miR-193b-Regulated Signaling Networks Serve as Tumor Suppressors in Liposarcoma and Promote Adipogenesis in Adipose-Derived Stem Cells.

Well-differentiated and dedifferentiated liposarcomas (WDLS/DDLS) account for approximately 13% of all soft tissue sarcoma in adults and cause substantial morbidity or mortality in the majority of patients. In this study, we evaluated the functions of miRNA (miR-193b) in liposarcoma in vitro and in vivo Deep RNA sequencing on 93 WDLS, 145 DDLS, and 12 normal fat samples demonstrated that miR-193b was significantly underexpressed in DDLS compared with normal fat. Reintroduction of miR-193b induced apoptosis in liposarcoma cells and promoted adipogenesis in human adipose-derived stem cells (ASC). Integrative transcriptomic and proteomic analysis of miR-193b-target networks identified novel direct targets, including CRK-like proto-oncogene (CRKL) and focal adhesion kinase (FAK). miR-193b was found to regulate FAK-SRC-CRKL signaling through CRKL and FAK. miR-193b also stimulated reactive oxygen species signaling by targeting the antioxidant methionine sulfoxide reductase A to modulate liposarcoma cell survival and ASC differentiation state. Expression of miR-193b in liposarcoma cells was downregulated by promoter methylation, resulting at least in part from increased expression of the DNA methyltransferase DNMT1 in WDLS/DDLS. In vivo, miR-193b mimetics and FAK inhibitor (PF-562271) each inhibited liposarcoma xenograft growth. In summary, miR-193b not only functions as a tumor suppressor in liposarcoma but also promotes adipogenesis in ASC. Furthermore, this study reveals key tyrosine kinase and DNA methylation pathways in liposarcoma, some with immediate implications for therapeutic exploration. Cancer Res; 77(21); 5728-40. ©2017 AACR.

Tunneling nanotubes: an alternate route for propagation of the bystander effect following oncolytic viral infection.

Tunneling nanotubes (TNTs) are ultrafine, filamentous actin-based cytoplasmic extensions which form spontaneously to connect cells at short and long-range distances. We have previously described long-range intercellular communication via TNTs connecting mesothelioma cells in vitro and demonstrated TNTs in intact tumors from patients with mesothelioma. Here, we investigate the ability of TNTs to mediate a viral thymidine kinase based bystander effect after oncolytic viral infection and administration of the nucleoside analog ganciclovir. Using confocal microscopy we assessed the ability of TNTs to propagate enhanced green fluorescent protein (eGFP), which is encoded by the herpes simplex virus NV1066, from infected to uninfected recipient cells. Using time-lapse imaging, we observed eGFP expressed in infected cells being transferred via TNTs to noninfected cells; additionally, increasing fluorescent activity in recipient cells indicated cell-to-cell transmission of the eGFP-expressing NV1066 virus had also occurred. TNTs mediated cell death as a form of direct cell-to-cell transfer following viral thymidine kinase mediated activation of ganciclovir, inducing a unique long-range form of the bystander effect through transmission of activated ganciclovir to nonvirus-infected cells. Thus, we provide proof-of-principle demonstration of a previously unknown and alternative mechanism for inducing apoptosis in noninfected recipient cells. The conceptual advance of this work is that TNTs can be harnessed for delivery of oncolytic viruses and of viral thymidine kinase activated drugs to amplify the bystander effect between cancer cells over long distances in stroma-rich tumor microenvironments.

Recombinant vaccinia virus GLV-1h68 is a promising oncolytic vector in the treatment of cholangiocarcinoma.

Although early stage cholangiocarcinoma (CC) can be cured by surgical extirpation, the options for treatment of advanced stage CC are very few and suboptimal. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) is a promising new strategy to treat human cancers. The ability of oncolytic VACV GLV-1h68 to infect, replicate in, and lyse three human CC cell lines was assayed in vitro and in subcutaneous flank xenografts in athymic nude mice. In this study, we have demonstrated that GLV-1h68 effectively infects and lyses three CC cell lines (KMC-1, KMBC, and KMCH-1) in vitro. Expression of the viral marker gene ruc-gfp facilitated real-time monitoring of infection and replication. Furthermore in athymic nude mice, a single dose of GLV-1h68 significantly suppressed tumor growth. The treatment was well tolerated in all animals. Recombinant VACV GLV-1h68 has significant oncolytic ability against CC both in vitro and in vivo. GLV-1h68 has the potential to be used clinically as a therapeutic agent against CC.

Oncolytic gene therapy with recombinant vaccinia strain GLV-2b372 efficiently kills hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) commonly presents at a late stage when surgery is no longer a curative option. As such, novel therapies for advanced HCC are needed. Oncolytic viruses are a viable option for cancer therapy owing to their ability to specifically infect, replicate within, and kill cancer cells. In this study, we have investigated the ability of GLV-2b372, a novel light-emitting recombinant vaccinia virus derived from a wild-type Lister strain, to kill HCC. METHODS: Four human HCC cell lines were assayed in vitro for infectivity and cytotoxicity. Viral replication was quantified via standard viral plaque assays. Flank HCC xenografts generated in athymic nude mice were treated with intratumoral GLV-2b372 to assess for tumor growth inhibition and viral biodistribution. RESULTS: Infectivity occurred in a time- and concentration-dependent manner with 70% cell death in all cell lines by day 5. All cell lines supported efficient viral replication. At 25 days after infection, flank tumor volumes decreased by 50% whereas controls increased by 400%. Tumor tissue demonstrated substantial GLV-2b372 infection at 24 hours, 48 hours, and 2 weeks. CONCLUSION: We demonstrate that GLV-2b372 efficiently kills human HCC in vitro and in vivo and is a viable treatment option for patients with HCC.

Gene therapy using therapeutic and diagnostic recombinant oncolytic vaccinia virus GLV-1h153 for management of colorectal peritoneal carcinomatosis.

BACKGROUND: Peritoneal carcinomatosis (PC) is a terminal progression of colorectal cancer (CRC). Poor response to cytoreductive operation and chemotherapy coupled with the inability to reliably track disease progression by the use of established diagnostic methods, make this a deadly disease. We examined the effectiveness of the oncolytic vaccinia virus GLV-1h153 as a therapeutic and diagnostic vehicle. We believe that viral expression of the human sodium iodide transporter (hNIS) provides both real-time monitoring of viral therapy and effective treatment of colorectal peritoneal carcinomatosis (CRPC). METHODS: Infectivity and cytotoxic effect of GLV-1h153 on CRC cell lines was assayed in vitro. Viral replication was examined by standard viral plaque assays. Orthotopic CRPC xenografts were generated in athymic nude mice and subsequently administered GLV-1h153 intraperitoneally. A decrease in tumor burden was assessed by mass. Orthotopic tumors were visualized by single-photon emission computed tomography/computed tomography after Iodine ((131)I) administration and by fluorescence optical imaging. RESULTS: GLV-1h153 infected and killed CRC cells in a time- and concentration-dependent manner. Viral replication demonstrated greater than a 2.35 log increase in titer over 4 days. Intraperitoneal treatment of orthotopic CRPC xenografts resulted in a substantial decrease in tumor burden. Infection of orthotopic xenografts was therapeutic and facilitated monitoring by (131)I-single-photon emission computed tomography/computed tomography via expression of hNIS in infected tissue. CONCLUSION: GLV-1h153 kills CRC in vitro effectively and decreases tumor burden in vivo. We demonstrate that GLV-1h153 can be used as an agent to provide accurate delineation of tumor burden in vivo. These findings indicate that GLV-1h153 has potential for use as a therapeutic and diagnostic agent in the treatment of CRPC.

Oncolytic immunotherapy using recombinant vaccinia virus GLV-1h68 kills sorafenib-resistant hepatocellular carcinoma efficiently.

BACKGROUND: Sorafenib is the standard systemic therapy for unresectable or recurrent hepatocellular carcinoma (HCC) but adds minimal increase in survival. Therefore, there is a great need to develop novel therapies for advanced or recurrent HCC. One emerging field of cancer treatment involves oncolytic viruses that specifically infect, replicate within, and kill cancer cells. In this study, we examined the ability of GLV-1h68, a recombinant vaccinia virus derived from the vaccine strain that was used to eradicate smallpox, to kill sorafenib-resistant (SR) HCC cell lines. METHODS: Four SR HCC cell lines were generated by repeated passage in the presence of sorafenib. Median inhibitory concentration was determined for all cell lines. The infectivity, viral replication, and cytotoxicity of GLV-1h68 were assayed for both parental and SR HCC cells. RESULTS: Infectivity increased in a time and concentration-dependent manner in all cell lines. All cell lines supported efficient replication of virus. No difference between the rates of cell death between the parental and SR cell lines was observed. CONCLUSION: Our results demonstrate that the oncolytic vaccinia virus GLV-1h68 kills both parental and SR HCC cell lines efficiently. This study indicates that patients who have failed treatment with sorafenib remain viable candidates for oncolytic therapy.

A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter.

BACKGROUND: Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with (99m)Tc pertechnetate scintigraphy and ¹²4I positron emission tomography (PET). METHODS: GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. (99m)Tc pertechnetate scintigraphy and ¹²4I microPET imaging were performed. RESULTS: GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK- 45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by (99m)Tc pertechnetate scintigraphy and ¹²4I microPET imaging 2 days after treatment. CONCLUSIONS: GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings.

An oncolytic vaccinia virus expressing the human sodium iodine symporter prolongs survival and facilitates SPECT/CT imaging in an orthotopic model of malignant pleural mesothelioma.

BACKGROUND: The purpose of this work was to examine the ability of an oncolytic vaccinia virus expressing the human sodium iodine transporter (hNIS) to provide real time monitoring of viral therapy and effective treatment of malignant pleural mesothelioma (MPM). METHODS: Infectivity and cytotoxic effects of GLV-1h153 on mesothelioma cell lines of all histologic subtypes were assayed in vitro. Viral replication was examined by standard viral plaque assay. Orthotopic MPM xenografts were generated in athymic nude mice, treated with intrapleural GLV-1h153, and assessed for effect on tumor burden and survival. Orthotopic tumors were also imaged on single photon emission computed tomography (SPECT)/computed tomography (CT) after (131)I administration. RESULTS: GLV-1h153-infected and killed all cell lines in a time- and concentration-dependent manner. Viral replication demonstrated a >2.5-log increase in titer over 4 days. Intrapleural treatment of orthotopic MPM xenografts resulted in a significant decrease in tumor burden 1 week after treatment and an improvement in survival. Infection of orthotopic xenografts was both therapeutic and facilitated monitoring by (131)I-SPECT/CT via expression of hNIS in infected tissue. CONCLUSION: Our results suggest that GLV-1h153 may be a promising therapeutic agent for MPM and warrants further investigation.

Irreversible electroporation ablation of the liver can be detected with ultrasound B-mode and elastography.

BACKGROUND: Irreversible electroporation (IRE) is a novel ablation technique that induces permanent membrane permeability and cell death. We are interested in ultrasound B-mode and elastography to monitor IRE ablation in the liver. METHODS: Yorkshire pigs underwent IRE ablation of the liver and were imaged with ultrasound B-mode and elastography. Histologic evaluation of cell death by triphenyltetrazolium chloride and hematoxylin and eosin staining was performed. RESULTS: Elastography showed that liver ablated by IRE exhibited increased tissue stiffness with a peak strain ratio of 2.22. The IRE lesion had a discrete border without bubble artifact, and the lesion size significantly correlated with area of cell death on histology. IRE ablation was unaffected by presence of large blood vessels or bile ducts. CONCLUSION: IRE ablation led to increased tissue stiffness that was detectable by elastography and indicative of cell death. Elastography may complement B-mode ultrasonography to monitor IRE ablation of the liver.

A vaccinia virus encoding the human sodium iodide symporter facilitates long-term image monitoring of virotherapy and targeted radiotherapy of pancreatic cancer.

UNLABELLED: To assess therapeutic response and potential toxicity of oncolytic virotherapy, a noninvasive, deep-tissue imaging modality is needed. This study aimed to assess the feasibility, parameters, and determining factors of serial imaging and long-term monitoring of virotherapy and the therapeutic response of pancreatic cancer xenografts treated with a vaccinia virus carrying the human sodium iodide symporter GLV-1h153. METHODS: Pancreatic cancer xenografts (PANC-1) in nude mice were treated systemically or intratumorally with GLV-1h153 and serially imaged using (124)I PET at 1, 2, 3, and 5 wk after viral injection. Signal intensity was compared with tumor therapeutic response and optical imaging, and tumors were histologically analyzed for morphology and the presence of virus. Autoradiography was performed using technetium-pertechnetate and γ-scintigraphy to assess determining factors for radiouptake in tumors. Finally, the enhanced therapeutic effect of combination therapy with GLV-1h153 and systemic radioiodine was assessed. RESULTS: GLV-1h153 successfully facilitated serial long-term imaging of virotherapy, with PET signal intensity correlating to tumor response. GLV-1h153 colonization of tumors mediated radioiodine uptake at potentially therapeutic doses. Successful radiouptake required the presence of virus, adequate blood flow, and viable tissue, whereas loss of signal intensity was linked to tumor death and necrosis. Finally, combining systemically administered GLV-1h153 and (131)I led to enhanced tumor kill when compared with virus or (131)I alone (P < 0.01). CONCLUSION: GLV-1h153 is a promising oncolytic agent for the treatment, long-term imaging, and monitoring of therapeutic response in a xenograft model of pancreatic cancer. GLV-1h153 provided insight into tumor biologic activity and facilitated enhanced tumor kill when combined with systemic targeted radiotherapy. These results warrant further investigation into parameters and potential synergistic effects of combination therapy.