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 and compared 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 MDCKMDR1 (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.
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), engineered them to express optical reporters and different therapeutic gene products, and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSC 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 that were nestin and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSC rapidly migrated to human GBM cells and penetrated human GBM spheroids, a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSC delivery of the proapoptotic agent tumor necrosis factor-α-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 thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSC-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-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 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.
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 (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.
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.
particularly in pluripotent stem cells, limiting their utility for tracking and eventual clinical applications. Targeted integration into genomic "safe harbors" offer a promising alternative approach to mark target cells, potentially circumventing these issues. The adeno-associated virus integration site 1 (AAVS1) has been proposed as a suitable safe harbor for human cells, and we now investigate its utility in our rhesus macaque NHP iPSC model. We have efficiently knocked-in both a truncated CD19 (hΔCD19) marker gene a non-immunogenic and clinical relevant marker, or green fluorescent protein (GFP) at the homologous AAVS1 site in rhesus iPSCs (RhiPSCs) using the clustered regularly interspaced short palindromic repeats/CRISPRassociated nuclease 9 (CRISPR-Cas9) system. PCR and Southern blot analyses demonstrated highly efficient knock-in into the AAVS1 locus, with over one third of clones screened containing only targeted but not random integrations. (Table 1). Edited RhiPSC-GFP/hΔCD19 clones retained a normal karyotype and pluripotency -as shown by teratoma formation. Directed differentiation of these clones to neutrophils, hepatocytes or cardiomyocytes was not hindered by the knock-in of marker genes into the AAVS1 sites. Notably, transgene expression was stable in undifferentiated RhiPSCs and differentiated cell types derived from the RhiPSC (Figure 1), in contrast to prior experience with viral vector delivery. We have established a computational platform to assess off-target effects of guide RNAs in the rhesus genome. Genetically marked RhiPSCs afford a unique opportunity to develop clinically relevant models for iPSC-based cell therapies.
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