- Title
- Investigation of coxsackievirus A21 as a potential treatment for pancreatic cancer
- Creator
- Smith, Lincoln
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Coxsackievirus A21 (CVA21) is a human specific, non-enveloped, 28 nm icosahedral, group C enterovirus with a single strand positive sense RNA genome. CVA21 causes common cold symptoms upon infection in humans. CVA21 utilises intercellular adhesion molecule 1 (ICAM-1) and decay accelerating factor (DAF) for cell surface binding and cell entry of target cells. Cancer cells commonly overexpress ICAM-1 making them susceptible to CVA21 infection. Upon invasion of a host cell, viral progeny is produced through hijacking cellular machinery. Host cell lysis occurs, and virus is able to infect nearby and distant cells through systemic spread. Furthermore, cancer cell oncolysis may lead to recognition of specific tumour cell epitopes by the host’s immune system and generation of an anti-tumour immune response. CVA21 is a potentially efficacious anti-cancer agent for multiple cancer types in preclinical studies and is undergoing investigation in clinical trials as treatments for a range of cancer types with promising early stage results. Investigation of CVA21 as a potential treatment for pancreatic cancer in clinical trials will hopefully be initiated in the near future. Pancreatic cancer (PC) is the most lethal of all cancer types. PC has the worst survival rates of any cancer type and incidence continues to increase annually. Even though the genomic aberration profiles and molecular pathogenesis of PC are well documented there are still no early detection mechanisms for PC. Pancreatic cancer is characterised by PC cells supported by a dense desmoplastic reaction, composed primarily of pancreatic stellate cells (PSCs), that is highly tumour promoting, with rapid progression. Currently, there are no treatments that can adequately control the disease. Furthermore, present treatment options provide unsatisfactory toxicity and quality of life to suffering patients. Efficacious treatment options are rapidly needed. The oncolytic virus, CVA21, has great potential as a treatment for PC. The capacity to directly lyse tumour cells as well as prompt an adaptive anti-tumour immune response while proving to exert minimal levels of host adverse events makes CVA21 an ideal anti-cancer agent. Investigation of CVA21 as a potential treatment for PC in the preclinical setting was the major aim of this study. The key hypothesis for this project was that CVA21 would be an effective anti-cancer agent against pancreatic cancer due to high expression of viral entry receptors, ICAM-1 and/or DAF on PC and PSCs. Furthermore, CVA21 would have enhanced anti-tumour activity in combination with chemotherapeutic or immunotherapeutic agents. Such questions were addressed through specific in vitro tissue culture and immunohistochemical analyses, and in vivo mouse models. Initially the study determined the expression levels of CVA21 cell entry receptors, ICAM-1 and DAF, on the surface of PC and PSCs and compared these levels to normal pancreatic cells. Evaluation of expression levels were deduced through quantitative real-time PCR and flow cytometric analyses of a panel of PC, PSCs, and normal pancreatic ductal epithelial cells. Furthermore, ICAM-1 cell surface expression on ex vivo patient PC tissues was monitored via immunohistochemical analyses. The second aim of the project was to screen the sensitivity of human PC cells and PSCs to CVA21 in comparison to normal pancreatic cells through viral infectivity and viral growth kinetic assays. Overall, findings presented demonstrate CVA21 to be a potentially efficacious and suitable treatment for PC. ICAM-1 and DAF, gene, and cell receptor expression showed that PC and PSCs, in general, overexpressed ICAM-1 and/or DAF compared to normal pancreatic ductal epithelial cells. A convincing cell lysis effect of CVA21 on PC and PSCs was observed. Thus, the hypothesis that CVA21 will be a potential effective anti-cancer agent against PC due to overexpression of ICAM-1 and/or DAF was confirmed. Moreover, minimal oncolytic activity of CVA21 on normal pancreatic ductal epithelial cells was observed and is promising when considering translation to a clinical setting. Furthermore, CVA21 was observed to target both PC and PSCs alone, and when co-cultured, implicating CVA21 as a strong candidate for oncolysis of PC cells and the major stromal desmoplastic reaction observed in native PC. Finally, observation of a stepwise increase in ICAM-1 cell surface expression over the progression of PC from immunohistochemically stained patient samples attested CVA21 as a specific and potential candidate for the treatment of PC. The third aim of the study was to determine the synergistic or antagonistic relationship between CVA21 and conventional chemotherapy on human PC and PSCs, compared to normal pancreatic cells. The sensitivity of the panel of pancreatic cell lines to gemcitabine was investigated through in vitro dose-response assays. Checkerboard assays employing the respective experimentally determined concentrations of CVA21 and gemcitabine required to reduce cell viability by 50% were titrated in combination against each cell line to observe the relationship. Computational analyses based off the Chou-Talalay method of drug calculation using the experimentally recorded values enabled the synergistic or antagonistic relationship between the two agents to be empirically calculated. The fourth aim of the study was to establish an orthotopic mouse model of human PC and investigate CVA21 as a treatment, alone, and in combination with conventional chemotherapy. In vivo investigations utilising an athymic nude mouse model of orthotopic PC generated from human Panc-1-luc cells was successfully conducted and the efficacy of CVA21 as a treatment for PC evaluated. Taken together, the results further enforced the hypothesis that CVA21 is a potential anti-cancer candidate against PC. Furthermore, the findings clearly illustrated the significant high-grade toxicity of gemcitabine as a treatment for PC. Dose-response assays of gemcitabine on a panel of PC, pancreatic stellate, and normal pancreas cells indicated normal pancreatic ductal epithelial cells to be incredibly susceptible, PSCs to be almost entirely refractive, and varying degrees of sensitivity on PC cells. Combination checkerboard assays of CVA21 and gemcitabine on a panel of cell lines revealed a synergistic relationship between the two agents when calculated from accurate experimental data. Thus, the hypothesis that CVA21 in combination with a chemotherapeutic agent would have a synergistic effect was validated in some instances. However, in many examples the calculated concentrations of gemcitabine were not clinically achievable. Moreover, there was a lack of sensitisation of stellate cells to gemcitabine, and increased toxicity towards normal pancreas cells. Therefore, caution should be displayed if using CVA21 and gemcitabine combination treatment in the clinical setting. The athymic nude mouse model of orthotopic PC generated from Panc-1-luc cells demonstrated CVA21 to be an efficacious and well tolerated treatment for PC when administered intratumourally. Neutralising antibodies for CVA21 were profiled over the course of the study and found to increase after consecutive treatments with CVA21. Furthermore, there was a statistically significant increase in survival time of mice treated with CVA21 compared to saline, or gemcitabine treated mice (P = 0.0021). Gemcitabine proved ineffective at controlling PC either as a single agent, or when combined with CVA21. Moreover, the profound toxic effects of gemcitabine reduced the welfare of mice such that the majority had to be euthanised due to weight loss. Additionally, there was a diminishing effect of gemcitabine towards the anti-viral immune response generated towards CVA21 when considering the neutralising antibody profiles of CVA21 treated, and CVA21 plus gemcitabine treated mice. The final aim of this project was to establish an immune competent mouse model of orthotopic, human ICAM-1 expressing, PC and investigate CVA21 as a treatment, alone, and in combination with immunotherapeutic agents. Generation of the model proved difficult and due to time constraints a reliable and well characterised model was not finalised. As such, investigations of CVA21 in combination with immune checkpoint inhibitors were not undertaken. Findings from three pilot mouse models, indicated C57BL/6 mice were the optimal strain for generation of an orthotopic PC model that showed hallmark metastatic characteristics of native PC; for example, metastases to liver, spleen, and lung. Secondly, both the surgical procedures to generate orthotopic PC and administration of CVA21 were well tolerated by mice. Finally, immune cell recovery was observed late in pilot model #2 while tumours were maintained and progressed in C57BL/6 mice. In this model, mice were administered 200 µg/antibody/mouse three days prior to tumour inoculation to suppress NK1.1, CD4, and CD8 cells. In contrast, in pilot model #3, when mice were administered 50 µg/antibody/mouse three days prior to tumour inoculation, immune cell recovery occurred earlier in the model and subsequent spontaneous remission of tumours in mice was observed. In conclusion, the presented data indicate CVA21 as a potential anti-cancer treatment for pancreatic cancer that is generally well tolerated in several mouse models. Further investigations are required to overcome immune-competent animal model limitations and optimise experimental protocols.
- Subject
- coxsackievirus A21; CVA21; pancreatic cancer; oncolytic virotherapy
- Identifier
- http://hdl.handle.net/1959.13/1391435
- Identifier
- uon:33232
- Rights
- Copyright 2018 Lincoln Smith
- Language
- eng
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