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.

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.

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.

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.

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.

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.

Oncolytic HSV1 targets different growth phases of breast cancer leptomeningeal metastases.

Leptomeningeal metastasis is a fatal complication of breast cancer which results when cancer cells seed in the meninges. Currently there is no cure, limiting survival to less than four months. Treatment options are palliative. We studied a replication conditional Herpes simplex virus 1 (HSV1) in this regard and present the therapeutic efficacy of oncolytic HSV1 on different stages of breast cancer leptomeningeal metastases growth, namely the lag, intermediate, and exponential phases. These phases characterized in a murine model represent the early, intermediate, and late stages of leptomeningeal disease in patients. In this model, virus was introduced into the ventricular system by stereotactic surgery, the same path cancer cells were introduced to create leptomeningeal metastases. Tumor growth was measured with Gd-MRI and virus replication was assessed by FHBG-PET and Fluc bioluminescence. Imaging results were correlated with H&E and HSV-TK immunohistochemical staining. A remarkable growth inhibition was observed when the lag phase was targeted which was associated with multiple virus replication cycles. The onset of debilitating symptoms was delayed, and survival was lengthened by nearly 2 weeks. A growth inhibition similar to the lag phase was observed when the intermediate phase was targeted, associated with robust virus replication. The regression of existing tumor led to a reversal of neurological symptoms, extending survival by nearly one week. A modest response was observed when the lag phase was targeted lengthening survival by 3 days. Oncolytic HSV1 presents a novel treatment option for breast cancer leptomeningeal metastases with potential for targeting different disease stages where virus replication and tumor response can be monitored with molecular imaging techniques that are in the clinic.

Bifunctional cancer cell-based vaccine concomitantly drives direct tumor killing and antitumor immunity.

The administration of inactivated tumor cells is known to induce a potent antitumor immune response; however, the efficacy of such an approach is limited by its inability to kill tumor cells before inducing the immune responses. Unlike inactivated tumor cells, living tumor cells have the ability to track and target tumors. Here, we developed a bifunctional whole cancer cell-based therapeutic with direct tumor killing and immunostimulatory roles. We repurposed the tumor cells from interferon-ß (IFN-ß) sensitive to resistant using CRISPR-Cas9 by knocking out the IFN-ß-specific receptor and subsequently engineered them to release immunomodulatory agents IFN-ß and granulocyte-macrophage colony-stimulating factor. These engineered therapeutic tumor cells (ThTCs) eliminated established glioblastoma tumors in mice by inducing caspase-mediated cancer cell apoptosis, down-regulating cancer-associated fibroblast-expressed platelet-derived growth factor receptor ß, and activating antitumor immune cell trafficking and antigen-specific T cell activation signaling. This mechanism-based efficacy of ThTCs translated into a survival benefit and long-term immunity in primary, recurrent, and metastatic cancer models in immunocompetent and humanized mice. The incorporation of a double kill-switch comprising herpes simplex virus-1 thymidine kinase and rapamycin-activated caspase 9 in ThTCs ensured the safety of our approach. Arming naturally neoantigen-rich tumor cells with bifunctional therapeutics represents a promising cell-based immunotherapy for solid tumors and establishes a road map toward clinical translation.

Establishment and immune phenotyping of patient-derived glioblastoma models in humanized mice.

Glioblastoma (GBM) is the most aggressive and common type of malignant brain tumor diagnosed in adults. Preclinical immunocompetent mouse tumor models generated using mouse tumor cells play a pivotal role in testing the therapeutic efficacy of emerging immune-based therapies for GBMs. However, the clinical translatability of such studies is limited as mouse tumor lines do not fully recapitulate GBMs seen in inpatient settings. In this study, we generated three distinct, imageable human-GBM (hGBM) models in humanized mice using patient-derived GBM cells that cover phenotypic and genetic GBM heterogeneity in primary (invasive and nodular) and recurrent tumors. We developed a pipeline to first enrich the tumor-initiating stem-like cells and then successfully established robust patient-derived GBM tumor engraftment and growth in bone marrow-liver-thymus (BLT) humanized mice. Multiplex immunofluorescence of GBM tumor sections revealed distinct phenotypic features of the patient GBM tumors, with myeloid cells dominating the immune landscape. Utilizing flow cytometry and correlative immunofluorescence, we profiled the immune microenvironment within the established human GBM tumors in the BLT mouse models and showed tumor infiltration of variable human immune cells, creating a unique immune landscape compared with lymphoid organs. These findings contribute substantially to our understanding of GBM biology within the context of the human immune system in humanized mice and lay the groundwork for further translational studies aimed at advancing therapeutic strategies for GBM.

Target receptor identification and subsequent treatment of resected brain tumors with encapsulated and engineered allogeneic stem cells.

Cellular therapies offer a promising therapeutic strategy for the highly malignant brain tumor, glioblastoma (GBM). However, their clinical translation is limited by the lack of effective target identification and stringent testing in pre-clinical models that replicate standard treatment in GBM patients. In this study, we show the detection of cell surface death receptor (DR) target on CD146-enriched circulating tumor cells (CTC) captured from the blood of mice bearing GBM and patients diagnosed with GBM. Next, we developed allogeneic "off-the-shelf" clinical-grade bifunctional mesenchymal stem cells (MSCBif) expressing DR-targeted ligand and a safety kill switch. We show that biodegradable hydrogel encapsulated MSCBif (EnMSCBif) has a profound therapeutic efficacy in mice bearing patient-derived invasive, primary and recurrent GBM tumors following surgical resection. Activation of the kill switch enhances the efficacy of MSCBif and results in their elimination post-tumor treatment which can be tracked by positron emission tomography (PET) imaging. This study establishes a foundation towards a clinical trial of EnMSCBif in primary and recurrent GBM patients.

The Potential of the Gut Microbiome to Reshape the Cancer Therapy Paradigm: A Review.

Importance: The gut microbiome, home to the vast kingdom of diverse commensal bacteria and other microorganisms residing within the gut, was once thought to only have roles primarily centered on digestive functions. However, recent advances in sequencing technology have elucidated intricate roles of the gut microbiome in cancer development and efficacy of therapeutic response that need to be comprehensively addressed from a clinically translational angle. Observations: This review aims to highlight the current understanding of the association of the gut microbiome with the therapeutic response to immunotherapy, chemotherapy, radiotherapy, cancer surgery, and more, while also contextualizing possible synergistic strategies with the microbiome for tackling some of the most challenging tumors. It also provides insights on contemporary methods that target the microbiota and the current progression of findings being translated from bench to bedside. Conclusions and Relevance: Ultimately, the importance of gut bacteria in cancer therapy cannot be overstated in its potential for ushering in a new era of cancer treatments. With the understanding that the microbiome may play critical roles in the tumor microenvironment, holistic approaches that integrate microbiome-modulating treatments with biological, immune, cell-based, and surgical cancer therapies should be explored.