Fig 1: SEC-seq measuring the transcriptome and VEGF-A secretion of normoxic or hypoxic MSCs.a, Schematic of the detection of secreted VEGF-A protein and corresponding global gene expression for individual MSCs using the SEC-seq method. b, UMAP dimensionality reduction based on transcriptomes from SEC-seq experiments on normoxic and hypoxic MSCs in nanovials. Cells are labeled according to the culture condition. c, UMAP displaying VEGF-A secretion level, shown as log transformation of the UMI collapsed anti-VEGF-A oligo-barcode reads per cell. d, UMAP displaying VEGFA transcript levels, shown as normalized transcripts per cell. e, Distribution of VEGF-A secretion for cell-loaded nanovials in normoxic and hypoxic conditions, detected by FACS using a fluorescent anti-VEGF-A antibody (top) or by the SEC-seq experiment in (b) using the oligo-barcoded anti-VEGF-A antibody (middle). The last plot shows distribution of VEGFA transcript levels from the SEC-seq experiment cells in (b) (bottom). f, UMAP displaying cluster assignment. g, Violin plots by cluster showing VEGFA transcripts and VEGF-A secretion levels for all cells in the normoxic clusters (N1-N5, red shades), hypoxic clusters (H1–4, blue shades), and mixed clusters (M1–3, green shades) from (f). For mixed clusters, the levels are shown separately for normoxic and hypoxic cells. The dashed line represents the mean across all cells for each plot. Data below this threshold are lightened to highlight differences. h, Scatter plots showing the relationship between VEGFA transcript and VEGF-A secretion levels for individual normoxic (left) and hypoxic (right) cells from the experiment in (b). Best fit regression lines and Pearson correlation coefficients are shown. i, Plot showing the ranking of all detected genes based on the correlation of their transcript levels to the VEGF-A secretion level per cell for normoxic (top) and hypoxic (bottom) MSCs. The rank of the VEGFA gene is highlighted, and the top three genes per sample are also noted.
Fig 2: Characterization of the high-VEGF-A secreting MSC subpopulation.a, Scatter plot of the transcript to VEGF-A secretion correlation for all genes from SEC-seq experiments for normoxic and hypoxic MSCs from Figure 3. The 10 most highly correlating genes based on both experiments are labeled. b, Table giving the ranking (based on average correlation), gene name, correlation to secretion in normoxic and hypoxic cells, and the average of those two values for the ten top genes from (a). c, UMAPs showing VEGF-A secretion levels and expression of 5 select correlated genes from (b) in normoxic and hypoxic MSCs from Figure 3. The VEGF-A secretion UMAP is given from Fig. 3c for comparison. d, As in (c), for a separate SEC-seq experiment performed on MSCs in the normoxic culture condition. e, Cell clusters projected onto the UMAP of the replicate SEC-seq experiment f, Heatmap of the top 10 differentially expressed genes from each cluster (indicated on top) of the SEC-seq experiment in (c,d) (rows=genes, columns=individual cells). Displayed at the top are the log transformed VEGF-A secretion and VEGFA transcript levels. Right: top 3 genes differentially expressed gene for each cluster. g, Heatmap of the top GO terms found for all of the differentially expressed genes from the clusters in (e). The (–logP) value indicates if the term was enriched for a given cluster. h, Venn diagram showing the overlap of differentially expressed genes from the highly secreting cluster in 3 SEC-seq experiments (top left: normoxic MSCs from (3b), top right: hypoxic MSCs from (3b), bottom: normoxic MSCs from (4e)). Overlapping genes form the Vascular Regenerative Signal (VRS). i, Gene ontology analysis for VRS genes from (h). Similar terms were collapsed. j, Average of the normalized transcripts level of VRS genes per cell, displayed for the SEC-seq experiment in (e) and (3b). k, As in (j), for MSCs loaded in oligo-barcoded nanovials (see Fig. 2j-l). l, As in (j), for a standard scRNA-seq experiment on unsorted, suspended MSCs. m, Comparison of gene type classification for VRS genes and genes differentially expressed in all clusters in (e) except for those from cluster c5. n, Enrichment of possible TF regulators of the VRS genes based on the TRRUST database. o, Consensus rank of VEGF-A secretion to gene correlation based the SEC-seq experiments used in (h), with red dots displaying all VRS genes. p, Schematic depicting the heterogeneity of VEGF-A secretion in MSCs under normoxic and hypoxic conditions, highlighting the importance of the VRS genes for marking high VEGF-A secretion.
Fig 3: Chemotherapy modulates the pro-angiogenic properties of bCAFs through NOXA-mediated MCL-1 degradation and subsequent activation of NF-κB signaling.A (Left) Representative experiments of proteins expression level (MCL-1, BCL-xL, and NOXA) in bCAFs after 18 h of chemotherapy treatment (5-Fluorouracil 22 µM, Cisplatin 11 µM, and Doxorubicin 1 µM) or not evaluated using western blots, Actin expression was used as loading control. (Right) Quantification of the amounts of MCL-1 and BCL-xL protein band relative to Actin in bCAFs after chemotherapy treatment (n = 3), results are expressed as a ratio relative to untreated bCAFs. One sample t-test, **P < 0.01, ns non-significant. B RT-qPCR of VEGF-A, FGF2, and ANGPT1 mRNA in bCAFs after 18 h of chemotherapy treatment or not normalized on RPLPO mRNA expression. Mean and SEM of three independent experiments are represented as relative quantity of mRNA. Student t-test, *P < 0.05, ns not significant. C VEGF-A was analysed by ELISA in bCAFs CM after 18 h of chemotherapy. Student t-test, **P < 0.01. D bCAFs were treated or not with chemotherapy for 18 h. The treatment was removed and cells were rinsed and cultured for additional 72 h in EGM2 medium supplemented with 1% of FBS. After 72 h, conditioned media (CM) were recovered, and a portion was incubated with VEGF inhibitor (BVZ, 2 mg/mL) for 2 h −37 °C before adding on HUVECs for tubulogenesis assay. E Representative images of tubulogenesis assay of HUVECs cultured in conditioned media from bCAFs treated with chemotherapy or not +/− BVZ (2 mg/mL). F Quantification of meshes, master junctions, and master segments of HUVECs cultured in conditioned media from bCAFs treated or not with chemotherapy +/− BVZ (2 mg/mL) (n = 3). Two-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns non-significant. G (Left) Representative pictures of engrafted tumours on CAM at ED17, tumours are composed of T47D cells and bCAFs pre-treated or not with chemotherapy for 18 h before xenograft (Top). The tumours were treated with VEGF inhibitor (bevacizumab, BVZ 100 µg/CAM) every 2 days during one week until ED17 (Bottom). (Right) Quantification of blood vessels density around the tumours (within a 5 mm radius of the tumour). Two-way ANOVA, **P < 0.01, *P < 0.05, ns non-significant. H MCL-1, BCL-xL, and NOXA proteins expression levels in bCAFs, surexpressing MCL-1 (CAFpLvxMCL-1) or BCL-xL (CAFpLvxBCL-xL) or not (CAFpLvxCTRL) after 18 h of chemotherapy were evaluated using Western blots analysis. Actin expression was used as loading control. I VEGF-A secretion was analysed by ELISA in bCAFs CM in the same conditions as (H) (n = 3) Two-way ANOVA, ****P < 0.0001; ns non-significant. J MCL-1 and NOXA proteins expression levels in bCAFs expressing NOXA (CAFsgCTRL) or not (CAFsgNOXA) after 18 h of chemotherapy were evaluated using Western blots analysis. Actin expression was used as loading control. K Quantification (percentage of cells positive for nuclear p65) (right) and confocal image (left) of p65 staining (red) in bCAFs expressing NOXA (CAFsgCTRL) or not (CAFsgNOXA) and treated with chemotherapy (1 µM) for 18 h or not. The nuclei were counterstained with DAPI (blue) (n = 3). Scale bar = 50 μm. Two-way ANOVA, ****P < 0.0001, *P < 0.05, ns not significant. L VEGF-A secretion was analysed by ELISA in bCAFs CM in the same conditions as (J). M qRT-PCR of VEGFA, CXCL8, IL-1β, and CXCL1 mRNA in bCAFs treated with chemotherapy (1 µM) for 18 h or not in combination with an IKKβ inhibitor (AS602868, 10 µM) or not normalized on RPLPO mRNA expression (n = 3). Two-way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns non-significant.
Fig 4: VEGF-A secretion induced by MCL-1 targeting is mediated by NF-kB in bCAFs.A Volcano plot showing differential expressed proteins between bCAFs after MCL-1 gene silencing (bCAFsgMCL-1) or not (bCAFsgCTRL) (n = 4). The red dots represent the upregulated expressed proteins; the blue dots represent the proteins whose expression is downregulated. B A hierarchically clustered heatmap per sample showing differential expressed proteins associated to their biological process (Gene Ontology). Orange and blue represent up and downregulated expression in bCAFs after MCL-1 gene silencing (sgMCL-1) or not (sgCTRL). Color density indicating proteins intensity levels. Log2FC of each protein was indicated on bar plot. C Bar plot based on TRANSFAC and JASPAR PWMs databases (Enrichr) of the major transcription factors implicated in the transcription of the upregulated proteins after MCL-1 gene silencing in bCAFs. Combined score = −log(odds.ratio) × p-value. D (Left) Gene set enrichment analysis with HALLMARK_TNFA_SIGNALING_VIA_NFKB on single cell RNA sequencing data of bCAFs expressing MCL-1 (sgCTRL) or not (sgMCL-1) and (right) violin plot showing signature score in each cluster under all conditions. E, F Quantification (percentage of cells positive for nuclear p65) (right) and confocal image (left) of p65 staining (red) in (E) bCAFs treated or not with S63845 500 nM for 18 h or (F) bCAFs sgCTRL or sgMCL-1. The nuclei were counterstained with DAPI (blue) (n = 3). Scale bar = 50 μm. Student t-test, ***P < 0.001. G, H qRT-PCR of VEGFA, CXCL8, IL-1β, and CXCL1 mRNA in bCAFs (G) after S63845 500 nM for 18 h (n = 3) or (H) sgCTRL and sgMCL1 (n = 4), in presence or not with an IKKβ inhibitor (AS602868, 10 µM) normalized on RPLPO mRNA expression. Two-way ANOVA, ****P < 0.0001, ***P < 0.001, ns non-significant.
Supplier Page from BioLegend for ELISA MAX™ Deluxe Set Human VEGF