Abstract 3442: Multi-omics comparison of early vs late-onset colorectal cancer reveals distinct tumor phenotype of early-onset patients

Abstract Though incidence and mortality of colorectal cancer (CRC) in individuals older than 50 (late-onset, LO-CRC) have been declining over the last 30 years, they have been increasing among adults less than 50 (early onset, EO-CRC), constituting a significant public health concern. Detailed molecular comparison of EO-CRC to LO-CRC tumors has thus far been limited to whole-exome sequencing and confounded by the complex tumor immune microenvironment (TME), with significant presence of non-tumor cells. Here, we present integrated comparison of EO- and LO-CRC by single-cell RNA-Sequencing (scRNA-Seq) and multi-plex immuno-fluorescence (mIF). We combine public data with in-house clinical samples to comprise a rich resource of scRNA-Seq on 43, 000 cells from 11 EO-CRC tumors and 19 LO-CRC tumors and perform mIF on a tissue microarray of 50 EO- and 50 LO-CRC samples, focusing on major immune cell subsets. Our ScRNA-Seq analysis utilizes an algorithm that leverages gene regulatory networks for Virtual Inference of Protein Activity by Enriched Regulon (VIPER) to cluster cell populations in the TME, identify cell types over-represented in EO-CRC (by Fisher’s Exact Test), and characterize regulatory proteins aberrantly activated in those cell types. By this approach, we have found small, but statistically significant elevation in fibroblasts (p=0.042), endothelial cells (p=0.025), and B-cells (p=0.042) in EO-CRC compared with LO-CRC, though no difference in T-cells. Further, scRNA-Seq VIPER analysis of tumor cells demonstrates a sub-phenotype conserved across patients and significantly over-represented in EO-CRC (p=0.028), marked by up-regulation of TLR4, C5AR2, LILRB4, PTPRE, and CCR5 (proteins known to play a role in immune signaling and inflammation) – and with specific activity of druggable proteins EPHB4, FGFR4, and IKBKB. Overall, we find that EO-CRC is transcriptionally similar to LO-CRC and is composed of a similar cellular milieu. mIF validates no differences in T-cell subsets but is unable to confirm increase in endothelial cells in EO-CRC found in our scRNA-Seq data set. Our finding of similar T-cell density is of particular interest and helps dispel the notion that EO-CRC is more immune “cold” than sporadic CRC in older cohorts. Most strikingly, we find that EO-CRC is marked by a particular cluster of TLR4-high tumor cells. It has previously been found that high fat diets can lead to increased CRC growth in a TLR4-dependent manner in murine models – thus suggesting a link between a known environmental risk factor and EO-CRC. Work is ongoing to validate TLR4-high tumor cells in human tissues and patient-derived organoids (PDOs). Further studies will utilize PDO as an in vitro model for drug screens targeting aberrantly active druggable protein targets in EO- CRC tumors and to further explore the mechanisms that contribute to development of EO-CRC. Citation Format: Multi-omics comparison of early vs late-onset colorectal cancer reveals distinct tumor phenotype of early-onset patients. Multi-omics comparison of early vs late-onset colorectal cancer reveals distinct tumor phenotype of early-onset patients [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 3442.