真菌来源的蛋白结合多糖通过抑制血管生成细胞因子的产生和促使肿瘤相关巨噬细胞从 M2 型向 M1 型转化，从而破坏乳腺癌细胞与巨噬细胞之间的串扰。

Protein-Bound Polysaccharides from Fungus Disrupt the Crosstalk Between Breast Cancer Cells and Macrophages through Inhibition of Angiogenic Cytokines Production and Shifting Tumour-Associated Macrophages from the M2 to M1 Subtype.

机构信息

Department of Immunology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, Torun, Poland,

Department of Immunology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, Torun, Poland.

出版信息

Cell Physiol Biochem. 2020 Jun 20;54(4):615-628. doi: 10.33594/000000244.

DOI:10.33594/000000244

PMID:32559360

Abstract

BACKGROUND/AIMS: The tumour microenvironment is rich in multiple cells that influence cancer development. Among them, macrophages are the most abundant immune cells, which secrete factors involved in carcinogenesis. Since protein-bound polysaccharides (PBP) from the fungus are believed to inhibit the growth of cancers, in the present study, we investigated whether these PBP influence crosstalk between triple-negative 4T1 breast cancer cells and RAW 264.7 macrophages.

METHODS

4T1 cells were cultured in conditioned media (CM) collected after: stimulation of the macrophages with PBP (CM-PBP) or incubation of non-treated macrophages (CM-NT). A co-cultured model of both cell lines was also employed to investigate the crosstalk between the cells. Cell viability was measured using the MTT assay. The levels of cytokines and chemokines were determined by ELISA methods. Commercial assay kits were used to assess the activity of both arginase 1 and inducible nitric oxide synthase (iNOS) and the level of cell migration.

RESULTS

The results revealed that CM-NT promotes proliferation and migration of 4T1 cells, and increases the secretion of pro-angiogenic factors (VEGF, MCP-1) by cancer cells. In contrast, CM-PBP inhibits 4T1 cell growth and migration, decreases the secretion of pro-angiogenic factors (VEGF, MCP-1) and upregulates the production of pro-inflammatory mediators (IL-6, TNF-α) with certain anti-tumoral properties Moreover, PBP-treated CM significantly decreases the level of M2 macrophage markers (arginase 1 activity, IL-10 and TGF-β concentrations), but upregulates iNOS activity and IL-6 and TNF-α production, which are M1 cell markers.

CONCLUSION

The results suggest that PBP suppress the favourable tumour microenvironment by inhibiting the crosstalk between 4T1 cells and macrophages through the regulation of production of angiogenic and inflammatory mediators, and modulating the M1/M2 macrophage subtype.

摘要

背景/目的：肿瘤微环境富含多种影响癌症发展的细胞。其中，巨噬细胞是最丰富的免疫细胞，它们分泌参与致癌的因子。由于真菌来源的蛋白结合多糖（PBP）被认为能抑制癌症的生长，因此在本研究中，我们研究了这些 PBP 是否影响三阴性 4T1 乳腺癌细胞与 RAW 264.7 巨噬细胞之间的串扰。

方法

将 4T1 细胞培养在条件培养基（CM）中，该培养基是在：巨噬细胞用 PBP 刺激（CM-PBP）或未处理的巨噬细胞孵育（CM-NT）后收集的。还采用了两种细胞系的共培养模型来研究细胞之间的串扰。使用 MTT 测定法测量细胞活力。通过 ELISA 方法测定细胞因子和趋化因子的水平。使用商业检测试剂盒评估精氨酸酶 1 和诱导型一氧化氮合酶（iNOS）的活性以及细胞迁移水平。

结果

结果表明，CM-NT 促进了 4T1 细胞的增殖和迁移，并增加了癌细胞分泌的促血管生成因子（VEGF、MCP-1）。相比之下，CM-PBP 抑制了 4T1 细胞的生长和迁移，减少了促血管生成因子（VEGF、MCP-1）的分泌，并上调了具有某些抗肿瘤特性的促炎介质（IL-6、TNF-α）的产生。此外，用 PBP 处理的 CM 显著降低了 M2 巨噬细胞标志物（精氨酸酶 1 活性、IL-10 和 TGF-β 浓度）的水平，但上调了 iNOS 活性和 IL-6 和 TNF-α 的产生，这些都是 M1 细胞标志物。