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Molecular analysis of circulating and disseminated tumor cells (CTCs/DTCs) has great potential as a means for continuous evaluation of prognosis and treatment efficacy in near-real time through minimally invasive liquid biopsies. To realize this potential, however, methods for molecular analysis of these rare cells must be developed and validated. Here, we describe the integration of imaging mass cytometry (IMC) using metal-labeled antibodies as implemented on the Fluidigm Hyperion Imaging System into the workflow of the previously established High Definition Single Cell Analysis (HD-SCA) assay for liquid biopsies, along with methods for image analysis and signal normalization. Using liquid biopsies from a metastatic prostate cancer case, we demonstrate that IMC can extend the reach of CTC characterization to include dozens of protein biomarkers, with the potential to understand a range of biological properties that could affect therapeutic response, metastasis and immune surveillance when coupled with simultaneous phenotyping of thousands of leukocytes.

The activome can be considered as a subset of the proteome that contains enzymes in their catalytically active form and can be interrogated by using probes targeted towards individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activitybased probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, simultaneous analysis becomes limited.To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry. Using lanthanide-based tags instead of fluorophores gives us the ability to monitor the activity of multiple proteases in parallel. As proof of concept we developed a panel of cathepsin and legumain specific probes and showed that we were able to identify an activome of these proteases in two cell lines and peripheral blood mononuclear cells, providing a framework for the use of mass cytometry for multiplexed enzyme activity detection.

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Glioblastoma (GBM) is a highly infiltrative brain tumor in which cells with properties of stem cells, called glioblastoma stem cells (GSCs), have been identified. In general, the dominant view is that GSCs are responsible for the initiation, progression, invasion and recurrence of this tumor. In this study, we addressed the question whether the differentiation status of GBM cells is associated with their invasive capacity. For this, several primary GBM cell lines were used, cultured either as neurospheres known to enrich for GSCs or in medium supplemented with 10% FCS that promotes differentiation. The differentiation state of the cells was confirmed by determining the expression of stem cell and differentiation markers. The migration/invasion potential of these cells was tested using in vitro assays and intracranial mouse models. Interestingly, we found that serum-induced differentiation enhanced the invasive potential of GBM cells, which was associated with enhanced MMP9 expression. Chemical inhibition of MMP9 significantly reduced the invasive potential of differentiated cells in vitro. Furthermore, the serum-differentiated cells could revert back to an undifferentiated/stem cell state that were able to form neurospheres, although with a reduced efficiency as compared to non-differentiated counterparts. We propose a model in which activation of the differentiation program in GBM cells enhances their infiltrative potential and that depending on microenvironmental cues a significant portion of these cells are able to revert back to an undifferentiated state with enhanced tumorigenic potential. Thus, effective therapy should target both GSCs and differentiated offspring and targeting of differentiation-associated pathways may offer therapeutic opportunities to reduce invasive growth of GBM.

Little is known about the complexity and plasticity of circulating tumor cell (CTC) biology in different compartments of the fluid microenvironment during tumor metastasis. Here we integrated phenomics, genomics, and targeted proteomics to characterize CTC phenotypic and genotypic heterogeneity in paired peripheral blood (PB) and bone marrow aspirate (BMA) from a metastatic prostate cancer patient following the rapid disease progression, using the High-Definition Single Cell Assay 3.0 (HDSCA3.0). Uniquely, we identified a subgroup of genetically clonal CTCs that acquired a mesenchymal-like state and its presence was significantly associated with one subclone that emerged along the clonal lineage. Higher CTC abundance and phenotypic diversity were observed in the BMA than PB and differences in genomic alterations were also identified between the two compartments demonstrating spatial heterogeneity. Single cell copy number profiling further detected clonal heterogeneity within clusters of CTCs (also known as microemboli or aggregates) as well as phenotypic variations by targeted proteomics. Overall, these results identify epithelial and mesenchymal CTCs in the clonal lineage of an aggressive prostate cancer case and also demonstrate a single cell multi-omic approach to deconvolute the heterogeneity and association of CTC phenotype and genotype in multi-medium liquid biopsies of metastatic prostate cancer.

The developmental program of epithelial to mesenchymal transition (EMT) can be activated in tumor cells and is implicated in the metastatic spread of tumor cells. EMT leads to loss of cell adhesion and increased motility of cells and can be detected by down regulation of epithelial makers such as E-cadherin and gain of mesenchymal ones such as vimentin and fibronectin. Interestingly, EMT has also been implicated in enhancing stemness properties of tumor cells and in causing chemotherapy resistance. The aim of this study is to examine the invasive, stem-ness and chemotherapy sensitive properties of mesenchymal and epithelial Non-small cell lung cancer (NSCLC) cells. In particular we use a TGF beta-inducible EMT model of A549 cells. A pannel of NSCLC cell lines, A549, H460, H322, SW1573, H1650 and H1299 were explored for epithelial (EpCAM. E-cadherin) and mesenhymal markers (N- cadherin, Vimentin and Fibronectin). Only A549 cells exhibit epithelial characteristics while remaning cell lines were either mesenchymal in nature or weekly expressing epithelial markers. Treatment of A549 cells with TGFbeta induced EMT as confirmed by loss of epithelial and gain of mesenchymal markers by FACS and WB analysis. This process was independent of Src since knockdown of Src did not effect EMT activation. Other kinases are currently evaluated for mediating EMT in this model. A549 mesenchymal cells (M) cells were somewhat more resistant to cisplatin and paclitaxel compared to parental A549 cells, whereas no marked difference in the response to proapoptotic TRAIL was found. Furthermore, using wound healing assays A549M cells showed higher rates of migration. Possible effects of EMT on stem cell properties in A549 cells were studied next. A549M cells displayed enhanced spheroid formation potential when cultured in serum free Neurobasal media (NBM) that was associated with an increase in Sox-2 expression. To further explore stemness of A549(M) cells, currently the expression of stem cell markers such as CD133, CD24, CD44 is examined. In conclusion, our results thus far suggest that in A549 NSCLC cells, induction of EMT by TGF beta results in chemoresistance, higher migratory potential and increased stem cell properties. The in-vitro results observed will be extended by in-vivo assays. For this purpose we are establishing orthotopic NSCLC mice models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2402. doi:1538-7445.AM2012-2402