Abstract 5492: Spatial organization and cellular composition of immunity hubs in human colorectal cancer

Abstract Background: Among colorectal cancer (CRC) tumors, mismatch repair deficient (MMRd) tumors have more immunogenic neo-antigens, hence more cytotoxic T-cell mediated anti-tumor immunity than mismatch repair proficient (MMRp) tumors. Previously1, we transcriptionally profiled over 300,000 cells from 62 MMRd and MMRp CRC tumors and found an MMRd-unique network of immunologically active transcriptional states enriched in interferon-stimulated genes (ISGs). With 4-color imaging, we showed that this program arises from spatially organized hubs of at least 3 cell types: IFNã-secreting T and interferon-responsive CXCL9/10/11+ myeloid and tumor cells. To characterize the breadth of cells involved in tumor immunity hubs, we must simultaneously measure the expression of 100s of genes in situ. Methods: To describe the cellular composition, structural organization and intercellular spatial interactions of these immunity hubs, we profiled primary untreated resection specimens from 17 CRC donors (n=12 MMRd, 5 MMRp) using multiplexed error-robust FISH (MERFISH) mRNA microscopy. Efforts were made to sample both the tumor body and the invasive border of the tumor. We designed a panel of 479 genes to capture key transcriptional programs from our single-cell atlas1. We wrote analytical pipelines to segment transcripts into cells, select high-quality cells, and organize cells into distinct zones in tissue. To leverage the rich information in the scRNA-seq atlas1, we designed a robust label transfer analysis to assign cell state labels to MERFISH cells by comparison to scRNAseq cells. Results: We profiled 7.5 million high quality MERFISH cells across 21 CRC tissue sections and labeled the cells into 7 major lineages: 63.19% epithelial, 22.52% stromal, 7.75% myeloid, 3.72% T/NK/ILCs, 1.45% plasma, 1.2% B, and 0.17% mast cells. We visualized the spatial location of these cell types. T cells aggregated into 3 distinct regions of tissue: tertiary lymphoid structures (TLS) outside the tumor, and smaller clusters near the invasive border and in the tumor center. We identified immunity hubs defined by colocalized CXCL9/10/11 expressing cells and T cells near the invasive border and in the center of MMRd tumors. As expected, we found enrichment of CXCL13, IFNG, and ISGs such as CD74 and MHCII genes at these locations. In contrast, we found evidence of CCL17+CCL19+LAMP3+ activated dendritic cells and B cells enriched in the TLS. These results support a distinct composition and localization of immunity hubs in tumors, in contrast to TLS outside tumors. Conclusions: Our study defines the composition, spatial organization, and intercellular interactions of immunity hubs, shedding light on the anti-tumor immune response in CRC. Ethics Approval: Approved by the DF/HCC Institutional Review Board (protocol #02-240).(1) Pelka, K. et al. Spatially organized multicellular immune hubs in human colorectal cancer. Cell 184, 4734-4752.e20 (2021) Citation Format: Mukta G. Palshikar, Maxwell Spurrell, Jack Demaray, Han Chen, Scott Engels, Roopa Madhu, Milan Parikh, Izabella Gazmora, Arnav Mehta, Nicolas Fernandez, Colles Price, Jiang He, Sonia Cohen, Karin Pelka, Nir Hacohen, Jonathan Chen, Ilya Korsunsky. Spatial organization and cellular composition of immunity hubs in human 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(6_Suppl):Abstract nr 5492.