Abstract 141: Utilizing multiplex imaging to characterize the extracellular matrix in cancer

Abstract The tumor microenvironment (TME) is known to be an essential component in the response to multiple therapeutics, including immunotherapies. The TME is comprised of the tumor itself, infiltrating immune cells, and the extracellular matrix (ECM). ECM has been shown to play a critical role in tumorigenesis, affect the efficacy of certain treatments, and regulate anti-tumor immunity. However, methods to study ECM have typically been limited to single-marker studies or have used dissociated methods which lose spatial context. Additionally, fluorescence-based approaches to study the ECM are challenging due to the auto-fluorescence properties of multiple ECM components. We chose to utilize the multiplex capabilities of Imaging Mass Cytometry (IMC) for its ability to detect up to 40 different proteins via metal-tagging and absence of auto-fluorescence. We selected and validated 40 markers which we believe play an integral role in ECM, including structural proteins as well as immune markers and metabolic enzymes. This 40-plex panel was validated using a tissue microarray (TMA) comprised of various healthy and diseased tissue samples such as Lung, Colon, Breast, Prostate, Kidney, and Liver and their respective tumor counterparts. After staining, our results underwent rigorous analysis through a multi-step image comparison of our staining results to those in the current literature, ensuring our staining was specific. We found significant differences in the protein expression of various ECM markers between the healthy tissue and their tumor counterpart. We found structural variations of multiple ECM proteins between the normal tissue and tumor tissue. However, the expression patterns of these ECM elements were not uniform between tumor types, with overexpression of HAPLN1 in breast cancer while decreased in colon cancer. The included immune markers allow for spatial studies to examine the role of ECM in regulating immune responses, such as CD8+ T-cell infiltration. Additionally, we found that tumors from non-small cell lung cancer (NSCLC) patients exhibit significant ECM heterogeneity. We used a patch-based analysis approach to examine the ECM phenotypes of a cohort of 35 NSCLC patients. Preliminary assessment demonstrates unique clusters of ECM phenotypes have a utility for predicting patient outcomes. Further analysis of this cohort will be presented. Overall, we present a highly-multiplexed IMC panel which allows for detailed assessment of the ECM. Citation Format: Anton Villamejor, Joseph Lownik, Ben Falk, Larry Milshteyn, Akil Merchant. Utilizing multiplex imaging to characterize the extracellular matrix in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 141.