Abstract 4953: Single cell signaling analysis reveals circulating tumor cell markers of drug susceptibility and tumor heterogeneity

Abstract

Complexity of cancer requires advancements of tools for intervention and diagnostics. Rare circulating tumor cells (CTCs) from patient blood could be used for real-time monitoring of solid cancer. We recently developed a microfluidic device, the CTC-iChip, which enriches well-preserved CTCs in solution through removal of the cellular components (red and white blood cells) and platelets of whole blood. Protein expression analysis in CTCs has been limited to immunofluorescent staining for a few well-characterized tumor markers (including EpCAM, HER2, PSA and Keratins) due to the limitations inherent to immunofluorescence labels and their precise resolution through microscopy. The scarcity of cells precludes large-scale proteomic analysis such as mass spectrometry of CTCs to effectively evaluate changes in total as well as phospho-proteins, which serve as excellent surrogate markers to monitor in real time the rapid alterations in signaling pathways within cells. This approach would be particularly important since the PI3K, mTOR, Akt, MAPK signaling pathways are aberrantly activated in many cancers and are promising therapeutic targets. In this work, we applied inductively-coupled plasma mass spectrometry (ICP-MS) measurement of cells (mass cytometry) that can measure up to ~40 proteins in millions of single cells due to quantitative, non-overlapping mass signals acquired from antibodies tagged with purified lanthanides. Sample preparation for mass cytometry was modified to minimize the loss of cells using our microfluidic platform in lieu of washing via centrifugation. Mass cytometry allowed identification of tumor cells simultaneous analysis of markers that are associated with proliferation, response or resistance to pathway inhibition due to administered therapeutic agent. We quantified the expression of twelve phospho-proteins to monitor changes multiple signaling nodes within single tumor cells. This approach allowed assay and compare the target proteome within single CTCs as well as individual tumor cells derived from primary and metastatic tumors. We found that some protein levels of CTCs represent primary tumor protein levels well. When targeted therapeutic was administered two of these epitopes were found to be significantly changed in both CTCs and primary tumor cells. We also found a portion of cancer cells in primary tumor were more susceptible to the targeted therapeutic than the rest of the tumor, showing a heterogeneity in response within the tumor. Overall, single cell multiplexed molecular analysis revealed tumor molecular heterogeneity and circulating markers of drug susceptibility. Citation Format: N Murat Karabacak, Yu Zheng, Erin Emmons, Michael Koulopoulos, Daniel A. Haber, Mehmet Toner, Shyamala Maheswaran. Single cell signaling analysis reveals circulating tumor cell markers of drug susceptibility and tumor heterogeneity [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 4953. doi:10.1158/1538-7445.AM2017-4953