Abstract 2665: Single-cell analysis and cytokines modulation of intra-tumor subpopulations in colorectal cancer tumoroids

Abstract Colorectal cancer (CRC) is still one of the most commonly diagnosed cancers worldwide. Treatment strategies include chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Despite the availability of such a diverse set of therapeutic strategies, around a million people still die from this disease each year worldwide. Phenotypic intra-tumor heterogeneity, arising from both genetic and non-genetic origins, has become a significant focus of study due to its impact on treatment response. Among the various subpopulations within a tumor, the stem-like cells have been extensively investigated, as they are recognized as tumor initiators. In contrast, the subpopulations identified by the expression of genes associated with differentiation has received comparatively less attention. An interesting hypothesis is that differentiated populations might plastically give rise to stem cell populations fueling tumor regrowth. Here we examine phenotypic intra-tumor heterogeneity in patient-derived metastatic CRC (mCRC) tumoroids. Our goal was to decipher the unknown role of differentiated cells and to relate it to growth and therapeutic response. In particular, we sought to determine whether manipulating exogenously the proportions of differentiated cells within CRC tumoroids could alter the drug response. We analysed one KRAS wild type (wt) model and one mutant (kras) model by scRNAseq. Our data confirmed that tumoroids present two main subpopulations, one associated to stemness (with high expression of LGR5), and the other associated to differentiation genes (with high expression of KRT20), coherently with reported studies. First we identified two novel cell surface markers associated to the KRT20+ subpopulation: GABRA2 and GPRC5C. Using FACS, we confirmed that our models present a double-positive subpopulation at the protein level. RT-qPCR confirmed that the expression of both genes increased over time. We next studied the effect of different cytokines on the modulation of this subpopulation. We found that exogenously adding IL6 and TGF-b1 alters the proportion of our markers, with a stronger effect in the wt sample. For both models, ELISA assays did not indicate any significant secretion into the culture medium of these cytokines. Interestingly, we found a release of IL6 receptor (IL6R) in the medium, particularly from the wt sample, likely as a results of the previously characterized cleavage of its membrane-bound form. Furthermore, RT-qPCR showed that TGF-b1 induced its own expression, a phenomenon known as autoinduction. Our preliminary results using the wt sample indicate that pre-treatment with these cytokines affected the response to Oxaliplatin, making it more sensitive to the chemotherapeutic reagent. Overall, our results indicate that cytokines released in the tumor microenvironment might reshape the intra-tumor phenotypic heterogeneity impacting drug-response. Citation Format: Sabrina Johanna Fletcher, Irene Catalano, Elena Grassi, Sofia Borgato, Barbara Peracino, Luca Primo, Livio Trusolino, Andrea Bertotti, Alberto Puliafito. Single-cell analysis and cytokines modulation of intra-tumor subpopulations in colorectal cancer tumoroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2665.