Mapping lymphoid responses to tumor growth and lymph node metastasis: multiomic spatial analysis of pancreatic ductal adenocarcinoma reveals tertiary lymph node structures

Introduction : Within the next five years, pancreatic ductal adenocarcinoma (PDAC) will become the second-highest cause of cancer death in the United States; even with surgical resection and chemotherapy, median overall survival remains less than three years. Spatial transcriptomic and proteomic analysis is one pathway toward therapeutic target discovery for PDAC. While previous spatial transcriptomic studies have uncovered remarkable intratumoral heterogeneity in primary and metastatic samples, most lack subcellular resolution and multiomic scale. Methods : Using the Singular G4X platform, we performed spatial multiomic analysis of 31 regions of interest in formalin-fixed, paraffin-embedded matched primary tumor and metastatic lymph nodes from eight patients. All patients had a pathological diagnosis of PDAC and received neoadjuvant chemotherapy at a single cancer center, and 7/8 patients showed pathological treatment effect with partial response to therapy. Data were analyzed in python. Results : After quality control, 2.4 million cells were integrated. Major cell types were manually annotated: acinar cells ( CPB1 + GATM +), adipocytes ( ADIPOQ + PLIN1 +), B cells ( MS4A1 + HLA-DRA +), cancer-associated fibroblasts ( COL1A1 + LUM +), plasma cells ( JCHAIN + IGHA1 / IGHM +), macrophages ( CD163 + CD68 +), mast cells ( KIT +), neuroendocrine cells ( NRXN1 + NCAM1 +), pericytes/endothelial cells ( PECAM1 + RGS5 +), smooth muscle cells ( TAGLN + MYH11 +), and T cells ( CD3D + IL7R +). Tumor cells formed three distinct clusters: KRT19 + MUC1 + ductal cells, PIGR + GATM + ductal cells, and STAT1 + CD44 + cells (from one patient with sarcomatoid dedifferentiation). The predominant cell type in the tumor microenvironment, cancer-associated fibroblasts (CAFs) could be subclustered into inflammatory CAF ( CXCL12 -high IL6 -high), mechanoresponsive CAF ( PDGFRB -high ACTA2 -high), and steady-state CAF phenotypes. Although we did not select regions of interest explicitly with tertiary lymphoid structures (TLSs) in mind, we found that 7/8 patients and 9/19 tumor sections contained putative TLSs, with 4 sections containing more than one TLS area. TLS morphology appeared similar to that of lymphoid aggregates in tumor-positive lymph nodes. A predominance of CAFs was also noted in the involved lymph nodes. B cells within TLSs stained positive for CD20 protein and expressed CXCR5 transcripts; they were surrounded by T cells that stained positive for CD3 and CD4/CD8 proteins. CXCR5 colocalized closely with its binding partner CXCL13, a known trigger of TLS formation. Conclusion : In a cohort of PDAC patients with lymph node metastasis, multiomic spatial analysis revealed TLSs in nearly all patients, suggestive of immune potential within the PDAC stroma. The CXCR5-CXCL13 axis should remain a target of active investigation in TLS formation.