Abstract 4624: Deciphering the tumor microenvironment at single-cell resolution using a workflow combining RNA transcript and protein detection with Brightplex®, a sequential chromogenic multiplex assay
Authors: Trinh Alex , Olive Marion , Collignon Aurélie , Chamourin Maité , Culley Georgia , Jaume Clemence , Leca Vanina , Benchaaben Assil , Bussotti Giovanni , Kassambara Alboukadel , Galon Jerome , Fieschi Jacques 
Published: April 2023

Abstract
Tumor-infiltrating immune cells play an important role against cancer and are critical to control tumor growth and spread. Immunotherapy and immune checkpoint blockade, which aim to reinvigorate exhausted T cells have revolutionized cancer therapy. Despite inducing long-term response in many cancer types, these therapies remain ineffective for a majority of the patients. A better understanding of the tumor microenvironment, and in particular the characterization of immune cells and other stromal cells, along with their abundance and distribution, may help to better stratify patients and understand mechanisms of resistance to immunotherapy. Brightplex® is a chromogenic multiplex technology allowing the detection of several biomarkers on a single FFPE slide to identify and quantify complex cell populations such as T-cells expressing immune checkpoints, M1 and M2 macrophages, Treg cells and myeloid-derived suppressor cells. However, the exquisite characterization of some cell types within the tumor microenvironment or their activation status may require the identification of soluble proteins that cannot be detected by IHC. For example, the detection of secreted proteins such as cytokines or activation factors would allow one to determine the activation status of immune cells or cancer-associated fibroblasts (CAF). In that case, the detection of RNA transcripts corresponding to soluble biomarkers by In-Situ hybridization can be used as a substitute to protein detection.Here, we propose a new multiplex platform combining In Situ Hybridization (ISH) and immunohistochemistry (IHC) using Brightplex® technology. This platform allows the detection of several biomarkers that can be either RNA transcripts or proteins on a single FFPE tissue section. Thanks to that technique it is possible to: 1) detect and quantify TGFb in cancer associated fibroblasts, or IL-10 in immunosuppressive cells, 2) Quantify the level of RNA expression by individual cells, 3) assess the spatial distribution of activated cells based on complex phenotypes within the tumor or other regions of interest. This new workflow combining RNA transcript detection and proteins detection using Brightplex® is automated on Bond RX platform. Following staining and slide digitization, images are fused to create a virtual multi-channel image where cells of interest are detected by digital pathology (DP). Following cell detection, their densities and spatial distribution are obtained using R-studio software.Integrated into an Immunogram, an analytics platform which integrates multi-omics datasets from Veracyte Biopharma Atlas, this new tool could be a powerful solution to understand the tumor landscape and predict response to immunotherapy and patient outcome.
Citation Format: Alex Trinh, Marion Olive, Aurélie Collignon, Maité Chamourin, Georgia Culley, Clemence Jaume, Vanina Leca, Assil Benchaaben, Giovanni Bussotti, Alboukadel Kassambara, Jerome Galon, Jacques Fieschi. Deciphering the tumor microenvironment at single-cell resolution using a workflow combining RNA transcript and protein detection with Brightplex®, a sequential chromogenic multiplex assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4624.