ABSTRACT
Objective To assess the therapeutic effect of plum-blossom needle tapping combined with topical imiquimod immunotherapy on cutaneous squamous cell carcinoma (SCC) in SKH-1 mice,and to explore the immunological mechanism.Methods A total of 40 SKH-1 mice with ultraviolet light-induced cutaneous SCC were randomly and equally divided into 4 groups:control group receiving no treatment,plum-blossom needle group receiving plum-blossom needle tapping on all the tumors once a day,imiquimod group topically treated with imiquimod 5％ cream at a dose of 1.2 g/kg once a day,combination group firstly treated with plum-blossom needle tapping on all the tumors,and after the stop of bleeding topically treated with imiquimod 5％ cream at the same dose as the imiquimod group once a day.All the mice were treated for 30 days.Morphological changes of tumors in all groups were photographed and recorded every day.The tumor size was measured once every three days,and changes of total tumor volume and survival rate of the mice were compared among the 4 groups.At the end of treatment,tunor tissues were resected,and histopathological changes were compared among the 4 groups.Real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure the mRNA expression of interferon-α (IFN-α),IFN-β,interleukin-1β (IL-1β),tumor necrosis factor-α (TNF-o) and IL-12 in tumor tissues.Results In the combination group,tumors on the back of mice grew slowly,and some even regressed.However,tumors grew fast in the control group,plum-blossom needle group and imiquimod group,and grew more slowly in the plum-blossom needle group and imiquimod group than in the control group.Before the treatment,there was no significant difference in the total tumor volume among the 4 groups (F =0.90,P ＞ 0.05).After 24-day treatment,the total tumor volume significantly differed among the 4 groups (F =5.16,P ＜ 0.05).The LSD-t test showed that the total tumor volume significantly decreased in the combination group compared with the control group (P ＜ 0.01),but no significant difference was observed among the other groups (P ＞ 0.05).Log-rank test revealed that survival curves significantly differed among the 4 groups (x2 =8.32,P ＜ 0.05).The survival rate was significantly higher in the combination group than in the control group (x2 =4.62,P =0.03),but did not differ between the plum-blossom needle group or imiquimod group and the control group or combination group (all P ＞ 0.05).Histopathological examination showed atypical cells arranged closely,a large number of tumor cells and some keratin pearls in the control group and plumblossom needle group,few dead tumor cells in the imiquimod group,and plenty of dead tumor cells,mild nuclear atypia and increased keratinization in the combination group.qRT-PCR revealed that the relative mRNA expression levels of IFN-α,IFN-β,IL-12,IL-1β and TNF-α were significantly higher in the combination group than those in the control group,plum-blossom needle group and imiquimod group (P ＜ 0.05).The imiquimod group showed significantly higher mRNA expression of IL-1β than the control group (P ＜ 0.01),but no significant differences were observed among the other groups (P ＞ 0.05).Conclusion Plum-blossom needle tapping can effectively enhance the anti-SCC activity and immunological effects of imiquimod in SKH-1 mice.
ABSTRACT
Objective To evaluate the antineoplastic effect of laser immunotherapy in mice with multiple cutaneous squamous cell carcinoma (CSCC).Methods Normal immunocompetent hairless SKH-1 mice were consecutively irradiated by ultraviolet rays to establish mouse models of multiple CSCC.Then,20 CSCC-bearing mice were randomly and equally divided into 4 groups:laser group irradiated with an 808-nm near-infrared laser (1 W/cm2,10 minutes) once every two weeks for a total of 3 sessions,laser immunotherapy group irradiated with an 808-nm nearinfrared laser as above and topically treated with imiquimod cream once a day for 3 days before and after each session of irradiation,imiquimod group treated with imiquimod cream as in the laser immunotherapy group,and control group receiving no treatment.The volume of tumors was calculated,and their morphology as well as the survival of mice were observed after the treatment.The t test and Mantel-Cox log-rank test were used to compare the volume of tumors on the dorsum and survival rates of mice,respectively,on days 27 and 60 after the start of treatment.Another 12 CSCC-bearing mice were randomly and equally divided into the 4 groups mentioned above,and tumor tissues were resected from these mice for histopathological examination on day 5 after the first treatment.Results On day 27,the volume of tumors significantly increased in the control group and imiquimod group (both P ＜ 0.05),but experienced no significant increase in either of the other two groups compared with that before the start of treatment (both P ＞ 0.05).Moreover,the volume of tumors in the control group was slightly larger than that in the imiquimod group (P ＞ 0.05),but significantly larger than that in the laser group and laser immunotherapy group (both P ＜ 0.05).The volume of tumors in the laser group increased,and was significantly larger than that in the laser immunotherapy group on day 60 (P ＜ 0.05).Mice all died within 40 days in the control group,and within 50 days in the imiquimod group;one mouse died on days 52 and 53 separately,and the other mice all survived longer than 60 days in the laser group;no mice died within 60 days in the laser immunotherapy group.By day 60,the survival time of mice was slightly longer in the imiquimod group (P ＞ 0.05),significantly longer in the laser group and laser immunotherapy group compared with the control group (both P ＜ 0.05),but similar between the laser group and laser immunotherapy group (P ＞ 0.05).Histopathological examination showed that 5-day topical application of imiquimod cream did not cause death of tumor cells,but numerous tumor cells died in the laser group and laser immunotherapy group.Multiple inflammatory cells were observed around the tumors,and the number of the inflammatory cells was obviously larger in both the laser group and laser immunotherapy group,especially in the latter group,compared with the control group.Conclusion Laser immunotherapy is a new effective and safe treatment for CSCC in mice,and has potential value especially in the treatment of multiple or metastatic CSCC.