Abstract 3988: Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics

Abstract The most well-described mechanism of immune evasion in PDAC is exclusion of immune cells by a desmoplastic stroma, but such immune exclusion is not uniform. Through single-nucleus RNA-seq and whole-transcriptome digital spatial profiling of patient tumors, we previously reported marked heterogeneity in the immune composition of PDAC. Here, we developed a novel multi-omics workflow integrating single cell spatial proteomics (96-plex, Akoya Phenocycler®-Fusion) and transcriptomics (990-plex, Nanostring CosMx) in primary patient PDAC tumors to systematically study spatial relationships and functional states of malignant, stromal, and immune cells. Consecutive sections from four independent tumors enabled whole slide proteomic assessment of >3.5M cells, and transcriptomic assessment of >200K cells. Strikingly, we observed an abundance of immune cell aggregates in all tissue sections, up to 57 within a 1 cm2 tumor section. Aggregates were diverse in composition with representation of myeloid cells, CD8+ T cells, and stromal cells, including fibroblasts and nerves. B/T lymphoid aggregates were well vascularized with co-localized CD31+CD34+ endothelial cells and appeared at various phases of maturation towards putative tertiary lymphoid structures (TLSs) containing CD38+ B cells, CD4+ T cells, and CD21+ follicular DCs. These tissues were enriched for interferon stimulated genes in T cells and type 1 interferon response signatures in malignant cells and cancer associated fibroblasts (CAFs) forming inflammatory multicellular hubs that have been associated with improved response to immune checkpoint blockade. Surprisingly, B cells, in addition to macrophages and CD8+ T cells, were major producers of interferon gamma (B cells: 13.7%, macrophages: 36.6%, CD8+ T cells: 12.1% of IFNG+ cells). Finally, to gain insight into the necessary factors for TLS formation in PDAC, we quantified the expression of chemokines and adhesion molecules associated with TLS initiation. Activated dendritic cells expressed CCL19 and CCL21, endothelial cells expressed CCL21, and in concert with inflammatory CAFs (iCAFs), CXCL12. Malignant cells expressed CXCL13 and CCL20 and displayed high levels of cell adhesion molecules ICAM1, VCAM1, and ICAM2. Together, our data suggests that the functions of lymphoid tissue organizer cells can be substituted by endothelial cells, dendritic cells, iCAFs, and, surprisingly, malignant cells. Our spatial immunophenotyping study of pancreatic cancer thus highlights the marked immune heterogeneity of the disease and suggests a central role for malignant and stromal cells in mediating diverse mechanisms of immune evasion and engagement. Functional studies will be conducted to investigate these mechanisms of tumor-immune crosstalk and identify novel immunotherapeutic vulnerabilities for this deadly disease. Citation Format: Dennis Gong, Jingyi Cao, Peter Wang, Niyati Jhaveri, Yue Hou, Bassem B. Cheikh, HaYeun Ji, Mark Gregory, Jason Reeves, Michael Patrick, Carina Shiau, Jennifer Su, Jimmy A. Guo, Joseph M. Beechem, Martin Hemberg, William L. Hwang, Ryan Park. Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics [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 3988.