Characterization and Clinical Relevance of Endometrial CAFs: Correlation between Post-Surgery Event and Resistance to Drugs

Abstract

Cancer-associated fibroblasts (CAFs) within a solid tumor can support the progression of cancer. We studied the identification and characterization of patient-derived endometrial CAFs in the context of their clinical relevance in endometrial cancers. We established patient-derived primary cultures of CAFs from surgically resected tumors (TCAF) and tumor-adjacent normal (NCAF) tissues in 53 consented patients with success rates of 97.7% and 75%, respectively. A passage of CAF was qualified by the (1) absence of CK 8,18,19, EpCAM, CD45, and CD31, and (2) presence of SMAalpha, S100A4, CD90, FAP, TE-7, CD155, PD-L1, TGFB, PDGFRA (qRT-PCR, flow cytometry, Western blot, ICC). Out of the 44 established CAFs, 31 were aggressive (having an early, i.e., 4–7 week, establishment time and/or >3 passages) compared to 13 which were non-aggressive. A post-surgery-event (PSE) was observed in 7 out of 31 patients bearing aggressive CAFs, 2 of whom were also positive for CTCs, while none of the 13 patients bearing non-aggressive CAFs had events. A positive correlation was found between patients with grade 3 (p = 0.025) as well as stage 3/4 diseases (p = 0.0106) bearing aggressive CAFs and the PSE. Finally, aggressive TCAFs from patients with PSE resisted the effects of paclitaxel and lenvatinib on the growth of HUVEC and endometrial tumor cells. Our study is the first to report a correlation between the PSE and the aggressive nature of CAFs in endometrial cancers and provides an undeniable reason to study the in-depth mechanism of CAF function towards the development of treatment resistance in endometrial cancers.

Endometrial CAFs Resist Effect of Antitumor Drugs In A Patient-Derived 2-Cell Hybrid Co-Culture Model

“…The treatment with paclitaxel decreased the growth of AN3CA cells in a single-cell AN3CA-only culture. Since we have previously reported the effect of paclitaxel on 3D Matrigel growth of AN3CA cells plated on CAF [9], the validity of the HyCC formats here was confirmed by testing the effect CAF on the effect of paclitaxel (Figure 3) as an internal validation control of drug effect as compared to the non-treated vehicle control (Figure 2) at day 7.…”

“…Our cohort consisted predominantly of patients with endometrioid adenocarcinoma, with fewer patients with high-grade serous carcinomas of both papillary and mixed types (see Table 1 of [9]). Paclitaxel is routinely used (weekly) as a single agent to patients with recurrent or metastatic disease is the most commonly practiced approach in the clinics [12] (https://www.cancer.org/).…”

“…We established and characterized the primary culture of CAF from the resected tumor tissues from patients (TCAF here off used interchangeably with CAF) [9]. As mentioned, we established a novel matrigel-based On-Top 2-cell HyCC model [8].…”

“…Our cohort involved a total of 53 patients with endometrial cancers who participated in the study and provided informed consent. The information about the patients included in the study has been presented earlier [9]. In short, CAFs were established from samples received at the time of surgery.…”

“…Characterization and validation of the primary culture of TCAF in the ex vivo culture were performed based on expression (ICC, qRT-PCR, WB, and flow cytometry) of positive and negative markers, as mentioned earlier [9]. In short, TCAFs were positive markers of CAF, including SMA, FAP, TE-7, and S100A4, , while they were always negative for EpCAM and CK 8,18 (a positive marker of epithelial tumor cells), CD31 (endothelial marker) and CD45 (LCA marker for leucocytes).…”