Abstract 1709: Improved understanding of the biology and pathophysiology of the tumor microenvironment in PDAC samples revealed by InSituPlex, Imaging Mass Cytometry, and advanced image processing
Authors: Quong Andrew , Nieto Jordan , Quong Derek , Esch Amanda , Maclean Kirsteen , Rees Mark , Fleury Devan , Chatterjee Gourab , Wharton Keith , Thagaard Jeppe , Schneider Fabian , Winkowski Dan , Mansfield James 
Published: June 2022

Abstract
Pancreatic cancer remains a deadly disease due to difficulties hindering its early diagnosis, giving way to metastasis of the tumor and resulting in poor prognosis. While there are many neoplasms of the pancreas, pancreatic invasive ductal adenocarcinoma (PDAC) is the most common, and treatment options are few, with poor overall survival. Recently, several publications have demonstrated improved outcomes with the inclusion of immunotherapy to cytotoxic drug combinations in some patients. However, optimally selecting patients as candidates for immunotherapy-chemotherapy combinations remains a critical challenge.
The complexities of the tumor microenvironment (TME) have been implicated in the failure of chemotherapy, radiation therapy, and immunotherapy. The tumor microenvironment of PDAC is especially rich with multiple interactions between pancreatic epithelial/cancer cells, stromal cells, immune cells, and the extracellular matrix (ECM). PDACs are characterized by a complex ECM of desmoplastic reaction consisting of an extensive and dense fibrotic stroma that surrounds and infiltrates clusters of malignant epithelial cells, together with the loss of basement membrane integrity and an abnormal vasculature. PD-L1 is also expressed in PDACs, and its overexpression has been associated with a poor prognosis.
In the present study we demonstrate a unique tissue phenotyping workflow combining complementary methods that can unravel the complexity of the tumor microenvironment. We highlight how a workflow combining multiple elements provides utility, robustness, and an ability to derive biological insights in PDAC samples. An InSituPlex® PD-L1 multiplex immunofluorescence assay (4 markers, CD8, CD68, PD-L1, Pan CK/Sox10) was used on whole slides to identify areas of high, medium, and low PD-L1 expression. Imaging Mass Cytometry™ (IMC™, 40 markers) was performed on selected regions of interest from each slide. Advanced tissue and cell segmentation followed by multiplex cellular phenotyping and spatial analyses were performed on both whole-slide InSituPlex and IMC data. These methods combine to give a detailed readout of the location and bio-distribution of specific cell phenotypes in situ in the TME of PDAC. Four-plex imaging and analysis of whole slides gives an overview of the immune status of the section, while 40-plex imaging and analysis gives a comprehensive and multiparametric exploration of cells present. These methods combine to reveal an exceptional view of the PDAC TME at the single-cell level.
Citation Format: Andrew Quong, Jordan Nieto, Derek Quong, Amanda Esch, Kirsteen Maclean, Mark Rees, Devan Fleury, Gourab Chatterjee, Keith Wharton, Jeppe Thagaard, Fabian Schneider, Dan Winkowski, James Mansfield. Improved understanding of the biology and pathophysiology of the tumor microenvironment in PDAC samples revealed by InSituPlex, Imaging Mass Cytometry, and advanced image processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1709.