Evaluating novel stem cell based therapies for brain tumors

The recognition that both adult and embryonic stem (ES) cell derived neural stem cells (NSC) selectively home to gliomas [1], has unveiled new roles for their use as on-site therapeutic delivery agents for the brain tumor treatment. In prior research, we have established that: a) therapeutically engineered adult neural stem cells migrate extensively to tumors and to infiltrating deposits in the brain (2) and have apoptotic effects when transplanted into mouse models of glioma (3-5); and b) the dynamics of targeted anti-tumor therapies and fate of stem cells can be visualized in real time in vivo (5-6). In order to circumvent, the ethical and isolation problems associated with employing adult and ES derived NSC, we and others (7) have recently generated NSC from induced pluripotent stem cells (ipNSC) that were previously derived from mouse fibroblasts. The long term goal of this proposal is to develop therapeutic ipNSC to study stem cell based anti-angiogenic and combined anti-angiogenic and cytotoxic therapies in different brain tumor models.

Recent phase II clinical studies provide evidence that delivery of anti-angiogenic drugs through vasculature transiently normalize the abnormal structure and function of the blood vessels and result in reduction of tumor-associated vasogenic brain edema resulting in clinical benefit in most patients (8-9). Evidence from a number of pre-clinical models suggest that anti-angiogenic agents, when used judiciously, 'normalize' tumor vessels and enhance the delivery and efficacy of concurrently administered cytotoxic agents (8,10,11). However, recent studies have shown that vessel normalization in the tumor bearing brains in mice result in the restoration of blood brain barrier and consequential inefficient delivery of cytotoxic drugs to brain tumors (12,13) thus disputing the existing rationale on the use of anti-angiogenic agents in combination with cytotoxic agents in pre-clinical and clinical settings. These results have raised fundamental questions about how anti-angiogenic agents and cytotoxic drugs can be used in combination in the clinic to 1) normalize tumor vasculature; and 2) enhance the outcome of cytotoxic therapy within the normalization window for brain tumors?

A number of studies provide evidence that upregulation of endogenous TSP-1 decreases vessel permeability (8,10,14) and its expression is upregulated in intact p53 tumor cells [15]. Based on the observations that short biological half-life of many of the currently used antiangiogenic inhibitors and the impaired intratumoral blood flow create logistical difficulties for effective normalization/inhibition of tumor-induced neovascularization, (8) we have recently cloned and characterized the anti-angiogenic fragment of TSP-1 (comprising of the regions of 3 type 1 repeats, where most of the peptides used in clinical settings have originated (16-17)) and shown its anti-angiogenic effect in vitro and anti-tumor effect in vivo. In this proposal, we will initially engineer mouse ipNSC with antiangiogenic, aaTSP-1 and test them for their vascular normalization potential in mouse models of glioma using a panel of both established cell lines (p53 wt and mutated) and invasive primary glioma cells from patients. The vascular normalization potential of on-site delivery of aaTSP-1 by ipNSC in vivo will be compared with systemic delivery of clinically approved known normalization agent, receptor tyrosine kinase inhibitor AZD2171 (8,18). We hypothesize that sustained on-site delivery of aaTSP-1 via ipNSC in the tumor micro-environment will overcome the lack of endogenous TSP-1 production in p53 mutant tumors and ultimately lead to prolonged vascular normalization and delay glioma growth in mouse glioma models. A close integration of: a) genetically engineered imaging markers; b) angiogenic probes; and c) bioluminescence imaging (BLI), intravital microscopy (IVM) and magnetic resonance imaging (MRI); and extensive neuropathology will allow us to assess fate and pharmacokinetics of ipNSC-aaTSP-1 and ultimately the aaTSP-1 mediated normalization process.

To circumvent the delivery of cytotoxic agents through vasculature, we will further engineer ipNSC with regulatable bi-functional EGFR_nb-TRAIL consisting of secretable version of epidermal growth factor receptor (EGFR) targeted nanobody (nb) (20) fused to tumor necrosis factor apoptosis inducing ligand (TRAIL) (3,21). In an ongoing study in close collaboration with Henegouwen lab (Utrecht University, Netherlands), we have characterized EGFR_nb-TRAIL and shown that it simultaneously targets cell proliferation and death pathways and induces apoptosis in both TRAIL resistant and sensitive cells glioma cells. The therapeutic effect of EGFR_nb-TRAIL within the normalization window created by aaTSP-1 will be tested in both malignant and invasive glioma models. We hypothesize that regulatable release of EGFR_nb-TRAIL from ipNSC will give us a direct measure of the cytotoxic effect of EGFRnb-TRAIL during aaTSP-1 mediated vessel normalization and lead to enhanced eradication of primary tumor mass. Furthermore, we hypothesize that therapeutic ipNSC will track micro-invasive tumor cells escaping the primary tumor mass. In an effort to simulate a clinical scenario of surgical resection of glioma, we will ultimately test the efficacy of therapeutic stem cells grown in biocompatible scaffolds in our recently developed glioma resection model in vivo.

Once validated, these studies can be easily translated into clinics for different cancer types using patients own reprogrammed stem cells. In next 4-6 years, we envision a therapeutic modality in which at the time of brain tumor surgery, the main tumor mass will be removed and genetically engineered therapeutic cells will be introduced and allowed to target the remaining tumor cells and micro-invasive tumor deposits in the brain thus eliminating the risk of recurrence. This will have a major impact in saving the lives of many brain cancer patients.