Combination Of Bevacizumab And Adoptive Cytokine Induced Killer Cells Therapy In Non Small Cell Lung Cancer

Background and objective:Cytokine-induced killer cells (CIK cells) are a heterogeneous population of cells generated from peripheral blood mononuclear cells (PBMC), which share phenotypic and functional properties with both natural killer (NK) and T cells. CIK cells therapy, as an adoptive immunotherapy with strong anti-tumor activity in vitro, represents a promising approach for the treatment of a broad array of malignant tumors. However, clinical trials in CIK cells therapy did not show more noticeable improvement as anticipated in cure rates or long term survival. Possible explanations are that abnormal tumor vasculature and hypoxic microenvironment may highly limit the therapeutic benefits of CIK cells therapy. We hypothesized that antiangiogenesis therapy could enhance the antitumor efficacy of CIK cells by normalizing tumor vasculature and modulating tumor hypoxic microenvironment. The purpose of this study was to investigate the antitumor effect and mechanism of antiangiogenesis combined with CIK cells therapy in animal models.Materials and Methods:1. A549 and SPC-A1 lung aderiocarcinoma xenograft were established subcutaneously in BALB/c nude mice respectively and then randomly divided into 4 groups. The detailed groups were as follows:(1) Group NS, treated with normal saline, as control group; (2) Group CIK, treated with CIK cells alone; (3) Group BEV, treated with bevacizumab alone; (4) Group BEV+CIK, treated with bevacizumab followed by transfusion of CIK cells. The dose of bevacizumab was intravenous injection of 5 mg/kg at dl, intravenous transfusion of CIK cells at d3 (1×106 cells per dose in a total volume of 100μl). Tumor volumes were recorded every other day throughout the experiment, and tumor growth curves were recorded.2. The structure of blood vessel of A549 lung carcinoma showed by Evans blue through intravital microscopy examination.3. Flow cytometry and immunohistochemistry was performed to observe the intratumoral infiltrated CIK cells after tumor samples were harvested.4. After treated by the combination therapy, the hypoxyprobeTM-1 kit and mouse IgGl monoclonal antibody were used to detected hypoxia area in tumor tissue. Continuous section slides were made and stained for hypoxia and tumor infiltrated CIK cells respectively to illustrate the relationship between them.Results:1. In A549 lung cancer models, tumor volumes growth curve showed thatCIK cells monotherapyhad nosignificant antitumor activity compared with NS group (968.52±93.42 vs.1158.51±81.18mm3,p=0.185>0.05). Bevcizumab limited tumor growth compared with NS controls,and reachedstatistical significance (647.17±37.35 vs.1158.51±81.18 mm3, p=0.021<0.05). The combination therapy (441.46±18.92mm3) hadsignificant antitumor activity compared with other groups. EA=0.44, EB=0.16, EA+B=0.66 and q=1.25>1.15, which confirms the assessment ofsynergism effect. In SPC-A1 lung cancer models, tumor volumes growth curve showed CIK cells monotherapy also had no significant antitumor activity compared with NS group (2822.64±637.26 vs.3876.55±853.46 mm3, p=0.105>0.05). Bevcizumab limited tumor growth compared with NS controls, and reached statistical significance (1877.73±539.93 vs.3876.55±853.46 mm3, p=0.005<0.05). The combination therapy had significant antitumor activity compared with other groups. EA=0.51, EB=0.26, EA+B=0.74 and q=1.17>1.15, which confirms the assessment of synergism effect.2. A549 tumor-bearing mice were treated with bevacizumab (5mg/kg, i.v.).24 hours later, intravital microscopy was performed to clarify tumor vessel normalization. Tumor vessels were less tortuous and less dilated in the bevacizumab group compared with NS group.3. Bevacizumab could decrease hypoxia area, as determined by pimonidazole in A549 lung carcinoma compared with the group NS.4. The study showed that hypoxia impeded the recruitment of CIK cells into tumor.5. Bevacizumab augments the homing of CIK cells in the tumor in A549 tumor-bearing mice.Conclusion:1. Combination of bevacizumab and CIK cells therapy synergistic inhibits the growth of lung cancer in mice models.2. Bevacizumab could normalize tumor vasculature and decrease tumor hypoxic area, and then enhanced more CIK cell infiltrated into tumor.