Cytostatic hypothermia and its impact on glioblastoma and survival.

Patients with glioblastoma (GBM) have limited options and require novel approaches to treatment. Here, we studied and deployed nonfreezing "cytostatic" hypothermia to stunt GBM growth. This growth-halting method contrasts with ablative, cryogenic hypothermia that kills both neoplastic and infiltrated healthy tissue. We investigated degrees of hypothermia in vitro and identified a cytostatic window of 20° to 25°C. For some lines, 18 hours/day of cytostatic hypothermia was sufficient to halt division in vitro. Next, we fabricated an experimental tool to test local cytostatic hypothermia in two rodent GBM models. Hypothermia more than doubled median survival, and all rats that successfully received cytostatic hypothermia survived their study period. Unlike targeted therapeutics that are successful in preclinical models but fail in clinical trials, cytostatic hypothermia leverages fundamental physics that influences biology broadly. It is a previously unexplored approach that could provide an additional option to patients with GBM by halting tumor growth.

Enriching neural stem cell and anti-inflammatory glial phenotypes with electrical stimulation after traumatic brain injury in male rats.

Traumatic brain injury (TBI) by an external physical impact results in compromised brain function via undesired neuronal death. Following the injury, resident and peripheral immune cells, astrocytes, and neural stem cells (NSCs) cooperatively contribute to the recovery of the neuronal function after TBI. However, excessive pro-inflammatory responses of immune cells, and the disappearance of endogenous NSCs at the injury site during the acute phase of TBI, can exacerbate TBI progression leading to incomplete healing. Therefore, positive outcomes may depend on early interventions to control the injury-associated cellular milieu in the early phase of injury. Here, we explore electrical stimulation (ES) of the injury site in a rodent model (male Sprague-Dawley rats) to investigate its overall effect on the constituent brain cell phenotype and composition during the acute phase of TBI. Our data showed that a brief ES for 1 hr on day 2 of TBI promoted anti-inflammatory phenotypes of microglia as assessed by CD206 expression and increased the population of NSCs and Nestin+ astrocytes at 7 days post-TBI. Also, ES effectively increased the number of viable neurons when compared to the unstimulated control group. Given the salience of microglia and neural stem cells for healing after TBI, our results strongly support the potential benefit of the therapeutic use of ES during the acute phase of TBI to regulate neuroinflammation and to enhance neuroregeneration.

Immuno-suppressive hydrogels enhance allogeneic MSC survival after transplantation in the injured brain.

Traumatic brain injury (TBI) triggers multiple biochemical and cellular processes that exacerbate brain tissue damage through a secondary injury. Therapies that prevent or limit the evolution of secondary injury could significantly reduce the neurological deficits associated with TBI. Mesenchymal stem cell (MSC) transplantation after TBI can ameliorate neurological deficits by modulating inflammation and enhancing the expression of neurotrophic factors. However, transplanted MSCs can be actively rejected by host immune responses, such as those mediated by cytotoxic CD8+ T cells, thereby limiting their therapeutic efficacy. Here, we designed an agarose hydrogel that releases Fas ligand (FasL), a protein that can induce apoptosis of cytotoxic CD8+ T cells. We studied the immunosuppressive effect of this hydrogel near the allogeneic MSC transplantation site and its impact on the survival of transplanted MSCs in the injured brain. Agarose-FasL hydrogels locally reduced the host cytotoxic CD8+ T cell population and enhanced the survival of allogeneic MSCs transplanted near the injury site. Furthermore, the expression of crucial neurotrophic factors was elevated in the injury penumbra, suggesting an enhanced therapeutic effect of MSCs. These results suggest that the development of immunosuppressive hydrogels for stem cell delivery can enhance the benefits of stem cell therapy for TBI.

Evaluation of M2-like macrophage enrichment after diffuse traumatic brain injury through transient interleukin-4 expression from engineered mesenchymal stromal cells.

BACKGROUND: Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities for the millions of those inflicted annually. In TBI, cellular mediators of inflammation, including macrophages and microglia, possess a range of phenotypes relevant for an immunomodulatory therapeutic approach. It is thought that early phenotypic modulation of these cells will have a cascading healing effect. In fact, an anti-inflammatory, "M2-like" macrophage phenotype after TBI has been associated with neurogenesis, axonal regeneration, and improved white matter integrity (WMI). There already exist clinical trials seeking an M2-like bias through mesenchymal stem/stromal cells (MSCs). However, MSCs do not endogenously synthesize key signals that induce robust M2-like phenotypes such as interleukin-4 (IL-4). METHODS: To enrich M2-like macrophages in a clinically relevant manner, we augmented MSCs with synthetic IL-4 mRNA to transiently express IL-4. These IL-4 expressing MSCs (IL-4 MSCs) were characterized for expression and functionality and then delivered in a modified mouse TBI model of closed head injury. Groups were assessed for functional deficits and MR imaging. Brain tissue was analyzed through flow cytometry, multi-plex ELISA, qPCR, histology, and RNA sequencing. RESULTS: We observed that IL-4 MSCs indeed induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression after TBI. However, here we demonstrate that acute enrichment of M2-like macrophages did not translate to improved functional or histological outcomes, or improvements in WMI on MR imaging. To further understand whether dysfunctional pathways underlie the lack of therapeutic effect, we report transcriptomic analysis of injured and treated brains. Through this, we discovered that inflammation persists despite acute enrichment of M2-like macrophages in the brain. CONCLUSION: The results demonstrate that MSCs can be engineered to induce a stronger M2-like macrophage response in vivo. However, they also suggest that acute enrichment of only M2-like macrophages after diffuse TBI cannot orchestrate neurogenesis, axonal regeneration, or improve WMI. Here, we also discuss our modified TBI model and methods to assess severity, behavioral studies, and propose that IL-4 expressing MSCs may also have relevance in other cavitary diseases or in improving biomaterial integration into tissues.

Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates.

Treatment of neuroepithelial cancers remains a daunting clinical challenge, particularly due to an inability to address rampant invasion deep into eloquent regions of the brain. Given the lack of access, and the dispersed nature of brain tumor cells, we explore the possibility of electric fields inducing directed tumor cell migration. In this study we investigate the properties of populations of brain cancer undergoing electrotaxis, a phenomenon whereby cells are directed to migrate under control of an electrical field. We investigate two cell lines for glioblastoma and medulloblastoma (U87mg & DAOY, respectively), plated as spheroidal aggregates in Matrigel-filled electrotaxis channels, and report opposing electrotactic responses. To further understand electrotactic migration of tumor cells, we performed RNA-sequencing for pathway discovery to identify signaling that is differentially affected by the exposure of direct-current electrical fields. Further, using selective pharmacological inhibition assays, focused on the PI3K/mTOR/AKT signaling axis, we validate whether there is a causal relationship to electrotaxis and these mechanisms of action. We find that U87 mg electrotaxis is abolished under pharmacological inhibition of PI3Kγ, mTOR, AKT and ErbB2 signaling, whereas DAOY cell electrotaxis was not attenuated by these or other pathways evaluated.

Engineering Controlled Peritumoral Inflammation to Constrain Brain Tumor Growth.

Brain tumors remain a great clinical challenge, in part due to their capacity to invade into eloquent, inoperable regions of the brain. In contrast, inflammation in the central nervous system (CNS) due to injuries activates microglia and astrocytes culminating in an astroglial scar that typically "walls-off" the injury site. Here, the hypothesis is tested that targeting peritumoral cells surrounding tumors to activate them via an inflammatory stimulus that recapitulates the sequelae of a traumatic CNS injury, could generate an environment that would wall-off and contain invasive tumors in the brain. Gold nanoparticles coated with inflammatory polypeptides to target stromal cells in close vicinity to glioblastoma (GBM) tumors, in order to activate these cells and stimulate stromal CNS inflammation, are engineered. It is reported that this approach significantly contains tumors in rodent models of GBM relative to control treatments (reduction in tumor volume by over 300% in comparison to controls), by the activation of the innate and adaptive immune response, and by triggering pathways related to cell clustering. Overall, this report outlines an approach to contain invasive tumors that can complement adjuvant interventions for invasive GBM such as radiation and chemotherapy.

Immunoengineering nerve repair.

Injuries to the peripheral nervous system are major sources of disability and often result in painful neuropathies or the impairment of muscle movement and/or normal sensations. For gaps smaller than 10 mm in rodents, nearly normal functional recovery can be achieved; for longer gaps, however, there are challenges that have remained insurmountable. The current clinical gold standard used to bridge long, nonhealing nerve gaps, the autologous nerve graft (autograft), has several drawbacks. Despite best efforts, engineering an alternative "nerve bridge" for peripheral nerve repair remains elusive; hence, there is a compelling need to design new approaches that match or exceed the performance of autografts across critically sized nerve gaps. Here an immunomodulatory approach to stimulating nerve repair in a nerve-guidance scaffold was used to explore the regenerative effect of reparative monocyte recruitment. Early modulation of the immune environment at the injury site via fractalkine delivery resulted in a dramatic increase in regeneration as evident from histological and electrophysiological analyses. This study suggests that biasing the infiltrating inflammatory/immune cellular milieu after injury toward a proregenerative population creates a permissive environment for repair. This approach is a shift from the current modes of clinical and laboratory methods for nerve repair, which potentially opens an alternative paradigm to stimulate endogenous peripheral nerve repair.

Engineering challenges for brain tumor immunotherapy.

Malignant brain tumors represent one of the most devastating forms of cancer with abject survival rates that have not changed in the past 60years. This is partly because the brain is a critical organ, and poses unique anatomical, physiological, and immunological barriers. The unique interplay of these barriers also provides an opportunity for creative engineering solutions. Cancer immunotherapy, a means of harnessing the host immune system for anti-tumor efficacy, is becoming a standard approach for treating many cancers. However, its use in brain tumors is not widespread. This review discusses the current approaches, and hurdles to these approaches in treating brain tumors, with a focus on immunotherapies. We identify critical barriers to immunoengineering brain tumor therapies and discuss possible solutions to these challenges.

Bacterial Carriers for Glioblastoma Therapy.

Treatment of aggressive glioblastoma brain tumors is challenging, largely due to diffusion barriers preventing efficient drug dosing to tumors. To overcome these barriers, bacterial carriers that are actively motile and programmed to migrate and localize to tumor zones were designed. These carriers can induce apoptosis via hypoxia-controlled expression of a tumor suppressor protein p53 and a pro-apoptotic drug, Azurin. In a xenograft model of human glioblastoma in rats, bacterial carrier therapy conferred a significant survival benefit with 19% overall long-term survival of >100 days in treated animals relative to a median survival of 26 days in control untreated animals. Histological and proteomic analyses were performed to elucidate the safety and efficacy of these carriers, showing an absence of systemic toxicity and a restored neural environment in treated responders. In the treated non-responders, proteomic analysis revealed competing mechanisms of pro-apoptotic and drug-resistant activity. This bacterial carrier opens a versatile avenue to overcome diffusion barriers in glioblastoma by virtue of its active motility in extracellular space and can lead to tailored therapies via tumor-specific expression of tumoricidal proteins.