Cancer-Associated Fibroblasts Provide a Stromal Niche for Liver Cancer Organoids That Confers Trophic Effects and Therapy Resistance

Abstract

We have successfully established murine and human 3dimensional co-culture models of primary liver tumorderived organoids with cancer-associated fibroblasts. This model system enables the study of the interactions between tumor cells and the stromal compartment and the response to anticancer drugs.BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play a key role in the cancer process, but the research progress is hampered by the paucity of preclinical models that are essential for mechanistic dissection of cancer cell-CAF interactions. Here, we aimed to establish 3-dimensional (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 Transwell culture systems were established in vitro. A xenograft model was used to investigate the cell-cell interactions in vivo. Gene expression analysis of CAF markers in our hepatocellular carcinoma 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 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 a Transwell system via paracrine signaling. Vice versa, cancer cells secrete 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 anticancer drugs including sorafenib, regorafenib, and 5-fluorouracil in the presence of CAFs, or the conditioned medium of CAFs. CONCLUSIONS:We successfully established murine and human 3D co-culture models and have shown robust effects of CAFs in liver cancer nurturing and treatment resistance.

Patient-Derived Cancer Organoids as Predictors of Treatment Response

“…Novel approaches for pancreatic cancer organoids include the co-culture with cancer-associated fibroblasts (CAFs) and using matched pancreatic ductal organoids. CAFs were shown to increase treatment resistance which shows the importance of tumor micro-environmental aspects on treatment efficacy ( 68 ). Using matched pancreatic ductal organoids and pancreatic cancer organoids the lack of therapeutic response of dual MEK-AKT inhibition observed in a clinical phase II trial ( 69 ) was investigated.…”

“…Importantly, hypoxia, a common characteristic of the tumor micro-environment of solid tumors which greatly contributes to malignant behavior and chemo- and radioresistance does develop in organoids once they reach a certain size ( 52 ). The importance of the tumor micro-environment has also been shown by incorporating cancer-associated fibroblasts in pancreatic cancer organoids which led to increased resistance towards treatment ( 68 ).…”

Current understanding of epigenetics mechanism as a novel target in reducing cancer stem cells resistance

“…Other studies also have suggested that one of the leading roles of CAFs is to produce niches to preserve CSC. CSCs produce CAFs through signaling pathways, and in contrast, CAFs maintain the spread and survival of CSCs by producing niches [ 92 , 93 ]. CAFs caused drug resistance in cancer by maintaining stemness in CSCs through secreting NRG1 and activating NF-κB, Wnt, and Notch3 signaling pathways [ 7 ].…”

3D Culture Systems for Exploring Cancer Immunology

“…Thus, 3D fibroblast cultures more accurately reflect cancer-associated fibroblast biology than 2D cultures. In 3D cultures, fibroblasts enhance organoid growth, invasion, and therapy resistance via both direct cell-cell contact as well as via paracrine signaling [ 47 , 67 , 68 , 69 ].…”