Reprogramming of Normal Fibroblasts into Ovarian Cancer-Associated Fibroblasts Via Non-Vesicular Paracrine Signaling Induces an Activated Fibroblast Phenotype

Introduction: Cancer-associated fibroblasts (CAFs) are key contributors to tumor progression and therapeutic resistance through dysregulation of the tumor extracellular matrix (ECM) composition and structure. CAFs may be derived from several cell types including normal fibroblasts, mesenchymal stem cells or epithelial cells through activation and reprogramming by cancer cells, recruitment or epithelial to mesenchymal transition. Ovarian cancer (OC) is one of the deadliest forms of cancer in women, where more than 80% of patients develop chemotherapy resistance, resulting in advanced recurrence and eventually death. Treatment barriers in ovarian cancer have been increasingly attributed to stromal reprogramming and activation, ECM remodeling, angiogenesis, and drug delivery. Thus, CAFs are considered to be notable players in promoting many aspects of tumor function and a promising targeted therapeutic option. While primary CAFs can be isolated from tumors and cultured in vitro, their lifespan is finite with limited expansion capacity, making it difficult to conduct reproducible studies of their role in OC chemoresistance. Therefore, there is a need for translational based approaches that allow for the reprogramming of normal fibroblasts into CAFs.

The overall objective of this investigation is to reprogram normal fibroblasts from both human uterine fibroblasts and ovarian fibroblasts origin into CAFs using ovarian tumor-derived conditioned media, followed by phenotypic and functional characterization of the conditioned CAFs. In the absence of reproducible and functionally characterized protocols to engineer and reprogram CAFs in culture, the development of effective anticancer therapies that mitigate CAF-mediated chemotherapy resistance in OC will likely remain difficult.

Materials and

Methods: Human primary uterine fibroblasts (HUFs) and immortalized normal ovarian fibroblasts (NOF151-hTERT, NOFs) were reprogrammed into CAFs using tumor-derived conditioned media from two OC cell lines (KURAMOCHI and SKOV-3). HUFs, NOFs, and uterine and ovarian-derived conditioned CAFs (uCAFs and oCAFs) were monitored routinely for morphological changes over passages. Expansion capacity was assessed in HUFs, NOFs, conditioned CAFs and compared to primary OC-CAFs (positive control). Both conditioned CAFs were phenotypically characterized by analyzing fibroblast markers (CD90, CD73, vimentin), CAF markers (FAP, α-SMA), and negativity for epithelial and immune markers (CD326 and CD45), as well as their cytokine secretion. Moreover, both conditioned CAFs were functionally characterized by collagen-contraction assay, ALDH activity, promotion of tumor growth (in 3D), and enhancement of drug resistance to standard of care carboplatin treatment (in 3D) in comparison to HUFs, NOFs, and primary CAFs. ECM signatures of the uterine-derived CAFs were assessed through transcriptomic analysis. Uterine CAFs were functionally and phenotypically characterized after the removal of tumor-derived conditioned media to demonstrate stable reprogramming. To investigate the activation of CAFs via non-vesicular paracrine signaling, conditioned media was subjected to ultracentrifugation to use conditioned media either free of extracellular vesicles (EVs) or containing only the EVs. Both HUFs and NOFs were cultured in these different media and evaluated for collagen contractility, ALDH activity, tumor growth promotion, and cytokine profile. CAFs were assessed until passage 15, including retrieval and the experiments were validated with different cell lots and at least 3 biological replicates per tumor-derived conditioned media.

Results, Conclusions, and Discussions: All stromal cells including HUFs, NOFs, uCAFs, oCAFs, and primary CAFs retained their fibroblast-like morphology in culture. Regrettably, cell surface marker expression was not reliable and just an indicator of activation through culture and exposure to serum as normal fibroblasts were positive for the same stromal and CAF-like markers than the conditioned CAFs. Therefore, we relied upon functional assays to more accurately confirm that the conditioned CAFs display a greater activation status when compared to normal fibroblasts. While HUFs and primary CAFs have limited expansion capacity and short lifespan, uCAFs have a higher expansion rate and were able to be kept in culture for longer passages (Figure A).Also, uCAFs had a significantly higher secretory profile (Figure B), higher collagen contractility (Figure C), and higher ALDH activity (Figure D) than HUFs and had similar results to primary OC-CAFs, revealing a clear reprogramming into a CAF-like phenotype. Promotion of cancer growth in 3D cultures revealed that while cancer co-culture with normal fibroblasts does not significantly increase tumor growth neither change drug sensitivity, uCAFs significantly promoted tumor growth and significantly enhanced drug resistance (Figure E). Moreover, a CAF-like transcriptional signature with involvement of the ECM/matrisome was identified in uCAFs (Figure F). Retrieval of conditioned media did not alter either the uCAF phenotype or functionality. By culturing the uCAFs in EVs and EVs free conditioned media, it was found that non-vesicular paracrine signaling was responsible for fibroblast activation and validated by tumor promotion (Figure G), ALDH activity (Figure H), and collagen contractility (Figure I). The same results were corroborated for NOF and oCAFs (data not shown). Critically, these results suggest that uterine and ovarian fibroblasts can be reprogrammed into CAFs using tumor-derived conditioned media.

In conclusion, our results present a reproducible and functionally characterized protocol to reprogram normal fibroblasts into CAFs using ovarian tumor-derived conditioned media. Moreover, our study is expected to have an important positive impact because it will provide strong evidence for further development of therapeutics that possess potentiality and specificity towards CAF/ECM-mediated chemotherapy resistance in OC and a translatable-based engineering approach that could be applied to different cancer cell types.

Acknowledgements (Optional): This project is supported by NIH/NIGMS Center for Cancer Biology 5P20GM103548 and NIH/NCI R21CA259158.