Gastrointestinal
Molecular determinants of susceptibility to oncolytic vesicular stomatitis virus in pancreatic adenocarcinoma

https://doi.org/10.1016/j.jss.2013.10.032Get rights and content

Abstract

Background

M protein mutant vesicular stomatitis virus (M51R-VSV) has oncolytic properties against many cancers. However, some cancer cells are resistant to M51R-VSV. Herein, we evaluate the molecular determinants of vesicular stomatitis virus (VSV) resistance in pancreatic adenocarcinoma cells.

Methods

Cell viability and the effect of β-interferon (IFN) were analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Gene expression was evaluated via microarray analysis. Cell infectability was measured by flow cytometry. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV.

Results

Four of five pancreatic cancer cell lines were sensitive to M51R-VSV, whereas Panc 03.27 cells remained resistant (81 ± 3% viability 72 h after single-cycle infection). Comparing sensitive MiaPaCa2 cells with resistant Panc 03.27 cells, significant differences in gene expression were found relating to IFN signaling (P = 2 × 10−5), viral entry (P = 3 × 10−4), and endocytosis (P = 7 × 10−4). MiaPaCa2 cells permitted high levels of VSV infection, whereas Panc 03.27 cells were capable of resisting VSV cell entry even at high multiplicities of infection. Extrinsic β-IFN overcame apparent defects in IFN-mediated pathways in MiaPaCa2 cells conferring VSV resistance. In contrast, β-IFN decreased cell viability in Panc 3.27 cells, suggesting intact antiviral mechanisms. VSV-treated xenografts exhibited reduced tumor growth relative to controls in both MiaPaCa2 (1423 ± 345% versus 164 ± 136%; P < 0.001) and Panc 3.27 (979 ± 153% versus 50 ± 56%; P = 0.002) tumors. Significant lymphocytic infiltration was seen in M51R-VSV–treated Panc 03.27 xenografts.

Conclusions

Inhibition of VSV endocytosis and intact IFN-mediated defenses are responsible for M51R-VSV resistance in pancreatic adenocarcinoma cells. M51R-VSV treatment appears to induce antitumor cellular immunity in vivo, which may expand its clinical efficacy.

Introduction

Vesicular stomatitis virus (VSV) is among several oncolytic viruses currently being developed as anticancer therapies. VSV, the prototypical member of the family Rhabdoviridae, is a negative-stranded RNA virus whose genome encodes for five proteins: nucleocapsid (N), polymerase proteins (L and P), surface glycoprotein (G), and peripheral matrix protein (M). VSV is a potently cytolytic virus that selectively replicates in cancer cells that have downregulated their antiviral responses [1]. The selectivity of VSV for cancer cells can be enhanced by introducing mutations in the M protein, such as in the M51R variant of VSV (M protein mutant vesicular stomatitis virus [M51R-VSV]), which contains a single arginine for methionine amino acid substitution at position 51 in the M protein [2], [3]. The mutant M protein has a decreased ability to inhibit host cell antiviral mechanisms. As a result, normal cells are able to resist M51R-VSV infection [4], [5] by mounting antiviral defenses, such as interferon (IFN)-mediated antiviral signaling. In contrast, many cancer cells remain susceptible to M51R-VSV infection because they possess defects in antiviral pathways [2], [6]. Several reports have shown that M51R-VSV is more selective for tumor cells and causes oncolysis in a variety of cancer types, including prostate cancer [7], breast cancer [8], glioblastoma [9], colorectal cancer [5], malignant melanoma [10], and neuroendocrine tumors [11]. Several VSV variants have been developed that are similarly selective for cancers with defective antiviral responses [1], and one of these is currently in a phase 1 clinical trial for treatment of hepatocellular carcinoma (http://clinicaltrials.gov/show/NCT01628640).

Although these preliminary reports are encouraging, VSV is not universally oncolytic in all tumor subtypes, and significant variation in VSV sensitivity exists, even among cancers from the same anatomical site [6]. For example, both VSV-resistant and VSV-sensitive cell lines have been described in colorectal cancer [5], prostate cancer [7], breast cancer [8], malignant melanoma [10], malignant mesothelioma [12], and bladder cancer [13]. Based on the available data, VSV resistance may be as high as 36% in pancreatic adenocarcinoma and has been observed in cells from both primary and metastatic sites [14], [15]. Preserved intact antiviral mechanisms are thought to confer resistance in VSV-resistant cell lines, but additional mechanisms may also contribute to resistance [5], [7], [14], [15], [16].

Oncolytic virus therapy is a particularly attractive strategy for treatment of cancers for which current therapies are ineffective. As such, despite advances in many other malignancies, pancreatic adenocarcinoma remains a significant therapeutic challenge. The 5-y relative survival rate for patients with pancreatic cancer is 6%—the lowest among all cancers [17]. Clinical outcomes are so poor because most pancreatic cancer patients present with locally advanced or metastatic disease, and pancreatic adenocarcinoma is largely resistant to traditional systemic treatments. Clearly, innovative and effective therapies are critical to improving clinical outcomes in patients with pancreatic cancer.

In the work presented herein, we confirm the results of Murphy et al. [14] and Moerdyk-Schauwecker et al. [15] and extend their finding by analyzing the oncolytic effects of VSV in a panel of additional pancreatic adenocarcinoma cells with significant variability in their VSV susceptibility. Using microarray gene analysis, we explored the genetic differences between VSV-sensitive and VSV-resistant cell lines and thereby hypothesized that differences in IFN signaling and viral endocytosis are key molecular determinants of VSV susceptibility. In support of these hypotheses, we showed that resistant cells are capable of blocking VSV infection during the early stages of viral replication and possess intact IFN responses. We also found that the integrity IFN signaling can explain the VSV susceptibility seen in sensitive cell lines. In a murine xenograft model, we found that tumors from both sensitive and resistant cells responded to intratumoral M51R-VSV treatment. Histologic examination of treated tumor suggests that adaptive cellular immunity contributes to the oncolysis of the in vitro resistant xenografts. Collectively, these data establish that oncolytic VSV is a viable therapeutic option for pancreatic adenocarcinoma. More specifically, it identifies two significant molecular mechanisms of VSV resistance and provides a framework for further research into the immunologic responses to VSV.

Section snippets

Cells and viruses

Panc 1, MiaPaCa2, BxPC3, Panc 03.27, and Panc 10.05 cell lines were obtained from the American Type Culture Collection and were grown in DMEM (Panc 1 and MiaPaCa2; Manassas, VA) or RPMI 1640 (BxPC3, Panc 03.27, and Panc 10.05) supplemented with various additives depending on the cell line according to American Type Culture Collection's specifications. The recombinant VSV viruses, rwt-VSV and M51R-VSV, were isolated from infectious VSV complementary DNA clones, and virus stocks were prepared

Variable susceptibility of pancreatic adenocarcinoma cells to VSV

The oncolytic activity of VSV in pancreatic adenocarcinoma was evaluated using a panel of five cell lines (Panc 1, MiaPaCa2, BxPC3, Panc 03.27, and Panc 10.05). Susceptibility to VSV was evaluated using either wild type or M protein mutant virus (rwt-VSV and M51R-VSV, respectively). Comparing the two viruses reveals the effect of host cell responses to VSV because rwt-VSV suppresses, whereas M51R-VSV induces host cell responses [2]. At low MOIs, a small percentage of cells are initially

Discussion

The results presented here are consistent with those of Murphy et al. [14], who found significant heterogeneity in VSV susceptibility among 13 pancreatic cancer cells and described similar results in terms of IFN responsiveness [14]. Given the aggressiveness of pancreatic adenocarcinoma and its insensitivity to traditional chemotherapy, it is encouraging that most cell lines tested to date are sensitive to VSV. Our results support previous findings that VSV-sensitive cancer cells possess

Conclusion

M51R-VSV is a viable option for the future treatment of pancreatic adenocarcinoma. The integrity of IFN-mediated antiviral mechanisms explains VSV sensitivity, although there is evidence that different defects exist at various stages of IFN signaling that inhibit the cells' ability to resist VSV oncolysis. Although VSV-resistant pancreatic cancer cells appear to possess intact IFN-mediated pathways, these cells also block the early stages of viral replication likely by inhibiting viral

Acknowledgment

This work was supported from the National Cancer Institute (J.S.) by grant number K08-CA131482, Robert Wood Johnson Foundation Harold Amos Faculty Development Award (J.S.) by grant number 63527, National Institute of Allergy and Infectious Diseases (D.L.) by grant number R01-AI32983, and the Bradshaw Surgical Resident Research Endowment (A.B.).

The authors thank Hermina Borgerink (Department of Comparative Medicine, Wake Forest School of Medicine) for staining of tissue sections and Lou Craddock

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