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.

Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

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.

Meningeal lymphatics and their role in CNS disorder treatment: moving past misconceptions.

The central nervous system (CNS) was previously thought to lack lymphatics and shielded from the free diffusion of molecular and cellular components by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCB). However, recent findings have redefined the roles played by meningeal lymphatic vessels in the recruitment and drainage of lymphocytes from the periphery into the brain and the potentiation of an immune response. Emerging knowledge surrounding the importance of meningeal lymphatics has the potential to transform the treatment of CNS disorders. This review details the most recent understanding of the CNS-lymphatic network and its immunologic implications in both the healthy and diseased brain. Moreover, the review provides in-depth coverage of several exciting avenues for future therapeutic treatments that involve the meningeal lymphatic system. These therapeutic avenues will have potential implications in many treatment paradigms in the coming years.

Developing and characterizing a two-layered safety switch for cell therapies.

Gene edited and engineered cell-based therapies are a promising approach for treating a variety of disorders, including cancer. However, the ability of engineered cells to persist for prolonged periods along with possible toxicity raises concerns over the safety of these approaches. Although a number of different one-dimensional suicide systems have been incorporated into therapeutic cell types, the incorporation of a two-layered suicide system that allows controlled killing of therapeutic cells at different time points is needed. In this study, we engineered a variety of therapeutic cells to express two different kill switches, RapaCasp9 and HSV-TK and utilized Rapamycin and Ganciclovir respectively to activate these kill switches. We show that the function of both RapaCasp9 and HSV-TK molecules is preserved and can be activated to induce apoptosis detected early (24 h) and late (48 h) post-activation respectively, with no toxicity. In vivo, we show the eradication of a majority of cells after treatment in subcutaneous and orthotopic models. Furthermore, we demonstrate how both suicide switches work independently and can be activated sequentially for an improved killing, thus ensuring a failsafe mechanism in case the activation of a single one of them is not sufficient to eliminate the cells. Our findings highlight the reliability of the double suicide system, effective on a variety of cells with different biological characteristics, independent of their anatomic presence.

Natural history of non-functioning pituitary microadenomas: results from the UK non-functioning pituitary adenoma consortium.

OBJECTIVE: The optimal approach to the surveillance of non-functioning pituitary microadenomas (micro-NFPAs) is not clearly established. Our aim was to generate evidence on the natural history of micro-NFPAs to support patient care. DESIGN: Multi-centre, retrospective, cohort study involving 23 endocrine departments (UK NFPA consortium). METHODS: Clinical, imaging, and hormonal data of micro-NFPA cases between January, 1, 2008 and December, 21, 2021 were analysed. RESULTS: Data for 459 patients were retrieved [median age at detection 44 years (IQR 31-57)-152 males/307 females]. Four hundred and nineteen patients had more than two magnetic resonance imagings (MRIs) [median imaging monitoring 3.5 years (IQR 1.71-6.1)]. One case developed apoplexy. Cumulative probability of micro-NFPA growth was 7.8% (95% CI, 4.9%-8.1%) and 14.5% (95% CI, 10.2%-18.8%) at 3 and 5 years, respectively, and of reduction 14.1% (95% CI, 10.4%-17.8%) and 21.3% (95% CI, 16.4%-26.2%) at 3 and 5 years, respectively. Median tumour enlargement was 2 mm (IQR 1-3) and 49% of micro-NFPAs that grew became macroadenomas (nearly all >5 mm at detection). Eight (1.9%) patients received surgery (only one had visual compromise with surgery required >3 years after micro-NFPA detection). Sex, age, and size at baseline were not predictors of enlargement/reduction. At the time of detection, 7.2%, 1.7%, and 1.5% patients had secondary hypogonadism, hypothyroidism, and hypoadrenalism, respectively. Two (0.6%) developed hypopituitarism during follow-up (after progression to macroadenoma). CONCLUSIONS: Probability of micro-NFPA growth is low, and the development of new hypopituitarism is rare. Delaying the first follow-up MRI to 3 years and avoiding hormonal re-evaluation in the absence of tumour growth or clinical manifestations is a safe approach for micro-NFPA surveillance.

Fate and Efficacy of Engineered Allogeneic Stem Cells Targeting Cell Death and Proliferation Pathways in Primary and Brain Metastatic Lung Cancer.

Primary and metastatic lung cancer is a leading cause of cancer-related death and novel therapies are urgently needed. Epidermal growth factor receptor (EGFR) and death receptor (DR) 4/5 are both highly expressed in primary and metastatic non-small cell lung cancer (NSCLC); however, targeting these receptors individually has demonstrated limited therapeutic benefit in patients. In this study, we created and characterized diagnostic and therapeutic stem cells (SC), expressing EGFR-targeted nanobody (EV) fused to the extracellular domain of death DR4/5 ligand (DRL) (EVDRL) that simultaneously targets EGFR and DR4/5, in primary and metastatic NSCLC tumor models. We show that EVDRL targets both cell surface receptors, and induces caspase-mediated apoptosis in a broad spectrum of NSCLC cell lines. Utilizing real-time dual imaging and correlative immunohistochemistry, we show that allogeneic SCs home to tumors and when engineered to express EVDRL, alleviate tumor burden and significantly increase survival in primary and brain metastatic NSCLC. This study reports mechanistic insights into simultaneous targeting of EGFR- and DR4/5 in lung tumors and presents a promising approach for translation into the clinical setting.

Gene-edited and -engineered stem cell platform drives immunotherapy for brain metastatic melanomas.

Oncolytic virus therapy has shown activity against primary melanomas; however, its efficacy in brain metastases remains challenging, mainly because of the delivery and immunosuppressive nature of tumors in the brain. To address this challenge, we first established PTEN-deficient melanoma brain metastasis mouse models and characterized them to be more immunosuppressive compared with primary melanoma, mimicking the clinical settings. Next, we developed an allogeneic twin stem cell (TSC) system composed of two tumor-targeting stem cell (SC) populations. One SC was loaded with oncolytic herpes simplex virus (oHSV), and the other SC was CRISPR-Cas9 gene-edited to knock out nectin 1 (N1) receptor (N1KO) to acquire resistance to oHSV and release immunomodulators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). Using mouse models of brain metastatic BRAFV600E/PTEN-/- and BRAFV600E/wt/PTEN-/- mutant melanomas, we show that locoregional delivery of TSCs releasing oHSV and GM-CSF (TSC-G) activated dendritic cell- and T cell-mediated immune responses. In addition, our strategy exhibited greater therapeutic efficacy when compared with the existing oncolytic viral therapeutic approaches. Moreover, the TSCs composed of SC-oHSV and SCN1KO-releasing GM-CSF and single-chain variable fragment anti-PD-1 (TSC-G/P) had therapeutic efficacy in both syngeneic and patient-derived humanized mouse models of leptomeningeal metastasis. Our findings provide a promising allogeneic SC-based immunotherapeutic strategy against melanomas in the CNS and a road map toward clinical translation.

Engineered cell-based therapies in ex vivo ready-made CellDex capsules have therapeutic efficacy in solid tumors.

Encapsulated cell-based therapies for solid tumors have shown promising results in pre-clinical settings. However, the inability to culture encapsulated therapeutic cells prior to their transplantation has limited their translation into clinical settings. In this study, we created a wide variety of engineered therapeutic cells (ThC) loaded in micropore-forming gelatin methacryloyl (GelMA) hydrogel (CellDex) capsules that can be cultured in vitro prior to their transplantation in surgically debulked solid tumors. We show that both allogeneic and autologous engineered cells, such as stem cells (SCs), macrophages, NK cells, and T cells, proliferate within CellDex capsules and migrate out of the gel in vitro and in vivo. Furthermore, tumor cell specific therapeutic proteins and oncolytic viruses released from CellDex capsules retain and prolong their anti-tumor effects. In vivo, ThCs in pre-manufactured Celldex capsules persist long-term and track tumor cells. Moreover, chimeric antigen receptor (CAR) T cell bearing CellDex (T-CellDex) and human SC releasing therapeutic proteins (hSC-CellDex) capsules show therapeutic efficacy in metastatic and primary brain tumor resection models that mimic standard of care of tumor resection in patients. Overall, this unique approach of pre-manufactured micropore-forming CellDex capsules offers an effective off-the-shelf clinically viable strategy to treat solid tumors locally.

A multinational empirical study of perceived cyber barriers to automated vehicles deployment.

The digital transformation of Automated Vehicles (AVs) has raised concerns in the cyber realm among prospective AV consumers. However, there is a dearth of empirical research on how cyber obstacles may impact the operation of AVs. To address this knowledge gap, this study examines the six critical cyber impediments (data privacy, AV connectivity, ITS infrastructure, lack of cybersecurity regulations, AV cybersecurity understanding, and AV cyber-insurance) that influence the deployment of AVs. The impact of gender, age, income level, and individual AV and cybersecurity knowledge on these obstacles are statistically assessed using a sample of 2061 adults from the United States, the United Kingdom, New Zealand, and Australia. The research revealed intriguing empirical findings on all cyber barriers in the form of a trichotomy: participants' education level, understanding of AVs, and cybersecurity knowledge. As education levels increase, the significance of a cyber barrier to AV deployment decreases; however, as AV comprehension and cybersecurity knowledge increase, the perception of a cyber barrier becomes significantly more important. In addition, the study demonstrates differences in perceptions of cyber barriers and AV deployments based on gender, age, income, and geographic location. This study's findings on cyber barriers and AV deployment have implications for academia and industry.

SARS-CoV-2 RBD protein enhances the oncolytic activity of the vesicular stomatitis virus.

Despite recent advances in the research on oncolytic viruses (OVs), a better understanding of how to enhance their replication is key to improving their therapeutic index. Understanding viral replication is important to improve treatment outcomes based on enhanced viral spreading within the tumor milieu. The VSV-Δ51 oncolytic virus has been widely used as an anticancer agent with a high selectivity profile. In this study, we examined the role of the SARS-CoV-2 spike protein receptor-binding domain (RBD) in enhancing VSV-Δ51 viral production and oncolytic activity. To test this hypothesis, we first generated a novel VSV-Δ51 mutant that encoded the SARS-COV-2 RBD and compared viral spreading and viral yield between VSV-Δ51-RBD and VSV-Δ51 in vitro. Using the viral plaque assay, we demonstrated that the presence of the SARS-CoV-2 RBD in the VSV-Δ51 genome is associated with a significantly larger viral plaque surface area and significantly higher virus titers. Subsequently, using an ATP release-based assay, we demonstrated that the SARS-CoV-2 RBD could enhance VSV-Δ51 oncolytic activity in vitro. This observation was further supported using the B16F10 tumor model. These findings highlighted a novel use of the SARS-CoV-2 RBD as an anticancer agent.