Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases, but not lung and liver metastases, exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-MET protein. Suppression of c-MET impaired tumor growth in the bone. Intratumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities coexisting with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in patients with metastatic prostate and breast cancer. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single-cell proteomics indicate that microenvironment- and cell state–dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors. Significance: Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies.

Identification of candidate metastasis genes has traditionally resulted from comparison of primary and metastatic tumor specimens. However, Circulating Tumor Cells (CTCs) contain metastatic precursors that are present transiently in the bloodstream and their analysis may reveal additional pathways that are induced for a limited time, as they invade and survive within the vasculature. By comparing transcriptome profiles of CTCs from breast, prostate and lung cancers with their primary tumor of origin, we observed consistent and significant induction of the β-globin gene (HBB) within CTCs. In contrast, expression of α-globin, its binding partner within hematopoietic cells, is not coordinately upregulated. The tumor-specific origin of HBB was further confirmed by analysis of human xenografts-derived CTCs in mice, where human-specific HBB polymorphisms are readily distinguishable in the murine background. In cultured cancer cells, we further demonstrate that induction of HBB is triggered by exposure to reactive oxygen species (ROS), and we identify KLF-family transcriptional regulators that mediate this effect. To investigate the function of aberrant β-globin expression within CTCs, we performed shRNA-mediated knockdown of HBB in breast CTC-derived cultures. Cells with depleted HBB expression displayed elevated intracellular ROS levels, increased sensitivity to hydrogen peroxide, and impaired metastatic potential in mouse models. Taken together, these observations suggest that β-globin, a component of functional hemoglobin in red blood cells, is deregulated in disseminated tumor cells, where it may function as a ROS scavenger, reducing oxidative stress and facilitating cancer metastasis. Citation Format: Yu Zheng, David T. Miyamoto, Ben S. Wittner, James P. Sullivan, Nicola Aceto, Nicole Vincent Jordan, Min Yu, Nezihi Murat Karabacak, Valentine Comaills, Robert Morris, Rushil Desai, Niyati Desai, Erin Emmons, Richard J. Lee, Chin-Lee Wu, Lecia V. Sequist, Wilhelm Haas, David T. Ting, Mehmet Toner, Sridhar Ramaswamy, Shyamala Maheswaran, Daniel A. Haber. Induction of β-globin protects circulating tumor cells from oxidative stress during dissemination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2679.

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