Spatially defined enrichment of a neuronal-like malignant phenotype in pancreatic cancer after neoadjuvant treatment.
Authors: Hwang William L. , Jagadeesh Karthik , Guo Jimmy , Hoffman Hannah I. , Yadollahpour Payman , Reeves Jason , Divakar Prajan , Delorey Toni , Mehta Arnav , Barth Jaimie Lynn , Zelga Piotr , Hong Theodore S. , Jain Rakesh K. , Aguirre Andrew , Fernandez Del-Castillo Carlos , Mino-Kenudson Mari , Liss Andrew , Ting David Tsai , Regev Aviv , Jacks Tyler 
Published: January 2022

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Background: Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistance to chemotherapy and radiotherapy is a major obstacle to improving clinical outcomes. Hence, there is an urgent need to elucidate the gene expression programs, spatial context, and interactions among different cell types in residual disease after neoadjuvant treatment. Methods: We optimized and applied single-nucleus RNA-seq (snRNA-seq) to 43 frozen primary PDAC tumors. Eighteen were treatment-naïve, 14 received FOLFIRINOX followed by radiotherapy with 5-FU or capecitabine (CRT), and 5 were subjected to CRT combined with losartan on protocol (CRTL). We performed unsupervised clustering of single nucleus profiles and then annotated and quantified cell subsets. Malignant and fibroblast gene expression programs were identified by consensus non-negative matrix factorization (cNMF). We mapped our cell type signatures and expression programs onto the tumor architecture using whole-transcriptome digital spatial profiling (DSP) to uncover distinct multicellular spatial neighborhoods and intercellular interactions that compose PDAC and are remodeled by neoadjuvant treatment. Results: Consistent with treatment effect, the proportion of malignant cells was significantly lower in tumors treated with neoadjuvant therapy. Within the immune compartment, CRTL was associated with a higher fraction of CD8+ T cells and Tregs compared to untreated and CRT tumors. Differential expression analysis of CD8+ T cells revealed greater effector function (e.g., IL2, CCL4, CCL5) and reduced quiescence/dysfunction markers (e.g., TIGIT, TCF7, KLF2, LEF1) associated with CRTL. We discovered expression programs across malignant and fibroblast profiles that formed a refined molecular taxonomy, including a novel neuronal-like malignant program enriched in the neoadjuvant groups and associated with the worst prognosis in independent cohorts. Ex vivo treatment of organoids derived from an untreated PDAC with FOLFIRINOX chemotherapy and radiotherapy recapitulated enrichment of the neuronal-like program. Whole-transcriptome DSP revealed three distinct multicellular neighborhoods: classical, squamoid-basaloid, and treatment-enriched. The observed enrichment in post-treatment residual disease of multiple spatially-defined receptor-ligand interactions and a neighborhood featuring colocalization of the neuronal-like malignant program, neurotropic CAF program, and CD8+ T cells may open new opportunities for therapeutic targeting in PDAC. Conclusions: Our work provides a high-resolution molecular framework for understanding the inter- and intra-tumoral heterogeneity of pancreatic cancer, spatial organization into discrete multicellular communities, and treatment-associated reprogramming as a blueprint for exploring novel therapeutic strategies tailored to residual disease.