Fig 2: Representative photomicrographs showing subcellular localization of different positive markers of CAF, SMAalpha, S100A4, TE-7, and PD-L1, as well as negative markers, EpCAM and CK 8,18 of CAFs in established primary cultures of NCAF and TCAF samples from patients with endometrial cancers by ICC: Images show H&E (A) upper panel, and negative markers such as CK 8,18 and EpCAM (A) lower panel as well as expressions of positive markers including SMAalpha (B) upper panel, S100A (B) lower panel, TE-7 (C) upper panel, and PD-L1 (C) lower panel in NCAFs and TCAFs of tumor samples from different patients. Hematoxylin was used as the counterstain. For ICC, pictures were taken at 20× and 40× dry-objective of Olympus BX43 Microscope using cellSens 1.18 LIFE SCIENCE IMAGING SOFTWARE (OLYMPUS CORPORATION). Photomicrographs show the information stamps for magnifications and the scale bars. A three-color scale-based conditional formatting of the pattern of % expression of CAF markers of CK 8,18, EpCAM, SMAalpha, S100A4, and TE-7 (by ICC) in cultured TCAFs (early passages) from 29 representative patients sorted on the basis of histology of the disease with corresponding grades/stages (as presented with color codes and numbers) is presented as a heatmap (D). The heatmap displays the % expression of CK 8,18, EpCAM, SMAalpha, S100A4, and TE-7 in TCAFs from 29 out of a total of 31 patients as evaluated by ICC. A very similar pattern of the expression of markers in TCAFs between different histologies, endometrioid adenocarcinoma, and carcinosarcomas of endometrial cancers was observed, although the number of patients for the latter was much smaller in the cohort. Inset shows a positive correlation between the % expressions of S100A4 and PD-L1 in TCAFs in these 27 patients out of a total of 29 patients. Trendline demonstrated the Pearson correlation between S100A4 and PD-L1 by ICC in TCAFs from patients. p = 0.0373, n = 27. Created using GraphPad Prism Version 9.4.1. The beta-galactosidase stain of endometrial CAFs of late passage (P11) as compared to epithelial tumor cell lines of endometrial AN3CA cell line (P7): Endometrial CAFs stain positive in contrast to AN3CA endometrial cancer cell line (E). Inset shows a higher magnification of CAF (the scale bar is computer-generated), showing the subcellular distribution of the stain.

Fig 3: Cellular localization of proteins for CAF markers in the Patient-Derived primary culture of ovarian CAF from pre- and post-frozen passages established from the representative tumor sample, HU-A148-Ov2 of ovarian cancers: We tested the cellular localization of the expression of protein by ICC for SMA (B), TE-7 (C), PD-L1 (D), S100A4 (E), CK 8, 18 (F), and CD31 (G) in Patient-Derived CAFs (before and after thawing). The morphological features are presented by H&E stain (A). Insets represent photomicrographs in lower magnification.

Fig 4: Expression of mRNA for positive and negative CAF markers in the Patient-Derived primary culture of ovarian CAF established from the representative tumor sample of ovarian cancers: We tested the expression of mRNA for SMA, CD90, PDGFRA and FAP-A (A), PD-L1, S100A4, and CD44 (B), EpCAM, CD45, and CD31 (C) in the tumor-derived CAF from the patient with grade 1, stage IVA low-grade appendiceal mucinous neoplasm and SMA, FAP-A, and S100A4 (D), EpCAM, CD45, and CD31 (E) and TGFB1, TGFB2, TGFBR1 and TGFBR2 (F) in the tumor-derived CAF from the patient with stage IA adult granulosa cell tumor. Bars in red color represent CAF derived from the tumor tissue, bars in green color represent the RL-95-2 endometrial cancer cell line, bars in blue color represent HUF, bars in dark blue color represent the MDA-MB-231 breast cancer cell line, bars in mustard color represent HUVEC endothelial cell line, bars in ash color represents HCC1954 breast cancer cell line, bars in orange color represents human PBMC, bars in dark violet color represents SKOV3 ovarian cancer cell line, bars in black color represents AN3CA endometrial cancer cell line. The melting curves are presented as the inset in each bar diagram.

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).