1452 Imaging mass cytometry identifies novel natural killer cell interactions in the pancreatic ductal adenocarcinoma tumor microenvironment

BackgroundPancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 10%,1-3 which is due in part to its dense desmoplastic and immunosuppressive stroma, caused by cancer-associated fibroblasts (CAFs).4-6 T cell function has been extensively explored in many cancers; however, natural killer (NK) cells of the innate immune system are relatively understudied in PDAC. Interestingly, we have previously shown that fibroblasts express known NK cell ligands and hypothesized that CAFs interact with NK cells as a potential immunosuppressive mechanism in PDAC.7 To further examine immune-stromal cell interactions in the PDAC tumor microenvironment (TME), we utilized Imaging Mass Cytometry (IMC), a novel multiplex imaging system that uses up to 40 metal-conjugated antibody markers to gain information of both tissue structure and single-cell data to conduct higher order proteomic single cell analysis.8 This novel technology will provide a deeper understanding of the immune architecture in PDAC.MethodsWe designed a 34 metal-conjugated antibody panel for known immune, epithelial, and stromal cells, and various immune synapses, cytokines/chemokines, and activation markers. IMC was performed on a human PDAC tissue microarray generated at the Lombardi Comprehensive Cancer Center. IMC image data was pre-processed, segmented, and feature extracted to generate single-cell data,9 and downstream analysis was performed using R. Single cells were clustered using Seurat-based clustering approaches to identify known cell populations based on the immune, epithelial, and stromal cell antibody markers. Identified cell clusters were spatially annotated on IMC images using the cytomapper R package.9ResultsIMC images and single-cell analysis preliminarily identified that NK cells are: 1) present within the PDAC TME and 2) surprisingly, co-localize with malignant PDAC cells. This has not been previously reported.ConclusionsUsing IMC, we have identified potentially novel interactions between NK cells and malignant epithelial cells in PDAC. Downstream cell neighborhood analysis will reveal further details on immune-stromal interactions in the PDAC TME and identify potential cell populations in further exploit in PDAC.AcknowledgementsAndrew Quong and Eric Swanson (Standard BioTools Inc.), and the Lombardi Comprehensive Cancer Center‘s Shared Resources, including Histopathology and Tissue Shared Resource, Flow Cytometry Shared Resource, and the Mass Spectrometry and Analytical Pharmacology Shared Resource.ReferencesBalachandran VP, Beatty GL, Dougan SK. Broadening the impact of immunotherapy to pancreatic cancer: challenges and opportunities. Gastroenterology 2019;156(7):2056-72. Shi M, Yu DH, Chen Y, Zhao CY, Zhang J, Liu QH, et al. Expression of fibroblast activation protein in human pancreatic adenocarcinoma and its clinicopathological significance. World J Gastroenterol. 2012;18(8):840-6. Swayden M, Soubeyran P, Iovanna J. Upcoming revolutionary paths in preclinical modeling of pancreatic adenocarcinoma. Front Oncol. 2019;9:1443. Karamitopoulou E. Tumour microenvironment of pancreatic cancer: immune landscape is dictated by molecular and histopathological features. Br J Cancer. 2019;121(1):5-14. Phillips P. Pancreatic stellate cells and fibrosis. In: Grippo PJ, Munshi HG, editors. Pancreatic Cancer and Tumor Microenvironment. Trivandrum (India)2012. Yu Y, Yang G, Huang H, Fu Z, Cao Z, Zheng L, et al. Preclinical models of pancreatic ductal adenocarcinoma: challenges and opportunities in the era of precision medicine. J Exp Clin Cancer Res. 2021;40(1):8. Malchiodi ZX, Fitzgerald A, Galvano E, Weiner LM. Abstract 6169: Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions. Cancer Res 2022. Elaldi R, Hemon P, Petti L, Cosson E, Desrues B, Sudaka A, et al. High dimensional imaging mass cytometry panel to visualize the tumor immune microenvironment contexture. Front Immunol. 2021;12:666233. Windhager J, Bodenmiller B, Eling N. An end-to-end workflow for multiplexed image processing and analysis. bioRxiv. 2021:2021.11.12.468357.Ethics ApprovalThis study was approved by the Lombardi Comprehensive Cancer Center‘s Biospecimen Use Committee (approval number: GU21-0929).