Dissecting Small Cell Lung Carcinoma Heterogeneity And Chemotherapy Resistance With Mass Cytometry

Introduction: Small cell lung cancer (SCLC) is a high grade neuroendocrine carcinoma of the lung responsible for up to 25% of lung cancer deaths. Treatment in SCLC has not changed significantly in the last 20 years. SCLC initially responds well to chemotherapy, but inevitably recurs. Characterization of tumor heterogeneity and changes in SCLC cell signaling and phenotypes after chemotherapy could yield new insights and therapeutic options. Mass cytometry uses metal labeled antibodies to profile expression and phosphorylation of more than 40 proteins in single cells and offers the opportunity to identify new subpopulations including potential cancer stem cell populations as well as targets for novel therapies in SCLC. Methods: Nude mice with SCLC patient derived xenografts (PDXs) were treated with one cycle of carboplatin/etoposide or saline injection. Tumors were harvested at ~2000mm 3 , disaggregated, and cryopreserved. PDX samples were stained with a 22 marker panel and an intercalator dye to identify nucleated cells. This panel measured phospho-signaling, neuroendocrine, immune, and mesenchymal cell markers, and functional markers including ki67 and cleaved caspase 3. ViSNE analysis and biaxial gating were used to identify major subpopulations of interest. Results: PDX tumors released viable tumor and stromal cells suitable for cryopreservation and mass cytometry. ACK buffer and enzymatic dissociation yielded the best quality cells by depleting red blood cells. Mouse cells, including leukocytes, were excluded using mouse MHC1 gating and iridium intercalator was used to identify nucleated cells. Single cell protein expression and phosphorylation was analyzed using viSNE and yielded at least 9 distinct subpopulations based on density islands with neuroendocrine (CD56+) and non-neuroendocrine (CD56-) populations. Chemotherapy treated cells had dramatic changes in subpopulation distribution compared to matched mock treated tumor. This included 2-3 fold expansion of SOX2+, CD117+, and pSTAT3+ populations with chemotherapy treatment. A small CD44+ tumor subpopulation identified in the chemotherapy treated cells was not present in the matched mock treated tumor suggesting a potential chemotherapy resistant/ stem- like subpopulation. Kinase activity showed stable p-AKT overall, but increased p-S6 in the chemotherapy treated cells. Conclusions: Mass cytometry was able to identify multiple neuroendocrine and non-neuroendocrine cell populations from SCLC PDXs and characterize their signaling. Chemotherapy treated PDX had differential subpopulation distribution with enrichment of multiple stem-like signaling factors. This work demonstrates the utility of mass cytometry and viSNE as novel techniques to identify subpopulations associated with chemotherapy resistance for future targeting and demonstrates the feasibility of this technique for characterizing signaling heterogeneity in human SCLC tumors. Citation Format: Jonathan M. Lehman, Nalin Leelatian, Bradford Harris, Megan Hoeksema, Zou Yong, Deon B. Doxie, Jonathan M. Irish, Pierre P. Massion. Dissecting small cell lung carcinoma heterogeneity and chemotherapy resistance with mass cytometry [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 3935. doi:10.1158/1538-7445.AM2017-3935