Figure 4: In vivo effects of combination therapy using transcutaneous CO2 treatment and DOX on human MFH. Three days after MFH cell implantation, mice were randomly assigned to four groups: control group, CO2 group, DOX group, and combination (CO2 + DOX) treatment group. Each treatment was performed twice weekly for 2 weeks, and the tumor volume (A) and body weight (B) in mice were monitored until the end of the treatment. (A) At day 7, we observed a significant decrease in tumor volume in the combination group compared with the control group (*p < 0.05). At 14 d, the tumor volume in the CO2 or DOX groups was significantly decreased compared with the control group (**p < 0.01 for CO2 or DOX groups versus control group). The tumor volume in the combination group was significantly reduced compared with the other three groups (**p < 0.01). (B) We observed significant body weight loss in both DOX and combination treated groups compared to groups without DOX treatment (*p < 0.05, Figure 4B). Mice treated with transcutaneous CO2 did not develop any observable negative side effects. (C) Immunohistochemical staining of HIF-1a in treated tumors. The expression of HIF-1a (Red arrows) was observed in tumors in control and DOX groups, but not in CO2 and combination groups.