Abstract B052: Single-cell and spatial analysis of the immune landscape unveils a subset of potentially tumor-reactive T cells in patients with localized PDAC
Authors: Kanu Elishama N , Fletcher Ashley A , Krishna Sri , Lowery Frank J , Zhuang Haotian , Agritelley Ethan S , Bao Jiayin , Eckhoff Austin M , Comatas Karrie , Wang Tao , Hwang Bin-Jin , Lidsky Michael E , Zani Sabino , Blazer Dan G , Allen Peter J , Klemen Nicholas D , Ji Zhicheng , Nussbaum Daniel P , Crosby Erika J 
Published: September 2024

Abstract
Even in its localized stages, pancreatic ductal adenocarcinoma (PDAC) is virtually incurable, characterized by early dissemination and disease recurrence. The liver is the most common site of metastasis, and in most cases hepatic recurrences drive survival outcomes. This underscores the critical need to target and prevent liver metastases. We leveraged a novel biobanking strategy to perform paired single-cell RNA (scRNA-seq) and TCR sequencing (scTCR-seq) to profile multisite immunity in patients with localized PDAC across the tumor, peripheral blood, and premetastatic liver. This analysis of three patients included three tumor, three blood, and nine liver (three per patient from separate segments) samples, and generated 85,748 cells for downstream analysis. We evaluated populations of tumor-expanded T cells as potential indicators of antitumoral immunity, and utilized validated computational approaches to identify tumor-reactive T cells based on key markers of dysfunction and exhaustion. Gene set enrichment analysis was performed on tumor infiltrating lymphocytes (TILs) using signatures specific to tumor-reactive CD4 and CD8 T cells (“NeoTCR4” and “NeoTCR8,” respectively), and scored these using the AUCell package in R. Overall, 153 CD4 and 22 CD8 T cells fit candidate NeoTCR4 and NeoTCR8 signature states, representing 5.6% and 0.8% of the total TIL population respectively. These nominated T cells expressed key marker genes associated with tumor specificity such as CXCL13, TIGIT, PDCD1, ENTPD1, and TOX. Thirteen of these predicted tumor-reactive T cell clones identified within the tumor were also detected within our liver samples, suggestive of a potential antitumoral immune response in the premetastatic liver space. We next performed 10x Xenium in situ spatial transcriptomic analysis of primary tumor, normal adjacent pancreas, and premetastatic liver from two of these patients, using tissue microarrays to incorporate replicate 2mm core biopsies from each of these sites. A custom designed gene panel, guided by our scRNA-seq dataset, targeted tumor-specific mutations (e.g. KRAS G12V, G12D, G12R, and Q61) and immune related genes with a specific focus on markers of T cell activity and exhaustion. Leveraging these latter marker genes, which represent a subset of the NeoTCR4 and NeoTCR8 signatures, we identified a population of exhausted TILs within the tumor microenvironment, representing potentially tumor-reactive T cells whose interactions can now be explored at spatial resolution. Future directions are aimed at experimentally validating the tumor reactivity of the predicted neoantigen-specific TILs within our dataset and characterizing the immune neighborhoods that exist in both the tumor and liver of PDAC patients. This work establishes exciting opportunities for novel T-cell therapy in the treatment of PDAC, and we have already implemented these experimental findings in a forthcoming clinical trial investigating the applicability of this predictive signature score for the generation of TCR-engineered lymphocyte therapy in patients with PDAC.
Citation Format: Elishama N Kanu, Ashley A Fletcher, Sri Krishna, Frank J Lowery, Haotian Zhuang, Ethan S Agritelley, Jiayin Bao, Austin M Eckhoff, Karrie Comatas, Tao Wang, Bin-Jin Hwang, Michael E Lidsky, Sabino Zani, Dan G Blazer III, Peter J Allen, Nicholas D Klemen, Zhicheng Ji, Daniel P Nussbaum, Erika J Crosby. Single-cell and spatial analysis of the immune landscape unveils a subset of potentially tumor-reactive T cells in patients with localized PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B052.