Enteroviral mediated oncolysis of cancer: evaluation of efficacy and obstacles to therapeutic success

Research Doctorate - Doctor of Philosophy (PhD)A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy