The Study On Synergistic Antitumor Effect Of Radiofrequency Ablation Combinated With Cytokine-induced Killer Cells

Radiofrequency ablation(RFA) as common minimally invasive treatmentcan increase the exposure of tumor cell epitopes and promote the synthesis of heat shock protein and inflammatory cytokines to effectively activate antitumor immune response.But that antitumor immune response can not prevent recurrence and metastasis.Cytokine-induced killer(CIK) cells have been applied on many malignancies as adoptive immunotherapy.CIK cells are ex vivo expanded T lymphocytes endowed with potent MHC-independent antitumor activity.The important factor influencing effect of CIK cells is whether immune effector cells can reach the target organ, and directly contact with tumor cells, thereby effectively killing tumor cells.So we assume when CIK cells are used at right time after RFA,immune response of RFA can promote CIK cellsimmigrating to tumor site and proliferation which can augment immune response induced by RFA.Ultimately the synergistic antitumor effect of combination RFA with cytokine-induced killer cells can be obtained.This study assesses antitumor effect of radiofrequency ablation combinated with cytokine-induced killer cells communication treatment from preclinical research to clinical trail.Part I The Preclinical study on synergistic antitumor effect of radiofrequency ablation combinated withcytokine-induced killer cellsObjective The preclinical research aimed to evaluate the antitumor immunity of the combination treatment with radiofrequency ablation(RFA) and cytokine-induced killer cells(CIK).Methods CIK cells were got from spleen cells of tumor-bearing mouse which were culturedex vivo. Mice were inoculated with B16 melonoma or CT26 colon cancer cell line on bilateral flanks, and randomly divided into four groups in which RFA for tumor right flank, CIK cell infusion, RFA plus CIK and no intervention was administrated, respectively.The growth curve of the left tumor and survival curve of mice were recorded. The amount and subset of the lymphocytes infiltratinginto the tumor on the left flank were analyzed by flow cytometry in B16 tumor-bearing mice. In addition, the number and phenotype of adoptive CIK migrating into the tumor were tested. CXCL10 protein in distant tumor was distected at 1,3,6,9 and 12 days after RFA by ELISA while Real-Time PCR was performed to test level of CXCL10 m RNA in distant tumor after 3 and 12 days after RFA. C57/BL6 Cxcl10 KO mice were used to detect the role of CXCL10 in RFA to stimulate anti-tumor immune response and promote the migration of CIK cells.The relevance of the antitumor effect and CIK cells infusion time interval after RFA was evaluated.Results CIK cells highly expressed chemokine receptor CXCR3,CXCR4 and CCR5.A short-lived inhibition of tumor growth was observed in mice treated with RFA or CIK alone. Combination treatment with RFA and CIK significantly inhibited the growth of tumors on contralateral flank. The median survival of mice treated with RFA and CIK was 42±3.3d, which was significant longer than that in untreated mice(20.5±2.2d), RFA alone(26±2.2d), CIK alone(31.5±2.2d)(P<0.001). The frequency of immune cells, including CD8+, CD4+T, NK(CD3-NK1.1+) and NKT(CD3+ NK1.1+), infiltrating into the contralateral tumor was significantly increased in mice treated with RFA plus CIK as comparing to mice treated with RFA and CIK alone. An elevated CD8+/Treg(CD4+Fox P3+) ratio and decreased accumulation of myeloid-derived suppressor cells(MDSC) were observed in RFA plus CIK group. Compared to mice treated with surgery plus CIK, the number of adoptive CIK cells migrating into the contralateral tumor was increased in mice treated with RFA plus CIK. In addition,the expressions of Ki-67, inducible costimulator(ICOS) and Granzyme B on adoptive CIK cells were upregulated in RFA plus CIK-treated mice. The level of chemokines CXCL10 in the distant tumor was raised dynamically after RFA.The synergistic anti-tumor effect was observed in a very short period of time after RFA while in a long period of time after RFA, there were no synergistic antitumor effect. CXCL10 knockout weakened RFA-induced antitumor immune reactions and thus attenuated the migration of CIK cells. CIK cells infusion at 3 days after RFA could produce synergic effect while CIK cells infusion at 12 days after RFA could notproduce synergic effect.Conclusion Combination treatment with RFA and CIK induced a boosted immune response in tumor microenvironment out of ablation zone and resulted in a synergistic anti-tumor effect.RFA promoted adoptive CIK cells migrating into a distant tumor and enhanced the proliferation and activity of intratumoral CIK cells. RFA stimulated anti-tumor immune response, and promoted the migration of CIK cells dependent on chemokine CXCL10. But the interval time of CIK cells infusion after RFA influenced the antitumor effect which may be relevant to change of level of chemokine in tumor microenvironment out of ablation zone after RFA.Part II The clinical study of radiofrequency ablation combined with cytokine-induced killer cells treating colorectal liver metastasesObjective This phase II, non-randomized clinical trial was to determine the efficacy and safety of the combination of radiofrequency ablation(RFA) and cytokine-induced killer(CIK) cells transfusion for patients with colorectal liver metastases(CRLMs).Methods A total of 60 eligible patients with CRLMs were enrolled and divided into Group A(RFA alone, n = 30) and Group B(RFA plus CIK, n = 30), and followed enzyme-linked immunosorbent spot(ELISPOT) assay was performed in 8 patients with CEA > 50ng/m L pre-RFA and 7 days post-RFA and CIK treatment, respectively.Results The median PFS times of Group A and Group B were 18.5 months and 23 months, respectively. The 2-year progression-free rates were 26.7% in Group A and 41.9% in Group B, respectively. The 3-year progression-free rates were 13.3% in Group A and 20.3% in Group B, respectively. There was a statistically significant difference between the PFS times(P = 0.0336). The median OS time was 43 months in Group A, and not reached in Group B. The 3-year survival rates were 64.6% in Group A and 81.0% in Group B, respectively(P = 0.1187). Among the 8 patients with CEA > 50ng/m L, 6 had increase of circulating CEA-specific T cells after RFA(P = 0.010). After CIK cell therapy, the number of CEA-specific T cells increased in all the 8 patients comparing with that pre-treatment(P = 0.001) and in 7 patients comparing with that post-RFA(P = 0.028).Conclusion We firstly confirm the combination of RFA and CIK cell boosts CEA-specific T cell response and shows to be an efficacious and safe treatment modality for patients with CRLMs.To summarize, this study investigated the synergistic antitumor effect of radiofrequency ablation combined with CIK cells in preclinical experiments and clinical research. This study proved that Combination treatment with RFA and CIK induced a boosted immune response in tumor microenvironment out of ablation zone and resulted in a synergistic anti-tumor effect.RFA promoted adoptive CIK cells migrating into a distant tumor and enhanced the proliferation and activity of intratumoral CIK cells. RFA stimulated anti-tumor immune response, and promoted the migration of CIK cells dependent on chemokine CXCL10. But the interval time of CIK cells infusion after RFA influenced the antitumor effect which may be relevant to change of level of chemokine in tumor microenvironment after RFA. The combination of RFA and CIK cells showed to be an efficacious and safe treatment modality for patients with CRLMs,augmented CEA-specific T cell response.