TGF-β1-induced differentiation enhances chemotherapy response in metastatic colorectal cancer organoids

BackgroundIn metastatic colorectal cancer, systemic therapies frequently fail, partly due to underlying phenotypic plasticity rooted in pre-existing multi-type cell populations. Intratumoral lineage hierarchies within colorectal tumors require renovated efforts to decode growth principles, design rational therapeutic approaches, and accurately interpret drug response. Understanding the cell-state dynamics of untreated tumors and the degree of cell responsiveness to exogenous stimuli is therefore crucial to improving currently underwhelming therapeutic outcomes. MethodsHere, we leveraged patient-derived organoids established from hepatic metastases of colorectal cancer patients to deconstruct population hierarchies by combining single-cell transcriptomics with single-molecule RNA fluorescent in situ hybridization. Computational frameworks were used to identify independent gene modules. We then employed flow cytometry analysis to track cytokine-induced population shifts using a cell surface marker, validating our findings through bulk RNA analysis, functional assays, and viability assays in response to oxaliplatin. ResultsOur data substantiate the existence of a dual population configuration within untreated metastatic colorectal cancer organoids with diverse genetic backgrounds. Gene modules detected via single-cell transcriptomics delineate a stem-like (LGR5+) and a differentiated-like (KRT20+) population that fluctuate dynamically over time. Spatially and temporally resolved, single-cell level analysis through single molecule FISH captures the inherent stochasticity in cell fate decisions revealing surprising phenotypic variability even across different organoids derived from the same patient. By using GABRA2 as a surface marker we track the emergence of differentiated cells over the course of time and investigate the respective roles of TGF-{beta}1 and IL-6 in the differentiation of organoids. Our findings indicate that IL-6 exerts no major effect within our cell autonomous setting. In stark contrast, TGF-{beta}1 triggered cell cycle arrest and differentiation, while simultaneously reducing clonogenic capacity and significantly amplifying the cytotoxic potency of oxaliplatin. ConclusionsOur findings provide evidence of the dual Stem and Differentiated population hierarchy in metastatic colorectal cancer organoids, and demonstrate how this axis can be effectively hijacked by TGF-{beta}1 to suppress tumor growth. Our results suggest that further mechanistic exploitation of this cell-autonomous, tumor-suppressive arm of TGF-{beta}1 signalling could open unappreciated therapeutic windows in advanced colorectal cancer.