Justin W. Ady

Intercellular communication in malignant pleural mesothelioma: properties of tunneling nanotubes.

Malignant pleural mesothelioma is a particularly aggressive and locally invasive malignancy with a poor prognosis despite advances in understanding of cancer cell biology and development of new therapies. At the cellular level, cultured mesothelioma cells present a mesenchymal appearance and a strong capacity for local cellular invasion. One important but underexplored area of mesothelioma cell biology is intercellular communication. Our group has previously characterized in multiple histological subtypes of mesothelioma a unique cellular protrusion known as tunneling nanotubes (TnTs). TnTs are long, actin filament-based, narrow cytoplasmic extensions that are non-adherent when cultured in vitro and are capable of shuttling cellular cargo between connected cells. Our prior work confirmed the presence of nanotube structures in tumors resected from patients with human mesothelioma. In our current study, we quantified the number of TnTs/cell among various mesothelioma subtypes and normal mesothelial cells using confocal microscopic techniques. We also examined changes in TnT length over time in comparison to cell proliferation. We further examined potential approaches to the in vivo study of TnTs in animal models of cancer. We have developed novel approaches to study TnTs in aggressive solid tumor malignancies and define fundamental characteristics of TnTs in malignant mesothelioma. There is mounting evidence that TnTs play an important role in intercellular communication in mesothelioma and thus merit further investigation of their role in vivo.

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.

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.

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.

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 viral therapy for pancreatic cancer: current research and future directions

The development of targeted agents and chemotherapies for pancreatic cancer has only modestly affected clinical outcome and not changed 5-year survival. Fortunately the genetic and molecular mechanisms underlying pancreatic cancer are being rapidly uncovered and are providing opportunities for novel targeted therapies. Oncolytic viral therapy is one of the most promising targeted agents for pancreatic cancer. This review will look at the current state of the development of these self-replicating nanoparticles in the treatment of pancreatic cancer.

Introduction to Robotic Surgery

The history of robotic surgery started in the 1980s with the PUMA 560 robot. It was used in 1985 to increase the precision of neurosurgical biopsies. That same robot was then used later to perform a transurethral resection of the prostate. This lead to the development of PROBOT, a robot designed specifically for transurethral resection of the prostate. Around the same time another robot called ROBODOC was designed to machine the femur with greater precision in hip replacement surgery and eventually went on to become the first surgical robot approved by the FDA.

Abstract 929: Tunneling nanotube conduits facilitate the bystander effect after oncolytic viral infection

Background: Oncolytic viruses have come to the forefront of cancer therapeutics following FDA approval for treatment of metastatic melanoma in 2015. Efficacy of viral therapy is enhanced by the bystander effect, a cellular phenomenon that amplifies the effects of the virus following activation of the prodrug ganciclovir (GCV) by virally-expressed thymidine kinase (TK) and intercellular spread of GCV via gap junctions. In the complex and stroma-rich tumor microenvironment, gap junctions may not completely account for cell-to-cell communication. Tunneling nanotubes (TNTs) are a novel and recently characterized alternative form of direct cell-to-cell communication in the tumor matrix. TNTs are fine, long, F actin-based cell extensions that serve as short and long-range conduits for efficient transfer of cellular cargo. Here, we investigate the ability of TNTs to mediate a TK-based bystander effect after oncolytic viral infection and administration of GCV. Methods: We infected 3 mesothelioma cell lines with NV1066, a mutant replication-competent strain of herpes simplex virus-1 (HSV-1) that encodes viral eGFP and viral TK. Confocal microscopy and time-lapse imaging were performed 12-36 hours later. A modified Transwell assay was used to separate infected cells from uninfected cells to assess TNT propagation of eGFP-tagged NV1066. GCV was added to infected cells in the top chamber to assess TNT propagation of TK-activated GCV. Apoptosis was measured using TUNEL assay in the bottom chamber to quantify the extent of the bystander effect. Results: Confocal microscopy demonstrated effective intercellular transfer of GFP-tagged virus between cells via TNTs prior to oncolysis. Quantification of TUNEL-positive cells at 48 hours indicated that addition of NV1066 to the top chamber resulted in 33% of the initially uninfected cells in the bottom chamber dying by 48 hours. The addition of GCV to virally infected cells in the top chamber significantly increased apoptosis in recipient cells in the bottom chamber, from 33% to 71%, producing a 2.3-fold increase in cell killing attributed to TNT transfer of viral TK-activated GCV (p = 0.007). Thus, TNTs were shown to transfer viral TK-activated GCV to non-infected cells, leading to cell death via a long-range form of the bystander effect. Conclusions: Here we demonstrate that TNTs provide a previously unknown and alternative mechanism for the bystander effect in which viral TK-activated GCV is transferred via TNTs and induces apoptosis in non-infected recipient cells. Citation Format: Emil Lou, Justin Ady, Venugopal Thayanithy, Kelly Mojica, Joshua Carson, Prassanna Rao, Yuman Fong. Tunneling nanotube conduits facilitate the bystander effect after oncolytic viral infection. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 929.