Abstract There are currently many treatments available for colorectal cancer (CRC), yet around a million people still die from this disease each year. Recently, the presence of distinct phenotypic subpopulations within tumors has gained interest as a potential source of drug resistance. In addition to cancer stem-like cells, a subset of “differentiated” cells—usually considered post-mitotic—has been identified in CRC tumors. The role and potential plasticity of these “differentiated” cells remain poorly understood. Here, we studied the presence and dynamics of transcriptionally distinct subpopulations in CRC. To do this, we used a set of patient-derived metastatic CRC (mCRC) tumoroids. We analyzed intra-tumor phenotypic heterogeneity using scRNA-seq during tumor growth and under Cetuximab (CTX) treatment, a monoclonal antibody against EGFR. Under normal growth conditions, we found the two main subpopulations previously described: a stem-like cell subpopulation and a “differentiated” one. We identified GABRA2 as a novel cell surface marker for the “differentiated” subpopulation. Furthermore, incubation with IL-6 or TGF-β1—two cytokines typically secreted by cells in the tumor microenvironment—increased the percentage of “differentiated” cells, suggesting that the microenvironment may influence intra-tumor heterogeneity. In contrast, when organoids were treated with CTX, new subpopulations emerged. In particular, we identified a group of cells with high expression of WNT-related genes, including both inhibitors and activators such as APCDD1 and WNT6. We validated APCDD1 as a marker for this subpopulation. Interestingly, when these cells were sorted and re-cultured, they were able to proliferate, indicating that they may contribute, at least in part, to incomplete treatment responses. Our preliminary results expand the current knowledge on tumor heterogeneity and plasticity in CRC. A deeper understanding of these phenomena could help redirect targeted treatments toward specific subpopulations or cellular interactions. Citation Format: Fletcher Sabrina Johanna, Catalano Irene, Grassi Elena, Borgato Sofia, Pizzini Letizia, Peracino Barbara, Primo Luca, Trusolino Livio, Bertotti Andrea, Puliafito Alberto. Intra-tumor heterogeneity in colorectal cancer tumoroids during normal growth and under treatment abstract. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86 (13Suppl): Abstract nr P60.
Johanna et al. (Thu,) studied this question.