Parvinder Hothi

Expression and Functional Heterogeneity of Chemokine Receptors CXCR4 and CXCR7 in Primary Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of these tumors indicates a need to identify new therapeutic targets. Therapy resistance of GBMs is attributed to heterogeneity of the glioblastoma due to genetic alterations and functional subpopulations. Chemokine receptors CXCR4 and CXCR7 play important roles in progression of various cancers although the specific functions of the CXCL12−CXCR4−CXCR7 axis in GBM are less characterized. In this study we examined the expression and function of CXCR4 and CXCR7 in four primary patient-derived GBM cell lines of the proliferative subclass, investigating their roles in in vitro growth, migration, sphere and tube formation. CXCR4 and CXCR7 cell surface expression was heterogeneous both between and within each cell line examined, which was not reflected by RT-PCR analysis. Variable percentages of CXCR4+CXCR7− (CXCR4 single positive), CXCR4−CXCR7+ (CXCR7 single positive), CXCR4+CXCR7+ (double positive), and CXCR4−CXCR7− (double negative) subpopulations were evident across the lines examined. A subpopulation of slow cell cycling cells was enriched in CXCR4 and CXCR7. CXCR4+, CXCR7+, and CXCR4+/CXCR7+ subpopulations were able to initiate intracranial tumors in vivo. CXCL12 stimulated in vitro cell growth, migration, sphere formation and tube formation in some lines and, depending on the response, the effects were mediated by either CXCR4 or CXCR7. Collectively, our results indicate a high level of heterogeneity in both the surface expression and functions of CXCR4 and CXCR7 in primary human GBM cells of the proliferative subclass. Should targeting of CXCR4 and CXCR7 provide clinical benefits to GBM patients, a personalized treatment approach should be considered given the differential expression and functions of these receptors in GBM.

VEGFR inhibitors upregulate CXCR4 in VEGF receptor-expressing glioblastoma in a TGFβR signaling-dependent manner

The failure of standard treatment for patients diagnosed with glioblastoma (GBM) coupled with the highly vascularized nature of this solid tumor has led to the consideration of agents targeting VEGF or VEGFRs, as alternative therapeutic strategies for this disease. Despite modest achievements in survival obtained with such treatments, failure to maintain an enduring survival benefit and more invasive relapsing tumors are evident. Our study suggests a potential mechanism by which anti-VEGF/VEGFR therapies regulate the enhanced invasive phenotype through a pathway that involves TGFβR and CXCR4. VEGFR signaling inhibitors (Cediranib and Vandetanib) elevated the expression of CXCR4 in VEGFR-expressing GBM cell lines and tumors, and enhanced the in vitro migration of these lines toward CXCL12. The combination of VEGFR inhibitor and CXCR4 antagonist provided a greater survival benefit to tumor-bearing animals. The upregulation of CXCR4 by VEGFR inhibitors was dependent on TGFβ/TGFβR, but not HGF/MET, signaling activity, suggesting a mechanism of crosstalk among VEGF/VEGFR, TGFβ/TGFβR, and CXCL12/CXCR4 pathways in the malignant phenotype of recurrent tumors after anti-VEGF/VEGFR therapies. Thus, the combination of VEGFR, CXCR4, and TGFβR inhibitors could provide an alternative strategy to halt GBM progression.

TGFβ‐Responsive HMOX1 Expression Is Associated with Stemness and Invasion in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To date, no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations, justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry, here we present five cell‐surface markers HMOX1, SLC16A1, CADM1, SCAMP3, and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non‐GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM, substantiating the histopathological hallmarks of GBM. In a prospective study (N = 8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness, we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFβ. siRNA‐mediated silencing of HMOX1 impaired GBM invasion—a phenomenon related to poor prognosis. In addition, surgical resection of GBM tumors caused declines (18% ± 5.1SEM) in the level of plasma HMOX1 as measured by ELISA, in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016;34:2276–2289

TGFβ‐Responsive HMOX1 Expression is Associated with Stemness and Invasion in GBM

Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To date, no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations, justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry, here we present five cell‐surface markers HMOX1, SLC16A1, CADM1, SCAMP3, and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non‐GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM, substantiating the histopathological hallmarks of GBM. In a prospective study (N = 8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness, we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFβ. siRNA‐mediated silencing of HMOX1 impaired GBM invasion—a phenomenon related to poor prognosis. In addition, surgical resection of GBM tumors caused declines (18% ± 5.1SEM) in the level of plasma HMOX1 as measured by ELISA, in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016;34:2276–2289

Large-Scale Production of Human Glioblastoma-Derived Cancer Stem Cell Tissue in Suspension Bioreactors to Facilitate the Development of Novel Oncolytic Therapeutics

Krishna Panchalingam1, Wendy Paramchuk1, Parvinder Hothi2, Nameeta Shah2, Leroy Hood3, Greg Foltz2 and Leo A. Behie1 1Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, Calgary, Alberta 2Ben and Catherine Ivy Center for Advanced Brain Tumour Treatment, Swedish Neuroscience Institute, Seattle, Washington 3Institute for Systems Biology, Seattle, Washington 1Canada 2,3United States of America

Epigenetic Profiling for the Molecular Classification of Metastatic Brain Tumors

Optimal treatment of brain metastases is often hindered by limitations in diagnostic capabilities. To meet these challenges, we generated genome-scale DNA methylomes of the three most frequent types of brain metastases: melanoma, breast, and lung cancers (n=96). Using supervised machine learning and integration of multiple DNA methylomes from normal, primary, and metastatic tumor specimens (n=1,860), we unraveled epigenetic signatures specific to each type of metastatic brain tumor and constructed a three-step DNA methylation-based classifier (BrainMETH) that categorizes brain metastases according to the tissue of origin and therapeutically-relevant subtypes. BrainMETH predictions were supported by routine histopathologic evaluation. We further characterized and validated the most predictive genomic regions in a large cohort of brain tumors (n=165) using quantitative methylation-specific PCR. Our study highlights the importance of brain tumor-defining epigenetic alterations, which can be utilized to further develop DNA methylation profiling as a critical tool in the histomolecular stratification of patients with brain metastases.

Precision knockdown of EGFR gene expression using radio frequency electromagnetic energy

Electromagnetic fields (EMF) in the radio frequency energy (RFE) range can affect cells at the molecular level. Here we report a technology that can record the specific RFE signal of a given molecule, in this case the siRNA of epidermal growth factor receptor (EGFR). We demonstrate that cells exposed to this EGFR siRNA RFE signal have a 30–70% reduction of EGFR mRNA expression and ~60% reduction in EGFR protein expression vs. control treated cells. Specificity for EGFR siRNA effect was confirmed via RNA microarray and antibody dot blot array. The EGFR siRNA RFE decreased cell viability, as measured by Calcein-AM measures, LDH release and Caspase 3 cleavage, and increased orthotopic xenograft survival. The outcomes of this study demonstrate that an RFE signal can induce a specific siRNA-like effect on cells. This technology opens vast possibilities of targeting a broader range of molecules with applications in medicine, agriculture and other areas.

Abstract 1425: Impact of VEGFR pathway inhibition on the expression and function of CXCR4 in primary patient derived glioblastoma cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastoma (GBM) is one of the most malignant and aggressive forms of primary brain tumors. Current standard treatment options for patients diagnosed with GBM include surgical resection, radiation, and chemotherapy. Since GBM features an extensively vascularized solid tumor, anti-angiogenic agents have also been considered as a treatment regimen for GBM patients. Indeed, evidence from clinical trials has shown that Bevacizumab (Avastin®), an anti-VEGF monoclonal antibody, successfully reduces the development of tumor microvascular networks and prolongs the progression-free survival rate of GBM patients. In a subset of patients, however, tumors become resistant to this treatment and exhibit a more infiltrative phenotype after anti-angiogenic therapy. A recent published study (Lu et. al., Cancer Cell, 22:21; 2012) proposed that the HGF/c-MET signaling pathway may be involved in this enhanced invasive phenotype post anti-angiogenic therapy targeting VEGFRs. These data suggest that integrating VEGFR and c-MET inhibitors will prevent tumor recurrence from anti-angiogenic therapy. Because of the heterogeneity of the tumor microenvironment and complexity in signaling communication among cancer cells, we are interested in the contribution of chemokines and chemokine receptors in this phenomenon. Characterization of several primary patient-derived glioblastoma cell lines indicated heterogeneity in the level of expression of VEGFRs: some lines were VEGFR positive while others were VEGFR negative. VEGF and c-MET mRNAs were detected in all lines. Flow cytometry analysis of chemokine receptors indicated that levels of CXCR4 were elevated after VEGF/VEGFR inhibitor treatment (Bevacizumab, Cediranib or Vandetanib) only in VEGFR-expressing GBM cell lines, while CXCR7 was not affected by VEGFR pathway inhibition. In addition, VEGFR inhibitor treatment enhanced the migration of VEGFR-expressing lines to CXCL12. c-MET inhibitors did not impact VEGFR inhibitor-stimulated CXCR4 expression, suggesting that this regulation is independent of c-MET. Together, our data suggest that the CXCL12/CXCR4 axis may be an additional potential factor contributing to the invasive phenotype in recurrent tumors after anti-angiogenic therapy. Thus, the combination of VEGFR pathway inhibitors and CXCR4 antagonists may provide an alternative strategy to halt tumor progression and provide benefit to patients with GBM. Citation Format: Kien Pham, Defang Luo, Dietmar Siemann, Brent A. Reynolds, Parvinder Hothi, Gregory Foltz, Jeffrey K. Harrison. Impact of VEGFR pathway inhibition on the expression and function of CXCR4 in primary patient derived glioblastoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1425. doi:10.1158/1538-7445.AM2013-1425