Background: A hallmark of cancer is the presence of an immunosuppressive tumor microenvironment (TME). Immunosuppressive M2 macrophages (MΦs) in the TME facilitate escape from immune surveillance and promote tumor growth; therefore, TME-induced immunosuppression is a potent immunotherapeutic approach to treating cancer.Methods: Cancer cell-secreted proteins were detected by using liquid chromatography–mass spectrometry (LC-MS). Neutralizing antibodies (nAbs) were used to assess which proteins were involved in MΦs polarization and differentiation. The protein–protein interaction was characterized using co-immunoprecipitation and immunofluorescence assays. Cancer-secreted heat shock protein 70 (Hsp70) protein was quantified using an enzyme-linked immunosorbent assay (ELISA). MΦ polarization and tumor growth were assessed in vivo with subcutaneous LLC-GFP tumor models and toll-like receptor 2 (TLR2) knockout mice; in vitro assessments were conducted using TLR2 knockout and both LLC-GFP and LN227 lentiviral-mediated knockdown (KD) cells.Results: Cancer cells released a secreted form of Hsp70 that acted on MΦ TLR2 to upregulate Mer receptor tyrosine kinase (MerTK) and induce MΦ M2 polarization. Hsp70 nAbs led to a reduction in CD14 expression by 75% in THP-1 cells in response to Gli36 EMD-CM. In addition, neutralizing TLR2 nAbs resulted in a 30% and 50% reduction in CD14 expression on THP-1 cells in response to MiaPaCa-2 and Gli36 exosome/microparticle-depleted conditioned media (EMD-CMs), respectively. Hsp70, TLR2, and MerTK formed a protein complex. Tumor growth and intra-tumor M2 MΦs were significantly reduced upon cancer cell Hsp70 knockdown and in TLR2 knockout mice.Conclusions: Cancer-secreted Hsp70 interacts with TLR2, upregulates MerTK on MΦs, and induces immunosuppressive MΦ M2 polarization. This previously unreported action of secreted Hsp70 suggests that disrupting the Hsp70-TLR2-MerTK interaction could serve as a promising immunotherapeutic approach to mitigate TME immunosuppression in solid cancers.
背景：肿瘤微环境（TME）的免疫抑制特征是癌症的标志之一。TME中的免疫抑制性M2型巨噬细胞（MΦs）有助于肿瘤逃避免疫监视并促进其生长；因此，针对TME诱导的免疫抑制是一种有效的癌症免疫治疗策略。 方法：采用液相色谱-质谱联用技术（LC-MS）检测癌细胞分泌蛋白。通过中和抗体（nAbs）评估参与MΦs极化和分化的蛋白质。利用免疫共沉淀和免疫荧光实验分析蛋白质间相互作用。采用酶联免疫吸附试验（ELISA）定量检测癌细胞分泌的热休克蛋白70（Hsp70）。通过皮下LLC-GFP肿瘤模型和Toll样受体2（TLR2）基因敲除小鼠进行体内MΦ极化及肿瘤生长评估；体外实验则使用TLR2敲除细胞、LLC-GFP及LN227慢病毒介导的敲低（KD）细胞进行评估。 结果：癌细胞释放分泌型Hsp70，通过作用于MΦ的TLR2上调Mer受体酪氨酸激酶（MerTK）并诱导MΦ向M2型极化。Hsp70中和抗体使THP-1细胞在Gli36 EMD-CM刺激下的CD14表达降低75%。此外，TLR2中和抗体分别使THP-1细胞在MiaPaCa-2和Gli36外泌体/微粒耗竭条件培养基（EMD-CMs）刺激下的CD14表达降低30%和50%。Hsp70、TLR2与MerTK形成蛋白质复合体。在癌细胞Hsp70敲低及TLR2基因敲除小鼠中，肿瘤生长和瘤内M2型MΦs均显著减少。 结论：癌细胞分泌的Hsp70与TLR2相互作用，上调MΦs的MerTK表达，并诱导免疫抑制性M2型MΦ极化。这一尚未报道的分泌型Hsp70作用机制表明，破坏Hsp70-TLR2-MerTK相互作用可能成为缓解实体瘤TME免疫抑制的潜在免疫治疗策略。
肿瘤(癌症)患者之家