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Background Surgical resection is a universal component of glioma therapy. Little is known about the postoperative microenvironment due to limited preclinical models. Thus, we sought to develop a glioma resection and recurrence model in syngeneic immune-competent mice to understand how surgical resection influences tumor biology and the local microenvironment. Methods We genetically engineered cells from a murine glioma mouse model to express fluorescent and bioluminescent reporters. Established allografts were resected using image-guided microsurgery. Postoperative tumor recurrence was monitored by serial imaging, and the peritumoral microenvironment was characterized by histopathology and immunohistochemistry. Coculture techniques were used to explore how astrocyte injury influences tumor aggressiveness in vitro. Transcriptome and secretome alterations in injured astrocytes was examined by RNA-seq and Luminex. Results We found that image-guided resection achieved >90% reduction in tumor volume but failed to prevent both local and distant tumor recurrence. Immunostaining for glial fibrillary acidic protein and nestin showed that resection-induced injury led to temporal and spatial alterations in reactive astrocytes within the peritumoral microenvironment. In vitro, we found that astrocyte injury induced transcriptome and secretome alterations and promoted tumor proliferation, as well as migration. Conclusions This study demonstrates a unique syngeneic model of glioma resection and recurrence in immune-competent mice. Furthermore, this model provided insights into the pattern of postsurgical tumor recurrence and changes in the peritumoral microenvironment, as well as the impact of injured astrocytes on glioma growth and invasion. A better understanding of the postsurgical tumor microenvironment will allow development of targeted anticancer agents that improve surgery-mediated effects on tumor biology.

Exosomes have recently come into focus as "natural nanoparticles" for use as drug delivery vehicles. Our objective was to assess the feasibility of an exosome-based drug delivery platform for a potent chemotherapeutic agent, paclitaxel (PTX), to treat MDR cancer. Herein, we developed different methods of loading exosomes released by macrophages with PTX (exoPTX), and characterized their size, stability, drug release, and in vitro antitumor efficacy. Reformation of the exosomal membrane upon sonication resulted in high loading efficiency and sustained drug release. Importantly, incorporation of PTX into exosomes increased cytotoxicity more than 50 times in drug resistant MDCK MDR1 (Pgp+) cells. Next, our studies demonstrated a nearly complete co-localization of airway-delivered exosomes with cancer cells in a model of murine Lewis lung carcinoma pulmonary metastases, and a potent anticancer effect in this mouse model. We conclude that exoPTX holds significant potential for the delivery of various chemotherapeutics to treat drug resistant cancers. From the Clinical Editor Exosomes are membrane-derived natural vesicles of ~40 - 200 nm size. They have been under extensive research as novel drug delivery vehicles. In this article, the authors developed exosome-based system to carry formulation of PTX and showed efficacy in the treatment of multi-drug resistant cancer cells. This novel system may be further developed to carry other chemotherapeutic agents in the future.

Background Cytotoxic neural stem cells (NSCs) have emerged as a promising treatment for Medulloblastoma (MB), the most common malignant primary pediatric brain tumor. The lack of accurate pre-clinical models incorporating surgical resection and tumor recurrence limits advancement in post-surgical MB treatments. Using cell lines from two of the 5 distinct MB molecular sub-groups, in this study, we developed an image-guided mouse model of MB surgical resection and investigate intra-cavity NSC therapy for post-operative MB. Methods Using D283 and Daoy human MB cells engineered to express multi-modality optical reporters, we created the first image-guided resection model of orthotopic MB. Brain-derived NSCs and novel induced NSCs (iNSCs) generated from pediatric skin were engineered to express the pro-drug/enzyme therapy thymidine kinase/ganciclovir, seeded into the post-operative cavity, and used to investigate intra-cavity therapy for post-surgical MB. Results We found that surgery reduced MB volumes by 92%, and the rate of post-operative MB regrowth increased 3-fold compared to pre-resection growth. Real-time imaging showed NSCs rapidly homed to MB, migrating 1.6-fold faster and 2-fold farther in the presence of tumors, and co-localized with MB present in the contra-lateral hemisphere. Seeding of cytotoxic NSCs into the post-operative surgical cavity decreased MB volumes 15-fold and extended median survival 133%. As an initial step towards novel autologous therapy in human MB patients, we found skin-derived iNSCs homed to MB cells, while intra-cavity iNSC therapy suppressed post-surgical tumor growth and prolonged survival of MB-bearing mice by 123%. Conclusions We report a novel image-guided model of MB resection/recurrence and provide new evidence of cytotoxic NSCs/iNSCs delivered into the surgical cavity effectively target residual MB foci.

Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSC(TE)), engineered them to express optical reporters and different therapeutic gene products, and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSC(TE) in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSC(TE) that were nestin(+) and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSC(TE) rapidly migrated to human GBM cells and penetrated human GBM spheroids, a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSC(TE) delivery of the proapoptotic agent tumor necrosis factor–a–related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally, h-iNSC(TE) thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSC(TE)–TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy, h-iNSC(TE)–TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSC(TE) is a platform for creating tumor-homing cytotoxic cell therapies for cancer, where the potential to avoid carrier rejection could maximize treatment durability in human trials.

Transdifferentiation (TD) is a recent advancement in somatic cell reprogramming. The direct conversion of TD eliminates the pluripotent intermediate state to create cells that are ideal for personalized cell therapy. Here we provide evidence that TD-derived induced neural stem cells (iNSCs) are an efficacious therapeutic strategy for brain cancer. We find that iNSCs genetically engineered with optical reporters and tumouricidal gene products retain the capacity to differentiate and induced apoptosis in co-cultured human glioblastoma cells. Time-lapse imaging shows that iNSCs are tumouritropic, homing rapidly to co-cultured glioblastoma cells and migrating extensively to distant tumour foci in the murine brain. Multimodality imaging reveals that iNSC delivery of the anticancer molecule TRAIL decreases the growth of established solid and diffuse patient-derived orthotopic glioblastoma xenografts 230- and 20-fold, respectively, while significantly prolonging the median mouse survival. These findings establish a strategy for creating autologous cell-based therapies to treat patients with aggressive forms of brain cancer., Neural stem cells have a tropism for glioblastoma. Here the authors employ fibroblasts directly reprogrammed into induced neural stem cells and loaded with cytotoxic molecules to migrate to xenotransplanted brain tumours in mice, achieving tumour shrinkage and prolonged survival.

Engineered stem cell (SC)-based therapy holds enormous promise for treating the incurable brain cancer glioblastoma (GBM). Retaining the cytotoxic SCs in the surgical cavity after GBM resection is one of the greatest challenges to this approach. Here, we describe a biocompatible electrospun nanofibrous scaffold (bENS) implant capable of delivering and retaining tumor-homing cytotoxic stem cells that suppress recurrence of post-surgical GBM. As a new approach to GBM therapy, we created poly(l-lactic acid) (PLA) bENS bearing drug-releasing human mesenchymal stem cells (hMSCs). We discovered that bENS-based implant increased hMSC retention in the surgical cavity 5-fold and prolonged persistence 3-fold compared to standard direct injection using our mouse model of GBM surgical resection/recurrence. Time-lapse imaging showed cytotoxic hMSC/bENS treatment killed co-cultured human GBM cells, and allowed hMSCs to rapidly migrate off the scaffolds as they homed to GBMs. In vivo, bENS loaded with hMSCs releasing the anti-tumor protein TRAIL (bENS(sTR)) reduced the volume of established GBM xenografts 3-fold. Mimicking clinical GBM patient therapy, lining the post-operative GBM surgical cavity with bENS(sTR) implants inhibited the re-growth of residual GBM foci 2.3-fold and prolonged post-surgical median survival from 13.5 to 31 days in mice. These results suggest that nanofibrous-based SC therapies could be an innovative new approach to improve the outcomes of patients suffering from terminal brain cancer.

Engineered stem cell (SC)-based therapy holds enormous promise for treating the incurable brain cancer glioblastoma (GBM). Retention of the cytotoxic SCs within the GBM resection is one of the greatest challenges to this approach. Here, we describe a biocompatible polymeric bio-scaffold (bPBS) transplant strategy that is capable of delivering and retaining tumor-homing cytotoxic SCs to prevent recurrence of surgically resected GBM. As a new approach to GBM therapy, we created bPBS bearing drug-releasing human mesenchymal stem cells (hMSCs). Using unique models of GBM resection/recurrence in mice, we discovered that bPBS-based transplant stabilizes cytotoxic hMSCs in the surgical cavity, increasing SC retention 5-fold and prolonging persistence 3-fold compared to standard direct injection. Time-lapse kinetic imaging and analysis showed the pPBS still allowed hMSCs to rapidly migrate off the matrix as they homed to GBMs but not normal cells of the brain. 3-dimension co-culture assays showed cytotoxic hMSC/bPBS treatment reduced the viability of multiple human GBM cells 50-90%. In vivo, bPBS loaded with cytotoxic hMSCs releasing the anti-tumor protein TRAIL (bPBSsTR) transplanted over established human GBM xenografts reduced tumor volumes 3-fold. Mimicking clinical GBM patient therapy, lining the post-operative GBM surgical cavity with bPBSsTR implants reduced post-surgical GBM volumes 2.3-fold and prolonged post-surgical median survival from 13.5 to 31 days compared to control-treated mice. These results suggest that polymeric bio-scaffold SC therapies could be an innovative new approach to improve the outcomes of patients suffering from terminal brain cancer.

Tumor-homing cytotoxic stem cell (SC) therapy is a promising new approach for treating the incurable brain cancer glioblastoma (GBM). However, problems of retaining cytotoxic SCs within the post-surgical GBM resection cavity are likely to significantly limit the clinical utility of this strategy. Here, we describe a new fibrin-based transplant approach capable of increasing cytotoxic SC retention and persistence within the resection cavity, yet remaining permissive to tumoritropic migration. This fibrin-based transplant can effectively treat both solid and post-surgical human GBM in mice. Using our murine model of image-guided model of GBM resection, we discovered that suspending human mesenchymal stem cells (hMSCS) in a fibrin matrix increased initial retention in the surgical resection cavity 2-fold and prolonged persistence in the cavity 3-fold compared to conventional delivery strategies. Time-lapse motion analysis revealed that cytotoxic hMSCs in the fibrin matrix remain tumoritropic, rapidly migrating from the fibrin matrix to co-localize with cultured human GBM cells. We encapsulated hMSCs releasing the cytotoxic agent TRAIL (hMSC-sTR) in fibrin, and found hMSC-sTR/fibrin therapy reduced the viability of multiple 3-D human GBM spheroids and regressed established human GBM xenografts 3-fold in 11 days. Mimicking clinical therapy of surgically resected GBM, intra-cavity seeding of therapeutic hMSC-sTR encapsulated in fibrin reduced post-surgical GBM volumes 6-fold, increased time to recurrence 4-fold, and prolonged median survival from 15 to 36 days compared to control-treated animals. Fibrin-based SC therapy could represent a clinically compatible, viable treatment to suppress recurrence of post-surgical GBM and other lethal cancer types.

Background: Engineered neural stem cells (NSC) are a promising new approach to treating glioblastoma (GBM). In clinical trials, the ideal NSC drug carrier should be easily isolated and autologous to avoid immune rejection. Methods: As a new approach to personalized NSC therapy for cancer, we directly transdifferentiated (TD) human fibroblasts in induced neural stem cells (h-iNSCs). The h-iNSCs were engineered to express optical reporters and either the pro-apoptotic agent TRAIL or thymidine kinase. The tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSCs were then assessed in human-derived GBM models of solid and surgically resected disease. All statistical tests were two-sided. Results: Our new single-factor Sox2 strategy converted human skin fibroblasts into nestin+ h-iNSCs in only 4 days and the h-iNSCs survived in the brain of mice for 3 weeks. Time-lapse motion analysis showed h-iNSCs rapidly migrated to human GBMs cells and penetrated solid human GBM spheroids. h-iNSC delivery of TRAIL reduced solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days (P

Engineered stem cell (SC)-based therapy holds enormous promise for treating the incurable brain cancer glioblastoma (GBM). Retaining the cytotoxic SCs in the surgical cavity after GBM resection is one of the greatest challenges to this approach. In this study, we describe a biocompatible electrospun nanofibrous scaffold (bENS) implant capable of delivering and retaining tumor-homing cytotoxic stem cells that suppress recurrence of post-surgical GBM. As a new approach to GBM therapy, we created poly(L-lactic acid) (PLA) bENS bearing drug-releasing human mesenchymal stem cells (hMSCs). We discovered that bENS-based implant increased hMSC retention in the surgical cavity 5-fold and prolonged persistence 3-fold compared to standard direct injection using our mouse model of GBM surgical resection/recurrence. Time-lapse imaging showed cytotoxic hMSC/bENS treatment killed co-cultured human GBM cells, and allowed hMSCs to rapidly migrate off the scaffolds as they homed to GBMs. In vivo, bENS loaded with hMSCs releasing the anti-tumor protein TRAIL (bENSsTR) reduced the volume of established GBM xenografts 3-fold. Mimicking clinical GBM patient therapy, lining the post-operative GBM surgical cavity with bENSsTR implants inhibited the re-growth of residual GBM foci 2.3-fold and prolonged post-surgical median survival from 13.5 to 31 days in mice. These results suggest that nanofibrous-based SC therapies could be an innovative new approach to improve the outcomes of patients suffering from terminal brain cancer.View Large Image | Download PowerPoint Slide

BACKGROUND: Engineered neural stem cells (NSC) are a promising new approach to treating glioblastoma (GBM). As NSC therapy for GBM enters clinical trials, the ideal NSC drug carrier should be easily isolated and autologous to avoid immune rejection. METHODS: As a new approach to personalized NSC therapy for cancer, we switched the fate of human fibroblasts in induced neural stem cells (h-iNSCs) using a new stem cell technology termed transdifferentiation (TD). The h-iNSCs were engineered to express optical reporters and either the pro-apoptotic agent TRAIL or prodrug/enzyme therapy thymidine kinase. The tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSCs were then assessed in human GBM cell models of solid and surgically resected disease in mice. RESULTS: To develop the first TD method capable of generating h-iNSCs fast enough for clinical GBM therapy, we discovered a new TD strategy that converted human skin fibroblasts into nestin+ h-iNSCs in only 4 days. Non-invasive optical imaging and immunostaining showed h-iNSCs survived in the brain of mice for 3 weeks and remained nestin+. Time-lapse motion analysis of 3-dimensional co-cultures showed h-iNSCs rapidly migrated to human GBMs cells and penetrated solid human GBM spheroids. Investigating h-iNSC therapy, h-iNSC secretion of TRAIL reduced solid GBM intracranial xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. h-iNSC prodrug/enzyme therapy regressed human patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical h-iNSC therapy, intra-cavity h-iNSC thymidine kinase/ganciclovir therapy delayed the regrowth of post-surgical GBMs 3-fold and prolonged survival in mice from 46 to 60 days. CONCLUSIONS: Transdifferentiating human skin into h-iNSCs is a new platform for creating tumor-homing cytotoxic cell therapies for cancer. Translating this approach has the potential to avoid carrier rejection and maximize treatment durability in patient trials.

Transdifferentiation (TD) is an exciting new advancement in somatic cell reprogramming that eliminates the pluripotent intermediate stage to create cells that are ideal for personalized cell transplant therapy. Induced neural stem cells (iNSCs) are the newest cell type created by TD. Here we begin to define the efficacy of iNSC therapy for central nervous system disorders. We developed the first iNSC-based drug deliver vehicles, and show that engineered iNSCs are tumor-homing drug delivery vehicles with significant anti-cancer effects in models of glioblastoma (GBM). After genetically engineering iNSCs with tumoricidal or diagnostic transgenes, we observed that the modified iNSCs proliferated and differentiated into astrocytes and neurons with the same efficiency as unmodified cells. Non-invasive serial imaging revealed that engineered iNSCs implanted into the parenchyma of mice survived more than 1 month. In vitro time-lapse motion analysis showed iNSCs exhibit tumor-homing properties similar to brain-derived NSCs, while iNSCs implanted into the frontal lobe of mice migrated through the brain homing to invasive GBM cells. iNSCs engineered with the anti-cancer molecule TRAIL (iNSC-sTR) stably released the tumoricidal agent and killed co-cultured human GBM cells. We evaluated iNSC-sTR therapy in orthotopic mouse models of solid and patient-derived diffuse GBM. We found that iNSC-sTR therapy reduced GBM volumes 20- to 230-fold and doubled the survival of tumor-bearing mice compared to control mice. These data provide the first evidence that tumoricidal iNSCs can be used to efficaciously treat brain cancer, and provide a foundation for the continued development of iNSC-based therapies.