Fig 2: Flow cytometry analysis of pre-MACS and post-MACS sorted cells at day 5 of EPC differentiationSamples were examined for the fraction of CD31+ cells using a flow cytometer.

Fig 3: Functional properties of CHIR- and DMSO-treated endothelial cells (ECs).(A) Flow cytometry analysis of CD31 expression in Passage 1 ECs following the dextran internalization assay. CD31+ cells were gated for further analysis. (B) Flow cytometry analysis of 10 kDa dextran-Alexa Fluor 488 (AF488) abundance in CD31+ cells. Cells were treated with DMSO or CHIR for 6 days prior to the assay. Representative plots from cells incubated with dextran for 2 hr at 37°C are shown. (C) Quantification of 10 kDa dextran-AF488 geometric mean fluorescence intensity in CD31+ cells. Treatment and assay conditions were as described in (B). Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA. (D) Quantification of the coefficient of variation (CV) of 10 kDa dextran-AF488 fluorescence intensity in CD31+ cells. Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA. (E) Transendothelial electrical resistance (TEER) of Passage 3 ECs. The x-axis indicates the number of days after seeding cells on Transwell inserts. Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different shape. p-value: two-way ANOVA. (F) Permeability of Passage 3 ECs to sodium fluorescein. Points represent replicate wells from two independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA.

Fig 4: Expression of mRNA for marker proteins of CAF by qRT-PCR: Each graph represents the expression of a gene of interest presented as its relative ratio to GAPDH. Inset in each graph is a melting curve of the gene of interest. CAF samples and the positive and negative controls for each gene are color-coded and listed at the bottom of the figure. (A) presents expressions of three negative markers of CAF, endothelial marker (CD31), epithelial marker (EpCAM), and common leucocyte antigen (CD45), in NCAFs and TCAFs with respective validation controls. (B) presents expressions of four positive markers of CAF (FAP-A, S100A4, PDGFR-A, and FGFR-1) in NCAFs and TCAFs with respective validation controls. (C) presents expressions of PD-L2 and CD44 in NCAFs and TCAFs with respective validation controls. (D) compares the qRT-PCR expression of markers with the protein expression of the same markers (SMA and PD-L1) by WB (inset) from a representative non-aggressive early passage NCAF and TCAF. The expressions of CD44, TGFB, and S100A4 by WB are also presented with the corresponding densitometric analyses (bar diagrams) using Image J. Beta-actin was used as the loading control.

Fig 5: Expression of marker proteins of CAF by flow cytometry: Expression of the six positives (CD155, CD90, SMA-A, S100A4, FAP-A) and two negatives (EpCAM and CD31) markers of CAF in the established TCAFs and NCAFs from the T and TAN samples from two patients; one bearing non-aggressive CAFs (A,B) and bearing aggressive CAFs (C,D) by flow cytometry. (D) compares the flow cytometry expression of SMA and S100A with the WB expression (inset) of SMA and S100A from the same representative aggressive CAFs (TCAF and NCAF). The expressions of PD-L1, and TGFB, by WB, are also presented. The protein expression of the same markers (SMA and S100A4) by WB (inset) from a representative aggressive early passage NCAF and TCAF. The expressions of other CAF markers, such as PD-L1 and TGFB, by WB are also presented with the corresponding densitometric analyses (bar diagrams) using Image J (E). Beta-actin was used as the loading control. The isotype controls for the respective protein are marked as open black lines compared to the red area under curves for the proteins. A three-color scale-based conditional formatting of the pattern of % expression of CAF markers of EpCAM, SMA-A, S100A4, and FAP (by flow cytometry) in the pairs of cultured TCAFs and NCAFs (early passages) from 17 representative patients (as presented with color codes) is presented as a heatmap (F). Red bars represent TCAFs and green bars represent NCAFs (E).