O13.03 Cancer cell–intrinsic rewiring of the secretome in immune-excluded colorectal cancer

Background Despite the key role of secreted factors in coordinating anti-tumor immunity, our understanding of how cancer cells rewire their secretome to avoid immune destruction is still very poor. This is largely due to technological shortcomings that limit our ability to reliably and comprehensively profile the cancer cell-intrinsic secretome of human tumors.MethodsHere, we develop a click chemistry-based approach to selectively profile the secretome of patient-derived colorectal cancer (CRC) and healthy colon (HC) organoids with high resolution. We use THRONCAT (1) to label the nascent proteome through incorporation of the threonine analog β-ethynylserine. This allows selectively isolating newly synthesized secreted proteins followed by their characterization using mass spectrometry. Focusing on immunologically unresponsive microsatellite stable (MSS) cancers, we compare the secretome of CRC and HC organoids to determine how cancer cells rewire their secretome, in samples directly derived from patient tissues. We integrate our secretomics datasets with clinical bulk and single cell RNA sequencing data to identify CRC-specific secreted proteins that mark immune excluded tumors.Results We observe a major rewiring of the secretome in the transition to malignancy, with 12-27% of all secreted proteins significantly altered between HC and CRC organoids. Notably, the secretome is markedly distinct between the immunologically divergent microsatellite stable (MSS) and instable (MSI) CRC subtypes, with NOTUM emerging as the top differentiating secreted protein. Cross-referencing our secretomics data with clinical datasets reveals that NOTUM expression is associated with decreased T cell infiltration across four independent datasets. In addition, we identify AZGP1 as a CRC-specific secreted factor associated with decreased infiltration of gamma delta T cells.Conclusion Overall, our study provides an in-depth characterization of the cancer cell-intrinsic secretome of MSS-CRC, and nominates NOTUM and AZGP1 as secreted factors that mark immune excluded tumors. This provides a framework to investigate the cell-intrinsic and -extrinsic regulators of secreted factors of interest to reveal novel immunotherapeutic targets.Reference Ignacio BJ, Dijkstra J, Mora N, Slot EFJ, van Weijsten MJ, Storkebaum E, et al. THRONCAT: metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog. Nat Commun. 2023;14(1):3367.