Imaging mass cytometry (IMC) is a novel technology with the potential of multiplex proteomic profiling of rare circulating and disseminated tumor cells (CTCs and DTCs) and immune cells from liquid biopsies. The high-definition single-cell analysis (HD-SCA) identifies CTCs and DTCs by the virtue of morphometric and fluorescent staining with exact locations of all the cells, which could be further characterized using downstream genomics and proteomics platform. This study aims to develop and employ metal-labeled antibody panels for multiplex proteomic characterization of CTCs, DTCs and immune cells obtained from longitudinal liquid biopsies from cancer patients on the basis of their epithelial-to-mesenchymal transition (EMT) status, cancer stem cell (CSC), dormancy, proliferation and drug resistance markers. For every antibody panel, 6-10 biomarkers were chosen, and metal-labeled antibodies were processed in a three-stage validation program using known positive and negative control cell lines. In the first stage, basal level marker expression was evaluated in positive control cells seeded on a glass slide. In the second stage, cell lines were spiked in blood and slides were prepared as per the HD-SCA protocol, stored at -800 C, and expression of biomarkers was evaluated in IMC staining. In the third stage, cell line-spiked slides were first stained in three colors immunofluorescent staining in HD-SCA, mimicking the clinical setup followed by IMC staining with metal-labeled antibodies. Antibodies showing expressions in all three stages were included to build a panel targeting EMT, CSC, vasculature mimicry, dormancy, proliferation and drug resistance biomarkers. Out of 69 metal-labeled antibodies, 24 antibodies targeting cancer and immune cell-specific biomarkers have been validated for multiplex proteomic profiling in HD-SCA platform. This includes epithelial markers EpCAM and E-cadherin; mesenchymal markers vimentin and fibronectin; CSC markers CD44, CD24, ALDH and beta-catenin; prostate-specific markers PSA, PSMA, AR-C terminal, AR-N terminal; and immune cell markers CD45, CD66a, CD3, HLA-DR, CD45RA, CD8a, CD4, CD38, CD14 and CD31. Building and validating metal-labeled antibody panels for EMT, CSC, dormancy and drug resistance biomarkers could achieve multiplex proteomic characterization of the rare cell IMC. It would be further used in monitoring treatment efficacy, mutational switchovers, and immune surveillance from longitudinal liquid biopsies collected from cancer patients to better understand the biology of a disease.

Citation Format: Milind Pore, Erik Gerdtsson, Christopher Boldt, Sophia Wix, Preeya Katti, Mohan Singh, Akil Merchant, James Hicks, Peter Kuhn. Developing metal-labeled antibody panels for multiplex proteomic characterization of rare circulating tumor cells by imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5656.