Cancer-associated fibroblasts provide a stromal niche for liver cancer organoids that confers trophic effects and therapy resistance.

Abstract Background & Aims Cancer associated fibroblasts (CAFs) play a key role in cancer process, but the research progress is hampered by the paucity of preclinical models essential for mechanistic dissection of cancer cell-CAF interactions. Here, we aim to establish a 3D organotypic co-cultures of primary liver tumor-derived organoids with CAFs, and to understand their interactions and the response to treatment. Methods Liver tumor organoids and CAFs were cultured from murine and human primary liver tumors. 3D co-culture models of tumor organoids with CAFs and trans-well culture systems were established in vitro. A xenograft model was used to interrogate the cell-cell interactions in vivo. Gene expression analysis of CAF markers in our hepatocellular carcinoma (HCC) cohort and an online liver cancer database indicated the clinical relevance of CAFs. Results To functionally investigate the interactions of liver cancer cells with CAFs, we have successfully established murine and human 3D co-culture models of liver tumor organoids with CAFs. CAFs promoted tumor organoid growth in co-culture with direct cell-cell contact and in trans-well system via paracrine signaling. Vice versa, cancer cells secret paracrine factors regulating CAF physiology. Co-transplantation of CAFs with liver tumor organoids of mouse or human origin promoted tumor growth in xenograft models. Moreover, tumor organoids conferred resistance to clinically used anti-cancer drugs including Sorafenib, Regorafenib and Fluorouracil (5-FU) in the presence of CAFs, or the conditioned medium of CAFs. Conclusions We have successfully established murine and human 3D co-culture models and have revealed robust effects of CAFs in liver cancer nurturing and treatment resistance.