Pediatric central nervous system (CNS) tumors, specifically Diffuse Midline Gliomas (DMG) are a leading cause of cancer-related deaths in children presenting an unmet need for novel therapeutic development. In this study, we assessed the combined effects of resveratrol (RSV) and the oncolytic herpes simplex virus (oHSV), hrR3 in patient-derived diffuse intrinsic pontine glioma (DIPG) cell lines. RSV or hrR3 monotherapy resulted in a dose- and time-dependent reduction in cell viability. RSV pretreatment followed by hrR3 infection led to a significantly greater reduction in tumor cell viability than monotherapy. RSV pretreatment further enhanced the replication of hrR3, as evidenced by increased viral titers. Mechanistic analysis showed that the combination therapy was associated with reduced phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) signaling pathways, along with increased expression of apoptotic markers, including cleaved caspase-3 and Poly (ADP-ribose) polymerase (PARP). Together these findings indicate that RSV potentiates hrR3-mediated oncolysis in DIPG cells by enhancing viral replication, suppressing survival signaling and promoting apoptosis in DIPG cells in vitro.

Glioblastoma (GBM) is an aggressive form of brain cancer that affects over 12, 000 people each year in the United States. Current treatments such as surgery, chemotherapy, and radiation have limitations and can often fail to achieve full tumor remission, as the five-year survival rate for this disease is around 7%. Recent research has revealed miRNA (miR), small non-coding RNAs that regulate gene expression, as contributors to the suppression and progression of various cancers, including GBM. Exosomes, tiny vesicles released by cells, show great promise as a cancer therapeutic. Our research focuses on using exosomes enriched with microRNA (miR) to address miR dysregulation in as a delivery system for glioblastoma treatment, as these vessels can cross the blood-brain barrier to deliver the treatment. Our goal is to examine the effectiveness of stem cell-derived exosomes carrying miR-124 and miR-7 as a new approach for treating glioblastoma, as this cancer is known for having downregulation of such microRNAs. Adult human stem cells (SC) are modified with target miR plasmids and then grown in an exosome-depleted medium. The exosomes that carry the miR are then separated, purified, measured, and examined. These exosomes are used in all subsequent research. The effects of exosomes on established and patient derived primary GBM cells are studied using a combination of cell viability tests and microscopy. To understand the molecular mechanisms that underlie the effect of miR modulation on cell proliferation and cell death pathways, a time course immunoblot analysis is carried out. To assess the effects of miR modulation in vivo, orthotopic animal models of GBM will be used. Our research has shown that stem cell-derived exosomes can be an effective delivery system, asthey can evade the immune system and target specific cells. We have successfully transferred microRNA cargo to our target tumor cells using exosomes. By modulating miR, we have been able to halt tumor progression in vitro by targeting multiple pathways involved in GBM growth and progression. Although we are still awaiting the results of our ongoing animal studies, our preliminary data suggests that using stem cell-derived exosomes loaded with miR-124 and miR-7 is a promising approach for treating GBM. Monique Kanitz Ruschel, Isaiah Davis, Karthik Gourishetti, Christian Jacobsen, Deepak Bhere. Exosome delivered combinatorial therapeutic approaches for advanced brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6696.

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.

Abstract Glioblastoma (GBM), a World Health Organization (WHO) Grade IV astrocytoma, is a highly aggressive brain tumor with poor prognosis. Current therapeutic approaches, which include surgery, chemotherapy, and radiation therapy, are largely ineffective and often result in tumor recurrence. Several characteristics of the tumors make them challenging to treat, including their protection by the blood-brain barrier, which hinders effective treatment delivery. Our research addresses this issue by using exosomes, which are small cell-derived vesicles, as delivery vehicles for microRNA (miR) therapies. Dysregulation of miR expression has been implicated in tumorigenesis, and targeting these dysregulated miRs is emerging as a promising approach to tumor treatment. In particular, miR-7 and miR-124 have been identified as tumor-suppressive miRs that are downregulated in various tumors, including GBM. In this study, stem cell-derived exosomes loaded with miR-7 and miR-124 have been evaluated as a combinatorial approach for treating GBM. In our work, human adipose stem cells (ADSCs) were transduced with the target miR-7 and miR-124 plasmids and cultured in exosome-depleted FBS. The miR-containing exosomes from the ADSCs were then isolated, purified, quantified, and characterized for use in subsequent studies. Murine and human GBM cells as well as primary patient-derived primary GBM cells were treated with miR-7 and miR-124 exosomes, and the efficacy of exosome treatment was determined using cell viability assays and microscopy. The mechanisms of miR modulation of cell proliferation and cell death pathways were analyzed with time-course western blot analysis. Orthotopic animal models of GBM will be used to assess the effectiveness of our methods in vivo. Our results have demonstrated successful delivery of miR-7 and miR-124 to GBM cells via stem cell-derived exosomes. Data show that treatment with these tumor-suppressive miRs halts GBM growth in vitro by modulating multiple pathways involved in tumor growth and progression. While we await results of our in vivo studies, our preliminary data demonstrate that delivery of miR-7 and miR-124 via stem cell-derived exosomes is a promising therapeutic approach for treating GBM. Citation Format: Sydney Thomas, Karthik Gourishetti, Karthik Rangavajhula, Deepak Bhere. Exosome delivered combinatorial treatment for malignant brain tumors [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A021.

Abstract Pancreatic cancer is a devastating illness that affects numerous individuals each year. Unfortunately, pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of this disease that accounts for a disproportionate number of cancer-related deaths. Despite representing only 3.3% of all cancer cases in the United States, pancreatic cancer is responsible for a staggering 8.3% of cancer-related fatalities. In 2023, 64,050 patients were diagnosed with PDAC alone. Current treatment options, which are limited to surgery, radiation, and chemotherapy, have failed to improve clinical outcomes. Recent research has revealed miRNA (miR) as a contributor to the suppression and progression of various cancers, including PDAC. Specifically, dysregulation of miR-7 has been associated with PDAC. Downregulation of miR-7 is linked to more aggressive cases of the disease. Another potential therapeutic approach to treating cancer is the use of oncolytic viruses (OV). These viruses can replicate in cancerous cells while leaving healthy cells unharmed, making them an attractive treatment option. In this study, we utilized exosomes isolated from human Umbilical Cord stem cells (hUCs) enriched with miR-7 and combined with OHSV to examine their effect on established PDAC cell lines. We utilized cell viability assays, microscopy, and western blot analysis to evaluate the impact of the combinatorial approach on the underlying molecular mechanisms. Our findings suggest that the successful transfer of miR cargo and treatment with oHSV into the target PDAC cells arrested cell proliferation by targeting apoptotic pathways. Citation Format: Monique Kanitz Ruschel, Isaiah Davis, Hannah Taylor Mills, Karthik Gourishetti, Deepak Bhere. Combinatorial effects of viral oncolysis and miRNA modulation as a novel therapeutic of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7258.

Abstract Glioblastoma, a type of brain cancer known for its aggressive nature and resistance to traditional treatments, remains a significant challenge for the medical community. Despite advances in surgical, radiation, and chemotherapy strategies, patients with glioblastoma typically have a poor prognosis, with a median survival time of only 15 months. While temporary symptom relief may be possible for some patients, complete tumor remission is often not achieved. Given the complexity of this disease, effective treatment options for glioblastoma are highly sought after. In this study, we have evaluated the effectiveness of a combinatorial approach using stem cell-derived exosomes enriched with miR-124 cargo paired with viral oncolysis for treating glioblastoma. Human Umbilical Cord Stem cells (hUCs) are genetically modified with specific miR plasmids, and then cultivated in a specialized medium that has been depleted of exosomes. The exosomes (exo) that contain the target miR are subsequently extracted, purified, quantified, and characterized. These exosomes are then utilized for all subsequent investigations. Using a combination of cell viability assays and microscopy, the overall effect of the exo-miR-124 and oncolytic virus on both established and patient-derived primary GBM cells is analyzed. Additionally, a time course western blot analysis is conducted to comprehend the molecular mechanisms underlying the effects of the miR modification and viral oncolysis on cell proliferation and death pathways. Orthotopic animal models of GBM will be utilized to assess the effects of miR modulation and viral oncolysis in vivo. Our research has shown that stem cell-derived exosomes are an effective way to deliver microRNA to specific cells, including tumor cells. This is because they can evade the immune system and target cells directly. Our experiments have shown that combining miR modulation paired with viral oncolysis as a promising approach for treating glioblastoma. Specifically, we have found that miR modulation and viral oncolysis can halt tumor cell proliferation in vitro by targeting multiple pathways involved in GBM growth and progression. Citation Format: Elle Y. Magnan, Karthik Gourishetti, Sydney Thomas, Deepak Bhere. Dual targeting as an effective therapeutic strategy for malignant brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4621.

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.

STEAM cocktail staining specifically identifies GBM cells.

Unsupervised hierarchical clustering analysis of patient and xenograft single cell qRT-PCR expression data.

Clinical association of CTC frequency

DNA-FISH analysis of non-EGFR-amplified GBM CTCs.

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.

Immunohistologic and DNA-FISH analysis of tumor cells and CTCs from a patient with metastatic GBM.

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.

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 (MSC Bif ) expressing DR-targeted ligand and a safety kill switch. We show that biodegradable hydrogel encapsulated MSC Bif (EnMSC Bif ) 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 MSC Bif 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 EnMSC Bif in primary and recurrent GBM patients.