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The difficulty of glioblastoma treatment makes it a good candidate for novel therapies, such as oncolytic viruses. Vesicular stomatitis virus expressing Lassa virus glycoprotein (Lassa-VSV) showed significant promise in animal models using established glioblastoma cell lines. These experiments were to determine the susceptibility of low-passage, patient-derived cell lines to Lassa-VSV oncolysis. Four patient-derived glioblastoma cell lines were infected with Lassa-VSV that expresses green fluorescent protein (GFP) and analyzed by fluorescence microscopy, flow cytometry, and cell viability assays. Cells were also analyzed as tumorspheres containing primarily glioma stem-like cells. Three low-passage, patient-derived cells were further analyzed with RNA sequencing (RNA-seq). Individual cell lines varied somewhat in their levels of viral gene expression and time course of Lassa-VSV-induced cell death, but each was susceptible to Lassa-VSV. Brain Tumor Center of Excellence (BTCOE) 4765 cells had the highest level of expression of interferon-stimulated genes but were most susceptible to Lassa-VSV-induced cell death, indicating that more susceptible cells do not necessarily have lower interferon pathway activation. Cells cultured as tumorspheres and infected with Lassa-VSV also showed variable susceptibility to Lassa-VSV, but BTCOE 4765 cells were least susceptible. Thus, patient-derived brain tumor cells show variable responses to Lassa-VSV infection, but each of the lines was susceptible to VSV oncolysis., Graphical abstract, Vesicular stomatitis virus containing the glycoprotein of Lassa virus (Lassa-VSV) has oncolytic activity against well-established human glioblastoma (GBM) cell lines. The experiments here demonstrated the susceptibility to Lassa-VSV of patient-derived GBM cell lines that vary in their expression of GBM subtype-specific genes and also in glioma stem-like cells.

Ovarian cancer is the third most common female cancer, with poor survival in later stages of metastatic spread. We test a chimeric virus consisting of genes from Lassa and vesicular stomatitis viruses, LASV-VSV; the native VSV glycoprotein is replaced by the Lassa glycoprotein, greatly reducing neurotropism. Human ovarian cancer cells in immunocompromised nude mice were lethal in controls. Chemotherapeutic paclitaxel and cisplatin showed modest cancer inhibition and survival extension. In contrast, a single intraperitoneal injection of LASV-VSV selectively infected and killed ovarian cancer cells, generating long-term survival. Mice with human ovarian cancer cells in brain showed rapid deterioration; LASV-VSV microinjection into brain blocked cancer growth, and generated long-term survival. Treatment of immunocompetent mice with infected mouse ovarian cancer cells blocked growth of non-infected ovarian cancer cells peritoneally and in brain. These results suggest LASV-VSV is a viable candidate for further study and may be of use in the treatment of ovarian cancer.

Key points Excitatory glutamate neurons are sparse in the rostral hypothalamic arcuate nucleus (ARC), the subregion that has received the most attention in the past. In striking contrast, excitatory neurons are far more common (by a factor of 10) in the caudal ARC, an area which has received relatively little attention. These glutamate cells may play a negative role in energy balance and food intake. They can show an increase in phosphorylated Stat-3 in the presence of leptin, are electrically excited by the anorectic neuromodulator cholecystokinin, and inhibited by orexigenic neuromodulators neuropeptide Y, met-enkephalin, dynorphin and the catecholamine dopamine. The neurons project local axonal connections that excite other ARC neurons including proopiomelanocortin neurons that can play an important role in obesity. These data are consistent with models suggesting that the ARC glutamatergic neurons may play both a rapid and a slower role in acting as anorectic neurons in CNS control of food intake and energy homeostasis. Abstract Here we interrogate a unique class of excitatory neurons in the hypothalamic arcuate nucleus (ARC) that utilizes glutamate as a fast neurotransmitter using mice expressing GFP under control of the vesicular glutamate transporter 2 (vGluT2) promoter. These neurons show a unique distribution, synaptic characterization, cellular physiology and response to neuropeptides involved in energy homeostasis. Although apparently not previously appreciated, the caudal ARC showed a far greater density of vGluT2 cells than the rostral ARC, as seen in transgenic vGluT2-GFP mice and mRNA analysis. After food deprivation, leptin induced an increase in phosphorylated Stat-3 in vGluT2-positive neurons, indicating a response to hormonal cues of energy state. Based on whole-cell recording electrophysiology in brain slices, vGluT2 neurons were spontaneously active with a spike frequency around 2 Hz. vGluT2 cells were responsive to a number of neuropeptides related to energy homeostasis; they were excited by the anorectic peptide cholecystokinin, but inhibited by orexigenic neuropeptide Y, dynorphin and met-enkephalin, consistent with an anorexic role in energy homeostasis. Dopamine, associated with the hedonic aspect of enhancing food intake, inhibited vGluT2 neurons. Optogenetic excitation of vGluT2 cells evoked EPSCs in neighbouring neurons, indicating local synaptic excitation of other ARC neurons. Microdrop excitation of ARC glutamate cells in brain slices rapidly increased excitatory synaptic activity in anorexigenic proopiomelanocortin neurons. Together these data support the perspective that vGluT2 cells may be more prevalent in the ARC than previously appreciated, and play predominantly an anorectic role in energy metabolism.

Given that the Ebola virus (EBOV) infects a wide array of organs and cells yet displays a relative lack of neurotropism, we asked whether a chimeric vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) might selectively target brain tumors. The mucin-like domain (MLD) of the EBOV GP may enhance virus immune system evasion. Here, we compared chimeric VSVs in which EBOV GP replaces the VSV glycoprotein, thereby reducing the neurotoxicity associated with wild-type VSV. A chimeric VSV expressing the full-length EBOV GP (VSV-EBOV) containing the MLD was substantially more effective and safer than a parallel construct with an EBOV GP lacking the MLD (VSV-EBOVΔMLD). One-step growth, reverse transcription-quantitative PCR, and Western blotting assessments showed that VSV-EBOVΔMLD produced substantially more progeny faster than VSV-EBOV. Using immunodeficient SCID mice, we focused on targeting human brain tumors with these VSV-EBOVs. Similar to the findings of our previous study in which we used an attenuated VSV-EBOV with no MLD that expressed green fluorescent protein (GFP) (VSV-EBOVΔMLD-GFP), VSV-EBOVΔMLD without GFP targeted glioma but yielded only a modest extension of survival. In contrast, VSV-EBOV containing the MLD showed substantially better targeting and elimination of brain tumors after intravenous delivery and increased the survival of brain tumor-bearing mice. Despite the apparent destruction of most tumor cells by VSV-EBOVΔMLD, the virus remained active within the SCID mouse brain and showed widespread infection of normal brain cells. In contrast, VSV-EBOV eliminated the tumors and showed relatively little infection of normal brain cells. Parallel experiments with direct intracranial virus infection generated similar results. Neither VSV-EBOV nor VSV-EBOVΔMLD showed substantive infection of the brains of normal immunocompetent mice. IMPORTANCE The Ebola virus glycoprotein contains a mucin-like domain which may play a role in immune evasion. Chimeric vesicular stomatitis viruses with the EBOV glycoprotein substituted for the VSV glycoprotein show greater safety and efficacy in targeting brain tumors in immunodeficient mice when the MLD was expressed within the EBOV glycoprotein than when EBOV lacked the mucin-like domain.