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.

MicroRNA (miR)-124: A Promising Therapeutic Gateway for Oncology.

MicroRNA (miR) are a class of small non-coding RNA that are involved in post-transcriptional gene regulation. Altered expression of miR has been associated with several pathological conditions. MicroRNA-124 (miR-124) is an abundantly expressed miR in the brain as well as the thymus, lymph nodes, bone marrow, and peripheral blood mono-nuclear cells. It plays a key role in the regulation of the host immune system. Emerging studies show that dysregulated expression of miR-124 is a hallmark in several cancer types and it has been attributed to the progression of these malignancies. In this review, we present a comprehensive summary of the role of miR-124 as a promising therapeutic gateway in oncology.

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.

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.

Oncolytic Herpes Simplex Virus-Based Therapies for Cancer.

With the increased worldwide burden of cancer, including aggressive and resistant cancers, oncolytic virotherapy has emerged as a viable therapeutic option. Oncolytic herpes simplex virus (oHSV) can be genetically engineered to target cancer cells while sparing normal cells. This leads to the direct killing of cancer cells and the activation of the host immunity to recognize and attack the tumor. Different variants of oHSV have been developed to optimize its antitumor effects. In this review, we discuss the development of oHSV, its antitumor mechanism of action and the clinical trials that have employed oHSV variants to treat different types of tumor.

Generation of TRAIL-resistant cell line models reveals distinct adaptive mechanisms for acquired resistance and re-sensitization.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumor cell-specific apoptosis, making it a prime therapeutic candidate. However, many tumor cells are either innately TRAIL-resistant, or they acquire resistance with adaptive mechanisms that remain poorly understood. In this study, we generated acquired TRAIL resistance models using multiple glioblastoma (GBM) cell lines to assess the molecular alterations in the TRAIL-resistant state. We selected TRAIL-resistant cells through chronic and long-term TRAIL exposure and noted that they showed persistent resistance both in vitro and in vivo. Among known TRAIL-sensitizers, proteosome inhibitor Bortezomib, but not HDAC inhibitor MS-275, was effective in overcoming resistance in all cell models. This was partly achieved through upregulating death receptors and pro-apoptotic proteins, and downregulating major anti-apoptotic members, Bcl-2 and Bcl-xL. We showed that CRISPR/Cas9 mediated silencing of DR5 could block Bortezomib-mediated re-sensitization, demonstrating its critical role. While overexpression of Bcl-2 or Bcl-xL was sufficient to confer resistance to TRAIL-sensitive cells, it failed to override Bortezomib-mediated re-sensitization. With RNA sequencing in multiple paired TRAIL-sensitive and TRAIL-resistant cells, we identified major alterations in inflammatory signaling, particularly in the NF-κB pathway. Inhibiting NF-κB substantially sensitized the most resistant cells to TRAIL, however, the sensitization effect was not as great as what was observed with Bortezomib. Together, our findings provide new models of acquired TRAIL resistance, which will provide essential tools to gain further insight into the heterogeneous therapy responses within GBM tumors. Additionally, these findings emphasize the critical importance of combining proteasome inhibitors and pro-apoptotic ligands to overcome acquired resistance.

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.

Shattering barriers toward clinically meaningful MSC therapies.

More than 1050 clinical trials are registered at FDA.gov that explore multipotent mesenchymal stromal cells (MSCs) for nearly every clinical application imaginable, including neurodegenerative and cardiac disorders, perianal fistulas, graft-versus-host disease, COVID-19, and cancer. Several companies have or are in the process of commercializing MSC-based therapies. However, most of the clinical-stage MSC therapies have been unable to meet primary efficacy end points. The innate therapeutic functions of MSCs administered to humans are not as robust as demonstrated in preclinical studies, and in general, the translation of cell-based therapy is impaired by a myriad of steps that introduce heterogeneity. In this review, we discuss the major clinical challenges with MSC therapies, the details of these challenges, and the potential bioengineering approaches that leverage the unique biology of MSCs to overcome the challenges and achieve more potent and versatile therapies.

Simultaneous downregulation of miR-21 and upregulation of miR-7 has anti-tumor efficacy.

Dysregulation of miRNA expression has been implicated in cancer. Numerous strategies have been explored to modulate miR but sub-optimal delivery and inability to concurrently target multiple pathways involved in tumor progression have limited their efficacy. In this study, we explored the potential co-modulation of upregulated miR-21 and downregulated miR-7 to enhance therapeutic outcomes in heterogenic tumor types. We first engineered lentiviral (LV) and adeno-associated viral (AAV) vectors that preferentially express anti-sense miR against miR-21(miRzip-21) and show that modulating miR-21 via miRzip extensively targets tumor cell proliferation, migration and invasion in vitro in a broad spectrum of cancer types and has therapeutic efficacy in vivo. Next, we show a significantly increased expression of caspase-mediated apoptosis by simultaneously downregulating miR-21 and upregulating miR-7 in different tumor cells. In vivo co-treatment with AAV-miRzip-21 and AAV-miR-7 in mice bearing malignant brain tumors resulted in significantly decreased tumor burden with a corresponding increase in survival. To our knowledge, this is the first study that demonstrates the therapeutic efficacy of simultaneously upregulating miR-7 and downregulating miR-21 and establishes a roadmap towards clinical translation of modulating miRs for various cancer types.

Stem Cell Therapies: A Way to Promising Cures.

Stem cells carry the remarkable ability to differentiate into different cell types while retaining the capability to self-replicate and maintain the characteristics of their parent cells, referred to as potency. Stem cells have been studied extensively to better understand human development and organogenesis. Because of advances in stem cell-based therapies, regenerative medicine has seen significant growth. Ophthalmic conditions, some of which are leading causes of blindness worldwide, are being treated with stem cell therapies. Great results have also been obtained in the treatment of oral and maxillofacial defects. Stem-cell-based therapies have great potential in the treatment of chronic medical conditions like diabetes and cardiomyopathy. The unique property of stem cells to migrate towards cancer cells makes them excellent vectors for the transportation of bioactive agents or for targeting cancer cells, both primary and metastatic. While these therapeutic strategies are extremely promising, they are not without limitations. Failure to completely eradicate the tumor and tumor relapse are some of those concerns. Stem cells share some characteristics with cancer stem cells, raising concerns for increasing the risk of cancer occurrence. Ethical concerns due to the fetal origin of stem cells and cost are other major obstacles in the large-scale implementation of such therapies.

A model of breast cancer meningeal metastases: characterization with in vivo molecular imaging.

Meningeal metastasis is a fatal complication of breast cancer which affects 8-15% of patients who experience severe neurological complications of cranial nerves, cerebrum, and spinal cord. Survival once diagnosed is less than 4 months. Currently there is no cure. Aggressive multimodal radiation, intra-CSF, or systemic chemotherapy is palliative. Investigation of urgently needed new treatment modalities is hindered by the lack of suitable animal models to effectively study tumor growth kinetics. We present a model of meningeal metastases where tumor growth and associated neurological symptoms have been characterized over 3 weeks by sequential molecular imaging, tumor growth kinetics, and histopathology. Meningeal metastases were induced by stereotaxic injection of human breast cancer cells (MDA-MB-231-Rluc) into the lateral ventricle. Tumor identified by Gd-MRI and Rluc-bioluminescence depict growth in 3 phases, namely lag, exponential, and plateau phase. Invasive tumor growth was highlighted by changes in contrast distribution in the meninges, ventricle and brain compartments over time where moderate contrast uptake in the early growth phase gave rise to a heavy tumor burden in the base of the brain in the latter phases. Tumor growth was accompanied with debilitating neurological symptoms and change in body mass. Tumor was confirmed by ex vivo histology. The reliability of the model to study novel therapeutics was confirmed by oncolytic virus delivered into the lateral ventricle showed potential for treatment. This effective and reliable model resembles human disease progression and is ideally suited to investigate novel treatments.

CRISPR-enhanced engineering of therapy-sensitive cancer cells for self-targeting of primary and metastatic tumors.

Tumor cells engineered to express therapeutic agents have shown promise to treat cancer. However, their potential to target cell surface receptors specific to the tumor site and their posttreatment fate have not been explored. We created therapeutic tumor cells expressing ligands specific to primary and recurrent tumor sites (receptor self-targeted tumor cells) and extensively characterized two different approaches using (i) therapy-resistant cancer cells, engineered with secretable death receptor-targeting ligands for "off-the-shelf" therapy in primary tumor settings, and (ii) therapy-sensitive cancer cells, which were CRISPR-engineered to knock out therapy-specific cell surface receptors before engineering with receptor self-targeted ligands and reapplied in autologous models of recurrent or metastatic disease. We show that both approaches allow high expression of targeted ligands that induce tumor cell killing and translate into marked survival benefits in mouse models of multiple cancer types. Safe elimination of therapeutic cancer cells after treatment was achieved by co-engineering with a prodrug-converting suicide system, which also allowed for real-time in vivo positron emission tomography imaging of therapeutic tumor cell fate. This study demonstrates self-tumor tropism of engineered cancer cells and their therapeutic potential when engineered with receptor self-targeted molecules, and it establishes a roadmap toward a safe clinical translation for different cancer types in primary, recurrent, and metastatic settings.

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.

microRNA-7 upregulates death receptor 5 and primes resistant brain tumors to caspase-mediated apoptosis.

Background: MicroRNAs (miRs) are known to play a pivotal role in tumorigenesis, controlling cell proliferation and apoptosis. In this study, we investigated the potential of miR-7 to prime resistant tumor cells to apoptosis in glioblastoma (GBM). Methods: We created constitutive and regulatable miR-7 expression vectors and utilized pharmacological inhibition of caspases and genetic loss of function to study the effect of forced expression of miR-7 on death receptor (DR) pathways in a cohort of GBM with established resistance to tumor necrosis factor apoptosis inducing ligand (TRAIL) and in patient-derived primary GBM stem cell (GSC) lines. We engineered adeno-associated virus (AAV)-miR-7 and stem cell (SC) releasing secretable (S)-TRAIL and utilized real time in vivo imaging and neuropathology to understand the effect of the combined treatment of AAV-miR-7 and SC-S-TRAIL in vitro and in mouse models of GBM from TRAIL-resistant GSC. Results: We show that expression of miR-7 in GBM cells results in downregulation of epidermal growth factor receptor and phosphorylated Akt and activation of nuclear factor-kappaB signaling. This leads to an upregulation of DR5, ultimately priming resistant GBM cells to DR-ligand, TRAIL-induced apoptotic cell death. In vivo, a single administration of AAV-miR-7 significantly decreases tumor volumes, upregulates DR5, and enables SC-delivered S-TRAIL to eradicate GBM xenografts generated from patient-derived TRAIL-resistant GSC, significantly improving survival of mice. Conclusions: This study identifies the unique role of miR-7 in linking cell proliferation to death pathways that can be targeted simultaneously to effectively eliminate GBM, thus presenting a promising strategy for treating GBM.

TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation.Significance: TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. Cancer Discov; 7(11); 1336-53. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growth and Self-Renewal in Embryonal Rhabdomyosarcoma.

Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.