Use of FLOSEAL® as a scaffold and its impact on induced neural stem cell phenotype, persistence, and efficacy.

Induced neural stem cells (iNSCs) have emerged as a promising therapeutic platform for glioblastoma (GBM). iNSCs have the innate ability to home to tumor foci, making them ideal carriers for antitumor payloads. However, the in vivo persistence of iNSCs limits their therapeutic potential. We hypothesized that by encapsulating iNSCs in the FDA-approved, hemostatic matrix FLOSEAL®, we could increase their persistence and, as a result, therapeutic durability. Encapsulated iNSCs persisted for 95 days, whereas iNSCs injected into the brain parenchyma persisted only 2 weeks in mice. Two orthotopic GBM tumor models were used to test the efficacy of encapsulated iNSCs. In the GBM8 tumor model, mice that received therapeutic iNSCs encapsulated in FLOSEAL® survived 30 to 60 days longer than mice that received nonencapsulated cells. However, the U87 tumor model showed no significant differences in survival between these two groups, likely due to the more solid and dense nature of the tumor. Interestingly, the interaction of iNSCs with FLOSEAL® appears to downregulate some markers of proliferation, anti-apoptosis, migration, and therapy which could also play a role in treatment efficacy and durability. Our results demonstrate that while FLOSEAL® significantly improves iNSC persistence, this alone is insufficient to enhance therapeutic durability.

Cytotoxic Engineered Induced Neural Stem Cells as an Intravenous Therapy for Primary Non-Small Cell Lung Cancer and Triple-Negative Breast Cancer.

Converting human fibroblasts into personalized induced neural stem cells (hiNSC) that actively seek out tumors and deliver cytotoxic agents is a promising approach for treating cancer. Herein, we provide the first evidence that intravenously-infused hiNSCs secreting cytotoxic agent home to and suppress the growth of non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). Migration of hiNSCs to NSCLC and TNBC in vitro was investigated using time-lapse motion analysis, which showed directional movement of hiNSCs to both tumor cell lines. In vivo, migration of intravenous hiNSCs to orthotopic NSCLC or TNBC tumors was determined using bioluminescent imaging (BLI) and immunofluorescent post-mortem tissue analysis, which indicated that hiNSCs colocalized with tumors within 3 days of intravenous administration and persisted through 14 days. In vitro, efficacy of hiNSCs releasing cytotoxic TRAIL (hiNSC-TRAIL) was monitored using kinetic imaging of co-cultures, in which hiNSC-TRAIL therapy induced rapid killing of both NSCLC and TNBC. Efficacy was determined in vivo by infusing hiNSC-TRAIL or control cells intravenously into mice bearing orthotopic NSCLC or TNBC and tracking changes in tumor volume using BLI. Mice treated with intravenous hiNSC-TRAIL showed a 70% or 72% reduction in NSCLC or TNBC tumor volume compared with controls within 14 or 21 days, respectively. Safety was assessed by hematology, blood chemistry, and histology, and no significant changes in these safety parameters was observed through 28 days. These results indicate that intravenous hiNSCs-TRAIL seek out and kill NSCLC and TNBC tumors, suggesting a potential new strategy for treating aggressive peripheral cancers.

Personalized-induced neural stem cell therapy: Generation, transplant, and safety in a large animal model.

In this study, we take an important step toward clinical translation by generating the first canine-induced neural stem cells (iNSCs). We explore key aspects of scale-up, persistence, and safety of personalized iNSC therapy in autologous canine surgery models. iNSCs are a promising new approach to treat aggressive cancers of the brain, including the deadly glioblastoma. Created by direct transdifferentiation of fibroblasts, iNSCs are known to migrate through the brain, track down invasive cancer foci, and deliver anticancer payloads that significantly reduce tumor burden and extend survival of tumor-bearing mice. Here, skin biopsies were collected from canines and converted into the first personalized canine iNSCs engineered to carry TNFα-related apoptosis-inducing ligand (TRAIL) and thymidine kinase (TK), as well as magnetic resonance imaging (MRI) contrast agents for in vivo tracking. Time-lapse analysis showed canine iNSCs efficiently migrate to human tumor cells, and cell viability assays showed both TRAIL and TK monotherapy markedly reduced tumor growth. Using intraoperative navigation and two delivery methods to closely mimic human therapy, canines received autologous iNSCs either within postsurgical cavities in a biocompatible matrix or via a catheter placed in the lateral ventricle. Both strategies were well tolerated, and serial MRI showed hypointense regions at the implant sites that remained stable through 86 days postimplant. Serial fluid sample testing following iNSC delivery showed the bimodal personalized therapy was well tolerated, with no iNSC-induced abnormal tissue pathology. Overall, this study lays an important foundation as this promising personalized cell therapy advances toward human patient testing.

Developing Implantable Scaffolds to Enhance Neural Stem Cell Therapy for Post-Operative Glioblastoma.

Pre-clinical and clinical studies have shown that engineered tumoricidal neural stem cells (tNSCs) are a promising treatment strategy for the aggressive brain cancer glioblastoma (GBM). Yet, stabilizing human tNSCs within the surgical cavity following GBM resection is a significant challenge. As a critical step toward advancing engineered human NSC therapy for GBM, we used a preclinical variant of the clinically utilized NSC line HB1.F3.CD and mouse models of human GBM resection/recurrence to identify a polymeric scaffold capable of maximizing the transplant, persistence, and tumor kill of NSC therapy for post-surgical GBM. Using kinetic bioluminescence imaging, we found that tNSCs delivered into the mouse surgical cavity wall by direct injection persisted only 3 days. We found that delivery of tNSCs into the cavity on nanofibrous electrospun poly-l-lactic acid scaffolds extended tNSC persistence to 8 days. Modifications to fiber surface coating, diameter, and morphology of the scaffold failed to significantly extend tNSC persistence in the cavity. In contrast, tNSCs delivered into the post-operative cavity on gelatin matrices (GEMs) persisted 8-fold longer as compared to direct injection. GEMs remained permissive to tumor-tropic homing, as tNSCs migrated off the scaffolds and into invasive tumor foci both in vitro and in vivo. To mirror envisioned human brain tumor trials, we engineered tNSCs to express the prodrug/enzyme thymidine kinase (tNSCstk) and transplanted the therapeutic cells in the post-operative cavity of mice bearing resected orthotopic patient-derived GBM xenografts. Following administration of the prodrug ganciclovir, residual tumor volumes in mice receiving GEM/tNSCs were reduced by 10-fold at day 35, and median survival was extended from 31 to 46 days. Taken together, these data begin to define design parameters for effective scaffold/tNSC composites and suggest a new approach to maximizing the efficacy of tNSC therapy in human patient trials.

Delivery of Cytotoxic Mesenchymal Stem Cells with Biodegradable Scaffolds for Treatment of Postoperative Brain Cancer.

Engineered stem cells have recently entered clinical trials as therapeutic agents for treating glioblastoma foci that remain after primary brain tumor resection. However, efficient delivery of anti-cancer mesenchymal stem cells (MSCs) into the resection cavity remains a potential obstacle to therapeutic efficacy in humans. Direct injection quickly leads to significant stem cell loss and poor tumor killing. Recent reports have shown that biodegradable scaffolds improve MSC persistence and restore therapeutic potential. Here, we describe a method for the delivery of therapeutic MSCs on biodegradable fibrin scaffolds into the resection cavity to treat postoperative brain cancer.

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNFα-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Sustained Delivery of Doxorubicin via Acetalated Dextran Scaffold Prevents Glioblastoma Recurrence after Surgical Resection.

The primary cause of mortality for glioblastoma (GBM) is local tumor recurrence following standard-of-care therapies, including surgical resection. With most tumors recurring near the site of surgical resection, local delivery of chemotherapy at the time of surgery is a promising strategy. Herein drug-loaded polymer scaffolds with two distinct degradation profiles were fabricated to investigate the effect of local drug delivery rate on GBM recurrence following surgical resection. The novel biopolymer, acetalated dextran (Ace-DEX), was compared with commercially available polyester, poly(l-lactide) (PLA). Steady-state doxorubicin (DXR) release from Ace-DEX scaffolds was found to be faster when compared with scaffolds composed of PLA, in vitro. This increased drug release rate translated to improved therapeutic outcomes in a novel surgical model of orthotopic glioblastoma resection and recurrence. Mice treated with DXR-loaded Ace-DEX scaffolds (Ace-DEX/10DXR) resulted in 57% long-term survival out to study completion at 120 days compared with 20% survival following treatment with DXR-loaded PLA scaffolds (PLA/10DXR). Additionally, all mice treated with PLA/10DXR scaffolds exhibited disease progression by day 38, as defined by a 5-fold growth in tumor bioluminescent signal. In contrast, 57% of mice treated with Ace-DEX/10DXR scaffolds displayed a reduction in tumor burden, with 43% exhibiting complete remission. These results underscore the importance of polymer choice and drug release rate when evaluating local drug delivery strategies to improve prognosis for GBM patients undergoing tumor resection.

Neural stem cell therapy for cancer.

Cancers of the brain remain one of the greatest medical challenges. Traditional surgery and chemo-radiation therapy are unable to eradicate diffuse cancer cells and tumor recurrence is nearly inevitable. In contrast to traditional regenerative medicine applications, engineered neural stem cells (NSCs) are emerging as a promising new therapeutic strategy for cancer therapy. The tumor-homing properties allow NSCs to access both primary and invasive tumor foci, creating a novel delivery platform. NSCs engineered with a wide array of cytotoxic agents have been found to significantly reduce tumor volumes and markedly extend survival in preclinical models. With the recent launch of new clinical trials, the potential to successfully manage cancer in human patients with cytotoxic NSC therapy is moving closer to becoming a reality.