Abstract 4004: Molecular characterization of circulating glioblastoma cells identifies a mesenchymal-like tumor cell subpopulation
Authors: Sullivan James P. , Nahed Brian V. , Chi Andrew S. , Madden Marissa N. , Oliveira Samantha M. , Springer Simeon , Wakimoto Hiroaki , Bhere Deepak , Shah Khalid , Spuhler Phil , Shah Ajay M. , Louis David N. , Toner Mehmet , Maheswaran Shyamala , Haber Daniel A. 
Published: May 2015

Abstract
Glioblastoma (GBM), the most common and aggressive form of glioma, is often heterogeneous, diffusely invasive and hypervascularized. Despite being aggressive, extracranial metastases are rare, suggesting constraints on hematologic dissemination or distant survival of primary GBM cells. We recently developed a novel microfluidic device, termed the CTC-iChip, for efficient, antigen agnostic capture of rare circulating tumor cells (CTCs). Using the CTC-iChip, we set out to determine whether GBM cells can be detected in the blood of mice using two orthotopic xenograft models representing diffuse (GBM8) and focal (GBM24) GBM. CTCs were identified in 5/11 and 2/5 mice bearing GBM8 and GBM24 xenografts, respectively. CTC numbers did not correlate with tumor size or latency. We then expanded our analysis to patients and identified CTCs in 12/38 GBM patients with a median 5.3 cells per ml (range: 0 to 48.2 cells per ml). To understand the molecular characteristics of GBM CTCs, we analyzed the expression of 49 GBM-specific and stem cell transcripts in single GBM CTCs from both mouse xenografts models as well as GBM patients. The expression of mesenchymal gene transcripts including VIM, TGFBR2, TGFB, and SERPINE1 was elevated in CTCs compared with cultured cells and matched primary tumor cells. Moreover, CTCs exhibited lower proliferative indices and decreased neural lineage transcripts. To determine the pathophysiologic significance of this expression profile and the prospective origin of GBM CTCs, RNA-ISH was performed on xenografts and patient tumor samples. In the highly migratory GBM8 xenograft model, broad tumor cell expression of CTC/mesenchymal transcripts was observed. However, in the GBM24 focal tumor growth model, CTC/mesenchymal gene transcripts were predominantly expressed in regions of tumor invasion, migration along fiber tracts, and around regions of necrosis. Similarly, RNA-ISH analysis of 6 GBM patient tumors revealed prominent expression of CTC/mesenchymal genes in peri-necrotic and migratory pseudopalisading cells. Finally, RNA-ISH analysis of a rare case of metastatic GBM revealed a significant enrichment of GBM/mesenchymal gene expressing tumor cells within lung and lymph node metastases. Taken together, these data provide the first evidence for the hematologic circulation of GBM cells and reveal the involvement of mesenchymal features in the pathophysiology of malignant brain tumors.
Citation Format: James P. Sullivan, Brian V. Nahed, Andrew S. Chi, Marissa N. Madden, Samantha M. Oliveira, Simeon Springer, Hiroaki Wakimoto, Deepak Bhere, Khalid Shah, Phil Spuhler, Ajay M. Shah, David N. Louis, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Molecular characterization of circulating glioblastoma cells identifies a mesenchymal-like tumor cell subpopulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4004. doi:10.1158/1538-7445.AM2014-4004