High-dimensional deconstruction of pancreatic ductal adenocarcinoma identifies tumor microenvironmental communities associated with survival

Bulk and single-cell analyses of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) have revealed a largely immunosuppressive milieu. Thus far, efforts to utilize insights from TME features to facilitate more effective therapeutics have largely failed. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy samples (n=22) and surgical samples (n=6), integrated with 3 public datasets (n=49), resulting in ∼140,000 individual cells from 77 patients. Based on expression markers assessed by Seurat v3 and differentiation status assessed by CytoTRACE, we divided the resulting tumor cellular clusters into 5 molecular subtypes based on expression of previously reported marker genes: Basal, Mixed Basal/Classical, Classical Low, Classical High, and ADEX. We then queried these 5 tumor cell profiles, along with 15 scRNA-seq-derived tumor microenvironmental cellular profiles, in 391 bulk expression samples from 4 published datasets of localized PDAC with associated clinical metadata using CIBERSORTx. Through unsupervised clustering analysis of these 20 cell state fractions representing tumor, leukocyte and stromal cells, we identified 7 unique clustering patterns representing combinations of tumor cellular and microenvironmental cell states present in PDAC tumors. We termed these cell state patterns communities, and found them to correlate with overall survival, tumor ecotypes, and tumor cellular differentiation status. The community associated with worst overall survival contained basal tumor cells, exhausted CD4 and CD8 T cells, and was enriched for fibroblasts. In contrast, the highest overall survival was associated with a community high in immune cell enrichment. The differentiation state of tumor cells (assessed by CytoTRACE) was also correlated with survival in a dose-dependent fashion. Further, we identified a subset of PDAC samples that were significantly enriched for CD8 T and plasma cells that achieved a 2-year overall survival rate of 71%, suggesting we can identify PDAC patients with significantly improved prognoses and, potentially, higher sensitivity to immunotherapy. In summary, we identified novel tumor microenvironmental communities from high-dimensional analysis of PDAC RNA sequencing data that reveal new connections between tumor microenvironmental composition and patient survival that could lead to better upfront risk stratification and more personalized clinical decision-making. ### Competing Interest Statement E.S. and A.A.C. have patent filings related to cancer biomarkers. F.Q. has stock options in Centene, Gilead, and Horizon Therapeutics. L.E.H and H.K. have received research funding, travel accommodations and honoraria from Varian Medical Systems, and from ViewRay. L.E.H. has done speakers bureaus for ViewRay, and for Varian Medical Systems. H.K. has consulted for Varian Medical Systems. W.G.H. is a member of the board of directors for Accuronix Therapeutics. A.A.C. has licensed technology to Droplet Biosciences, Tempus Labs, and to Biocognitive Labs. A.A.C. has served as a consultant/advisor to Roche, Tempus, Geneoscopy, NuProbe, Daiichi Sankyo, AstraZeneca, AlphaSights, and Guidepoint. A.A.C. has received honoraria from Roche, Foundation Medicine, and Dava Oncology. A.A.C. has stock options in Geneoscopy, research support from Roche and Tempus Labs, and ownership interests in Droplet Biosciences and LiquidCell Dx. No potential conflicts of interest were disclosed by the other authors. ### Funding Statement This work was supported by the National Institutes of Health (NIH) National Cancer Institute (NCI) Washington University SPORE in Pancreatic Cancer under award number 5P50CA196510 (D.G.D., R.C.F. and W.A.H.) including a Career Enhancement Program subaward to A.A.C. This work was also supported by NIH under award number U2CCA233303 (L.D., R.C.F., W.E.G). This work was also supported by the NCI Washington University Participant Engagement and Cancer Genomic Sequencing Center under award number 1U2CCA252981 (L.D., R.C.F., A.A.C.), a NCI K08 career development award under award number K08CA238711 (A.A.C.), the Cancer Research Foundation Young Investigator Award (A.A.C.), the Washington University Alvin J. Siteman Cancer Research Fund (A.A.C.), and the V Foundation for Cancer Research V Scholar Award (A.A.C.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Washington University School of Medicine gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors