Fig. 1: Isolated NAFs and CAFs exhibit stable phenotypic differences in vitro.

a Principal component analysis of the bulk RNA-seq data of cultured CAFs (n = 6; #1–6) and NAFs (n = 3; #2–4). b Volcano plot depicting the significantly deregulated genes (p < 0.05, fold change >1.5) between CAFs and NAFs. Highly expressed (HEX) NAF and CAF differentiation markers are indicated. c Bar plot displaying the negative log10 false discovery rate (FDR) and number of differentially expressed genes (NDE) of the most significantly upregulated KEGG2020 terms enriched in isolated and cultured CAFs according to conducted bulk RNA-seq analysis. d Assessment of paired CAFs and NAFs to remodel a rat tail collagen 1 plug depicted by plug shrinkage after 24 h (n = 5; #3, #7, #8, #9, #10; paired t-test, **P < 0.01). e High expression of the top CAF differentiation markers is associated with poorer overall survival (left) in colorectal cancer, whereas the expression of the HEX CAF markers is not prognostic for OS (right). High expression was defined using the upper quartile of the total cohort. f Validation of HEX NAF and CAF markers by independent RT-qPCR. Comparison of the log2fc in CAFs (n = 6; #1–6) vs NAFs (n = 3; #2–4) measured by RT-qPCR (black) and bulk RNA-seq (gray) is displayed. g Validation of HEX NAF and CAF markers on protein level by Western Blot. h Quantitative evaluation of Western blot results in (g). Signal intensity was normalized to the respective GADPH bands in CAFs (n = 6; #1–6) compared to NAFs (n = 3; #2–4) with the average log2fc displayed. i Immunocytochemical detection of ITGA3 (left) and ADH1B (right) representing HEX-markers for NAFs and CAFs, respectively. For quantitative analyses total cell count and number of positive cells were determined using ImageJ and averaged among three random areas per sample in paired NAFs and CAFs (n = 5, #2, #3, #8, #11, #12). Scalebar represents 150 μm (paired two-tailed t-test, ***P < 0.001). #, patient numbers as detailed in Supplementary Tables S1, S2.