Abstract 2920: Characterizing human melanoma treatment response in vivo using single-cell mass cytometry analysis of longitudinal tumor biopsies
Authors: Doxie Deon B. , Greenplate Allison R. , Diggins Kirsten E. , Maier Caroline E. , Sosman Jeffrey A. , Kelley Mark C. , Irish Jonathan M. 
Published: July 2017

Abstract
Introduction: Metastatic melanoma that harbor BRAFV600E mutations are treated with inhibitors that inhibit MAPK signaling downstream of BRAF. Although these treatments improve patient survival, the majority of tumors develop resistance. It is increasingly clear that the presence and emergence of cell subsets in the tumor microenvironment shapes treatment responses. Mass cytometry is a single cell platform capable of measuring &gt;35 proteins on millions of tumor cells. Here, mass cytometry was used to characterize matched tumor samples obtained before, during, and after therapy in order to quantify the impact of therapy on tumor cell populations.
Methods: Melanoma tumors from adults with metastases were biopsied or surgically resected from 12 patients with consent. Patients received two weeks of BRAFV600E inhibitor dabrafenib followed by two weeks of dabrafenib and MEK inhibitor trametinib. Tumors were enzymatically digested and cryopreserved with protocols for mass cytometry (Leelatian and Doxie et al., Cytometry B 2016). Samples were stained with a 30+ antibody panel focused on melanoma identity and markers of lineage and trafficking for leukocytes, fibroblast, and endothelial cells. Viable nucleated cells were identified by total histone H3 and rhodium dye exclusion. Expert biaxial gating and viSNE analysis were used to identify, computationally map, and track populations and their subsets over time. Tissue microarrays were also analyzed by histology to confirm findings observed by mass cytometry.
Results: More than 99% of cells were characterized and fell into one of the four major populations. Melanoma phenotypes included loss of MHC class I and/or expression of one or more markers of identity including SOX10, SOX2, nestin, S100β, and MCAM. Significant decreases in nestin, MCAM, MHC I and S100β (p &lt; 0.01, p &lt; 0.05, p &lt; 0.05, and p&lt;0.05, respectively) were observed in cells after treatment. Decreases in nestin and S100β populations identified by mass cytometry were observed in histology analysis of TMAs from matched tumors. Analysis of pre- and post-therapy melanoma cells and BRAF mutated cell lines indicated pre-therapy tumor cells had phenotypes most similar to cell lines. Strikingly, 6hrs or 1 day of treatment of cell lines with dabrafenib and trametinib did not result in decrease in nestin expression. The nestin low post-therapy phenotype of melanoma tumor cells was present in all melanoma cell lines at a low abundance (average of 7.0% +/- 3.9% in cell lines, N = 7).
Conclusions: Single cell analysis and longitudinal monitoring of patient’s revealed tumor ecosystems experience rapid changes in cellular diversity during targeted therapy. Lower nestin expression distinguished treated melanoma tumor cells. The in vitro results suggest that in vivo remodeling of the tumor microenvironment results from a shift in host-tumor cellular interactions and not a direct response to inhibitors.
Citation Format: Deon B. Doxie, Allison R. Greenplate, Kirsten E. Diggins, Caroline E. Maier, Jeffrey A. Sosman, Mark C. Kelley, Jonathan M. Irish. Characterizing human melanoma treatment response in vivo using single-cell mass cytometry analysis of longitudinal tumor biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2017-2920