Anti-tumor Effects Of Granulocyte-macrophage Colony-stimulating Factor Combined With Metronomic Paclitaxel In The Treatment Of Lewis Lung Carcinoma Transplanted In Mice

Objective:Metronomic chemotherapy in combination with immunotherapy is an attractive method in cancer treatment.The purpose of the presentstudywastoinvestigatetheanti-tumoreffectof granulocyte-macrophage colony-stimulating factor?GM-CSF?in combination with metronomic paclitaxel on Lewis lung carcinoma transplanted in mice and its associated anti-tumor mechanisms.Methods:A model of Lewis lung cancer subcutaneous transplanted tumor was established.As long as the tumor volume was about 100 mm³?About 12 days after inoculation?,tumor-bearing mice were randomized to six groups?18 for each group?:control group?intraperitoneal injection with 0.9%saline solution at the dose of 0.1 ml every day,d1-d10?,metronomic paclitaxel group?intraperitoneal injection with paclitaxel at the dose of 3 mg/kg every other day,d1,d3,d5,d7,d9?,conventional paclitaxel group?intraperitoneal injection with paclitaxel at the dose of 15 mg/kg at the first day,d1?,GM-CSF group?subcutaneous injection with GM-CSF at the dose of 5?g/kg every day,d1-d10?,GM-CSF+conventional paclitaxel group?subcutaneous injection with GM-CSF at the dose of 5?g/kg every day,d1-d10+intraperitoneal injection with paclitaxel at the dose of 15 mg/kg at the first day,d1?,GM-CSF+metronomic paclitaxel group?subcutaneous injection with GM-CSF at the dose of 5?g/kg every day,d1-d10+intraperitoneal injection with paclitaxel at the dose of 3 mg/kg every other day,d1,d3,d5,d7,d9?.In the present study,tumor volume and survival were recorded dynamically.The day after the end of the treatment,the peripheral blood of mice was obtained by removing the eyeball,and using a cytometer to test the white blood cell count.Micro ¹⁸F-FDG PET/CT(¹⁸F-Fluorodeoxyglucose positron emission tomography/computed tomography)was used to obtain tumor SUVmax values,HE?hematoxylin and eosin?staining was used to detect important organs?heart,liver,kidney,lung?pathological change.Microvessel density and tumor cell proliferation were determined by immunohistochemistry,while apoptosis was examined by TUNEL?Terminal deoxynucleotidyl transferase-mediated nick end labeling?assay.The number and maturation of dendritic cell in spleen and tumor tissue were determined by flow cytometry.Results:The tumor in the control group and GM-CSF group grew faster than that in the other groups,and the difference between the two groups was not significant,suggesting that GM-CSF alone could not effectively inhibit tumor growth.Metronomic paclitaxel group,conventional paclitaxel group,GM-CSF+conventional paclitaxel group and GM-CSF+metronomic paclitaxel group all showed inhibition of tumor growth?P<0.05 vs control group?.In addition,GM-CSF+metronomic paclitaxel group showed the smallest tumor volume among all groups?all P<0.05?.The tumor volume inhibition rate in the metronomic paclitaxel group,conventional paclitaxel group,GM-CSF+conventional paclitaxel group and GM-CSF+metronomic paclitaxel group were 39.91%,37.04%,48.03%and 66.85%,separately.GM-CSF in combination with metronomic paclitaxel effectively prolonged the survival of tumor-bearing mice with a median survival time of 48 days?P<0.05,vs the other groups?.When paclitaxel was administrated by metronomic chemotherapy,the white blood cell was?10.14+0.99?×10⁹/L,while the conventional paclitaxel group was?8.37+1.09?×10⁹/L,and the difference between the two groups was significant?P<0.05?.These results suggested that metronomic chemotherapy was able to reduce the level of leukocyte reduction caused by conventional chemotherapy.The white blood cell in the control group was?12.18±1.11?×10⁹/L,while GM-CSF+metronomic paclitaxel group was?12.87±1.04?×10⁹/L,and no significant difference was observed between the two groups,which indicating that the combined regimen could effectively reduce white blood cell reduction.In addition,HE stained sections of important targeted organs including heart,liver,lung and kidney of each group were observed and that no obvious pathological changes were found.To sum up,GM-CSF combined with metronomic paclitaxel is an effective and safe treatment regimen.Metronomic paclitaxel could effectively reduce the microvessel density of tumor tissues?P<0.05,compared to control group?,indicating that metronomic chemotherapy had an antiangiogenic effect.When GM-CSF combined with metronomic paclitaxel,the microvessel density was further reduced,and the difference was significant compared to the other five groups?P<0.05?.Conventional paclitaxel could reduce the number and maturity of dendritic cells in the spleen and tumor tissues compared to the control group?P<0.05?.In contrast to this result,metronomic paclitaxel could increase the number and maturity of dendritic cells both in the spleen and tumor tissues compared to the control group?P<0.05?.GM-CSF combined with metronomic paclitaxel could further increase the number of dendritic cells and their maturity in the spleen and tumor tissue,and the differences were significant compared to the control group,metronomic paclitaxel group,conventional paclitaxel group,GM-CSF group and GM-CSF+conventional paclitaxel group?P<0.05?.Conclusion:1.GM-CSF combined with metronomic paclitaxel could effectively inhibit tumor growth and prolong the survival;2.GM-CSF combined with metronomic paclitaxel could effectively avoid white blood cell reduction without important targeted organs damage,indicating that this combination therapy was safe;3.GM-CSF combined with metronomic paclitaxel could induce anti-tumor immune effect through recruiting tumor dendritic cells,the enhanced anti-tumor effect mechanisms of this combination therapy were associated with the antiangiogenic effect and apoptosis induction.