The Mechanism Of Interleukin 12 Combined With Traditional Chemotherapy In The Treatment Of Lung Cancer

Lung cancer is one of the most commonly diagnosed cancers and the leading cause of cancer deaths globally,causing more than 1.4 million deaths annually.Paclitaxel and Cisplatin doublet chemotherapy is one of the first-line treatments for patients with NSCLC and SCLC.Although the chemotherapy exhibits certain efficacy,drug-related toxic effects such as vomiting,hair loss,weight loss cannot be ignored and survival remains dismal.Therefore,novel therapeutic approaches,such as immunotherapies,are urgently needed.Interleukin 12(IL-12)is a pro-inflammatory cytokine that was originally identified as an NK cell stimulatory factor(NKSF)and a cytotoxic lymphocyte maturation factor.Physiologically,IL-12 has been implicated in the stimulation of NK and T cell proliferation,the enhancement of NK and CD8+T cell cytolytic activity and the induction of cytokine production,particularly Interferon gama(IFN-?).Furthermore,IL-12 promotes the differentiation of T helper 1(Thl)cells,thereby bridging innate and adaptive immunity,which might reflect the antitumor immunity induced through IL-12.The combination of immunity and chemotherapy forms an technology,which is expected to provide new solutions for tumor treatment.We assumed that tumor cells would be killed by low-dose chemotherapy,and then IL-12 would be used to stimulate the immune system in vivo to further exert the immune attack effect,thereby improving the anti-tumor effect.Based on this principle,we constructed mouse lung cancer models,treated with paclitaxel(PTX)and cisplatin(CDDP),and then stimulated with IL-12.The results are as follows:1.Mouse orthotopic lung cancer models were successfully constructedIn order to simulate the real situation of human lung cancer,we abandoned the commonly used method of subcutaneous transplantation in tumor models and chose to establish orthotopic lung cancer models.Through multiple attempts and explorations,two different mouse orthotopic lung cancer models were successfully established by tail vein injection of CT26 tumor cells and intrapleural injection of LLC tumor cells.The results showed that the lung surface of the two models were filled with tumor nodules after 20 days of inoculation of tumor cells,and the model of LLC tumor cells combined with pleural effusion.No tumor nodules were found in other organs.Pathological section results showed that normal lung tissue was infiltrated by tumor cells.The mouse lung cancer model can simulate the real situation of lung cancer.2.IL-12 shows positive therapeutic effect on lung cancerBy administering different drug combinations to tumor-bearing mice,we found that PTX+CDDP+recombinant mouse interleukin-12(rmIL-12)or rmIL-12 alone could significantly improve the survival time of tumor bearing mice.The number of tumor nodules in rmIL-12 or PTX+CDDP+rmIL-12 treatment group was significantly reduced by pathological section technique.At the same time,the tumor cell lines stably expressing luciferase reporter gene was constructed,and the imaging observation of tumor bearing mice was carried out by using the method of in vivo animal imaging.The results showed that compared with the control group,the lung tumor nodules of the mice were significantly reduced after the PTX+CDDP+rmIL-12 combination treatment3.IL-12 activates the immune system and rapidly stimulates NK cells to secrete IFN-?We found that spleens of tumor-bearing mice treated with IL-12 or PTX+CDDP+IL-12 were markedly enlarged and spleens of tumor-bearing mice treated with PTX+CDDP alone were slightly shrunken compared with the control.The mononuclear cell numbers from the spleens of tumor-bearing mice treated with IL-12 or PTX+CDDP+IL-12 were significantly increased compared with the PTX+CDDP-treated mice and the control.The serum IFN-? levels in tumor-bearing mice treated with IL-12 or PTX-CDDP+IL-12 were much higher than those in the control.The expression of intracellular IFN-? using flow cytometry were examined.Interestingly,the production of IFN-? from NK cells was markedly and rapidly increased following IL-12 or PTX+CDDP+IL-12 treatment.These data suggest that IL-12 can rapidly activate NK cells to produce IFN-?.4.NK cells and IFN-? are essential for the anti-tumor effects of IL-12Nfil3-/-,CD4-/-,CD8-/-,IFN-?-/-.and WT mice were injected with LLC tumor cells to establish lung cancer models.The tumor-bearing mice were treated with IL-12 or NS and tumor growth was monitored through whole body imaging.Remarkably,tumor growth in Nfil3-/-mice or in IFN-?-/-mice was significantly faster than that in the WT,CD4-/-and CD8-/-mice.Furthermore,IL-12 treatment inhibited tumor growth in the WT mice,but not in the Nfil3-/-and IFN-?-/-mice.These results were also confirmed by survival analyses,showing that tumor-bearing WT mice exhibited significant long-term survival compared with tumor-bearing Nfil3-/-and IFN-?-/-mice after treatment with IL-12.These data indicate that NK cells and IFN-? play essential roles in the anti-tumor effect of IL-12 in the lung cancer models.5.IL-12 via mediation of IFN-y suppresses tumor angiogenesisIFN-y-/-mice and WT mice were tumor-beared and then administered rmIL-12,PTX+CDDP,and vehicle control.The results of immunofluorescence experiments showed that rmIL-l2 greatly inhibited the expression of vascular markers CD31,CD 105 and VEGFR3 in lung tumors,but in tumor-bearing IFN-y-/-mice,even the administration of rmIL-12 could not inhibit the expression of CD31,CD 105 and VEGFR3.However,the expressions of CD31,CD 105 and VEGFR3 in the PTX+CDDP treatment group were not inhibited.The above results indicated that IL-12 via mediation of IFN-? suppresses tumor angiogenesis.In summary,the combination of IL-12 and traditional chemotherapy can induce obvious antitumor effect and prolong the survival time of mice with lung cancer.This mechanism is dependent on the rapid production of IFN-y in activated NK cells,resulting in the inhibition of tumor angiogenesis.Thus,our research might provide a new strategy for the treatment of lung cancer.