Analysis of Immunological Treatment Effects of Virotherapy in Tumor Tissue.

In addition of being directly tumoricidal, oncolytic viruses have emerged as potent partners for established and investigational immunotherapies due to their immune-stimulatory effects. The shifting focus on virus-mediated immune modulation calls for a comprehensive analysis of the tumor microenvironment (TME) and the factors orchestrating the antiviral and antitumor immune response. The oncolytic VSV-GP studied in our lab is a safe and potent antitumor agent with a fast replication cycle and killing of a broad range of different cancer types. It induces a robust local inflammatory conversion of the TME and drives a strong adaptive immune response toward the tumor. Here we present our multidisciplinary approach to study VSV-GP treatment effects in tumors by assessing both immune cells (tumor-infiltrating lymphocytes and tumor-associated macrophages) and immune-regulatory factors (cytokines) as well as characterizing immune signatures using an immune-targeted NanoString gene expression system.

In Vivo Bioimaging for Monitoring Intratumoral Virus Activity.

In vivo studies are the mainstay of translational immune-oncology and virotherapy research. In general oncology, bioluminescence imaging provides a convenient and reliable tool to visualize disseminated tumors and monitor growth kinetics or treatment effects. Unique aspects of this method in the field of oncolytic viruses are tracing the process of tumor-specific targeting, assessing potential off-target replication, and visualizing intratumoral spread. In addition, the longitudinal monitoring of virus activity kinetics over time is a very powerful feature supporting the subsequent, often elaborate, preclinical biodistribution and pharmtox program. Here we present a step-by-step standard imaging protocol used in our group for both tumor and virus monitoring, along with background information and general principles that should allow the reader to modify and adapt the protocol according to their needs.

The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion.

The L-protein of vesicular stomatitis virus (VSV) is a single-chain multi-domain RNA-dependent RNA polymerase. Previously reported attempts of intramolecular insertions of fluorescent proteins into the L-protein resulted in temperature-sensitive and highly attenuated polymerase activity. Here, we describe a novel insertion site that was selected based on in silico prediction. Of five preselected locations, insertion of the fluorescent protein mCherry in the VSV polymerase between amino acids 1620 and 1621 preserved polymerase function even after extended passaging and showed only mild attenuation compared to wildtype VSV polymerase. High magnification fluorescence imaging revealed a corpuscular cytosolic pattern for the L-protein. To confirm that the insertion site tolerates inclusion of proteins others than mCherry, we cloned mWasabi into the same position in L, generating a VSV-LmWasabi, which was also functional. We also generated a functional dual-color-dual-insertion VSV construct with intramolecularly labeled P and L-proteins. Together, our data present an approach to tag VSV polymerase intramolecularly without perturbing enzymatic activity. This L fusion protein might enable future tracing studies to monitor intracellular location of the VSV transcription and replication machinery in real-time life-imaging studies.

The lytic activity of VSV-GP treatment dominates the therapeutic effects in a syngeneic model of lung cancer.

BACKGROUND: Oncolytic virotherapy is thought to result in direct virus-induced lytic tumour killing and simultaneous activation of innate and tumour-specific adaptive immune responses. Using a chimeric vesicular stomatitis virus variant VSV-GP, we addressed the direct oncolytic effects and the role of anti-tumour immune induction in the syngeneic mouse lung cancer model LLC1. METHODS: To study a tumour system with limited antiviral effects, we generated interferon receptor-deficient cells (LLC1-IFNAR1-/-). Therapeutic efficacy of VSV-GP was assessed in vivo in syngeneic C57BL/6 and athymic nude mice bearing subcutaneous tumours. VSV-GP treatment effects were analysed using bioluminescent imaging (BLI), immunohistochemistry, ELISpot, flow cytometry, multiplex ELISA and Nanostring® assays. RESULTS: Interferon insensitivity correlated with VSV-GP replication and therapeutic outcome. BLI revealed tumour-to-tumour spread of viral progeny in bilateral tumours. Histological and gene expression analysis confirmed widespread and rapid infection and cell killing within the tumour with activation of innate and adaptive immune-response markers. However, treatment outcome was increased in the absence of CD8+ T cells and surviving mice showed little protection from tumour re-challenge, indicating limited therapeutic contribution by the activated immune system. CONCLUSION: These studies present a case for a predominantly lytic treatment effect of VSV-GP in a syngeneic mouse lung cancer model.

Sprouty2 enhances the tumorigenic potential of glioblastoma cells.

Background: Sprouty2 (SPRY2), a feedback regulator of receptor tyrosine kinase (RTK) signaling, has been shown to be associated with drug resistance and cell proliferation in glioblastoma (GBM), but the underlying mechanisms are still poorly defined. Methods: SPRY2 expression and survival patterns of patients with gliomas were analyzed using publicly available databases. Effects of RNA interference targeting SPRY2 on cellular proliferation in established GBM or patient-derived GBM stemlike cells were examined. Loss- or gain-of-function of SPRY2 to regulate the tumorigenic capacity was assessed in both intracranial and subcutaneous xenografts. Results: SPRY2 was found to be upregulated in GBM, which correlated with reduced survival in GBM patients. SPRY2 knockdown significantly impaired proliferation of GBM cells but not of normal astrocytes. Silencing of SPRY2 increased epidermal growth factor-induced extracellular signal-regulated kinase (ERK) and Akt activation causing premature onset of DNA replication, increased DNA damage, and impaired proliferation, suggesting that SPRY2 suppresses DNA replication stress. Abrogating SPRY2 function strongly inhibited intracranial tumor growth and led to significantly prolonged survival of U87 xenograft-bearing mice. In contrast, SPRY2 overexpression promoted tumor propagation of low-tumorigenic U251 cells. Conclusions: The present study highlights an antitumoral effect of SPRY2 inhibition that is based on excessive activation of ERK signaling and DNA damage response, resulting in reduced cell proliferation and increased cytotoxicity, proposing SPRY2 as a promising pharmacological target in GBM patients.

Oncolytic activity of the rhabdovirus VSV-GP against prostate cancer.

Oncolytic viruses, including the oncolytic rhabdovirus VSV-GP tested here, selectively infect and kill cancer cells and are a promising new therapeutic modality. Our aim was to study the efficacy of VSV-GP, a vesicular stomatitis virus carrying the glycoprotein of lymphocytic choriomeningitis virus, against prostate cancer, for which current treatment options still fail to cure metastatic disease. VSV-GP was found to infect 6 of 7 prostate cancer cell lines with great efficacy. However, susceptibility was reduced in one cell line with low virus receptor expression and in 3 cell lines after interferon alpha treatment. Four cell lines had developed resistance to interferon type I at different levels of the interferon signaling pathway, resulting in a deficient antiviral response. In prostate cancer mouse models, long-term remission was achieved upon intratumoral and, remarkably, also upon intravenous treatment of subcutaneous tumors and bone metastases. These promising efficacy data demonstrate that treatment of prostate cancer with VSV-GP is feasible and safe in preclinical models and encourage further preclinical and clinical development of VSV-GP for systemic treatment of metastatic prostate cancer.

The Oncolytic Virus VSV-GP Is Effective against Malignant Melanoma.

Previously, we described VSV-GP, a modified version of the vesicular stomatitis virus, as a non-neurotoxic oncolytic virus that is effective for the treatment of malignant glioblastoma and ovarian cancer. Here, we evaluate the therapeutic efficacy of VSV-GP for malignant melanoma. All of the human, mouse, and canine melanoma cell lines that were tested, alongside most primary human melanoma cultures, were infected by VSV-GP and efficiently killed. Additionally, we found that VSV-GP prolonged the survival of mice in both a xenograft and a syngeneic mouse model. However, only a few mice survived with long-term tumor remission. When we analyzed the factors that might limit VSV-GP's efficacy, we found that vector-neutralizing antibodies did not play a role in this context, as even after eight subsequent immunizations and an observation time of 42 weeks, no vector-neutralizing antibodies were induced in VSV-GP immunized mice. In contrast, the type I IFN response might have contributed to the reduced efficacy of the therapy, as both of the cell lines that were used for the mouse models were able to mount a protective IFN response. Nevertheless, early treatment with VSV-GP also reduced the number and size of lung metastases in a syngeneic B16 mouse model. In summary, VSV-GP is a potent candidate for the treatment of malignant melanoma; however, factors limiting the efficacy of the virus need to be further explored.

Application of interferon modulators to overcome partial resistance of human ovarian cancers to VSV-GP oncolytic viral therapy.

Previously, we described an oncolytic vesicular stomatitis virus variant pseudotyped with the nonneurotropic glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, which was highly effective in glioblastoma. Here, we tested its potency for the treatment of ovarian cancer, a leading cause of death from gynecological malignancies. Effective oncolytic activity of VSV-GP could be demonstrated in ovarian cancer cell lines and xenografts in mice; however, remission was temporary in most mice. Analysis of the innate immune response revealed that ovarian cancer cell lines were able to respond to and produce type I interferon, inducing an antiviral state upon virus infection. This is in stark contrast to published data for other cancer cell lines, which were mostly found to be interferon incompetent. We showed that in vitro this antiviral state could be reverted by combining VSV-GP with the JAK1/2-inhibitor ruxolitinib. In addition, for the first time, we report the in vivo enhancement of oncolytic virus treatment by ruxolitinib, both in subcutaneous as well as in orthotopic xenograft mouse models, without causing significant additional toxicity. In conclusion, VSV-GP has the potential to be a potent and safe oncolytic virus to treat ovarian cancer, especially when combined with an inhibitor of the interferon response.