marker
expression raises the possibility that CAFS comprise a diverse group of cells made up of
several subtypes.[1]

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Objectives: The main goal of this experimental work is to identify the subtypes of CAFs,
at the cellular and functional level, and understand their role in the TME.
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Methods
We will use breast carcinoma cells. In the first place, we perform a Fluorescence-
activated cell sorting to have a specific cell population (CAFs).
FACS is a specialized type of flow cytometry that provides a method for sorting a
heterogeneous mixture of biological cells into containers, one cell at a time, based upon
the specific light scattering and fluorescent characteristics of each cell.
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How FACS works?
Very briefly, the cell suspension is dragged in the center of a narrow, rapidly flowing
stream of liquid. The flow is arranged so that there is a large separation between cells
relative to their diameter. Then, a vibrating mechanism causes the stream of cells to
break into individual droplets. The flow passes through a fluorescence measuring station
where the fluorescent character of interest of each cell is measured.
This fluorescence is obtained when we add to the cell suspension antibody specific for a
particular cell surface protein associated to a fluorescent molecule.
(https://www.sinobiological.com/category/fcm-facs-facs)
To obtain only CAFs, we will perfume a negative selection, where we will select cells that
are negative for these markers: EpCAM-/CD45-/CD31-/NG2. With this, we don’t select
epithelial cells, immune cells, endothelial cells and pericytes.
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After we have CAFs isolated, we perform a Single-cell RNA sequencing, where we
analyze the gene expression and can elucidate what genes are expressed by CAFs.
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How Single-cell RNA sequencing works?
Single-cell RNA sequencing provides transcriptional profiling of thousands of individuals
cells, which enables us to understand at the single-cell level what genes are expressed,
in what quantities, and how they differ across thousands of cells within a heterogeneous
sample.
Firstly, and after we isolate cells, we do cDNA using reverse transcription. Then, we
amplify cDNA by PCR and procced for the library preparation and consequently, we
sequence these libraries (via Next Generation Sequencing). Lastly, we perform
bioinformatic analyses.
Library is a collection of similarly sized DNA fragments with known adapter sequencer
added to the 5’ and 3’ ends.
After we know what genes are expressed in CAFs, we follow to study the function of
each subtype.
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To do this, we use a 3D microfluid platform (3D microfluid Cancer Microenvironment),
where we will do independent experiments for each subtype. A microfluidic device
consisted of four injection ports for filling collagen gel, a pair of side channels for driving
interstitial flow, and one central channel for loading cells (cancer cells and CAFs). The
interior of the channels is coated with ECM proteins for cell adhesion.

To study angiogenesis, we add the vascular endothelial growth factor (VEGF), and we
analyze it through confocal microscopy. However, we can collect the medium or cells in
channel to perform biochemical or PCR analysis.[2]
However, to study tumor migration, we first inject HUVECs for 2 days to allow the
formation of an endothelial monolayer in the central channel. Then we inject cancer
cells with CAFs. After 2 days, we count the number of cancer cells in the collagen gel
(invasive cell number), measure the area of propagated cells (invaded area) and
measure the maximum distance traveled from the primary site (penetrated distance).
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To do this, we do immunostaining, where we stained the CAF and the actin filaments
(Fibroblast-specific protein-1 antibody or anti-vimentin antibody or anti-alpha smooth
muscle actin antibody; Phalloidin-tetramethylrhodamine B isothiocyanate). We stained
the nuclei with DAPI.

The heterogeneity of the origin, phenotype, and function of CAFs poses a challenge to
the current research on CAFs.

However, the plasticity of CAFs offers promise concerning immunotherapy and targeted
therapy. Based on the aforementioned challenges and directions, researchers should
perform a multidirectional and multidimensional study on CAFs, discover specific
biomarkers of CAFs and corresponding targeted treatments, and explore the interaction
mechanisms between CAFs, tumor cells, immune cells, and other components of the
TME. These investigations may result in breakthroughs in the methods used for the
diagnosis, treatment, monitoring, and prevention of tumors.

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