Patient-Derived Primary Cancer-Associated Fibroblasts Mediate Resistance to Anti-Angiogenic Drug in Ovarian Cancers

Abstract

Ovarian cancers rank first in both aggressiveness and dismal prognosis among gynecological neoplasms. The poor outcome is explained by the fact that most patients present with late-stage disease and progress through the first line of treatment. Ovarian neoplasms, especially epithelial ovarian cancers, are diagnosed at advanced/metastatic stages, often with a high angiogenesis index, one of the hallmarks of ovarian cancers with rapid progression and poor outcome as resistance to anti-angiogenic therapy develops. Despite therapy, the metastatic progression of aggressive ovarian cancer is a spectacularly selective function of tumor cells aided and abetted by the immune, mesenchymal and angiogenic components of the tumor microenvironment (TME) that enforces several pro-metastatic event(s) via direct and indirect interactions with stromal immune cells, cancer-associated fibroblasts (CAFs), and vascular endothelial cells. Since transdifferentiation of tumor endothelium is one of the major sources of CAFs, we hypothesized that ovarian CAF plays a critical role in resisting anti-angiogenic effects via direct crosstalk with endothelium and hence plays a direct role in the development of resistance to anti-angiogenic drugs. To test the hypothesis, we set up a hybrid ex vivo model for co-culture comprising Patient-Derived ex vivo primary CAFs from ovarian tumor samples and human umbilical vein endothelial cells (HUVEC). Patient-Derived CAFs were characterized by the mRNA and protein expression of positive (SMA, S100A4, TE-7, FAP-A, CD90/THY1), negative (EpCAM, CK 8,18, CD31, CD44, CD45), functional (PDGFRA, TGFB1, TGFB2, TGFRA) and immunological markers (PD-L1, PD-L2, PD-1) associated with CAFs by qRT-PCR, flow cytometry, Western blot, and ICC. Data from our HUVEC-on-CAF ex vivo Hybrid Co-Culture (HyCC) study demonstrate the pro-angiogenic effect of Patient-Derived ovarian CAFs by virtue of their ability to resist the effect of anti-angiogenic drugs, thereby aiding the development of resistance to anti-angiogenic drugs. Ascertaining direct experimental proof of the role of CAFs in developing resistance to specific anti-angiogenic drugs will provide an opportunity to investigate new drugs for counteracting CAF resistance and "normalizing/re-educating" TME in aggressive ovarian cancers. Our data provide a unique experimental tool for the personalized testing of anti-angiogenic drugs, positively predicting the development of future resistance to anti-angiogenic drugs well before it is clinically encountered in patients.