Correction: Specific transfection of inflamed brain by macrophages: A new therapeutic strategy for neurodegenerative diseases.

[This corrects the article DOI: 10.1371/journal.pone.0061852.].

Combining the constitutive TRAIL-secreting induced neural stem cell therapy with the novel anti-cancer drug TR-107 in glioblastoma.

Tumor-homing neural stem cell (NSC) therapy is emerging as a promising treatment for aggressive cancers of the brain. Despite their success, developing tumor-homing NSC therapy therapies that maintain durable tumor suppression remains a challenge. Herein, we report a synergistic combination regimen where the novel small molecule TR-107 augments NSC-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy (hiNeuroS-TRAIL) in models of the incurable brain cancer glioblastoma (GBM) in vitro. We report that the combination of hiNeuroS-TRAIL and TR-107 synergistically upregulated caspase markers and restored sensitivity to the intrinsic apoptotic pathway by significantly downregulating inhibitory pathways associated with chemoresistance and radioresistance in the TRAIL-resistant LN229 cell line. This combination also showed robust tumor suppression and enhanced survival of mice bearing human xenografts of both solid and invasive GBMs. These findings elucidate a novel combination regimen and suggest that the combination of these clinically relevant agents may represent a new therapeutic option with increased efficacy for patients with GBM.

Auto-loaded TRAIL-exosomes derived from induced neural stem cells for brain cancer therapy.

Transdifferentiation (TD), a somatic cell reprogramming process that eliminates pluripotent intermediates, creates cells that are ideal for personalized anti-cancer therapy. Here, we provide the first evidence that extracellular vesicles (EVs) from TD-derived induced neural stem cells (Exo-iNSCs) are an efficacious treatment strategy for brain cancer. We found that genetically engineered iNSCs generated EVs loaded with the tumoricidal gene product TRAIL at nearly twice the rate as their parental fibroblasts, and the TRAIL produced by iNSCs were naturally loaded into the lumen of EVs and arrayed across their outer membrane (Exo-iNSC-TRAIL). Uptake studies in ex vivo organotypic brain slice cultures showed Exo-iNSC-TRAIL selectively accumulates within tumor foci, and co-culture assays showed that Exo-iNSC-TRAIL killed metastatic and primary brain cancer cells more effectively than free TRAIL. In an orthotopic mouse model of brain cancer, Exo-iNSC-TRAIL reduced breast-to-brain tumor xenografts around 3000-fold greater than treatment with free TRAIL, with all Exo-iNSC-TRAIL treated animals surviving through 90 days post-treatment. In additional in vivo testing against aggressive U87 and invasive GBM8 glioblastoma tumors, Exo-iNSC-TRAIL also induced a statistically significant increase in survival. These studies establish a new easily generated, stable, tumor-targeted EV to efficaciously treat multiple forms of brain cancer.

Auto-loaded TRAIL-exosomes derived from induced neural stem cells for brain cancer therapy.

Transdifferentiation (TD), a somatic cell reprogramming process that eliminates pluripotent intermediates, creates cells that are ideal for personalized anti-cancer therapy. Here, we provide the first evidence that extracellular vesicles (EVs) from TD-derived induced neural stem cells (Exo-iNSCs) are an efficacious treatment strategy for brain cancer. We found that genetically engineered iNSCs generated EVs loaded with the tumoricidal gene product TRAIL at nearly twice the rate of their parental fibroblasts, and TRAIL produced by iNSCs was naturally loaded into the lumen of EVs and arrayed across their outer membrane (Exo-iNSC-TRAIL). Uptake studies in ex vivo organotypic brain slice cultures showed that Exo-iNSC-TRAIL selectively accumulates within tumor foci, and co-culture assays demonstrated that Exo-iNSC-TRAIL killed metastatic and primary brain cancer cells more effectively than free TRAIL. In an orthotopic mouse model of brain cancer, Exo-iNSC-TRAIL reduced breast-to-brain tumor xenografts by approximately 3000-fold compared to treatment with free TRAIL, with all Exo-iNSC-TRAIL treated animals surviving through 90 days post-treatment. In additional in vivo testing against aggressive U87 and invasive GBM8 glioblastoma tumors, Exo-iNSC-TRAIL also induced a statistically significant increase in survival. These studies establish a novel, easily generated, stable, tumor-targeted EV to efficaciously treat multiple forms of brain cancer.

Trans-lesion synthesis and mismatch repair pathway crosstalk defines chemoresistance and hypermutation mechanisms in glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides an aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

Local administration of irinotecan using an implantable drug delivery device stops high-grade glioma tumor recurrence in a glioblastoma tumor model.

The treatment for Glioblastoma is limited due to the presence of the blood brain barrier, which restricts the entry of chemotherapeutic drugs into the brain. Local delivery into the tumor resection margin has the potential to improve efficacy of chemotherapy. We developed a safe and clinically translatable irinotecan implant for local delivery to increase its efficacy while minimizing systemic side effects. Irinotecan-loaded implants were manufactured using hot melt extrusion, gamma sterilized at 25 kGy, and characterized for their irinotecan content, release, and drug diffusion. Their therapeutic efficacy was evaluated in a patient-derived xenograft mouse resection model of glioblastoma. Their safety and translatability were evaluated using histological analysis of brain tissue and serum chemistry analysis. Implants containing 30% and 40% w/w irinotecan were manufactured without plasticizer. The 30% and 40% implants showed moderate local toxicity up to 2- and 6-day post-implantation. Histopathology of the implantation site showed signs of necrosis at days 45 and 14 for the 30% and 40% implants. Hematological analysis and clinical chemistry showed no signs of serious systemic toxicity for either implant. The 30% implants had an 80% survival at day 148, with no sign of tumor recurrence. Gamma sterilization and 12-month storage had no impact on the integrity of the 30% implants. This study demonstrates that the 30% implants are a promising novel treatment for glioblastoma that could be quickly translated into the clinic.

Utilizing induced neural stem cell-based delivery of a cytokine cocktail to enhance chimeric antigen receptor-modified T-cell therapy for brain cancer.

Chimeric antigen receptor (CAR)-modified T-cell therapy has shown enormous clinical promise against blood cancers, yet efficacy against solid tumors remains a challenge. Here, we investigated the potential of a new combination cell therapy, where tumor-homing induced neural stem cells (iNSCs) are used to enhance CAR-T-cell therapy and achieve efficacious suppression of brain tumors. Using in vitro and in vivo migration assays, we found iNSC-secreted RANTES/IL-15 increased CAR-T-cell migration sixfold and expansion threefold, resulting in greater antitumor activity in a glioblastoma (GBM) tumor model. Furthermore, multimodal imaging showed iNSC delivery of RANTES/IL-15 in combination with intravenous administration of CAR-T cells reduced established orthotopic GBM xenografts 2538-fold within the first week, followed by durable tumor remission through 60 days post-treatment. By contrast, CAR-T-cell therapy alone only partially controlled tumor growth, with a median survival of only 19 days. Together, these studies demonstrate the potential of combined cell therapy platforms to improve the efficacy of CAR-T-cell therapy for brain tumors.

Trans-Lesion Synthesis and Mismatch Repair Pathway Crosstalk Defines Chemoresistance and Hypermutation Mechanisms in Glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides a new aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define novel molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

Trans-Lesion Synthesis and Mismatch Repair Pathway Crosstalk Defines Chemoresistance and Hypermutation Mechanisms in Glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides a new aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define novel molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

Longitudinal 3-D Visualization of Microvascular Disruption and Perfusion Changes in Mice During the Evolution of Glioblastoma Using Super-Resolution Ultrasound.

Glioblastoma is an aggressive brain cancer with a very poor prognosis in which less than 6% of patients survive more than five-year post-diagnosis. The outcome of this disease for many patients may be improved by early detection. This could provide clinicians with the information needed to take early action for treatment. In this work, we present the utilization of a non-invasive, fully volumetric ultrasonic imaging method to assess microvascular change during the evolution of glioblastoma in mice. Volumetric ultrasound localization microscopy (ULM) was used to observe statistically significant ( ) reduction in the appearance of functional vasculature over the course of three weeks. We also demonstrate evidence suggesting the reduction of vascular flow for vessels peripheral to the tumor. With an 82.5% consistency rate in acquiring high-quality vascular images, we demonstrate the possibility of volumetric ULM as a longitudinal method for microvascular characterization of neurological disease.

A living ex vivo platform for functional, personalized brain cancer diagnosis.

Functional precision medicine platforms are emerging as promising strategies to improve pre-clinical drug testing and guide clinical decisions. We have developed an organotypic brain slice culture (OBSC)-based platform and multi-parametric algorithm that enable rapid engraftment, treatment, and analysis of uncultured patient brain tumor tissue and patient-derived cell lines. The platform has supported engraftment of every patient tumor tested to this point: high- and low-grade adult and pediatric tumor tissue rapidly establishes on OBSCs among endogenous astrocytes and microglia while maintaining the tumor's original DNA profile. Our algorithm calculates dose-response relationships of both tumor kill and OBSC toxicity, generating summarized drug sensitivity scores on the basis of therapeutic window and allowing us to normalize response profiles across a panel of U.S. Food and Drug Administration (FDA)-approved and exploratory agents. Summarized patient tumor scores after OBSC treatment show positive associations to clinical outcomes, suggesting that the OBSC platform can provide rapid, accurate, functional testing to ultimately guide patient care.

Cell-based therapies for glioblastoma: Promising tools against tumor heterogeneity.

Glioblastoma (GBM) is a highly aggressive tumor with a devastating impact on quality-of-life and abysmal survivorship. Patients have very limited effective treatment options. The successes of targeted small molecule drugs and immune checkpoint inhibitors seen in various solid tumors have not translated to GBM, despite significant advances in our understanding of its molecular, immune, and microenvironment landscapes. These discoveries, however, have unveiled GBM's incredible heterogeneity and its role in treatment failure and survival. Novel cellular therapy technologies are finding successes in oncology and harbor characteristics that make them uniquely suited to overcome challenges posed by GBM, such as increased resistance to tumor heterogeneity, modularity, localized delivery, and safety. Considering these advantages, we compiled this review article on cellular therapies for GBM, focusing on cellular immunotherapies and stem cell-based therapies, to evaluate their utility. We categorize them based on their specificity, review their preclinical and clinical data, and extract valuable insights to help guide future cellular therapy development.

Current approaches in enhancing TRAIL therapies in glioblastoma.

Glioblastoma (GBM) is the most prevalent, aggressive, primary brain cancer in adults and continues to pose major medical challenges due in part to its high rate of recurrence. Extensive research is underway to discover new therapies that target GBM cells and prevent the inevitable recurrence in patients. The pro-apoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted attention as an ideal anticancer agent due to its ability to selectively kill cancer cells with minimal toxicity in normal cells. Although initial clinical evaluations of TRAIL therapies in several cancers were promising, later stages of clinical trial results indicated that TRAIL and TRAIL-based therapies failed to demonstrate robust efficacies due to poor pharmacokinetics, resulting in insufficient concentrations of TRAIL at the therapeutic site. However, recent studies have developed novel ways to prolong TRAIL bioavailability at the tumor site and efficiently deliver TRAIL and TRAIL-based therapies using cellular and nanoparticle vehicles as drug loading cargos. Additionally, novel techniques have been developed to address monotherapy resistance, including modulating biomarkers associated with TRAIL resistance in GBM cells. This review highlights the promising work to overcome the challenges of TRAIL-based therapies with the aim to facilitate improved TRAIL efficacy against GBM.

Magnetic Control of Protein Expression via Magneto-mechanical Actuation of ND-PEGylated Iron Oxide Nanocubes for Cell Therapy.

Engineered cells used as smart vehicles for delivery of secreted therapeutic proteins enable effective treatment of cancer and certain degenerative, autoimmune, and genetic disorders. However, current cell-based therapies use mostly invasive tools for tracking proteins and do not allow for controlled secretion of therapeutic proteins, which could result in unconstrained killing of surrounding healthy tissues or ineffective killing of host cancer cells. Regulating the expression of therapeutic proteins after success of therapy remains elusive. In this study, a noninvasive therapeutic approach mediated by magneto-mechanical actuation (MMA) was developed to remotely regulate the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein, which is secreted by transduced cells. Stem cells, macrophages, and breast cancer cells were transduced with a lentiviral vector encoding the SGpL2TR protein. SGpL2TR comprises TRAIL and GpLuc domains optimized for cell-based applications. Our approach relies on the remote actuation of cubic-shape highly magnetic field responsive superparamagnetic iron oxide nanoparticles (SPIONs) coated with nitrodopamine PEG (ND-PEG), which are internalized within the cells. Cubic ND-PEG-SPIONs actuated by superlow frequency alternating current magnetic fields can translate magnetic forces into mechanical motion and in turn spur mechanosensitive cellular responses. Cubic ND-PEG-SPIONs were artificially designed to effectively operate at low magnetic field strengths (<100 mT) retaining approximately 60% of their saturation magnetization. Compared to other cells, stems cells were more sensitive to the interaction with actuated cubic ND-PEG-SPIONs, which clustered near the endoplasmic reticulum (ER). Luciferase, ELISA, and RT-qPCR analyses revealed a marked TRAIL downregulation (secretion levels were depleted down to 30%) when intracellular particles at 0.100 mg/mL Fe were actuated by magnetic fields (65 mT and 50 Hz for 30 min). Western blot studies indicated actuated, intracellular cubic ND-PEG-SPIONs can cause mild ER stress at short periods (up to 3 h) of postmagnetic field treatment thus leading to the unfolded protein response. We observed that the interaction of TRAIL polypeptides with ND-PEG can also contribute to this response. To prove the applicability of our approach, we used glioblastoma cells, which were exposed to TRAIL secreted from stem cells. We demonstrated that in the absence of MMA treatment, TRAIL essentially killed glioblastoma cells indiscriminately, but when treated with MMA, we were able to control the cell killing rate by adjusting the magnetic doses. This approach can expand the capabilities of stem cells to serve as smart vehicles for delivery of therapeutic proteins in a controlled manner without using interfering and expensive drugs, while retaining their potential to regenerate damaged tissue after treatment. This approach brings forth new alternatives to regulate protein expression noninvasively for cell therapy and other cancer therapies.

Injectable pH Thermo-Responsive Hydrogel Scaffold for Tumoricidal Neural Stem Cell Therapy for Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults and despite recent advances in treatment modalities, GBM remains incurable. Injectable hydrogel scaffolds are a versatile delivery system that can improve delivery of drug and cell therapeutics for GBM. In this report, we investigated an injectable nanocellulose/chitosan-based hydrogel scaffold for neural stem cell encapsulation and delivery. Hydrogels were prepared using thermogelling beta-glycerophosphate (BGP) and hydroxyethyl cellulose (HEC), chitosan (CS), and cellulose nanocrystals (CNCs). We evaluated the impact of neural stem cells on hydrogel gelation kinetics, microstructures, and degradation. Furthermore, we investigated the biomaterial effects on cell viability and functionality. We demonstrated that the incorporation of cells at densities of 1, 5 and 10 million does not significantly impact rheological and physical properties CS scaffolds. However, addition of CNCs significantly prolonged hydrogel degradation when cells were seeded at 5 and 10 million per 1 mL hydrogel. In vitro cell studies demonstrated high cell viability, release of TRAIL at therapeutic concentrations, and effective tumor cell killing within 72 h. The ability of these hydrogel scaffolds to support stem cell encapsulation and viability and maintain stem cell functionality makes them an attractive cell delivery system for local treatment of post-surgical cancers.

Spatiotemporal analysis of induced neural stem cell therapy to overcome advanced glioblastoma recurrence.

Genetically engineered neural stem cells (NSCs) are a promising therapy for the highly aggressive brain cancer glioblastoma (GBM); however, treatment durability remains a major challenge. We sought to define the events that contribute to dynamic adaptation of GBM during treatment with human skin-derived induced NSCs releasing the pro-apoptotic agent TRAIL (iNSC-TRAIL) and develop strategies that convert initial tumor kill into sustained GBM suppression. In vivo and ex vivo analysis before, during, and after treatment revealed significant shifts in tumor transcriptome and spatial distribution as the tumors adapted to treatment. To address this, we designed iNSC delivery strategies that increased spatiotemporal TRAIL coverage and significantly decreased GBM volume throughout the brain, reducing tumor burden 100-fold as quantified in live ex vivo brain slices. The varying impact of different strategies on treatment durability and median survival of both solid and invasive tumors provides important guidance for optimizing iNSC therapy.

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.

Next-generation Tumor-homing Induced Neural Stem Cells as an Adjuvant to Radiation for the Treatment of Metastatic Lung Cancer.

The spread of non-small cell lung cancer (NSCLC) to the leptomeninges is devastating with a median survival of only a few months. Radiation offers symptomatic relief, but new adjuvant therapies are desperately needed. Spheroidal, human induced neural stem cells (hiNeuroS) secreting the cytotoxic protein, TRAIL, have innate tumoritropic properties. Herein, we provide evidence that hiNeuroS-TRAIL cells can migrate to and suppress growth of NSCLC metastases in combination with radiation. In vitro cell tracking and post-mortem tissue analysis showed that hiNeuroS-TRAIL cells migrate to NSCLC tumors. Importantly, isobolographic analysis suggests that TRAIL with radiation has a synergistic cytotoxic effect on NSCLC tumors. In vivo, mice treated with radiation and hiNeuroS-TRAIL showed significant (36.6%) improvements in median survival compared to controls. Finally, bulk mRNA sequencing analysis showed both NSCLC and hiNeuroS-TRAIL cells showed changes in genes involved in migration following radiation. Overall, hiNeuroS-TRAIL cells +/- radiation have the capacity to treat NSCLC metastases.

Fibrin gel enhances the antitumor effects of chimeric antigen receptor T cells in glioblastoma.

Regional delivery of chimeric antigen receptor (CAR) T cells in glioblastoma represents a rational therapeutic approach as an alternative to intravenous administration to avoid the blood-brain barrier impediment. Here, we developed a fibrin gel that accommodates CAR-T cell loading and promotes their gradual release. Using a model of subtotal glioblastoma resection, we demonstrated that the fibrin-based gel delivery of CAR-T cells within the surgical cavity enables superior antitumor activity compared to CAR-T cells directly inoculated into the tumor resection cavity.

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.