title: Utilizing tumor hypoxia to enhance oncolytic viral therapy in colorectal metastases.
abstract: OBJECTIVE
  To determine the effects of hypoxia-induced ribonucleotide reductase (RR) production on herpes oncolytic viral therapy.
  
  
  BACKGROUND
  Hypoxia is a common tumor condition correlated with therapeutic resistance and metastases. Attenuated viruses offer a unique cancer treatment by specifically infecting and lysing tumor cells. G207 is an oncolytic herpes virus deficient in RR, a rate-limiting enzyme for viral replication.
  
  
  METHODS
  A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested by luciferase assay. 10xHRE was cloned upstream of UL39, the gene encoding the large subunit of RR (10xHRE-UL39). CT26 murine colorectal cancer cells were transfected with 10xHRE-UL39, incubated in hypoxia (1% O2) or normoxia (21% O2), and infected with G207 for cytotoxicity assays. CT26 liver metastases, with or without 10xHRE-UL39, were created in syngeneic Balb/C mice (n = 40). Livers were treated with G207 or saline. Tumors were assessed and stained immunohistochemically for G207.
  
  
  RESULTS
  10xHRE increased luciferase expression 33-fold in hypoxia versus controls (P < 0.001). In normoxia, 10xHRE-UL39 transfection did not improve G207 cytotoxicity. In hypoxia, G207 cytotoxicity increased 87% with 10xHRE-UL39 transfection versus nontransfected cells (P < 0.001). CT26 were resistant to G207 alone. Combining 10xHRE-UL39 with G207 resulted in a 66% decrease in tumor weights (P < 0.0001) and a 65% reduction in tumor nodules (P < 0.0001) versus G207 monotherapy. 10xHRE-UL39-transfected tumors demonstrated greater viral staining.
  
  
  CONCLUSIONS
  Hypoxia-driven RR production significantly enhances viral cytotoxicity in vitro and reduces tumor burden in vivo. G207 combined with RR under hypoxic control is a promising treatment for colorectal cancer, which would otherwise be resistant to oncolytic herpes virus alone.
authors.affiliation: Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Maura
authors.initials: M
authors.lastname: Reinblatt
authors.forename: Richard H
authors.initials: RH
authors.lastname: Pin
authors.forename: Howard J
authors.initials: HJ
authors.lastname: Federoff
authors.forename: Yuman
authors.initials: Y
authors.lastname: Fong
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D002470:Cell Survival; D015179:Colorectal Neoplasms / Q000473:pathology / Q000628:therapy* / Q000821:virology; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D000860:Hypoxia; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000556:secondary* / Q000628:therapy*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity