灵芝多糖微乳诱导结直肠癌肿瘤相关巨噬细胞M1极化及其联合PD-1抑制剂协同抗肿瘤研究

Ganoderma lucidum polysaccharide microemulsion induces M1 repolarization of colorectal cancer-associated macrophages and combined synergistic anti-tumor therapy with PD-1 inhibitor

目的:验证灵芝多糖微乳诱导结直肠癌肿瘤相关巨噬细胞(TAMs)M1极化能力,以及联合PD-1抑制剂协同抗肿瘤的可行性。方法:采用“一步成乳法”制备灵芝多糖微乳(GLP-M),并对其进行理化性质表征。借助RAW264.7细胞考察GLP-M诱导巨噬细胞极化的能力;通过Transwell小室法考察GLP-M处理巨噬细胞后对肿瘤细胞增殖、凋亡的影响,以及GLP-M处理肿瘤细胞后对巨噬细胞极化的影响;考察GLP-M诱导巨噬细胞产生活性氧(ROS)行为。建立荷MC38鼠源肠癌细胞的皮下移植瘤模型,以生理盐水和灵芝多糖为阴性对照和剂型对照组,设置GLP-M、PD-1抑制剂以及GLP-M+PD-1联合给药组;PD-1抑制剂每3天尾静脉注射1次,给药2次,其余制剂每天灌胃1次,给药6次。考察治疗期间肿瘤生长曲线、体质量变化、生存时间、病理切片等。结果:GLP-M粒径为(58.3±2.1) nm, Zeta电位为(-23.5±1.8) mV,多分散系数(PDI)为0.221±0.003,包封率为(82.5±3.2)%,TEM图显示GLP-M形态圆整、分散均匀。GLP-M干预IL-4诱导的RAW264.7细胞后CD86的表达量升高(33.2±1.9)%, ROS水平升高(39.4±2.5)%,差异均具有统计学意义。在RAW264.7与MC38细胞共培养模型中,GLP-M可以显著提高肿瘤细胞的增殖抑制率和凋亡率,促进巨噬细胞的CD86表达。体内抗肿瘤实验显示:GLP-M、PD-1以及GLP-M+PD-1的肿瘤抑制率分别为(44.3±4.2)%、(51.3±2.7)%和(63.7±4.3)%。GLP-M+PD-1组45 d生存率最高(25%),且该组肿瘤切片结果显示坏死明显,细胞增殖被显著抑制。结论:GLP-M可能通过干预TAMs M1极化的机制提高与PD-1抑制剂等免疫治疗协同抗结直肠癌治疗效能。

OBJECTIVE To verify the feasibility of Ganoderma lucidum polysaccharide microemulsion in M1 repolarization of colorectal cancer-associated macrophages(TAMs) and combined synergistic anti-tumor therapy with PD-1 inhibitor.METHODS Ganoderma lucidum polysaccharide microemulsion(GLP-M) was prepared by one-step emulsion method, and then characterized comprehensively. RAW264.7 cells were used to test the ability of GLP-M in inducing macrophages polarization and releasing reactive oxygen species(ROS). Transwell chambers-mediated cell coculture model was used to assay the proliferation and apoptosis of MC38 cells when RAW264.7 cells were treated with GLP-M, and the polarization of macrophages when MC38 cells were treated with GLP-M. MC38 tumor-bearing mice model was established to validate the advantages in anticancer efficacy. Saline and Ganoderma lucidum polysaccharide were used as negative control group and formulation control group, respectively, and GLP-M, PD-1 inhibitor and GLP-M+PD-1 combined administration groups were set as treatment groups. PD-1 inhibitor was injected into the tail vein once every three days for two times, and the other preparations were administered by intragastric administration once every day for 6 times. The tumor growth curve, body weight change, survival period, and pathological section during and after treatment were investigated.RESULTS The particle size of GLP-M was(58.3±2.1) nm, the Zeta potential was(-23.5±1.8) mV, the polydispersity index(PDI) was 0.221±0.003, and the encapsulation efficiency was(82.5±3.2)%. After treated with GLP-M, the expression of CD86 and released ROS in IL-4-pretreated RAW264.7 cells increased by(33.2±1.9)% and(39.4±2.5)%, respectively, which were both statistically higher than those in the PBS group. In the co-culture model of RAW264.7 and MC38 cells, GLP-M could significantly increase the anti-proliferation and apoptosis rate of tumor cells, and promote the expression of CD86 in macrophages. As for in vivo anti-tumor efficacy, the tumor inhibition rate of GLP-M, PD-1 and GLP-M+PD-1 were(44.3±4.2)%,(51.3±2.7)% and(63.7±4.3)%, respectively. The GLP-M+PD-1 group had the highest 45-day survival rate(25%), which resulted in the significant tumor necrosis and cell-proliferation blockade.CONCLUSION GLP-M can induce colorectal cancer TAMs M1 polarization. Such a feature might be used in combined therapy with PD-1 inhibitor to improve the efficacy of anti-colorectal cancer treatment.