Normal Uterine Fibroblast Are Reprogramed into Ovarian Cancer-Associated Fibroblasts by Ovarian Tumor-derived Conditioned Media

Abstract

SUMMARYCancer-associated fibroblasts (CAFs) are key contributors to ovarian cancer (OC) progression and therapeutic resistance through dysregulation of the extracellular matrix (ECM). CAFs are a heterogenous population derived from different cell types through activation and reprogramming. Current studies rely on uncharacterized heterogenous primary CAFs or normal fibroblasts that fail to recapitulate CAF-like tumor behavior. Here, we present a translatable-based approach for the reprogramming of normal uterine fibroblasts into ovarian CAFs using ovarian tumor-derived conditioned media to establish two well-characterized ovarian conditioned CAF lines. Phenotypic and functional characterization demonstrated that the conditioned CAFs expressed a CAF-like phenotype, strengthened proliferation, secretory, contractility, and ECM remodeling properties when compared to resting normal fibroblasts, consistent with an activated fibroblast status. Moreover, conditioned CAFs significantly enhanced drug resistance and tumor progression and resembled a CAF-like subtype associated with worse prognosis. The present study provides a reproducible, cost-effective, and clinically relevant protocol to reprogram normal fibroblasts into CAFs using tumor-derived conditioned media. Using these resources, further development of therapeutics that possess potentiality and specificity towards CAF-mediated chemoresistance in OC are further warranted.

Multicompartmentalized microvascularized tumor-on-a-chip to study tumor-stroma interactions and drug resistance in ovarian cancer

“…Importantly, the device allowed us to investigate tumor-stroma cellular crosstalk, compare normal or cancer-associated fibroblast’s role in modulating drug response, and further investigate targeted therapies that can overcome this induced drug resistance. This study demonstrates that CAF are key contributors in the OC-TME compartmentalization and influence drug response, which is in accordance with ours and other reports [28, 36-38]. This device grants drug screening under a faithful recapitulation of the OC-TME, which would provide strong evidence for allowing new opportunities to improve chemotherapy effectiveness and mitigating chemoresistance in ovarian cancer.…”

“…Moreover, it is known that in the TME cancer cells recruit stromal cells into their vicinity through tumor-stromal interactions and signaling molecules and activate them into cancer-like phenotype [36]. In this study, we have used cancer-associated fibroblasts, as activated fibroblasts, derived from primary human uterine fibroblasts by their exposure to conditioned media from specific OC cell line (KURAMOCHI or SKOV-3) that have been previously functionally characterized in terms of CAF-like phenotype, higher proliferation rate, contractility, ECM remodeling, tumor progression, and drug resistance [28]. CAF are the most important cell type in the OC-TME constituting a heterogeneous population of cells because they can be derived from normal fibroblasts, bone marrow mesenchymal stem cells, adipocytes, endothelial cells, or epithelial cells and they can be activated through different signaling pathways, exosomes, or cytokines such as TGF-β and also through ECM remodeling, although most of these CAF subtypes have not been fully characterized yet.…”

“…Moreover, human adipose-derived stem cells (ADSC) were used as a normal fibroblast model because of their high expansion ability and reproducibility suitable for high-throughput research, although they are not directly representative of normal tissue origin including fallopian tube or ovary [47]. Currently, the OC-stroma interaction studies use a variety of normal fibroblasts from different non-ovarian origins such are NIH3T3 fibroblasts from mouse embryos [28], lung fibroblasts [48], or dermal fibroblasts [49]. Unfortunately, the immortalized human normal ovarian fibroblast cell line (NOF151-hTERT) [50] is not commercially available.…”

“…Additional CAF- or ECM-targeting drugs or combination of drugs [66] should be further explored, as well as validation of TME compartmentalization with additional cell types present in the OC-TME such as adipocytes, mesothelial cells [46], and immune cells. Investigating tumor-immune interactions and immunotherapies could improve outcomes for OC patients, considering that high grade serous carcinoma was one of the first cancer types were the presence of tumor infiltrated lymphocytes was correlated with higher survival rates and CAF act as a barrier for immune infiltration and induce tumor inflammatory processes [28, 37].…”

“…Primary human adipose-derived stem cells (StemPro TM , R7788115, ThermoFisher Scientific) were used as a normal fibroblasts control (NF) and they were cultured in MesenPro RS TM Medium supplemented by MesenPro RS TM Supplement (12746012, Gibco). Cancer-associated fibroblasts (CAF) specific for each OC cell line (CAF-KURAMOCHI or CAF-SKOV3) were developed by growing primary human uterine fibroblasts (HUF, ATCC PCS-460-010) in presence of conditioned media from each OC cell line as previously described [28].…”