Prakash Rath

Stem Cells as Vectors to Deliver HSV/tk Gene Therapy for Malignant Gliomas

The prognosis of patients diagnosed with malignant gliomas including glioblastoma multiforme (GBM) is poor and there is an urgent need to develop and translate novel therapies into the clinic. Neural stem cells display remarkable tropism toward GBMs and thus may provide a platform to deliver oncolytic agents to improve survival. First we provide a brief review of clinical trials that have used intra-tumoral herpes simplex virus thymidine kinase (HSV/tk) gene therapy to treat brain tumors. Then, we review recent evidence that neural stem cells can be used to deliver HSV/tk to GBMs in animal models. While previous clinical trials used viruses or non-migratory vector-producing cells to deliver HSV/tk, the latter approaches were not effective in humans, primarily because of satellite tumor cells that escaped surgical resection and survived due to low efficiency delivery of HSV/tk. To enhance delivery of HSV/tk to kill gliomas cells, recent animal studies have focused on the ability of neural stem cells, transduced with HSV/tk, to migrate efficiently and selectively to regions occupied by GBM cells. This approach holds the promise of targeting GBM cells that have infiltrated the brain well beyond the original site of the tumor epicenter.

Isolation and characterization of a population of stem-like progenitor cells from an atypical meningioma

The majority of meningiomas are benign tumors associated with favorable outcomes; however, the less common aggressive variants with unfavorable outcomes often recur and may be due to subpopulations of less-differentiated cells residing within the tumor. These subpopulations of tumor cells have tumor-initiating properties and may be isolated from heterogeneous tumors when sorted or cultured in defined medium. We report the isolation and characterization of a population of tumor-initiating cells derived from an atypical meningioma. We identify a tumor-initiating population from an atypical meningioma, termed meningioma-initiating cells (MICs). These MICs self-renew, differentiate, and can recapitulate the histological characteristics of the parental tumor when transplanted at 1000 cells into the flank regions of athymic nude mice. Immunohistochemistry reveals stem-like protein expression patterns similar to neural stem and progenitor cells (NSPCs) while genomic profiling verified the isolation of cancer cells (with defined meningioma chromosomal aberrations) from the bulk tumor. Microarray and pathway analysis identifies biochemical processes and gene networks related to aberrant cell cycle progression, particularly the loss of heterozygosity of tumor suppressor genes CDKN2A (p16(INK4A)), p14(ARF), and CDKN2B (p15(INK4B)). Flow cytometric analysis revealed the expression of CD44 and activated leukocyte adhesion molecule (ALCAM/CD166); these may prove to be markers able to identify this cell type. The isolation and identification of a tumor-initiating cell population capable of forming meningiomas demonstrates a useful model for understanding meningioma development. This meningioma model may be used to study the cell hierarchy of meningioma tumorogenesis and provide increased understanding of malignant progression.

Developmental cues and persistent neurogenic potential within an in vitro neural niche

BackgroundNeurogenesis, the production of neural cell-types from neural stem cells (NSCs), occurs during development as well as within select regions of the adult brain. NSCs in the adult subependymal zone (SEZ) exist in a well-categorized niche microenvironment established by surrounding cells and their molecular products. The components of this niche maintain the NSCs and their definitive properties, including the ability to self-renew and multipotency (neuronal and glial differentiation).ResultsWe describe a model in vitro NSC niche, derived from embryonic stem cells, that produces many of the cells and products of the developing subventricular zone (SVZ) and adult SEZ NSC niche. We demonstrate a possible role for apoptosis and for components of the extracellular matrix in the maintenance of the NSC population within our niche cultures. We characterize expression of genes relevant to NSC self-renewal and the process of neurogenesis and compare these findings to gene expression produced by an established neural-induction protocol employing retinoic acid.ConclusionsThe in vitro NSC niche shows an identity that is distinct from the neurally induced embryonic cells that were used to derive it. Molecular and cellular components found in our in vitro NSC niche include NSCs, neural progeny, and ECM components and their receptors. Establishment of the in vitro NSC niche occurs in conjunction with apoptosis. Applications of this culture system range from studies of signaling events fundamental to niche formation and maintenance as well as development of unique NSC transplant platforms to treat disease or injury.

Regulation of glioblastoma multiforme stem-like cells by inhibitor of DNA binding proteins and oligodendroglial lineage-associated transcription factors.

Tumor‐initiating stem cells (also referred to as cancer stem cells, CSCs) are a subpopulation of cancer cells that play unique roles in tumor propagation, therapeutic resistance and tumor recurrence. It is increasingly important to understand how molecular signaling regulates the self‐renewal and differentiation of CSCs. Basic helix‐loop‐helix (bHLH) transcription factors are critical for the differentiation of normal stem cells, yet their roles in neoplastic stem cells are not well understood. In glioblastoma neurosphere cultures that contain cancer stem cells (GBM‐CSCs), the bHLH family member inhibitors of DNA binding protein 2 and 4 (Id2 and Id4) were found to be upregulated during the differentiation of GBM‐CSCs in response to histone deacetylase inhibitors. In this study, we examined the functions of Id2 and Id4 in GBM neurosphere cells and identified Id proteins as efficient differentiation regulators of GBM‐CSCs. Overexpression of Id2 and Id4 promoted the lineage‐specific differentiation of GBM neurosphere cells as evidenced by the induction of neuronal/astroglial differentiation markers Tuj1 and GFAP and the inhibition of the oligodendroglial marker GalC. Id protein overexpression also reduced both stem cell marker expression and neurosphere formation potential, a biological marker of cancer cell “stemness.” We further showed that Id2 and Id4 regulated GBM neurosphere differentiation through downregulating of another bHLH family member, the oligodendroglial lineage‐associated transcription factors (Olig) 1 and 2. Our results provide evidence for distinct functions of Id proteins in neoplastic stem cells, which supports Id proteins and their downstream targets as potential candidates for differentiation therapy in CSCs. (Cancer Sci 2012; 103: 1028–1037)

Crizotinib and erlotinib inhibits growth of c-Met+/EGFRvIII+ primary human glioblastoma xenografts

OBJECTIVES Receptor tyrosine kinases (RTK), such as c-Met and epidermal growth factor receptor (EGFR), are implicated in the malignant progression of glioblastoma. Studies show that RTK systems can co-modulate distinct and overlapping oncogenic downstream signaling pathways. EGFRvIII, a constitutively activated EGFR deletion mutant variant, leads to increased tumor growth and diminishes the tumor growth response to HGF: c-Met pathway inhibitor therapy. Conversely, activation of the c-Met pathway diminishes the tumor growth response to EGFR pathway inhibitors. Previously we reported that EGFRvIII and c-Met pathway inhibitors synergize to inhibit tumor growth in isogenic GBM cell lines engineered to express EGFRvIII. More recently, studies suggest that despite targeting RTK signaling in glioblastoma multiforme, a subpopulation of stem-like tumor-propagating cells can persist to replenish the tumor cell population leading to tumor recurrence. PATIENTS AND METHODS Mayo 39 and Mayo 59 xenograft lines were cultured and xenografts were maintained. Subcutaneous xenograft lines were serially passaged in nude mice to generate subcutaneous xenografts. Xenografts were implanted in 6-8 week old nude mice. Once tumors reached a substantial size (150 mm3), mice were randomly divided into 4 groups: 1) control vehicle, 2) Crizotinib (crizo), 3) Erlotinib (erlot), or 4) Crizotinib + Erlotinib, (n = 5 per group). RESULTS Crizotinib (c-Met pathway inhibitor) and Erlotinib (EGFR pathway inhibitor) in combination significantly inhibited tumor growth, phospho-EGFRvIII, phospho-Met, phospho-AKT, phospho-MAPK, and neurosphere growth in Mayo 39 and Mayo 59 primary GBM subcutaneous xenografts. The expression of the stem cell markers Nestin, Musashi, Olig 2 and Sox2 were also significantly down-regulated by c-Met inhibition, but no additive down-regulation was seen by co-treatment with Erlotinib. CONCLUSIONS These results are consistent with and corroborate our previous findings demonstrating that targeting these two parallel pathways with c-Met and EGFR inhibitor therapy provides substantial anti-tumor activity in glioblastoma models.

Abstract 4311: EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase

Glioblastoma multiforme (GBM) are the most frequent and aggressive primary brain tumors in adults. Uncontrolled proliferation and abnormal cell migration are two prominent spatially and temporally disassociated characteristics of GBMs. In this study, we investigated the role of the receptor tyrosine kinase EphB2 in controlling the proliferation/migration dichotomy of GBM. We studied EphB2 gain-of-function and loss-of function in glioblastoma-derived stem-like neurospheres (GBM-SCs), whose in vivo growth pattern closely replicates human GBM. EphB2 expression stimulated GBM neurosphere cell migration and invasion, and inhibited neurosphere cell proliferation in vitro. In parallel, EphB2 silencing increased tumor cell proliferation and decreased tumor cell migration. EphB2 was found to increase tumor cell invasion in vivo using an internally controlled dual-fluorescent xenograft model. Xenografts derived from EphB2 overexpressing GBM neurospheres also showed decreased cellular proliferation. The non-receptor tyrosine kinase focal adhesion kinase (FAK) was found to be co-associated with and highly activated by EphB2 expression and FAK activation facilitated focal adhesion formation, cytoskeleton structure change and cell migration in EphB2-expression GBM neurosphere cells. Taken together, our findings indicate that EphB2 has pro-invasive and anti-proliferative actions in GBM stem-like neurospheres mediated, in part, by interactions between EphB2 receptors and FAK. These novel findings suggest that tumor cell invasion can be therapeutically targeted by inhibiting EphB2 signaling and that optimal anti-tumor responses to EphB2 targeting may require the concurrent use of anti-proliferative agents. 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 4311. doi:1538-7445.AM2012-4311