Combination Of Argon-helium Cryoablation And In Vivo Loaded Dendritic Cell Vaccine For The Treatment Of Non-small Cell Lung Cancer

[Background]Release of tumor antigens following cryoablation makes the in vivo loading of dendritic cell vaccine feasible. The local cryoablated tissue is devided into the central necrosis zone, the periphery apoptosis zone and the normal tissue zone. Some studies have proved combined cryotherapy and intratumoral administration of dendritic cells （DCs） can enhance the antitumor immune response. While whether the administration site of DCs can affect the immune response remains unknown and it will be studied in the first part of this study.The immune adjuvant CpG-ODN has been proved to be efficient in enhancing the antitumor immune response in the protocol of co-administration of DCs and CpG-ODN. Immature DCs are potent in antigen uptake and are weak in antigen presentation. Mature DCs are potent in antigen presentation and are weak in antigen uptake. To capitalize on the antigen uptake function of DCs, the immature state of DCs should be maintained for a period of time until they uptake antigens in the preparation of dendritic cell vaccine. Whether the application chance of CpG-ODN can affect the antitumor-immune response needs to be further studied. This will be discussed in the second part of this study.Cryoablation is now mostly applied in the therapy of advanced stage NSCLC. Here, we prepared advanced stage Lewis lung cancer mouse models to evaluate the therapeutic effect of the combined protocol.Part Ⅰ Influence of Administration Site and CpG-ODN on the Homing of DCs[Objectives]To study the influence of administration site of DCs and CpG-ODN on the homing of DCs following cryoablation.[Methods]1、DCs were in vitro cultured and labeled by CFSE.2、DCs/DCs+CpG-ODN were administrated to the central zone, the periphery zone and the normal tissue zone respectively following cryoablation. DCs/DCs+CpG-ODN were administrated to the contralateral zone as control.3、CFSE labelled cells in the draining lymph nodes was assessed by flowcytometry （FCM）[Results]DCs were collected on the 6th day. CFSE-labeled DCs in the draining lymph nodes were tested by FCM. Number of DCs in the draining lymphonodes peaked on the third day. DCs/DCs+CpG-ODN administrated to the normal tissue area had a higher homing rate compared with other groups. CpG-ODN enhanced the homing rate of DCs.PartⅡ Effect of Application Chance of CpG-ODN on the in vivo Loaded DCs Vaccine Following Cryoablation of NSCLC[Objectives]To study the effect of application chance of CpG-ODN on the in vivo loaded DCs vaccine following cryoablation of NSCLC and select the best application chance of CpG-ODN.[Methods]The Lewis lung cancer （LLC）-bearing mice received cryoablation and injection of in vitro-cultured DCs into the peritumoral zone. Subsequently, CpG-ODN was administered to experimental animals 6 hours,12 hours, and 24 hours after DC injection. The mice in the control group received coadministration of DCs and CpG-ODN simultaneously. Therapeutic efects were evaluated by survival rates. The resistance to rechallenge of LLC cell was assessed by lung metastasis and in vitro cytotoxicity of splenocytes. Furthermore, T-cell subsets and multiple cytokines IL-4, IL-10, and-12; IFN-γ; TNF-α on the blood were assessed to elucidate the underlying mechanisms.[Results]Higher ratios of CD³⁺ CD⁴⁺ T and CD³⁺ CD⁸⁺ T cells and higher levels of IL-12, IFN-γ and TNF-α were found in the blood of the mice that received CpG-ODN therapy 12 h after DCs administration. The cytotoxicity potency of the splenocytes of these mice was signifcantly higher compared with the mice in other groups. Moreover, the mice receiving CpG-ODN therapy 12 h after DC injection showed signifcantly better resistance to rechallenge. Compared with the mice in other groups, the mice receiving CpG-ODN therapy 12 h after DC injection were superior in survival rates and antimetastatic effects.PartⅢ Therapeutic Effect of Combined Protocol of Cryoablation, DCs and CpG-ODN on the Advanced NSCLC[Objectives]To study the Therapeutic effect of combined protocol of cryoablation, DCs and CpG-ODN on the advanced NSCLC.[Methods]Advanced NSCLC models of C57BL/6 mouse were designed and the combined protocol of cryoablation+DCs+CpG-ODN （12h） was performed to the models. Survival, lung matastasis and distant matastasis were tested to evaluate the therapeutic effect with the cryoablation group as control.[Results]Advanced LLC-bearing mice that treated by the combined protocol, with a lower tumor volume of lung matastasis and distant matastasis, were improved in survival compared with those treated by cryotherapy.[Conclusion]Our study suggested that the therapeutic efficacy of the combined therapeutic protocol of cryoablation+DCs+CpG-ODN was closely associated with the administration site of DCs and application chance of CpG-ODN. Peritumoral zone was considered to be the best injection site and in situ administration of CpG-ODN 12 h after DCs injection is considered the optimum application.The combined protocol of cryoablation+DCs+CpG-ODN （12h） is effective for the treatment of advanced stage NSCLC, while the distant metastatic tumor can’t be eliminated by this protocol.