Abstract 5105: Unravelling the mystery of cancer-associated fibroblast populations in the tumor microenvironment by a fully automated sequential chromogenic multiplex assay

The tumor microenvironment (TME) is one of the driving factors of tumor progression and invasion. Inside this microenvironment, Cancer-Associated Fibroblasts (CAF) play a significant role in tumor modulation and in the development of drug resistance. This major stromal cell population has been highlighted as a significant prognostic factor in several tumors. CAF are characterized by an original, phenotypic and function heterogeneity, as well as a high plasticity. Several studies have demonstrated that this population contains different subtypes with the capacity to promote or inhibit cancer. Moreover, their biological role as allies or foes is dependent on many factors, including the cancer stage. There is yet no consensus on the molecular definition of these key TME players, but several markers have been used to identify them. However, many of these markers come with their own set of downsides, such as low specificity and inability to detect different subtypes within the wider CAF population. In this context, novel refined immunophenotyping approaches such as multiplex technology are vital for accurate identification and quantification of CAF. Here we assessed the presence, abundance and localization of CAF subsets within the pancreatic cancer microenvironment by using the Brightplex® solution, an automated sequential chromogenic multiplex assay. A unique combination of biomarkers (Fibroblast activation protein (FAP), Alpha smooth muscle actin (αSMA), Platelet derived growth factor receptor alpha/beta (PDGFRα/β), Fibroblast specific protein 1 (FSP-1), negative markers) was developed to assess CAF heterogeneity and plasticity on a single FFPE tumor tissue section by preserving tissue9s spatial contexture. Briefly, a tissue section was sequentially stained, digitized, unstained and re-stained with antibodies targeting the selected markers. Images of the whole slide were then analyzed by digital pathology and the detection of positive cells was performed for each marker independently. In addition, tissue segmentation tools were used to assess CAF densities in parenchyma, tumor stroma and invasive margin regions. This precise characterization of CAF subtypes has been performed on pancreatic ductal adenocarcinoma FFPE tissues. The several subtypes of CAF were resolved spatially and studied in relation to CD3+ and CD8+ T cell infiltration (Immunoscore®) at different stages of pancreatic cancer development. This new tool allows to decipher the heterogeneity and plasticity of CAF to evaluate their contribution to disease progression and help clinical researchers to understand mode of action of CAF-targeting drugs. This new Brightplex® panel will be integrated into our Immunogram to better understand the immune contexture of the tumor. Citation Format: Thomas Sbarrato, Mounia Filahi, Veronique Frayssinet, Anne-Laure Bailly, Caroline Lauge, Caroline Davin, Helene Girardi, Anna Martirosyan, jacques Fieschi. Unravelling the mystery of cancer-associated fibroblast populations in the tumor microenvironment by a fully automated sequential chromogenic multiplex assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5105.