In vivo experiments for validating the feasibility of the technique using rat models with orthotopic lung tumors, with four different treatment groups (herpes simplex virus [HSV] thymidine kinase [TK]/ganciclovir [GCV] plus radiofrequency hyperthermia [RFH], RFH alone, HSV-TK/GCV alone, and phosphate-buffered saline). (a, b) X-ray and optical imaging showed a smaller increase of mean bioluminescence signals (red-yellow) in the combination therapy group compared with the other three groups (HSV-TK/GCV plus RFH, 1.29 [95% confidence interval {CI}, 0.51, 2.06] vs RFH, 2.26 [95% CI: 1.51, 3.01]; HSV-TK/GCV, 1.76 [95% CI: 1.20, 2.31]; phosphate-buffered saline, 2.94 [95% CI: 2.29, 3.58]; P < .001). Error bars indicate the standard deviation. Subsequent pathologic evaluation showed that the smallest tumor size occurred in the combination therapy group. (c) Western blotting analysis of tumor tissues was used to confirm significantly enhanced green fluorescent protein (GFP) gene expression in tumors treated with RFH compared with tumors without RFH treatment (GFP/HSV-TK plus RFH, 1.57 [95% CI: 1.08, 2.06] vs GFP/HSV-TK, 1.00 [95% CI: 1.00, 1.00]; P < .05; six animals per group). Error bar indicates the standard deviation. GAPDH = glyceraldehyde 3-phosphate dehydrogenase. * = P < .05, ** = P < .01, and *** = P < .001.