Technology

SCENITH: A Flow Cytometry-Based Method to

Functionally Profile Energy Metabolism with Single-
Arguello et al., 2020, Cell Metabolism€
Cell Resolution 32, 1063–1075 December 1, 2020 ª
2020 Elsevier Inc.
https://doi.org/10.1016/j.cmet.2020.11.007
Authors
Rafael J. Arguello, Alexis J. Combes,€ Remy
Char, ..., Emeline Tabouret,
Matthew F. Krummel, Philippe Pierre

Correspondence
[email protected]

In Brief
Arguello et al. have developed a€ functional
assay to quantify metabolic dependencies
and capacities of multiple cell types with
single-cell resolution in parallel. Applying
SCENITH directly to whole blood and human
tumor samples, they identify metabolic
differences between tumor- and juxta-
tumorassociated immune cells.

d SCENITH is a simple method for complex metabolic profiling samples

ex vivo

d SCENITH
monitors rapid changes in protein translation upon
metabolic pathway inhibition

d Theprofile of translation inhibition reveals metabolic capacities and

dependencies

d SCENITHprofiles abundant and non-abundant cells in parallel in blood

and tumor samples
 2015). The presence of
ll effector immune cells in the ll
INTRODUCTION

Technology
SCENITH: A Flow Cytometry-Based
Method to Functionally Profile Energy
Metabolism with Single-Cell Resolution
Rafael J. Arguello,€ 1,9,11,* Alexis J. Combes,2,3,9 Remy Char,1 Julien-Paul Gigan,1 Ania I. Baaziz,1 Evens Bousiquot,1

Voahirana Camosseto, Bushra Samad,2,3 Jessica Tsui,2,3 Peter Yan,2,3 Sebastien Boissonneau,4
1,7

Dominique Figarella-Branger,5 Evelina Gatti,1,7,8 Emeline Tabouret,6 Matthew F. Krummel,2,3,10 and Philippe Pierre1,7,8,10
1Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
2Department of Pathology, University of California, San Francisco, San Francisco, CA, USA
3ImmunoX Initiative, University of California, San Francisco, San Francisco, CA, USA

4Aix-Marseille Univ, Institut de Neurosciences des Systems, Faculte´ de Medecine, Marseille, France

5Aix-Marseille Univ, APHM, CNRS, INP, Inst Neurophysiopathol, CHU Timone, Service d’Anatomie Pathologique et de Neuropathologie, Marseille,

France
6Aix-Marseille Univ, APHM, CNRS, INP, Inst Neurophysiopathol, CHU Timone, Service de Neurooncologie, Marseille, France

7Institute for Research in Biomedicine (iBiMED) and Ilidio Pinho Foundation, Department of Medical Sciences, University of Ave iro, 3810-193 Aveiro,

Portugal
8International Associated Laboratory (LIA) CNRS ‘‘Mistra’’, 13288 Marseille Cedex 9, France

9These authors contributed equally

10These authors contributed equally

11Lead Contact

*Correspondence: [email protected] https://doi.org/10.1016/j.cmet.2020.11.007

SUMMARY

Energetic metabolism reprogramming is critical for cancer and immune responses. Current methods to functionally profile
the global metabolic capacities and dependencies of cells are performed in bulk. We designed a simple method for
complex metabolic profiling called SCENITH, for single-cell energetic metabolism by profiling translation inhibition.
SCENITH allows for the study of metabolic responses in multiple cell types in parallel by flow cytometry. SCENITH is
designed to perform metabolic studies ex vivo, particularly for rare cells in whole blood samples, avoiding metabolic biases
introduced by culture media. We analyzed myeloid cells in solid tumors from patients and identified variable metabolic
profiles, in ways that are not linked to their lineage or their activation phenotype. SCENITH’s ability to reveal global
metabolic functions and determine complex and linked immune-phenotypes in rare cell subpopulations will contribute to
the information needed for evaluating therapeutic responses or patient stratification.
Energetic metabolism comprises a series of interconnected biochemical pathways target tissue is a predictive marker of response to
capable of using energy-rich molecules to produce ATP. Cells can produce ATP either cancer immunotherapy (Galon et al., 2006). The
by oxidative phosphorylation (OXPHOS) or by performing glycolysis. Aerobic success of immunotherapies is restricted to a relatively
glycolysis supports not only proliferation but also cell survival in hypoxic conditions. small proportion of patients, as it requires a functional
Immune cells are specially equipped to migrate into peripheral tissues and to adapt and metabolically competent immune system to
to the change in microenvironment. Their energetic metabolism profile is known to respond to treatment (Antonia et al., 2018; Wolchok et
correlate with their microanatomical localization, activation, proliferation, or al., 2017). Consequently, there is a great need for a
functional state (O’Sullivan et al., 2019; Russell et al., 2019). Activation of T cells is simple immuno-metabolic profiling method for
generally linked to a metabolic switch from OXPHOS to aerobic glycolysis (Pearce et complex samples that can be utilized to stratify
al., 2009; Roos and Loos, 1973; Warburg et al., 1958; Wieman et al., 2007). patients before treatment and to monitor responses
Competition for glucose within the tumor microenvironment influences cancer after immunotherapies.
progression and the anti-tumoral immune response by regulating metabolism and Current methods to determine metabolic profiles
function in both tumoral cells and tumor-infiltrating lymphocytes (TILs) (Chang et al., can be classified into three groups. The first group,
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epitomized by Seahorse (Agilent Technologies), uses oligomycin A
ll metabolic inhibitors (i.e., 2-deoxyd-glucose [DG] and [O]) while monitoring the extracellular acidification
rate (ECAR) and oxygen consumption rate

Cell Metabolism 32, 1063–1075, December 1, 2020 ª 2020 Elsevier Inc.

CyTOF (e.g.,
Method Met-Flow, scMEP) MSI Seahorse SCENITH

Output metabolic metabolomic metabolic capacities metabolic capacities and

phenotype profile (unbiased) and dependencies dependencies

Functional profile of the cells no no yes (4) yes (non-limited)

(no. of treatments)

Cell purification required no no yes no

Single-cell resolution yes yes no yes

Phenotypic analysis yes no no yes

Compatible with cell sorting no no no yesa

Ex vivo application yes yes no yes

Metabolic readout levels of markers metabolite levels changes in changes in protein

(min 10 channels) extracellular pH and synthesis levels (one
[O2] channel)

Time (Hs) from sampling 0–1 0–1 24 0–1

to profiling

No. cells required in subsets 500 200 1,000,000 2,000

Equipment needed CyTOF cytometer any imaging mass Seahorse any flow cytometerb
cytometer Analyzer
a
Not shown
b SCENITH also has the potential to be analyzed by CyTOF. MSI, microscopy using heavy metal coupled with oligonucleotide -labeled antibodies (not shown).

(OCR). This method requires a large amount of purified cells to be cell era (Artyomov and Van den Bossche, 2020) by developing a
incubated with special culture media for several hours and allows the method that functionally determines the overall metabolic capacities
user to establish different parameters and to determine global and dependencies of cells independent of their phenotype.
metabolic dependencies and capacities of the cells (Zhang et al., Approximatively half of the total energy that mammalian cells
2012). The second group quantifies the maximal activities of enzymes, produce by degrading glucose, amino acids, and/or lipids is
such as the maximal dehydrogenase activity, by adding high immediately consumed by the protein synthesis (PS) machinery
concentrations of substrates to single cells using histochemistry (Buttgereit and Brand, 1995; Lindqvist et al., 2018; Schimmel, 1993).
(Miller et al., 2017) or to cell lysates. Finally, the third group uses mass The tremendous energetic cost associated with this essential
spectrometry (MS) and MS imaging to measure the levels of metabolic process offers a methodological opportunity to determine
metabolites produced by metabolic pathways (Palmer et al., 2017). the PS levels as a measure of global metabolic activity. We took
Recently, three cytometry-based (FACS and CyTOF) phenotypic advantage of the drug puromycin (puro), whose incorporation is a
characterizations of cells, using a panel of anti-human or mouse reliable readout for measuring PS levels in vitro and in vivo (Andrews
antibodies recognizing transporters and metabolic enzymes, have and Tata, 1971; Aviner, 2020; Hidalgo San Jose and Signer, 2019;
been used in parallel with histology to correlate their expression with Miyamoto-Sato et al., 2000; Nemoto et al., 1999; Rangaraju et al.,
the microanatomical localization of T cells (Ahl et al., 2020; Hartmann 2019; Schmidt et al., 2009; Seedhom et al., 2016; Wool and Kurihara,
et al., 2020; Levine et al., 2020). 1967), combined with a novel antipuro monoclonal antibody, to
Cell sorting and incubation with cell culture media can change the develop a simple method for complex metabolic profiling with single-
metabolic activity of the cells (Llufrio et al., 2018), and thus they cell resolution based on PS levels as the readout. We termed this
cannot be applied to establish metabolic profiles of heterogenous and method SCENITH (singlecell energetic metabolism by profiling
scarce living cell populations obtained from human blood samples or translation inhibition), with reference to our previous SUnSET
biopsies, ex vivo (Tables 1 and S1). Predicting the state of global (Schmidt et al., 2009) and SunRiSE (Arguello et al., 2018€ ) methods
metabolism by phenotyping the level of certain metabolic markers for studying protein synthesis. SCENITH was used directly in whole
(Ahl et al., 2020; Hartmann et al., 2020; Levine et al., 2020; Wculek et blood, in primary and secondary lymphoid organs, and in human
al., 2019) or characterizing the maximal potential activity of a small tumor samples to deconvolve the complex functional energetics of
subset of enzymes in situ (Miller et al., 2017) is very challenging. We immune and stromal cells with single-cell resolution. Our results
aimed to complement the toolbox for metabolic studies in the single- demonstrate that our method is ideal for analyzing heterogenous
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samples, from which the details of metabolism, particularly among
rare immune cell subsets, have remained inaccessible.

DESIGN

Characterizing the Energetic Metabolism Profile by Monitoring

Changes in Protein Synthesis Levels in Response to Metabolic
Inhibitors
To test whether the kinetics of the levels of PS and ATP are tightly
coupled, we measured in mouse embryonic fibroblasts (MEFs) both
ATP and PS levels after blocking ATP production (Figure 1A).

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(legend on next page)

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To inhibit ATP production, we treated cells with a mix of inhibitors that 1F; STAR Methods). Conversely, FAO and
block both glycolysis and OXPHOS (Figure 1A). To optimize the signal- AAO cap is defined as the capacity to use fatty acids and amino acids
to-noise ratio of puro intracellular detection, we developed a novel as sources for ATP production when glucose oxidation is inhibited (i.e.,
monoclonal anti-puro antibody (clone R4743L-E8) specifically adapted glycolysis- and glucose-derived acetyl-CoA by OXPHOS) (Figures 1F
for intracellular flow cytometry. Both PS levels (Figures 1B and 1D) and and S1B).
ATP levels (Figure 1C) dropped within 5–10 min after blocking ATP
synthesis, with a strikingly similar slope, showing that changes in ATP RESULTS
levels and PS levels are tightly correlated (Figure 1E; r = 0.985; p <
0.0001). To increase the sensitivity of the translation measurement, SCENITH Recapitulates Seahorse Metabolic Profiling of Steady-State
the time of incubation with puro can be experimentally determined and Activated T Cells
and increased if the cells of interest have very low metabolic activity The metabolic switch of T cells to aerobic glycolysis upon activation
(i.e., naive T cells) (Figure S1; Table S2). Indeed, we tested the optimal was originally documented in the 1970s (Roos and Loos, 1973;
time of incubation for whole blood and determined that 40 min of Warburg et al., 1958) and recently confirmed using the Seahorse
puro treatment is optimal for detecting translation in T cells, technology (van der Windt et al., 2012, 2013). To benchmark our
monocytes, and neutrophils in whole-blood samples of mice (Figure method, we monitored the energy metabolism in isolated human
S1C). To test the relationship between ATP consumption and blood T cells at steady state or upon activation using Seahorse and
transcriptional or translational activities, we treated metabolically SCENITH in parallel (Figure 2A). Upon activation, an increase in the
active cells with the same inhibitors to block de novo ATP synthesis, glycolytic capacity of T cells was measured with both methods in
together with translation and/or transcription inhibitors. Altogether, excellent agreement (Figures 2B and 2C, respectively) (Spearman r2 =
our results confirmed that PS is one of the most energy-consuming 0.85, p < 0.01) (Figure 2D). We observed a statistically significant
metabolic activities (Buttgereit and Brand, 1995; Lindqvist et al., 2018) decrease in the spare respiratory capacity in bulk T cells upon
(Figure S1), and most importantly, it represents a stable and reliable activation with Seahorse (Figures S2A and S2B). Interestingly, an
readout to rapidly evaluate the impact of metabolic pathway increase in OCR by Seahorse was paralleled with an increase in the
inhibition on the cell. global level of PS measured by SCENITH, albeit to a larger extent
As both ATP and PS levels are tightly coupled, puro fluorescence (Figures 2E and 2F, respectively). Overall, the metabolic profiles of T
(measuring PS) can act as a surrogate for energetic pathway inhibition cells upon activation obtained by Seahorse and SCENITH were very
and can be monitored by flow cytometry. The principle of SCENITH is consistent. The level of translation (Figure 2F) correlated with the
thus to incubate a given sample in parallel with specific inhibitors of global metabolic activity of the cells, and changes in the response to
metabolic pathways. If a cell population is energetically dependent on inhibitors confirmed the metabolic switch toward aerobic glycolysis
the inhibited metabolic pathway, its ATP concentration will drop as that occurs upon T cell activation. However, SCENITH showed two
well as its PS levels. The latter will be established using puro main advantages over Seahorse measurements. First, the magnitude
incorporation detected by direct immuno-fluorescence (Figures 1F of the change in the glycolytic capacity and the standard error of the
and S1B). SCENITH allows metabolic profiles to be measured in measurements with SCENITH were superior (Figures 2A, 2C, 2E, and
heterogenous cell populations at single-cell resolution by combining 2F). Second, SCENITH analysis was performed with 10-fold fewer T
cell identification and puromycinylation detection by multiparametric cells. Moreover, SCENITH could incorporate a full spectrum of T cell
flow cytometry (FACS) (Figure 1G). Using PS levels, the level of glucose markers in the analysis, allowing us to study in parallel the CD3 + T cell
dependence is calculated to quantify the proportion of the PS, and subpopulations present in the bulk sample.
therefore of ATP/GTP production, dependent on glucose oxidation
(Figure 1F; STAR Methods). The mitochondrial dependence (mito Metabolic Deconvolution of Blood T Cell Subsets by SCENITH
dep), i.e., the proportion of PS dependent on OXPHOS, is similarly Identifies a Memory CD8+ T Cell Subset
established. Two additional derived parameters, ‘‘glycolytic capacity’’ Constitutively Displaying High Glycolytic Capacity We next applied
(Glyc. cap) and ‘‘fatty acid and amino acid oxidation capacity’’ (FAO SCENITH to mixed populations, which due to their heterogeneity are
and AAO cap), are also calculated. Glyc. cap is defined as the maximum relatively inaccessible to metabolic monitoring by Seahorse. We took
capacity to sustain PS when mitochondrial OXPHOS is inhibited (Figure advantage of CD45RA, IL7RA

Figure 1. SCENITH Design Based on Dynamic Changes in Protein Synthesis Levels upon Blockade of Different Metabolic Pathways
(A) Blocking ATP production and kinetics of ATP and translation levels.
(B) Visualization of protein synthesis after puro incorporation and staining with a new monoclonal anti-puro (clone R4743L-E8). Histogram PS level by flowcytometry in MEFs
after blocking both mitochondrial respiration and glucose oxidation for different amounts of time.
(C–E) Measurement in MEFs upon blocking ATP synthesis versus time of ATP levels (C), PS by flow cytometry (D), and correlation of both (E). Dot represents the means and
bar the SD (R = 0.985, p < 0.0001, N = 3).
(F) Schematic representation of a sample that contains three cell types with different metabolism profiles (aerobic glycolysis, glycolysis/OXPHOS, FAO, and
AAO/OXPHOS). Treating the mix of cells with specific drugs (DG or O) will affect each cell subset in a different way.
(G) Examples of metabolic monitoring using SCENITH. The glucose dependence and FAO and AAO capacity and the mitochondrial dependency and glycolyticcapacity
can be calculated from the MFI of puro in the different treatments following the formulas (STAR Methods).

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(H) Description of SCENITH procedure. Extract the sample, divide it, and treat each with the inhibitors (e.g., DG, O, DG+O, H) and puro. After staining and flowcytometry,
the profile of response of the different cell subsets is analyzed. The profile reveals the metabolic capacities and dependencies of the cells (i.e., high glucose dependence [‘‘pop
1’’] and high glycolytic capacity profile [‘‘pop 2’’]).

Figure 2. Parallel Seahorse and SCENITH Metabolic Analysis of Resting and Activated T Cells
(A) Scheme of the experiment for analysis of resting and activated T cells.
(B and C) Metabolic profile of T cells from three healthy donors (P1, P2, and P3) analyzed with Seahorse (B) and SCENITH (C). ECAR and translation levels of nonactivated and
activated T cells (P1) and glycolytic capacity from both methods are shown (*p < 0.05; **p < 0.01; ***p < 0.001; N = 3 each in triplicate). (D) Correlation between the changes
in glycolytic capacity of steady-state and activated T cells from three donors measured by Seahorse and SCENITH (Pearson r = 0.92; R 2 = 0.85; p < 0.01; N = 3).
(E) Basal OCR in non-activated (non-Act) and activated T cells. Each bar represents the mean of P1, P2, and P3 (in triplicate).
(F) Basal translation levels (anti-Puro gMFI) in non-activated (non-Act) and activated T cells (aCD3/CD28). Bars represent the mean of P1, P2, and P3. (G) SCENITH metabolic
profile of whole blood directly treated with inhibitors with or without pre-incubation (1:4 V/V) in DMEM 10% FCS for 3 h. Data represent pooled whole blood from three
mice (in duplicate) from three independent experiments. Two-way ANOVA, multiple comparisons.
(CD127), CCR7, CD45RO, CD57, PD1, and perforin expression to phenotypically distinct clusters/subpopulations (Figures 3A and 3B).
identify and analyze T cell subsets present in human blood draws. The metabolic profiles of non-activated naive T cells, as well as
Briefly, naive T cells show expression of both CD45RA and IL-7 memory (effector memory and central memory) CD4+ and highly
receptor alpha (CD127) and the chemokine receptor CCR7. The lack of differentiated CD8+ (HDE), showed a medium to high degree of
expression of CD45RA is a known marker of antigenic stimulation mitochondrial dependence (Figure 3C), consistent with previous
experience (memory and effector cells). Early effector memory (EEM) reports on their metabolic activity (Pearce et al., 2009). In contrast,
CD8+ T cells do not express CD45RA and CD57, and do not yet express the less abundant cell subsets such as EEM CD8 + T and natural killer
cytotoxic markers like perforin. Monitoring the expression levels of (NK) cells (a small fraction that co-purified with T cells) showed higher
these nine markers and analysis by dimensionality reduction, using glycolytic capacity. To determine if similar metabolic trends are
tdistributed stochastic neighbor embedding (t-SNE), yielded six observed in other species and preparations, we performed SCENITH
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on resting and activated mouse splenic T cells (Figures S3A and S3B) results consistently demonstrated a switch
and human blood central memory CD4+ T cell subsets (Figure S3C). Our toward high glycolytic capacity and high glucose dependence in both
A B

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Figure 3. Metabolism Profile of Resting Human Blood T Cells by SCENITH Identifies Different Metabolic Profile of Human T Cell
Subsets
For a Figure360 author presentation of Figure 3, see https://doi.org/10.1016/j.cmet.2020.11.007.
(A) SCENITH analysis pipeline of T cells purified from human blood (95% pure). Dimensionality reduction (t-SNE) based on phenotypic markers is performed on the
concatenated treated cells (Co, DG, O, and DGO).
(legend continued on next page)
mouse and human T cells upon activation (Figure S3). In bulk analysis, Metabolic Profiling of Mouse and Human Myeloid Cell Subsets
naive CD4+ and CD8+ T cells represented the most abundant subsets Compared to T cell subsets, the metabolic profile of myeloid cell
and thus likely dominated the energy metabolism monitoring subsets from human and mouse tissue origin has been far less studied
performed by Seahorse (Figures 2E and 3C). Consequently, Seahorse (Saha et al., 2017). Among myeloid cell subsets, dendritic cells (i.e.,
measurements indicate a rather low ‘‘mean’’ glycolytic rate/capacity DC1, DC2, DC3, and pDCs) are non-abundant professional antigen-
and high ‘‘mean’’ oxygen consumption rate (Figure 2B) and are thus presenting cells (APCs), which serve as sentinels for the immune
in accordance with the metabolism of naive T cells determined by system. Each subset expresses a particular set of microbial pattern
SCENITH (Figures 3C and 3D). However, the presence of CD8+ EEM, recognition receptors and is specialized in activation of CD8+ (i.e., DC1)
which display high glycolytic capacity but represent no more that 5% and CD4+ (i.e., DC2 and DC3) T cells and antiviral cytokine production
of the T cells present in the sample (2,000 cells), remained completely (pDCs). For instance, lipopolysaccharide (LPS) detection by TLR4 on
masked during Seahorse analysis. DC2 and DC3 results in changes in gene expression, membrane traffic,
Another feature of SCENITH’s resolutive power is the possibility to and energetic metabolism (Amiel et al., 2012; Everts et al., 2012;
characterize single-cell behaviors according to their sensitivity to Krawczyk et al., 2010). DCs patrol all tissues emanating from the
metabolic inhibitors independent of their phenotype. This allows for bloodstream, where they represent a very small fraction of the
the identification of functional metabolic heterogeneity first, and for peripheral blood mononuclear cells (PBMCs), making the isolation of
the determination of the phenotype or sorting cells afterward. As a millions of DC1s, DC2s, or pDCs from the same donor very challenging.
proof of concept, resting purified T cells were treated with oligomycin We therefore used SCENITH to profile the energy metabolism of
prior to translation monitoring. The histogram of translation levels human blood myeloid cell subsets from healthy donors as well as of
showed two T cell subpopulations upon mitochondrial inhibition, one mouse bone marrow- and spleen-derived DCs stimulated or not to
with high and one with low levels of translation (Figure 3D). The generate a detailed metabolic atlas of the myeloid cell populations.
population with blocked translation was labeled as ‘‘mitochondrial Following deconvolution, we first ranked myeloid cells by glucose
dependent’’ and the cells with a high level of translation were labeled dependence, finding that classical monocytes (Mono1, CD14 +CD16)
as ‘‘glycolytic’’ (Figure 3D). As shown in the t-SNE, the phenotype of were the most glucose dependent, whereas the DC precursors (DC5)
glycolytic and respiratory T cells recapitulated our previous results were the least (Figure 4A). These populations lay near the extremes of
(Figures 3A–3C) and showed that the expression of CD45RA, mostly mitochondrial dependence: DC5s display highest and Mono1 the
present in naive T cells, correlated well with the level of mitochondrial lowest. However, we also observed examples of cell subsets (e.g., pDC
dependence (Figure 3D). In conclusion, we found that SCENITH allows and Mono2) that were dependent on glucose and mitochondrial
for both the measurement of the metabolic profile of known non- respiration. In contrast, DC1 and DC2 displayed relatively high
abundant cell subsets of interest and the sorting and identification of glycolytic capacity and moderate glucose dependence, suggesting a
‘‘unknown’’ cells with specific metabolic dependencies present within certain degree of metabolic plasticity. As expected, cells ranked in
a heterogenous sample. opposite order for FAO and AAO capacity compared to glucose
Cellular energy metabolism is affected by nutrient availability, and dependence, consistent with the idea that cells with low glucose
Seahorse measurements typically require special unbuffered media dependence can sustain translation and energy production by free
and incubation in culture media over several hours. Comparatively, fatty and/or amino acid oxidation.
SCENITH can be performed directly on whole blood. To investigate the To test whether a rapid metabolic switch occurs upon TLR4
effect of media, we tested whether short-term incubation of blood activation in human myeloid cells, we used an antibody panel for
with cell culture media would impact the metabolic profile of immune analyzing Mono1, Mono2, DC1, DC2, and pDCs in PBMCs treated with
cells. Freshly isolated blood was directly treated with control, DG, O, LPS. While the Mono1 subset increased their global level of translation
or DGO and puro or treated after pre-dilution and incubation for 0 or (Figure S4A) and the DC1 subset showed a mild increase in glucose
3 h in DMEM 10% FCS. Incubation of blood for 3 h with culture media dependence and a moderate decrease in mitochondrial dependence
induced a statistically significant increase in the glycolytic capacity of (Figure S4A), all other subsets did not change their metabolism.
NK cells and monocytes (Figures 3E and S3D). In contrast, it had no We previously observed metabolic differences in human versus
impact on the metabolic parameters of both naive and effector CD4 + mouse blood monocytes. To gain insight into the
and CD8+ T cells, B cells, and neutrophils.

(B) Heatmap showing the level of expression of each marker (gMFI) in each cluster/subset from the t-SNE after dimensionality reduction.
(C) Metabolic profile of the T cell subsets identified (naive CD4 and CD8 T cells in green, memory CD4 and HDE CD8 T cells in or ange, EEM CD8 T cells in red, andNK cells in
blue) after SCENITH analysis. Representative translation level (anti-Puro gMFI) (P1) is shown (N = 3). Black line represents background level obtained after DG+O treatment.
(D) Two distinct metabolic profiles in human blood T cells after O treatment (left panel) revealing glycolytic and mitochondrial-dependent T cell subsets. Histogramshows the
level of translation in all T cells (light gray line) upon mitochondrial inhibition, indicating the presence of glycolytic c ell subsets (in red) and mitochondrialdependent cells

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(in blue). Gating them into the t-SNE plot (right panel) to identify the phenotype of glycolytic and mitochondrial-dependent cells (blue). The marker of antigen experience
CD45RA, lost in cells that have been previously exposed to TCR stimulations, correlates with the metabolic profile.
(E) Metabolic changes induced by short-term incubation of blood with cell culture media. Metabolic parameters of cell types when blood is pre-incubated withDMEM 10% FCS
(0 or 3 h) or directly incubated with the inhibitors (i.e., Co, DG, O, DGO, or Harringtonine) and puro. Data from pooled whole bloo d from three mice (in duplicate) from
three independent experiments are shown (N = 3). Statistical significance between both conditions by t test (**p < 0.005).

Figure 4. Metabolic Profile of Human Blood DCs and Monocytes and Mouse Bone Marrow-Derived DCs Using SCENITH
(A) Metabolic profile of human blood monocytes and DC subsets obtained by SCENITH (N = 5 independent healthy donor s). Statistical significance by two-way ANOVA
comparing all columns (*p < 0.05; **p < 0.005; ****p < 0.0001). The pDC, Mono2, or Mono1 showed statistically significant dif ferences against DC1 or DC5. (B) Metabolic
profile of mouse bone marrow-derived DCs (FLT3L-DC) obtained by SCENITH (N = 3) in non-treated versus LPS-treated cells; two-way ANOVA, *p < 0.05; **p < 0.005; ****p
< 0.0001.
(C) Metabolic profile of blood monocytes from human (N = 4) or mouse (N = 9, 3 mice pooled, in duplicate, three independent experiments). Statistical significance
between human and mouse monocytes by t test (*p < 0.05). metabolic profile of mouse DC populations, we analyzed bone
marrow-derived DC1, DC2, and pDC (FLT3L-BMDC) subsets at steady
state and upon activation with LPS (Figure 4B). As in human samples,

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the mouse DC2 subset showed the metabolic profiles toward lower mitochondrial dependence.
highest glycolytic capacity, followed by DC1 and pDC subsets. While However, only DC2 showed an increased global level of PS, probably
glucose dependence was high in both DC1 and DC2 subsets from reflecting the abundant TLR4 expression in this DC subset. Considering
human (40% and 55%) and mouse (80 and 100%) samples, a 4-fold that we analyzed DCs isolated from human blood and derived in vitro
lower glucose dependence was observed in mouse versus human from mouse bone marrow,
pDCs (15% versus 60%). LPS treatment shifted the DC1 and DC2

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Figure 5. Paralleled SCENITH and scRNA-Seq in Human Tumor and Juxtatumoral Samples Identify Conserved Metabolic Profiles

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(A and B) Myeloid subsets observed in the human meningioma tumor sample (A) and renal carcinoma (B). Myeloid cells gated on CD45+ /
CD3CD20CD19CD56/Live-dead/singlets. Number in the t-SNE represents the percentage of the population.

(legend continued on next page)

their SCENITH metabolic profiles are surprisingly comparable. These myeloid cells (Figure S5D). Then, we tested the expression (mRNA)
results confirm that SCENITH allows the user to identify cell of these glycolytic and respiratory metabolic genes in the tumor-
populations sharing similar metabolic profiles and that energy associated myeloid populations (CD45+LinHLA-DR+). Sorted cells
metabolism in DCs varies according to their state of activation. from the renal carcinoma and juxta-tumoral tissue were subjected to
scRNA-seq (Figures 5E and S5E). Analysis of 12,801 and 2,080 cells
Profiling the Metabolic State of Human TumorAssociated Myeloid for the tumor and for the juxta-tumoral tissue yielded 6 and 5
Cells clusters, respectively. To rigorously identify the populations, we used
Immunotherapies are a game changer in oncology, yet only a fraction well-known signatures (Villani et al., 2017) and numbered cellular
of patients show complete immune-mediated rejection of the tumor. identities in the t-SNE representations (Figure S5E). We focused on 5
The heterogeneous responses of patients to immunotherapies have Mono/Mac clusters expressing MAFB and/or CSF1R that were
created a strong need for understanding the functional state of present in both tumor and juxta-tumoral tissue (Figure S5F).
tumor-associated immune cells (Galon et al., 2006). We thus used Expression of classical surface markers such as CD16 and CD14
SCENITH to perform parallel phenotypic and metabolic profiling of (Figure S5G) confirmed that clusters 0 and 1 represent classical
blood and tumor samples, and to investigate the heterogeneity of monocytes (Mono1). Cluster 2 represents CD14CD16+ nonclassical
immune cell subsets by comparing tumors of diverse origins. In monocytes (Mono2), while co-expression of CD14 and CD16 for
particular, we analyzed PBMCs from healthy donors, explanted clusters 3 and 4 corresponds to macrophagelike populations. We
meningioma, brain metastasis (originated from a breast cancer), and performed unsupervised differential gene expression (DGE) analysis
renal carcinoma tumors and renal juxta-tumoral tissue. In the case of and generated heatmaps for the top 5 most differentially expressed
kidney tissues, both SCENITH and single-cell RNA sequencing (scRNA- in the tumor (Figure S6A, left) and the juxta-tumoral tissue (Figure
seq) analysis were performed in parallel on the same samples. S6A, right). In addition to highlighting key genes that contributed to
We observed eight myeloid subsets in meningioma and six in renal the unbiased segregation of these populations in both tissues, we
carcinoma, in which the phenotype and metabolic profile were confirmed the high expression of macrophage-specific genes by
determined (Figures 5A–5C, S5A, and S5B). Upon clustering of all the clusters 3 and 4, such as APOE, C1QC, and RGS1. We next overlaid
cell subsets in all tumors based on their metabolic profile, two groups the t-SNE plots with our two metabolic gene signatures. Highly
emerged, a ‘‘glycolytic cluster’’ and a ‘‘respiratory cluster’’ (Figures correlating with SCENITH profiles, monocyte clusters (0, 1, and 2)
5C and S5C). Mono1 and neutrophils displayed glycolytic metabolism presented an enrichment in glycolytic gene signature both in tumor
profiles in all blood samples and tumors tested (Figure 5D). In and juxta-tumoral tissue. Conversely, macrophages (cluster 3)
contrast, Mono2, DC1, and DC2 showed relatively high glycolytic showed high expression of the respiratory signature in the tumor,
capacity when isolated from kidney tumor and juxta-tumoral tissues, while, as predicted by SCENITH, this was not detectable in
while these subsets showed high respiratory metabolism profile in juxtatumoral tissue (Figure 5E). DCs, distinct from macrophages,
the two brain tumors. Conversely, tumor-associated macrophages presented an enrichment in a glycolytic gene signature both in tumor
(TAMs) showed high mitochondrial dependence, while juxta-tumoral and juxta-tumoral tissue (Figure S6B).
macrophages displayed higher glycolytic capacity (Figure 5D), Altogether, these results indicate that tumor micro-environment
suggesting that the tumor microenvironment modifies TAM specifically modifies the functional metabolism of macrophages by
metabolism. The decrease in the glycolytic capacity in TAMs as durably affecting metabolic gene expression. By correlating the results
compared to juxta-tumoral macrophages was previously associated of scRNA-seq analysis and SCENITH profiling on blood myeloid cell
with increased immunosuppression, tumor progression, and poor subsets (Figure 5F), we identified a functional gene signature (Figure
patient survival (Vitale et al., 2019). These SCENITH results reveal S5D) that can be used to describe the metabolic profile using gene
that the anatomical origin of the tumor could influence the expression.
metabolism of immune subsets, introducing an additional layer of
heterogeneity in the tumor environment.

Linking scRNA-Seq and Energetic Metabolism Profile in Tumor-

Associated Myeloid Cells
We decided to link the discriminative power of SCENITH with scRNA-
seq to support our results regarding the cellular complexity of the
renal carcinoma tumor environment and to correlate metabolic
profiling with gene expression. We compared the functional
metabolic profile obtained by SCENITH with the metabolic gene
expression pattern obtained by scRNAseq. We first identified
expression signatures of glycolytic and respiratory genes that tightly
correlated with the functional metabolism profiles of human blood

1074 Cell Metabolism 32, 1063–1075, December 1, 2020

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DISCUSSION monocytes and neutrophils in the blood and within the tumor. In a
similar fashion, the metabolic profile of naive and effector CD8 T cell
SCENITH is a simple method for complex immuno-metabolic profiling. subsets supports the idea that the metabolic profile of cells in the blood
It allows the user to simultaneously establish the phenotype and is pre-adapted and related to the migratory capacity and function of
extract the global metabolic profile of multiple cell types in parallel, ex the cells. This hypothesis will require further studies that may profit
vivo. This rapid and sensitive method is from SCENITH.

(C) Heatmap of the metabolic profile (columns) of each myeloid cell subset from each type of tissue (rows). Unsupervised hierarchal clustering of subsets bymetabolic
profile identifies respiratory (blue bar) and glycolytic (red bar) clusters.
(D) Reordering of the rows by cell type based on (B) to identify changes in metabolism profile in the same cell subset in blood, tumors, and juxta-tumoral tissue.(E)
Clusters of myeloid cells identified in the renal carcinoma and juxta-tumoral tissue by scRNA-seq (left panel). Expression of glycolytic and respiratory gene signatures in all
cells extracted from the tumor. Summary of the results obtained by SCENITH and scRNA-seq in tumor and juxta-tumoral myeloid cells. Populations named with numbers in
the t-SNE and described in table.
consistent and comparable to other established techniques, including Importantly, SCENITH can establish the metabolic profile of
Seahorse. As the treatments with inhibitory drugs are performed in circulating EEM CD8 T cells directly from 200 mL blood. EEM CD8 T cells
parallel, SCENITH can be used to monitor cellular responses to a represent around 5% of the total T cells (i.e., 500 cells per condition),
combination of multiple metabolites and inhibitors. Given that flow and we consistently measured their metabolic profile. Of note, the
cytometers are available in most research institutes and hospitals, same analysis would have required the purification of 1.2 million EEM
SCENITH represents an accessible method to study energy metabolism. (400,000 EEM in triplicates) to be analyzed with the Seahorse XF24,
Compared to other methods (Tables 1 and S1), its sensitivity, thus representing a gain of sensitivity of approximately 800-fold. This
accessibility, singlecell resolution, requirement for only one gain was even more dramatic when the metabolism profile of DC
fluorescent channel, stability of the readout, manipulation time, subsets was established from human tumor biopsies, in which they
compatibility with fixation, and sorting make SCENITH an unrivalled constituted approximately 0.5% of the myeloid cell population. These
approach for studying global metabolism in cells in tissue slices and results demonstrate the analytical capacity and discriminative power
complex populations ex vivo (Artyomov and Van den Bossche, 2020). of SCENITH and its potential in analyzing how the diet, age, and
The resolutive power of SCENITH highlighted variations among anatomical/tissue context could influence energetic metabolism of
subsets of the same cell populations. Our results suggest that changes immune cell subsets. The metabolic profile of innate and adaptive
in metabolism are embedded in the myeloid differentiation program immune cells correlates with the type of cytokines they produce; thus,
and that modulating metabolism in DC precursors might influence the the metabolic profile represents a universal readout for functional
generation of DC subsets with potential clinical relevance. One state (Buck et al., 2015). For these reasons, SCENITH analysis could be
surprising observation is the higher glycolytic capacity of human blood used to define the immune metabolic contexture and complement an
monocytes compared to the mouse blood counterpart (Figure 4C). This immunoscore that defines immune fitness of tumors and predicts and
may be an inherent difference between human and mouse, or stratifies patients for tailored therapies, aiming to manipulate
influenced by environmental factors, like circadian rhythms, diet, metabolic pathways to improve anti-tumoral immune effector
and/or microbiota, and other factors. Interestingly, mouse monocytes functions.
increased their glycolytic capacity upon incubation with cell culture
media, suggesting that the metabolic profile of these cells is sensitive Limitations of Study
to nutrient availability. As a consequence of having single-cell resolution, SCENITH does not
It has been previously shown by Marciano et al. that amplify the signal by increasing the number of total cells analyzed.
puromycinylation does not reveal a very strong decrease in PS levels in For this reason, one of the limitations of our method is that it is not
cell lines under mild conditions of starvation. This evidence led the suitable for studying cells with undetectable levels of PS (i.e., mature
authors to conclude that puro is not reliable to monitor translation in spermatozoids, quiescent stem cells, acutely stressed cells, etc.). Of
cells upon starvation conditions. In contrast, we could reliably detect note, the incubation time with inhibitors and puro can be increased
inhibition of PS upon treatment with metabolic inhibitors, reaching when cells with very low metabolic activity are studied (i.e.,
levels of inhibition (i.e., DGO) comparable to the treatment with an circulating naive T cells and others) (Figure S1C; Table S2). Another
inhibitor of translation initiation (Figure S3D). The results from limitation of SCENITH is that at steady state we assess one metabolic
Marciano et al. and our study might be interpreted as contradictory; readout (i.e., the global level of PS). However, SCENITH may
however, the experimental conditions of both studies are not potentially be combined with a larger panel of phenotypic markers,
comparable, the main difference being that they pulse cells with puro fluorescent lipids or sugars, and mitochondrial tracers. These
only after 3 h of mild starvation and at this late time point cell lines additions will need to be properly tested to ensure that they do not
might be simply recovering translation to some extent. interfere with the metabolic functions of the cells.
The metabolic profile of immune subsets in the blood correlates with SCENITH is based on a fixable readout detected by
migratory capacity and effector functions in the body. Neutrophils and immunohistology, and we envision the combination of our method
monocytes migrate into hypoxic/ damaged tissues and are already with ATAC-seq, Cite-seq, CyTOF, and MS imaging ex vivo and
engaged in aerobic glycolysis in the blood. We confirmed this in human potentially also in vivo (Tables 1 and S1). One limitation when
Cell Metabolism 32, 1063–1075, December 1, 2020 1075
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combining SCENITH with sequencing technologies is that fixation and supported by research funding from the Canceropoˆ le PACA, Institut National du
permeabilization are required for staining with antipuro. For this Cancer, and Re´ gion Sud to R.J.A. The P.P. laboratory is sponsored by la Fondation
de la Recherche Me´ dicale (FRM) grant DEQ20140329536. The study was partly
reason, the scRNA-seq, ATAC-seq, or imaging method to be used
supported by grants from l’Agence Nationale de la Recherche (ANR-FCT 12-ISV3-
needs to be compatible with this treatment (Attar et al., 2018; 0002-01), A*MIDEX project ‘‘CSI’’ (ANR-11-IDEX-0001-02), DCBIOL Labex (ANR-11-
Rosenberg et al., 2018). LABEX-0043), INFORM Labex (ANR-11-LABEX-0054) funded by the ‘‘Investissements
Translation levels by puromycinylation can be determined in vivo d’Avenir’’ French government program, the Ilı´dio Pinho Foundation, FCT (Fundac¸
(Hidalgo San Jose and Signer, 2019; Seedhom et al., 2016). However, a˜ o para a Cieˆ ncia e a Tecnologia), and Programa Operacional Competitividade e
Internacionalizac¸ a˜ o – Compete2020 (FEDER; POCI-01-0145FEDER-016768 and
performing SCENITH in vivo has been until now challenging, in
POCI-01-0145-FEDER-030882). We acknowledge financial support from France Bio
particular with regard to determining the time of treatment and Imaging no. ANR-10-INBS-04-01 and ImagImm CIML imaging. We thank Noella
concentrations of the inhibitors to ensure homogeneous distribution Lopes-Pappalardo and Alexandre Boissonnas for reading the manuscript and
throughout the different tissues of a whole animal (e.g., 2-DG is suggestions and Lionel Spinelli for useful statistical advice. We thank Marc Barad
rapidly excreted, and puro does not cross the blood-brain barrier). and Sylvain Bigot from the CIML Cytometry core facility and Alice Carrier and
Laurence Borge of the CRCM metabolomics core facility. We thank Pierre Golstein
The implementation of SCENITH in vivo will require further
for mentoring and correcting the manuscript.
investigation; however, we show here that SCENITH can be efficiently
applied to fresh whole blood (Figures S1 and 3E) and in fresh tissue AUTHOR CONTRIBUTIONS
slices (Figure 5), avoiding interferences coming from cell culture
conditions or cell isolation procedures. Our method allowed us to Conceptualization, R.J.A.; Method Development, R.J.A. and A.J.C.; Validation, R.J.A.,
A.J.C., R.C., J.-P.G., A.I.B., E.B., P.Y., and V.C.; Formal Analysis, R.J.A., A.J.C., and B.S.;
reveal the global metabolic capacities and dependencies of multiple
Investigation, R.J.A., A.J.C., M.F.K., and P.P.; Tissue Resources, E.T., D.F.-B., S.B., and
cell types in parallel within their physiological context (i.e., M.F.K.; Writing – Original Draft, R.J.A.; Writing, R.J.A., A.J.C., E.G., M.F.K., and P.P.;
physiological concentration of metabolites, growth factors, Writing – Review & Editing,
electrolytes, and cellular interactions). R.J.A., A.J.C., E.G., E.T., R.C., M.F.K., and P.P.; Visualization, R.J.A., A.J.C.,
STAR+METHODS E.G., M.F.K., and P.P.; Supervision, R.J.A., M.F.K., and P.P.; Funding Acquisition, R.J.A.,
M.F.K., and P.P.

Detailed methods are provided in the online version of this paper and
DECLARATION OF INTERESTS
include the following:
The authors declare no competing interests. There are restrictions to the commercial
d KEY RESOURCES TABLE d use of SCENITH due to a pending patent application (PCT/EP2020/ 060486).
RESOURCE AVAILABILITY
B Lead Contact Received: March 18, 2020
Revised: August 9, 2020
B Materials Availability
Accepted: November 11, 2020
B Data and Code Availability d EXPERIMENTAL MODEL
Published: December 1, 2020
AND SUBJECT DETAILS
B Mouse Experiments REFERENCES
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Cance´ ropoˆ le PACA ‘‘Emergence’’ grant ‘‘HiDi-Glio’’ to R.J.A. This study was partly
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STAR+METHODS
KEY RESOURCES TABLE

Antibodies

Anti-human Axl Alexa Fluor 488 (Clone: R&D Biosystems Cat#FAB145G

108724)

Anti-human BDCA1 PE-Cy7 (Clone: L161) Biolegend Cat#331516

Anti-human BDCA3 FITC (Clone: Miltenyi Biotec Cat#130-113-317

AD5-14H12)

Anti-human BDCA4 PE (Clone: 12C2) Biolegend Cat#354504

Anti-human CD1c PE (Clone: L161) Biolegend Cat#331506

Anti-human CD1c PE/Cy7 (Clone: L161) Biolegend Cat#331516

Anti-human CD3 BB700 (Clone: SK7) BD Bioscience Cat#566575

Anti-human CD3 BV711 (Clone: UCHT1) BD Bioscience Cat#563725

Anti-human CD3 BV510 (Clone: HIT3a) BD Bioscience Cat#564713

Anti-human CD4 PE-Dazzle 594 (Clone: Biolegend Cat#300548

RPA-T4)

Anti-human CD8a BV605 (Clone: RPA-T8) Biolegend Cat#301040

Anti-human CD11b PE-Cy7 (Clone: ICRF44) eBioscience Cat#25-0118-42

Anti-human CD11c A700 (Clone: 3.9) eBioscience Cat#56-0116-42

Anti-human CD11c APD-R700 (Clone: 3.9) BD Bioscience Cat#566610

Anti-human CD11c BUV385 (Clone: B-ly6) BD Bioscience Cat#563797

Anti-human CD14 BUV805 (Clone: M5E2) BD Bioscience Cat#612902

Anti-human CD14 BV711 (Clone: M5E2) BD Bioscience Cat#740773

Anti-human CD14 BV711 (Clone: M5E2) Biolegend Cat#301838

Anti-human CD16 BV605 (Clone: 3G8) BD Bioscience Cat#563172

Anti-human CD16 BV605 (Clone: 3G8) Biolgend Cat#302040

Anti-human CD16 BV650 (Clone: 3G8) BD Bioscience Cat#563692

Anti-human CD16/CD32 Human Biolegend Cat#422302

TruStain FcX

Anti-human CD19 BB700 (Clone: SJ25C1) BD Bioscience Cat#566396

Anti-human CD19 BV385 (Clone: HIB19) Biolegend Cat#302240

Anti-human CD19 BV510 (Clone: HIB19) BD Bioscience Cat#740164

Anti-human CD20 BB700 (Clone: 2H7) BD Bioscience Cat#745889

Anti-human CD20 BV785 (Clone: 2H7) Biolegend Cat#302355

Anti-human CD22 PE-Cy7 (Clone: HIB22) BD Bioscience Cat#563941

Anti-human CD25 APC (Clone: M-A251) BD Bioscience Cat#560987

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Anti-human CD38 Alexa Fluor 488 (Clone: Biolegend Cat#303512

HIT-2)

Anti-human CD38 APC-R700 (Clone: HIT-2) BD Bioscience Cat#564979

Anti-human CD38 BV786 (Clone: HIT-2) BD Bioscience Cat#563964

Anti-human CD44 BUV737 (Clone: G44-26) BD Bioscience Cat#564941

Anti-human CD45 APC-eFluor 780 eBioscience Cat#47-0459-42

(Clone: HI30)

Anti-human CD45RA BV785 (Clone: HI100) Biolegend Cat#304140

Anti-human CD45RO PE (Clone: UCHL1) Biolegend Cat#304206

Anti-human CD56 BB700 (Clone: BD Bioscience Cat#566573

NCAM16.2)
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REAGENT or RESOURCE SOURCE IDENTIFIER

Anti-human CD56 BUV737 (Clone: BD Bioscience Cat#612767

NCAM16.2)

Anti-human CD56 BV510 (Clone: B159) BD Bioscience Cat#740171

Anti-human CD64 BUV737 (Clone: 10.1) BD Biocience Cat#564425

Anti-human CD86 BUV737 (Clone: FUN-1) BD Bioscience Cat#564428

Anti-human CD90 PE (Clone: 5E10) Biolegend Cat#328110

Anti-human CD106 PE (Clone: 51-10C9) BD Bioscience Cat#555647

Anti-human CD117 BV785 (Clone: 104D2) Biolegend Cat#313238

Anti-human CD123 BV785 (Clone: 6H6) Biolegend Cat#306031

Anti-human CD127 BV650 (Clone: A019D5) Biolegend Cat#351326

Anti-human CD141 APC (Clone: 1A4) BD Bioscience Cat#564123

Anti-human CD163 AF647 (Clone: GHI/61) BD Bioscience Cat#562669

Anti-human CD163 BV650 (Clone: GHI/61) BD Bioscience Cat#563888

Anti-human CD197 BV421 (Clone: G043H7) Biolegend Cat#353208

Anti-human CD197 PE/Dazzle 594 (Clone: Biolegend Cat#353236

G043H7)

Anti-human CD206 FITC (Clone: 15-2) Biolegend Cat#321104

Anti-human CD213a1 PE/Cy7 Biolegend Cat#360407

(Clone: SS12B)

Anti-human CD326 (Ep-CAM) BV650 Biolegend Cat#324226

(Clone: 9C6)

Anti-human CD370/Clec9A PE (Clone: 3A4) BD Bioscience Cat#563488

Anti-human CTLA4 BV421 (Clone: BNI3) Biolegend Cat#369606

Anti-human FcεRIa BV421 (Clone: AER-37) Biolegend Cat#334624

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Anti-human FOXP3 PE-Cy7 (Clone: eBiocience Cat#25-4776-42

PCH101)

Anti-human HLA-DR BV605 (Clone: L243) Biolegend Cat#307640

Anti-human HLA-DR BUV395 (Clone: BD Bioscience Cat#564040

G46-6)

Anti-human ICOS BV711 (Clone: DX29) BD Bioscience Cat#563833

Anti-human Ki67 AF488 (Clone: B56) BD Bioscience Cat#558616

Anti-human PD-1 BV786 (Clone: EH12) BD Bioscience Cat#563789

Anti-human PD-L1 BV786 (Clone: MIH1) BD Bioscience Cat#563739

Anti-human PD-L2 BV650 (Clone: MIH18) BD Bioscience Cat#563844

Anti-mouse B220 BV421 (Clone: RA3-6B2) Biolegend Cat#103251

Anti-mouse CD3 BV421 (Clone: 145-2C11) Biolegend Cat#100335

Anti-mouse CD4 APC-eFluor 780 (Clone: eBioscience Cat#47-0042-82

RM4-5)

Anti-mouse CD8 APC (Clone: 53-6.7) eBioscience Cat#17-0081-33

Anti-mouse CD11c BB515 (Clone: N418) DB Biosience Cat#565586

Anti-mouse CD16/CD32 Mouse BD Fc BD Pharmigen Cat#553142

Block (Clone: 2.4G2)

Anti-mouse CD44 PE (Clone: IM-7) Biolegend Cat#103023

Anti-mouse CD62L BV785 (Clone: MEL-14) Biolegend Cat#10440

Anti-mouse CD80 PerCP-Cy5.5 (Clone: Biolegend Cat#104722

16-10A1)

Anti-mouse Ki67 PE-eFluor-610 (Clone: eBioscience Cat#61-5698-82

SolA15)

Anti-mouse Ly6C BV711 (Clone: HK1.4) Biolegend Cat#128037

Anti-mouse Ly6G PE-Cy7 (Clone: 1A8) Biolegend Cat#127618

Anti-mouse CD86 APC (Clone: GL1) BD Bioscience Cat#558703

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Anti-mouse MHC II Alexa Fluor 700 (Clone: eBioscience Cat#56-5321-82

M5/114.15.2)

Anti-mouse MHC II BUV805 (Clone M5/ BD Bioscience Cat#748844

114.15.2)

Anti-mouse NK1.1 BV510 (Clone PK136) Biolegend Cat#108373

Anti-Puromycin Alexa Fluor 647 (Clone: This paper RRID: AB_2827926

R4743L-E8)

Anti-Puromycin Alexa Fluor 488 (Clone: This paper RRID: AB_2827926

R4743L-E8)
Biological Samples

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Human whole blood IFS (Institut Francais du Sang) Cat#311

Human renal carcinoma and Juxta tumoral University of California, San Francisco Trial protocol number: 13-12246; UCSF IRB
tissue (UCSF) institutional review board approved protocol (UCSF IRB# 14-15342)
(IRB)approved protocol (UCSF Committee on
Human Research (CHR)

Human meningioma La Timone hospital. Tumor bank Authorization number AC-2018-31053

Human brain metastasis La Timone hospital. Tumor bank Authorization number B-0033-00097

Chemicals, Peptides, and Recombinant Proteins

Actinomycin D (ActD) Merck Cat#A1410-2MG

2-Deoxy-Glucose (2DG) Merck Cat#D6134-25G

B-mercaptoethanoel VWR Cat#0482-100ML

Cycloheximide (CHX) Merck Cat#01810-1G

Dimethyl sulfoxide (DMSO) Merck Cat#D8418-100ML

DNasel Merck Cat#11284932001

Extrapure LPS Invivogen Cat#tlrl-2216

FCCP Merck Cat#C2920-10MG

Gentamicin Merck Cat#G1272-10ML

Glucose Merck Cat#G7021

Harringtonine Abcam Cat#ab141941

Ionomycin Merck Cat#I-0634

L-glutamine Life technologies Cat#25030-024

Liberase TL research Grade Merck Cat#540102001

Oligomycin A Merck Cat#75351-5MG

PMA Merck Cat#P-8139

Poly I:C Invivogen Cat#tlrl-pic

Puromycin Merck Cat#P7255

Sodium pyruvate Life Technologies Cat#11360-039

Critical Commercial Assays

Brilliant Stain Buffer Plus BD Bioscience Cat#566385

CellTiter-Glo Luminescent Cell Promega Cat#G7570

Viability Assay

Chromium Single Cells 30 Library & Gel 10X genomic Cat#120237

Bead Kit v2

Chromium Single Cell A Chip Kit 10X Genomics Cat#120236

Chromium i7 Multiplex Kit 10X Genomics Cat#120262

Dynabeads Hum. T-Activator CD3/CD28 for Life Technologies Cat#11131D

T Cell Expansion and Activation

Fixation/Permeabilization Solution Kit BD Bioscience Cat#554714

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Foxp3 / Transcription Factor Staining eBioscience Cat#00-5323-00

Buffer Set
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LIVE/DEAD Fixable Aqua Dead Cell Thermo Fisher Cat#L34957

Stain (BV510)

RosetteSep Human T Cell Enrichment Stem Cell Technologies Cat#15021

Cocktail

Zombie UV Fixable Viability kit Biolegend Cat#423107

Zombie Yellow Fixable Viability Kit Biolegend Cat#BLE423103

Deposited Data

Single cell RNA-seq data This paper GEO: GSE159913

Experimental Models: Cell Lines

Mouse: MEF cells ATCC SCRC-1008

Experimental Models: Organisms/Strains

CellRanger (V3.02 and v3.1.0) 10X Genomics https://support.10xgenomics.com/singlecell-

gene-expression/software/downloads/ latest

Mouse: C57BL/6J The Jackson Laboratory Stock 000664

Software and Algorithms

FlowJo Treestar V10.3

FlowR Guillaume VOISSINE https://github.com/VoisinneG/flowR

Ggplot2 (v3.2.1, R package) Wickham, 2009 https://ggplot2.tidyverse.org

Seahorse Wave Desktop Agilent V2.6

Prism8 GraphPad V8.3

R (v3.6.1) Cran, The R foundation https://www.r-project.org

R software package Seurat Satija et al., 2015 http://satijalab.org/seurat

Seurat (v3.1.0, R package) Stuart et al., 2019 https://github.com/satijalab/seurat

RESOURCE AVAILABILITY

Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rafael J. Arguello
(€ [email protected]).

Materials Availability
All the reagents, including a full panel of inhibitors and the monoclonal antibody clone R4743L-E8, conjugated with Alexa Fluor 647 or Alexa
Fluor 488 (SCENITH kit) are available upon reasonable request. Further information by email to the lead contact and at http://
www.scenith.com/.

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Data and Code Availability

The accession number for the single cell RNA sequencing data reported in this paper is GEO: GSE159913 [NCBI tracking system #21365922]

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mouse Experiments
Wild type C57BL/6 mice were purchased from Jackson Laboratories and maintained in the animal facility of CIML under specific pathogen-free
conditions and maintained at 21C in a 12 h light-dark cycle with water and food ad libitum. This study was carried out in strict accordance with
the recommendations in the Guide for the Care and Use of Laboratory Animals the French Ministry of Agriculture and of the Eur opean Union.
Animals were housed in the CIML animal facilities accredited by the French Ministry of Agriculture to perform experiments on alive mice. All
animal experiments were approved by Direction De´ partementale des Services Ve´ te´ rinaires des Bouches du Rhoˆ ne (Approval number A13-
543). All efforts were made to minimize animal suffering.
To obtain blood, six to eight week male mice where euthanized by CO 2 and blood was collected by cardiac puncture in Heparin tubes. To
obtain splenocytes, six to eight weeks old wild type C57BL/6J male mice were euthanized by cervical dislocation and splenectomized. Mouse
splenocytes were cultured in DMEM containing 5% of Fetal Calf Serum (FCS) and 50 mM of 2-Mercaptoethanol (Mouse cells culture media,
MCCM) at 37C 5% of CO2. Single cells suspentions from the spleens were generated and cultured in MCCM.
For in vitro studies, FLT3L BM-derived DCs (FLT3L-bmDCs) were differentiated in vitro from the bone marrow of 6-8 week/old from the same
male mice. Bone marrow were kept from femur and tibia and plate at 1.5 3 106 cells/mL with 4mL/well in 6-well plates in RPMI (GIBCO), 10% of
Fetal Calf Serum (FCS) and 50 mM of 2-Mercaptoethanol (Mouse cells culture media, MCCM) during 6 days at 37C 5% of CO 2 culture.
Differeciation has been done by adding directly FLT3L in culture at day 0.

Human Experiments
The renal carcinoma patient enrolled in this study provided written and informed consent to tissue collection under a University of California,
San Francisco (UCSF) institutional review board (IRB)-approved protocol (UCSF Committee on Human Research (CHR) no. 13-12246). The
meningioma and brain meastasis patients enroled in this study provided written and informed consent in accordance with institutional, national
guidelines and the Declaration of Helsinki. This protocol was approved by institutional review board (AP-HM CRB-TBM tumor bank:
authorization number AC-2018-31053, B-0033-00097). The identity, age (between 30 and 60) and sex of the adult cancer patients and healthy
donors was kept confidential following the ethics comitee guidelines.
Mononuclear cell enriched from blood of healthy donors (P1-P5) (3 Female, 2 Male; age between 30 and 60; mean 43) was submitted to
Ficoll-paque plus (PBL Biomedical Laboratories). PBMCs and whole blood were cultured in the absense (non stimulated) or in the presence of
LPS for 4 h. Immune cell stimulations were performed in the absence (Control) or presence of 0,1 mg/mL of extrapure Lipopolysacharide
(Invivogen), 10 mg/mL Poly I:C (Invivogen) or PMA (5 ng/mL; Sigma) and ionomycin (500 ng/mL) over night for T cell stimulations and 4 h for
DCs. T cells from human donors (P1, P2, P3) were isolated using the RosetteSep negative isolation method and activated (using BD Human T
cell activator beads coated with anti-CD3 and anti-CD28) or not.

Cell Lines
Mouse Embryonic Fibroblast (MEF) cells derived from C57BL/6 background male and female sex mixed were used. For experiments, MEFs were
cultured in DMEM culture media supplemented with 10% FCS at 37C 5% of CO 2 culture.

METHOD DETAILS

ATP Measurement
2 3 104 MEFs were seeded in 100ul of 5% FCS DMEM culture media ON in opaque 96 well plates. Cells were incubated with the inhibitors for
the times indicated in the Figure. After, 100ul of Cell titer-Glo luminiscence ATP reconstituted buffer and substrate (Promega) was added to
each well and Luminiscence was measured after 10 min following manufacturer instructions. A standard curve with ATP was performed using
the same kit and following manufacturer instructions.

Metabolic Flux Analysis (Seahorse)

OCR and ECAR were measured with the XF24 Extracellular Flux Analyzer (Seahorse Bioscience). 4 3 105 cells with aCD3/aCD28 beads or not,
were placed in triplicates in XF medium (nonbuffered Dulbecco’s modified Eagle’s medium containing 2.5 mM glucose, 2 mM L-glutamine, and
1 mM sodium pyruvate) and monitored 25 min under basal conditions and in response to 10mM glucose, 1 mM oligomycin, 100 mM 2-Deoxy-
Glucose. Glycolytic capacity was measured by the difference between ECAR level after add oligomycin and before add glucose. O CR, ECAR and
SRC parameters was analyzed and extract from Agilent Seahorse Wave Desktop software. Glycolytic capacity was obtained by the difference
between ECAR level after add Oligomycin and before add Glucose.

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SCENITH
Fifty microliters of blood were seeded in 96 well plates for studying blood cells metabolism. Alternatively cells were plated at 1 3 106 cells/mL,
0.5 mL/well in 48-well plates. Experimental duplicates/triplicates were performed in all conditions. After differentiation, activation or harvesting
of human of cells, wells were treated during 30-45 min with Control, 2-Deoxy-D-Glucose (DG, final concentration 100mM), Oligomycin (Oligo,
final concentration 1 mM), or a sequential combination of the drugs at the final concentrations before mentioned. As negative control, the
translation initiation inhibitor Harringtonine was added 15 min before addition of puro (Harringtonine, 2 mg/mL). Puro (final concentration 10
mg/mL) is added during the last 15-45 min of the metabolic inhibitors treatment. After puro treatment, cells were washed in cold PBS and
stained with a combination of Fc receptors blockade and fluorescent cell viability marker, then primary conjugated antibodies against surface
markers during 25 min at 4C in PBS 1X 5% FCS, 2mM EDTA (FACS wash buffer). After washing, cells were fixed and permeabilized using FOXP3
fixation and permeabilization buffer (Thermofisher eBioscience) following manufacturer instructions. Intracellular staining of puro using our in
house produced fluorescently labeled anti-Puro monoclonal antibody with Alexa Fluor 647 to obtain up to 10 times better signal to noise ratio
than commercially available monoclonal antibodies was performed by incubating cells during 1 h at 4C diluted in permeabilization buffer. For
SCENITH troubleshooting see Table S2.

Processing of Human Solid Tumors SCENITH

0.2-0.4 g of solid tumor tissue was partially dissociated using chirurgical scissors or tissue chopper (McIlwain Tissue Chopper Standard plate) to
generate ‘‘tumor explant suspention.’’ Tissue explants suspention, containing tissue cubes of approximately 400 mm of cross section, were put
in suspention in complete RPMI media and incubated directly with control or metabolic inhibitors, and with puro following the SCENITH
protocol. Next, tumor explants were dissociated using Tissue Liberase and DNaseI with the help of a Gentle Macs (Miltenyi biotec) following
manufacturers instructions. Cell suspentions were washed, counted and 2-5 3 106 total cells were seed in triplicates before proceeding with
lived dead and FC block staining. Next, cells were stained for surface makers, fixed and permeabilized with FOXP3 fixation and permeabilization
kit and stained for nuclear and cytoplasmic markers as mentioned above.

Human Single Cell RNA-sequencing

Live CD3-CD19/20-CD56- cells were sorted from renal carcinoma tumor and juxta tumoral tissue using a BD FACSAria Fusion. After sorting, cells
were pelleted and resuspended at 1 3 103 cells/ml in 0.04% BSA/PBA and loaded onto the Chromium Controller (10X Genomics). Samples were
processed for single-cell encapsulation and cDNA library generation using the Chromium Single Cell 30 v2 Reagent Kits (10X Genomics). The
library was subsequently sequenced on an Illumina HiSeq 4000 (Illumina).

Single Cell Data Processing

Sequencing data was processed using 10X Genomics Cell Ranger V1.2 pipeline. The Cell Ranger subroutine mkfastq converted raw, Illumina bcl
files to fastqs which were then passed to Cell Ranger’s count, which aligned all reads using the aligner STAR (Dobin et al., 2013) against GRCh38
genomes for human cells. After filtering reads with redundant unique molecular identifiers (UMI), count generated a final gene-cellular barcode
matrix. Both mkfastq and count were run with default parameters.

Cellular Identification and Clustering of scRNaseq data

For each sample, the gene - barcode matrix was passed to the R (v. 3.6.0) software package Seurat (Satija et al., 2015) (http://
satijalab.org/seurat) (v3.1.1) for all downstream analyses. We then filtered on cells that expressed a minimum of 200 genes and required that
all genes be expressed in at least 3 cells. We also removed cells that contained > 5% reads associated with cell cycle genes (Kowalczyk et al.,
2015; Macosko et al., 2015). Count data was then log2 transformed and scaled using each cell’s proportion of cell cycle genes as a nuisance
factor (implemented in Seurat’s ScaleData function) to correct for any remaining cell cycle effect in downstream clustering and differential
expression analyses. For each sample, principal component (PC) analysis was performed on a set of highly variable genes defined by Seurat’s
FindVariableGenes function. Genes associated with the resulting PCs (chosen by visual inspection of scree plots) were then used for graph-
based cluster identification and subsequent dimensionality reduction using t-distributed stochastic neighbor embedding (t-SNE). Cluster-based
marker identification and differential expression were performed using Seurat’s FindAllMarkers for all between-cluster comparisons.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical Analysis
Statistical analysis was performed with GraphPad Prism software. When several conditions were to compare, we performed a oneway ANOVA,
followed by Tukey range test to assess the significance among pairs of conditions. When only two conditions were to test, we performed
Student’s t test or Welch t test, according the validity of homoscedasticity hypothesis (* p < 0.05, ** p < 0.01, *** p < 0.005).

Quantification and Meaning of SCENITH Derived Parameters

To quantify the energetic metabolism parameters that constitute the metabolic profile of a cell, such as pathways dependency, we used simple
algorithms that quantifiy the relative impact of inhibiting a given pathway compared to a complete inhibition of ATP synthesis (Figure 1F). While

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SCENITH allow the use of any combination of metabolic or signaling inhibitors, herein we focused on inhibitors of glycolysis and of mitochondrial
respiration to derive metabolic parameters. The percentual of glucose dependence (Gluc. dep) quantifies how much the translation levels are
dependent on glucose oxidation. Gluc. dep is calculated as the difference between PS levels in 2-Deoxy-D-Glucose (DG) treated cells compared
to control (Co), divided by the difference in PS upon complete inhibition of ATP synthesis (DG, first and then Oligomycin A, combined; treatment
DGO) compared to control cells (Figure 1F). In a similar fashion, percentual mitochondrial dependence (Mitoc. dep) quantifies how much
translation is dependent on oxydative phosphorylation. Mitoc. dep is defined as the difference in PS levels in Oligomycin A (‘‘O,’’ mitochondrial
inhibitor) treated cells compared to control relative to the decreased in PS levels upon full inhibition of ATP synthesis inhibition (treatment
DGO) also compared to control cells (Figure 1F). Two additional derived parameters, ‘‘glycolytic capacity’’ (Glyc. cap) and ‘‘fatty acids and amino
acids oxidation capacity’’ (FAO and AAO cap) were also calculated. Glycolytic capacity is defined as the maximum capacity to sustain protein
synthesis levels when mitochondrial OXPHOS is inhibited (Figure 1F; see Statistical Analysis). Converserly, FAO and AAO capacity is defined as
the capacity to use fatty acids and aminoacids as sources for ATP production in the mitochondria when glucose oxidation is inhibited (Glycolysis
and glucose-derived acetyl-CoA by OXPHOS) (Figures 1F and S1B). While the total level of translation correlates with the global metabolic activity
of the cells, the dependency parameters underline essential cellular pathways that cannot be compensated, while ‘‘capacity’’; as the inverse of
dependency, shows the maximun compensatory capacity of a subpopulation of cells to exploit alternative pathway/s when one is inhibited
(Figures 1F and S1C).
For standard deviation calculation of SCENITH in different cell types of one sample (Figures 3 and 4), we followed the propagation of error
that is required when the means of means are used into a formula. For standard deviation calculation:

Co = GeoMFI of anti-Puro-Fluorochrome upon Control treatment

DG = GeoMFI of anti-Puro-Fluorochrome upon 2-Deoxy-D-Glucose treatment
O = GeoMFI of anti-Puro-Fluorochrome upon Oligomycin A treatment
DGO = GeoMFI of anti-Puro-Fluorochrome upon DG+O treatment

100 Co DG
Glucose Dependence ðGluc: depÞ = ðCoð DGO ÞÞ

SDGlucdep = Co ½Gluc:dep:23 SDCo2ffi+s vDG ½Gluc:dep:23 SDDG2ffi+s vDGO ½Gluc:dep: 3 SDDGO ffi v
v v 2 2v

SDGlucdep =uutv 100ðDGO DG2 Þ23 SDCo2!ffi+sDGO 100 Co23 SDDG2ffi+vtuu 100CoðCoDGO DG2Þ23 SDDGO2!ffi ðCo DGOÞ

ð Þ

100 Co O
MitochondrialdependenceðMitoc:depÞ = ðCoð DGO ÞÞ

SDMitodep =vtuu 100ðDGO DG2 Þ23 SDCo2!ffi+sDGO 100 O23 SDO2ffi+vuut 100CoðCoDGO DG2Þ23 SDDGO2!ffi ðCo DGOÞ

ð Þ

100 Co O
GlycolyticcapacityðGlyco:capÞ = 100 ðCoð DGO ÞÞ

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SDGlyc:cap: =vutu 100CoðDGODGO DG2 Þ23 SDCo2!ffi+sCo 100DGO23 SDO2ffi+vuut 100DGOðDG CoCo2Þ23 SDDGO2!ffi ð

Þ ð Þ

100 A DG
FAOandAAOcapacityðFAOandAAOcapÞ = 100 ðA ð DGO ÞÞ

SDFAOandAAOcap: =utuv 100ðDGO DG2 Þ23 SDCo2!ffi+sCo 100DGO23 SDO2ffi+uutv 100DGOðDG CoCo2Þ23 SDDGO2!ffi ðCo

DGOÞ ð Þ

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