Abstract 3442: Multimodal mapping of the immune landscape in human pancreatic cancer

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the US. Unfortunately, recent clinical trials using immunotherapy targeting the highly immunosuppressive tumor microenvironment have showed disappointing results, as there is no method to predict which patients will respond to therapy. More recently, the development of single cell technology has allowed for in-depth profiling at the cellular level using small amounts of tissue, raising the potential to develop precision medicine tools at time of endoscopic fine needle biopsy. Results: We performed single-cell RNA sequencing (scRNAseq) on endoscopic fine needle biopsies from 10 PDAC tumors at time of diagnostic endoscopic biopsy or 6 surgically resected tissues. We also sequenced 3 adjacent or normal pancreas tissues. We captured 8,521 cells from 3 surgical normal adjacent samples and 46,244 cells from PDAC tumors. Mapping of putative interactions between ligands and receptors demonstrated upregulation of key signaling pathways, including Hedgehog, NOTCH, and chemokine signaling within myeloid, epithelial, T, and NK cells. Differential expression analysis in cytotoxic CD8 T cells of PDA patients revealed increased expression of genes involved in T cell activation (GZMB, GZMA), exhaustion (GZMK, EOMES) as well as immune checkpoint pathway upregulation when compared to cytotoxic T cells in adjacent normal pancreatic tissue. Among the most significantly increased genes in CD8 T cells of PDAC tumor was the immune checkpoint TIGIT. Upon further analysis of the CD8 T cells, we found TIGIT was almost exclusively expressed in exhausted CD8 T cells, while other checkpoints such as PD-1 and LAG3 were equally distributed across effector and exhausted T cell populations. Interestingly, we were able to capture patient-specific heterogeneity of gene expression in T cells, suggesting the possibility of individualized T cell gene signatures present in PDAC tumors. We used mass cytometry and immunostaining to validate our transcript-based findings. Conclusion: Overall, we have successfully performed robust in-depth profiling using single-cell sequencing of PDAC tumors from fine needle biopsies. TIGIT, but not other immune checkpoints, correlates with T cells exhaustion in tumors, revealing an important biological function of this relatively understudied checkpoint. Analysis of our results identified patient-specific heterogeneity of key signaling pathways in different cell compartments of PDAC tumors that have to potential to be leveraged for precision medicine. Citation Format: Nina Steele, Eileen Carpenter, Samantha Kemp, Veerin Sirihorachai, Stephanie The, Lawrence Delrosario, Jenny Lazarus, El-ad Amir, Valerie Gunchick, Carlos Espinoza, Samantha Bell, Lindsey Harris, Valerie Irizarry-Negron, Dan Paglia, Justin Macchia, Fatima Lima, Angel Ka Yan Chu, Heather Schofield, Erik Jan Wamsteker, Richard Kwon, Allison Schulman, Anoop Prabhu, Ryan law, Arjun Sondhi, Katelyn Donahue, Hari Nathan, Clifford Cho, Michelle Anderson, Vaibhav Sahai, Costas Lyssiotis, Benjamin Allen, Arvind Rao, Weiping Zou, Filip Bednar, Timothy Frankel, Marina Pasca di Magliano. Multimodal mapping of the immune landscape in human pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3442.

Lorazepam stimulates IL-6 production and is associated with poor survival outcomes in pancreatic cancer

“…Therefore, we hypothesized that LOR increases inflammatory signaling by promoting GPR68 activation in CAFs. To further support that GPR68 is preferentially expressed in CAFs, we analyzed human PDAC single cell sequencing data from Steele et al (20). As observed in the Peng et al (2019) dataset, GPR68 was most highly enriched in human PDAC CAFs (Fig.…”

“…S4D). In addition to being expressed on CAFs, reanalysis of the CAF cluster in the human PDAC single cell sequencing by Steele et al (20) indicated that Gpr68 is not highly expressed on pericytes (RGS5 marker), supporting that it is a fibroblast-specific marker (Supplemental Fig. S4E-S4G).…”

“…CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) between GPR68 expression and PDAC progression, we reanalyzed GPR68 expression by disease stage in the human PDAC single cell sequencing by Steele et al (20). GPR68 was not expressed strongly in the normal human pancreas but was expressed on PDAC primary tumors and PDAC metastases, supporting its likely role in disease pathogenesis (Supplemental Fig.…”

“…S4E-S4G). To determine the relationship between GPR68 expression and PDAC progression, we reanalyzed GPR68 expression by disease stage in the human PDAC single cell sequencing by Steele et al (20). GPR68 was not expressed strongly in the normal human pancreas but was expressed on PDAC primary tumors and PDAC metastases, supporting its likely role in disease pathogenesis (Supplemental Fig.…”

“…PCA clustering and UMAP visualization was performed to generate unbiased clusters. Populations were labeled based on established lineage markers(20, 21).…”