Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus.

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.

Allogeneic stem cells engineered to release interferon ß and scFv-PD1 target glioblastoma and alter the tumor microenvironment.

Highly malignant brain tumors, glioblastomas (GBM), are immunosuppressive, thereby limiting current promising immunotherapeutic approaches. In this study, we created interferon receptor 1 knockout allogeneic mesenchymal stem cells (MSC) to secrete dual-function pro-apoptotic and immunomodulatory interferon (IFN) ß (MSCKO-IFNß) using a single lentiviral vector CRISPR/Cas9 system. We show that MSCKO-IFNß induces apoptosis in GBM cells and upregulates the cell surface expression of programmed death ligand-1 in tumor cells. Next, we engineered MSCKO to release a secretable single-chain variable fragment (scFv) to block programmed death (PD)-1 and show the ability of MSCKO-scFv-PD1 to enhance T-cell activation and T-cell-mediated tumor cell killing. To simultaneously express both immune modulators, we engineered MSCKO-IFNß to co-express scFv-PD1 (MSCKO-IFNß-scFv-PD1) and show the expression of both IFNß and scFv-PD1 in vitro leads to T-cell activation and lowers the viability of tumor cells. Furthermore, to mimic the clinical scenario of GBM tumor resection and subsequent treatment, we show that synthetic extracellular matrix (sECM) encapsulated MSCKO-IFNß-scFv-PD1 treatment of resected tumors results in the increase of CD4+ and CD8+ T cells, mature conventional dendritic cells type II and activation of microglia as compared to the control treatment group. Overall, these results reveal the ability of MSCKO-IFNß-scFv-PD1 to shape the tumor microenvironment and enhance therapeutic outcomes in GBM.

Using System Dynamics Approach to Explore the Mode Shift between Automated Vehicles, Conventional Vehicles, and Public Transport in Melbourne, Australia.

With the increasing use of automated vehicles (AVs) in the coming decades, government authorities and private companies must leverage their potential disruption to benefit society. Few studies have considered the impact of AVs towards mode shift by considering a range of factors at the city level, especially in Australia. To address this knowledge gap, we developed a system dynamic (SD)-based model to explore the mode shift between conventional vehicles (CVs), AVs, and public transport (PT) by systematically considering a range of factors, such as road network, vehicle cost, public transport supply, and congestion level. By using Melbourne's Transport Network as a case study, the model simulates the mode shift among AVs, CVs, and PT modes in the transportation system over 50 years, starting from 2018, with the adoption of AVs beginning in 2025. Inputs such as current traffic, road capacity, public perception, and technological advancement of AVs are used to assess the effects of different policy options on the transport systems. The data source used is from the Victorian Integrated Transport Model (VITM), provided by the Department of Transport and Planning, Melbourne, Australia, data from the existing literature, and authors' assumptions. To our best knowledge, this is the first time using an SD model to investigate the impacts of AVs on mode shift in the Australian context. The findings suggest that AVs will gradually replace CVs as another primary mode of transportation. However, PT will still play a significant role in the transportation system, accounting for 50% of total trips by person after 2058. Cost is the most critical factor affecting AV adoption rates, followed by road network capacity and awareness programs. This study also identifies the need for future research to investigate the induced demand for travel due to the adoption of AVs and the application of equilibrium constraints to the traffic assignment model to increase model accuracy. These findings can be helpful for policymakers and stakeholders to make informed decisions regarding AV adoption policies and strategies.

BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the pleura, with few available treatment options. We recently reported that loss of function of the nuclear deubiquitinase BRCA1-associated protein 1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptor 4 (DR4) and death receptor 5 (DR5). Using tissue microarrays of tumor samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptor expression. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wt-BAP1, but not catalytically inactive BAP1 mutant, reduced promoter activities of DR4 and DR5, suggesting deubiquitinase activity is required for the regulation of gene expression. Co-immunoprecipitation studies demonstrated direct binding of BAP1 to the transcription factor Ying Yang 1 (YY1), and chromatin immunoprecipitation assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Knockdown of YY1 also increased DR4 and DR5 expression and sensitivity to TRAIL. These results suggest that BAP1 and YY1 cooperatively repress transcription of TRAIL receptors. Our finding that BAP1 directly regulates the extrinsic apoptotic pathway will provide new insights into the role of BAP1 in the development of MPM and other cancers with frequent BAP1 mutations.

Mesenchymal stem cell immunomodulation: In pursuit of controlling COVID-19 related cytokine storm.

The coronavirus disease 2019 (COVID-19) pandemic has grown to be a global public health crisis with no safe and effective treatments available yet. Recent findings suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus pathogen that causes COVID-19, could elicit a cytokine storm that drives edema, dysfunction of the airway exchange, and acute respiratory distress syndrome in the lung, followed by acute cardiac injury and thromboembolic events leading to multiorgan failure and death. Mesenchymal stem cells (MSCs), owing to their powerful immunomodulatory abilities, have the potential to attenuate the cytokine storm and have therefore been proposed as a potential therapeutic approach for which several clinical trials are underway. Given that intravenous infusion of MSCs results in a significant trapping in the lung, MSC therapy could directly mitigate inflammation, protect alveolar epithelial cells, and reverse lung dysfunction by normalizing the pulmonary microenvironment and preventing pulmonary fibrosis. In this review, we present an overview and perspectives of the SARS-CoV-2 induced inflammatory dysfunction and the potential of MSC immunomodulation for the prevention and treatment of COVID-19 related pulmonary disease.

Engineered stem cells targeting multiple cell surface receptors in tumors.

Multiple stem cell types exhibit inherent tropism for cancer, and engineered stem cells have been used as therapeutic agents to specifically target cancer cells. Recently, stem cells have been engineered to target multiple surface receptors on tumor cells, as well as endothelial and immune cells in the tumor microenvironment. In this review, we discuss the rationales and strategies for developing multiple receptor-targeted stem cells, their mechanisms of action, and the promises and challenges they hold as cancer therapeutics.

Stem cell-released oncolytic herpes simplex virus has therapeutic efficacy in brain metastatic melanomas.

The recent Food and Drug Administration approval of immunogenic oncolytic virus (OV) has opened a new era in the treatment of advanced melanoma; however, approximately 50% of patients with melanoma develop brain metastasis, and currently there are no beneficial treatment options for such patients. To model the progression of metastases seen in patients and to overcome the hurdles of systemic delivery of OV, we developed melanoma brain metastasis models in immunocompromised and immunocompetent mice, and tested the fate and efficacy of oncolytic herpes simplex virus (oHSV)-armed mesenchymal stem cells (MSCs). Using brain-seeking patient-derived melanoma cells and real-time in vivo imaging, we show a widespread distribution of micrometastases and macrometastases in the brain, recapitulating the progression of multifoci metastases seen in patients. We armed MSCs with different oHSV variants (MSC-oHSV) and found that intracarotid administration of MSC-oHSV, but not of purified oHSV alone, effectively tracks metastatic tumor lesions and significantly prolongs the survival of brain tumor-bearing mice. In a syngeneic model of melanoma brain metastasis, a combination of MSC-oHSV and PD-L1 blockade increases IFNγ-producing CD8+ tumor-infiltrating T lymphocytes and results in a profound extension of the median survival of treated animals. This study thus demonstrates the utility of MSCs as OV carriers to disseminated brain lesions, and provides a clinically applicable therapeutic platform to target melanoma brain metastasis.

Stem Cells Engineered During Different Stages of Reprogramming Reveal Varying Therapeutic Efficacies.

Stem cells are emerging as promising treatment strategies for several brain disorders and pathologies. In this study, we explored the potential of creating induced pluripotent stem cell-derived neural stem cells (ipNSC) by using either unmodified or gene-modified somatic cells and tested their fate and therapeutic efficacies in vitro and in vivo. We show that cells engineered in somatic state lose transgene-expression during the neural induction process, which is partially restored by histone deacetylase inhibitor treatment whereas cells engineered at the ipNSC state have sustained expression of transgenes. In vivo, bimodal mouse and human ipNSCs engineered to express tumor specific death-receptor ligand and suicide-inducing therapeutic proteins have profound anti-tumor efficacy when encapsulated in synthetic extracellular matrix and transplanted in mouse models of resected-glioblastoma. This study provides insights into using somatic cells for treating CNS disorders and presents a receptor-targeted cancer therapeutic approach for brain tumors. Stem Cells 2018;36:932-942.

Therapeutic targeting of chemoresistant and recurrent glioblastoma stem cells with a proapoptotic variant of oncolytic herpes simplex virus.

Temozolomide (TMZ) chemotherapy, in combination with maximal safe resection and radiotherapy, is the current standard of care for patients with glioblastoma (GBM). Despite this multimodal approach, GBM inevitably relapses primarily due to resistance to chemo-radiotherapy, and effective treatment is not available for recurrent disease. In this study we identified TMZ resistant patient-derived primary and previously treated recurrent GBM stem cells (GSC), and investigated the therapeutic activity of a pro-apoptotic variant of oHSV (oHSV-TRAIL) in vitro and in vivo. We show that oHSV-TRAIL modulates cell survival and MAP Kinase proliferation signaling pathways as well as DNA damage response pathways in both primary and recurrent TMZ-resistant GSC. Utilizing real time in vivo imaging and correlative immunohistochemistry, we show that oHSV-TRAIL potently inhibits tumor growth and extends survival of mice bearing TMZ-insensitive recurrent intracerebral GSC tumors via robust and selective induction of apoptosis-mediated death in tumor cells, resulting in cures in 40% of the treated mice. In comparison, the anti-tumor effects in a primary chemoresistant GSC GBM model exhibiting a highly invasive phenotype were significant but less prominent. This work thus demonstrates the ability of oHSV-TRAIL to overcome the therapeutic resistance and recurrence of GBM, and provides a basis for its testing in a GBM clinical trial.

Measuring impact of telephone triage in Acute Medicine.

The Society for Acute Medicine's Benchmarking Audit (SAMBA) was undertaken for the 5th time in June 2016. For the first time, data on telephone triage calls prior to admission to Acute Medical Units were collected: 1238 patients were referred from Emergency Departments, 925 from General Practitioners (GPs), 52 from clinics and 147 from other sources. Calls from Emergency Departments rarely resulted in admission avoidance. Calls from Primary Care resulted in avoidance of an admission in 115 (12%) patients; the percentage of avoided admissions was highest if the call was taken by a Consultant. Consultant triage might result in admission avoidance but the impact of local context on the effectiveness is not clear.

Combination of systemic chemotherapy with local stem cell delivered S-TRAIL in resected brain tumors.

Despite advances in standard therapies, the survival of glioblastoma multiforme (GBM) patients has not improved. Limitations to successful translation of new therapies include poor delivery of systemic therapies and use of simplified preclinical models which fail to reflect the clinical complexity of GBMs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in tumor cells and we have tested its efficacy by on-site delivery via engineered stem cells (SC) in mouse models of GBM that mimic the clinical scenario of tumor aggressiveness and resection. However, about half of tumor lines are resistant to TRAIL and overcoming TRAIL-resistance in GBM by combining therapeutic agents that are currently in clinical trials with SC-TRAIL and understanding the molecular dynamics of these combination therapies are critical to the broad use of TRAIL as a therapeutic agent in clinics. In this study, we screened clinically relevant chemotherapeutic agents for their ability to sensitize resistant GBM cell lines to TRAIL induced apoptosis. We show that low dose cisplatin increases surface receptor expression of death receptor 4/5 post G2 cycle arrest and sensitizes GBM cells to TRAIL induced apoptosis. In vivo, using an intracranial resection model of resistant primary human-derived GBM and real-time optical imaging, we show that a low dose of cisplatin in combination with synthetic extracellular matrix encapsulated SC-TRAIL significantly decreases tumor regrowth and increases survival in mice bearing GBM. This study has the potential to help expedite effective translation of local stem cell-based delivery of TRAIL into the clinical setting to target a broad spectrum of GBMs.

Engineering toxin-resistant therapeutic stem cells to treat brain tumors.

Pseudomonas exotoxin (PE) potently blocks protein synthesis by catalyzing the inactivation of elongation factor-2 (EF-2). Targeted PE-cytotoxins have been used as antitumor agents, although their effective clinical translation in solid tumors has been confounded by off-target delivery, systemic toxicity, and short chemotherapeutic half-life. To overcome these limitations, we have created toxin-resistant stem cells by modifying endogenous EF-2, and engineered them to secrete PE-cytotoxins that target specifically expressed (interleukin-13 receptor subunit alpha-2) or overexpressed (epidermal growth factor receptor) in glioblastomas (GBM). Molecular analysis correlated efficacy of PE-targeted cytotoxins with levels of cognate receptor expression, and optical imaging was applied to simultaneously track the kinetics of protein synthesis inhibition and GBM cell viability in vivo. The release of IL13-PE from biodegradable synthetic extracellular matrix (sECM) encapsulated stem cells in a clinically relevant GBM resection model led to increased long-term survival of mice compared to IL13-PE protein infusion. Moreover, multiple patient-derived GBM lines responded to treatment, underscoring its clinical relevance. In sum, integrating stem cell-based engineering, multimodal imaging, and delivery of PE-cytotoxins in a clinically relevant GBM model represents a novel strategy and a potential advancement in GBM therapy.

Targeting breast to brain metastatic tumours with death receptor ligand expressing therapeutic stem cells.

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study, we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging, we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next, we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain, we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective, potent and secretable variant of a TRAIL, S-TRAIL, and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore, the incorporation of pro-drug converting enzyme, herpes simplex virus thymidine kinase, into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications.

Encapsulated stem cells loaded with hyaluronidase-expressing oncolytic virus for brain tumor therapy.

Despite the proven safety of oncolytic viruses (OV) in clinical trials for glioblastoma (GBM), their efficacy has been hindered by suboptimal spreading within the tumor. We show that hyaluronan or hyaluronic acid (HA), an important component of extracellular matrix (ECM), is highly expressed in a majority of tumor xenografts established from patient-derived GBM lines that present both invasive and nodular phenotypes. Intratumoral injection of a conditionally replicating adenovirus expressing soluble hyaluronidase (ICOVIR17) into nodular GBM, mediated HA degradation and enhanced viral spread, resulting in a significant antitumor effect and mice survival. In an effort to translate OV-based therapeutics into clinical settings, we encapsulated human adipose-derived mesenchymal stem cells (MSC) loaded with ICOVIR17 in biocompatible synthetic extracellular matrix (sECM) and tested their efficacy in a clinically relevant mouse model of GBM resection. Compared with direct injection of ICOVIR17, sECM-MSC loaded with ICOVIR17 resulted in a significant decrease in tumor regrowth and increased mice survival. This is the first report of its kind revealing the expression of HA in GBM and the role of OV-mediated HA targeting in clinically relevant mouse model of GBM resection and thus has clinical implications.

Antiangiogenic variant of TSP-1 targets tumor cells in glioblastomas.

Three type-1 repeat (3TSR) domain of thrombospondin-1 is known to have anti-angiogenic effects by targeting tumor-associated endothelial cells, but its effect on tumor cells is unknown. This study explored the potential of 3TSR to target glioblastoma (GBM) cells in vitro and in vivo. We show that 3TSR upregulates death receptor (DR) 4/5 expression in a CD36-dependent manner and primes resistant GBMs to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced caspase-8/3/7 mediated apoptosis. We engineered human mesenchymal stem cells (MSC) for on-site delivery of 3TSR and a potent and secretable variant of TRAIL (S-TRAIL) in an effort to simultaneously target tumor cells and associated endothelial cells and circumvent issues of systemic delivery of drugs across the blood-brain barrier. We show that MSC-3TSR/S-TRAIL inhibits tumor growth in an expanded spectrum of GBMs. In vivo, a single administration of MSC-3TSR/S-TRAIL significantly targets both tumor cells and vascular component of GBMs, inhibits tumor progression, and extends survival of mice bearing highly vascularized GBM. The ability of 3TSR/S-TRAIL to simultaneously act on tumor cells and tumor-associated endothelial cells offers a great potential to target a broad spectrum of cancers and translate 3TSR/TRAIL therapies into clinics.

Multifunctional receptor-targeting antibodies for cancer therapy.

During the past decade, monospecific antibodies targeting cell-surface receptors in different tumour types have achieved substantial success and have been at the forefront of cancer treatment. However, redundant signalling and crosstalk between different pathways within tumour cells and between tumour cells and their microenvironment can limit the efficacy of receptor-targeted monospecific-based therapies. Advances in antibody engineering technologies have enabled strategies that simultaneously target multiple receptors to circumvent the limitations of conventional monospecific therapies and achieve enhanced therapeutic efficacy. In the past 5 years, a range of multifunctional, receptor-targeting, antibody-based molecules have emerged, which allow targeting of multiple surface receptors on tumour cells and endothelial or immune cells in the tumour microenvironment. In this Review, we discuss the rationales and strategies for the use of multifunctional receptor-targeting antibodies, their mechanisms of action, and the promises and challenges they hold as cancer therapeutics. This knowledge provides opportunities to improve current targeted therapy outcomes for patients with cancer.