Engineering Ovarian Cancer-Associated Fibroblasts using tumor conditioned media

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 and have limited expansion capacity, making it difficult to conduct a reproducible study of their role in OC chemoresistance. Therefore, there is a need to establish protocols that allow for the reprogramming of normal fibroblasts using tumor-conditioned media. The overall objective of this investigation is to present a protocol to reprogram human uterine fibroblasts into CAFs using tumor-derived conditioned media, followed by phenotypic and functional characterization of the CAFs in vitro. 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) were reprogrammed into CAFs using tumor-derived conditioned media from two OC cell lines (Kuramochi and SKOV3). Tumor-derived conditioned media was functionally characterized by cytokine/chemokine array. HUFs and conditioned-CAFs were monitored routinely for morphological changes over passages. Expansion capacity was assessed in HUFs, conditioned CAFs and compared to primary OC-CAFs (positive control). Conditioned CAFs were phenotypically characterized by analyzing fibroblast markers (CD90, CD73, vimentin), CAF markers (FAP, α-SMA), and negativity for epithelial markers (EpCAM, Cytokeratin and PAX8), as well as their cytokine secretion and ECM deposition capabilities. Moreover, conditioned CAFs were functionally characterized by collagen-contraction assay, promotion of tumor growth, spheroid formation, and enhancement of drug resistance to standard of care carboplatin treatment in comparison to HUFs and primary CAFs. Specifically, tumor growth and enhancement of drug resistance were assessed using physiologically relevant substrates (fibrinogen and collagen hydrogels) where the stromal cells were co-cultured with their corresponding OC cell line, which significantly improved cell attachment, growth, cell-cell interactions and cell-ECM interactions. Retrieval of tumor-derived conditioned media was also investigated and conditioned-CAFs were re-assessed phenotypically and functionally including marker expression, collagen contractility and tumor growth. CAFs were assessed until passage 15 including retrieval and the protocol was validated with different HUFs lots and at least 3 biological replicates per tumor-derived conditioned media.


Results, Conclusions, and Discussions:: All stromal cells including HUFs, conditioned CAFs and primary CAFs retained their fibroblast-like morphology in culture (Figure A). While as expected HUFs and primary CAFs have limited expansion capacity and short lifespan (about 10 doublings), conditioned CAFs have higher expansion rate about 3-4-fold and were able to be kept in culture for longer passages (Figure B). Phenotypically, conditioned CAFs were positive for stromal and CAF markers and negative for epithelial markers (selected markers are included in Figure C). Cytokine secretion capabilities of conditioned CAFs was similar to primary CAFs (Figure D). Functionally, conditioned CAFs have significantly higher collagen contractility compared to HUFs (Figure E). Moreover, conditioned CAFs significantly enhanced tumor growth about 2.5-3-fold (Figure F), enhanced drug resistance by reduced cell killing (Figure G) and promoted better spheroid formation with larger spheroids and more intact in shape (Figure H) when compared to HUFs, as expected in a CAF-like phenotype. Retrieval of conditioned media did not alter either their phenotype or functionality. Critically, these results suggest that uterine fibroblast can be reprogramed into CAFs using tumor-derived conditioned media. Further characterization of the matrisome as a key contributor to ECM remodeling will allow us to further refine the phenotype of the conditioned CAFs. In conclusion, our results present a reproducible and functionally characterized protocol to reprogram HUFs into CAFs using ovarian tumor-derived conditioned media. Moreover, our results are expected to have an important positive impact because they will provide strong evidence for further development of therapeutics that possess potentiality and specificity towards CAF-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.


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