Multiplex Immunohistochemistry protocol in formalin-fixed, paraffin embedded Colorectal Liver Metastases patient tissues and Machine Learning- based Tissue Segmentation and Cell Phenotyping analysis 

Abstract Colorectal cancer liver metastasis (CLM) is the most of common cause of death in patients with colorectal cancer (CRC) worldwide. There are various immune-suppressive and tumor-promoting mechanisms, which contribute to the unresponsiveness associated with current check point immunotherapy in CRC. Techniques such as Multiplex immunohistochemistry (mIHC) and automated image quantitation analysis, enable a deeper understanding of the diversity of the host’s anti-tumor immune response and could identify potential targets for immunotherapy. Herein, we present a 7-plex OPALTM protocol used to assess the composition and spatial distribution of T cell markers CD3, CD8, Foxp3 and CD103 and the epithelial to mesenchymal transition (EMT) markers alpha smooth muscle actin (α-SMA) and E-cadherin. The protocol has been manually optimized and validated in two independent cohorts of formalin-fixed, paraffin embedded CLM patient tissues (n=42) using well-established antibodies, single spectral library, negative controls and biological controls for corroborating staining pattern of α-SMA+ (sclerosed hemangioma samples) and T-cell infiltrates (benign liver). The accurate profiling of T- cell composition, location and phenotypic characterization could reveal important insights about the influence of T lymphocytes on prognosis after liver metastasectomy.