Utilizing Tumor Hypoxia to Enhance Oncolytic Viral Therapy in Colorectal Metastases

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Abstract
Objective: To determine the effects of hypoxia-induced ribonucleotide reductase (RR) production on herpes oncolytic viral therapy. Summary Background Data: 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.