Abstract 6946: Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids

Abstract Colorectal cancer (CRC) is one of the most diagnosed cancers worldwide. Though several treatment strategies are available today, around a million people die of this disease each year worldwide. Thus, there is still a pivotal need for new and more effective treatments. Genetic and non-genetic intra-tumor heterogeneity has emerged lately as an intrinsic feature of malignancies and as one of the factors responsible for therapy resistance or tolerance. In particular, the nature and role that phenotypically distinct, yet genetically indistinguishable, subpopulations have on tumor biology is far from being understood. Here we investigate phenotypic heterogeneity in CRC by identifying specific transcriptionally well defined cell subpopulations. We aim at deciphering the interactions between populations in terms of differentiation and plasticity, tumor growth and potentially exposed targetable vulnerabilities. To this end, we used patient-derived metastatic CRC (mCRC) organoids, including KRAS wild type (wt) and mutant (kras) models. We performed in total scRNAseq for 7 mCRC samples (4 wt; 3 kras) in different growth media: normal growth medium (supplemented with EGF) (n=1) ; medium without EGF (noEGF) (n=7) ; or treated with Cetuximab (CTX, a clinically approved monoclonal antibody against EGFR) (n=7). We found that the wt sample grown in EGF presents 2 distinct subpopulations: one with stemness characteristics (LGR5 high) and the other with a more differentiated phenotype (KRT20 high), in agreement with the current bibliography. Interestingly, in EGF deprived cultures new subpopulations appear. In particular, we were able to identify a subpopulation with distinct expression of WNT-related genes (APCDD1, WNT6, WIF1). This subpopulation is also present, and in cases increasing in fraction, when organoids are treated with CTX (in 3 out of 7 samples). Interestingly, a fraction of the WNT positive subpopulation seems to be able to proliferate under CTX treatment (Ki67 positive). We identified the negative regulator of the WNT pathway, APCDD1, as a potential marker to track this subpopulation. We took one wt and one kras sample as models to validate these findings by flow cytometry. Furthermore, sorting of APCDD1 positive and negative cells, and further RT-qPCR analysis, confirmed good correlation between antibody staining and gene expression. Moreover, we found that both types of cells are able to re-grow as organoids after re-culturing. This suggests that this WNT-associated subpopulation could be contributing, at least in part, to CTX tolerance or resistance. Our results show promising potential to help unravel basic features of intra-tumor heterogeneity both in basal conditions and upon treatment, and possibly to find new therapeutic targets. Citation Format: Sabrina J. Fletcher, Irene Catalano, Elena Grassi, Sofia Borgato, Barbara Peracino, Elena Piretto, Luca Alessandri, Raffaele Calogero, Luca Primo, Livio Trusolino, Andrea Bertotti, Alberto Puliafito. Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6946.