Dual stromal and tumoral origins of TWEAK drive CSC maintenance in ovarian cancer relapse

Serous ovarian cancer (OC) has one of the highest relapse rates of all cancers, with around 85% of patients relapsing following chemotherapy. Evidence suggests that cancer stem-like cells (CSCs) exhibit chemoresistance, sustained self-renewal, and tumor-initiating potential to drive recurrence. We have previously identified TWEAK, an inflammatory cytokine enriched in OC tumors post-chemotherapy, promotes CSC maintenance and survival. Furthermore, antagonism of TWEAK-Fn14 signaling following chemotherapy in a murine relapse model significantly prolonged survival. Because Fn14 is broadly expressed within the tumor microenvironment, we sought to identify sources of TWEAK. Conventionally, TWEAK is secreted by macrophages, and although single cell sequencing of treatment-naive human OC tumors suggests that infiltrating tumor associated macrophages (TAMs) primarily express TWEAK, recent work in relapsing pancreatic cancer identified cancer cells as an additional source of TWEAK. We therefore hypothesized that TWEAK in ovarian cancer is derived from multiple sources, including macrophages and cancer cells. Since macrophages are the predominant immune cell type found in OC tumors, we used an interperitoneal xenograft nude mouse bearing human CAOV4 OC cells to model relapse. Inhibition of macrophage recruitment using a CSF-R1 inhibitor BLZ945 during chemotherapy significantly reduced CSC expression and improved survival, compared to chemotherapy alone. Using a 14-marker flow cytometry panel, we profiled TWEAK expression in relapsed tumors across macrophage (CD45, F4-80, CD11b, LyVe1, CD80, CD206, CD273, MHC Class II), endothelial (CD31), fibroblast (PDGFRa), and CSCs (CD117) populations. We identified 12 distinct cell populations, representing four TAM (CD45+/F4-80+/CD11b+) clusters including two high in MHC Class II, one tissue-resident macrophage (TRM) cluster (CD45+/F4-80+/LyVe1+), two fibroblast clusters, one endothelial cluster, and four cancer cell clusters. Four clusters expressed TWEAK, included one TAM, TRM, fibroblast, and OC cluster. The highest baseline TWEAK expression was in the TAM and TRM clusters; however, these decreased after chemotherapy. The OC cluster, comprised of CSCs, significantly increased (twofold) TWEAK expression following chemotherapy and were BLZ945 responsive, suggesting their chemotherapy-dependent TWEAK expression was also macrophage-dependent. Preliminary in vitro studies of gene expression, imaging, and flow cytometry further indicate that OC-derived TWEAK increases in response to chemotherapy. Future studies will investigate how macrophages regulate OC-derived TWEAK and the contribution of this signaling to relapse. Defining the stromal and tumoral cell sources of TWEAK and delineating their roles in TWEAK-Fn14 mediated tumor recurrence may facilitate development of targeted therapies for patients at risk of recurrence.