title: Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection.
abstract: BACKGROUND
  In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms--e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV).
  
  
  METHODS
  Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice.
  
  
  RESULTS
  Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis.
  
  
  CONCLUSIONS
  Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.
authors.affiliation: Department of Biochemistry, Biocenter, University of Wuerzburg, D-97074 Wuerzburg, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Weibel
authors.forename: Viktoria
authors.initials: V
authors.lastname: Raab
authors.forename: Yong A
authors.initials: YA
authors.lastname: Yu
authors.forename: Andrea
authors.initials: A
authors.lastname: Worschech
authors.forename: Ena
authors.initials: E
authors.lastname: Wang
authors.forename: Francesco M
authors.initials: FM
authors.lastname: Marincola
authors.forename: Aladar A
authors.initials: AA
authors.lastname: Szalay
mesh_terms: D000818:Animals; D001808:Blood Vessels / Q000473:pathology / Q000503:physiopathology; D002199:Capillary Permeability / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D007962:Leukocytes / Q000378:metabolism / Q000473:pathology; D008325:Mammary Neoplasms, Experimental / Q000473:pathology / Q000628:therapy* / Q000821:virology*; D051379:Mice; D008819:Mice, Nude; D050130:Oncolytic Virotherapy / Q000379:methods*; D050504:Oncolytic Viruses / Q000502:physiology; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D014183:Transplantation, Heterologous; D014616:Vaccinia virus / Q000502:physiology*; D014664:Vasodilation
chemical_list: D019408:Platelet Endothelial Cell Adhesion Molecule-1