Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide. A key feature that has recently gained attention as a potential driver of treatment resistance is intra-tumor phenotypic heterogeneity. This heterogeneity suggests the presence of subpopulations within tumors that may persist or resist therapy, potentially through adaptive mechanisms. During CRC progression, at least two distinct cellular subpopulations have been documented: one associated with stemness, typically characterized by high expression of LGR5, and another displaying a more differentiated state, marked by elevated KRT20 expression. The latter population is generally considered to be non-proliferative.
Here, we investigated the presence and dynamics of transcriptionally distinct subpopulations in patient-derived CRC tumoroids using single-cell RNA sequencing and RNAScope. We examined intra-tumor heterogeneity both during normal tumor development and under treatment with Cetuximab (CTX), a monoclonal antibody targeting EGFR that is currently used as a targeted therapy. In addition, we identified cell surface markers for subpopulations of interest, enabling their tracking and isolation by FACS analysis.
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 cell surface marker for this subpopulation. Interestingly, a fraction of these cells exhibited high Ki67 expression, indicating that they were capable of proliferating even under treatment. This suggests that they may contribute, at least in part, to incomplete treatment responses.
Our preliminary results reveal that intra-tumor phenotypic heterogeneity is a defining feature of CRC with direct implications for treatment response. Unraveling these dynamics could pave the way for novel therapeutic strategies that specifically target resistant subpopulations or their interactions.