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Abstract Background: Molecular profiling of colorectal cancer (CRC) has demonstrated marked heterogeneity between tumors that appear histologically similar. Although existing classification systems such as the Consensus Molecular Subtypes (CMS) have shown clinical utility, these bulk sequencing-based methods fail to adequately address the intra-tumoral heterogeneity of CRC. To disentangle the complex landscape and identify clinically-relevant properties of CRC we generated a single cell sequencing (scRNASeq) database from 225 tumor samples derived from resection of primary colon or liver metastases. Our analysis defines cell-type-specific gene programs significantly associated with relapse and survival and suggests interventions which may abrogate their effect. Methods: Utilizing a bio-repository of CRC specimens with an average of 5 years of clinical follow-up, we performed scRNASeq on 225 patient samples comprising ~ 1, 000, 000 total cells and 2. 4 billion gene expression measurements. Following traditional clustering approaches, we then inferred gene expression programs through consensus non-negative matrix factorization (cNMF) to characterize cell activities. Gene program activity was explored on a cell-type-specific level and associated with clinical outcomes such as relapse. Treatment effects were additionally explored. Results: We identified 56 cell-type specific gene programs significantly associated with relapse in adult CRC patients. Interestingly these programs spanned all cellular compartments with the majority found in stromal or immune, as opposed to tumor cells. Program activity such as the non-canonical Granulocyte Chemotaxis program in B Cells was protective of relapse (relapse median expression 0% activity vs non-relapse activity 40% pAdj. 05 × 1050) whereas the Epithelial Mesenchymal Transition (EMT) program in Cancer Associated Fibroblasts (CAF) was strongly associated with relapse (relapse median expression 16% activity vs non-relapse activity 0% pAdj. 05 × 1010). Cumulative program usage enabled stratification on a patient level to provide an estimate of comparative disease free survival (DFS) and overall survival (OS) ; gene-programs were significantly associated with outcomes such as the CAF-EMT program which was associated with a DFS HR of 3 (pAdj. 0005). Responses to chemotherapy and treatment effect were also explored; prior treatment with Bevacizumab was associated with a significant reduction in the CAF-EMT program (11% activity in non-exposed patients versus 0% activity in exposed patients pAdj. 05 × 1010). Conclusions: Analysis of the largest CRC scRNASeq cohort to date identifies a number of previously undescribed cell-type specific gene programs significantly associate with relapse after surgery. These programs provide insights into the underlying mechanisms and suggest that the TME plays an important role in relapse. Citation Format: Nicholas J. Hornstein, Mingshuang Wang, Abdelrahman Yousef, Saikat Chowdhury, Fadl Zeineddine, Mohammad Zeineddine, Mahmoud Yousef, Shuangjie You, Betul Gunes, David Menter, Scott Kopetz, John Paul Shen. Cell-type-specific gene programs suggest immune and stromal drivers of relapse in colorectal cancer 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 881.
Hornstein et al. (Fri,) studied this question.