背景  近年来，肿瘤免疫治疗（Tumor immunotherapy）发展迅速，是目前最为热门的研究领域之一。它已成为继手术、放化疗和靶向治疗后的第四类肿瘤治疗方法，被认为是最有潜力治愈肿瘤的方法。其中，免疫检查点阻断疗法（Immune checkpoint blockage therapy）在肿瘤免疫治疗中发挥关键作用。常见的免疫检查点包括程序性死亡配体1（Programmed death-ligand 1，PD-L1）和其配体程序性死亡分子1（Programmed death 1，PD-1）、CD47及其受体信号调节蛋白α （signal-regulatoryprotein，SIRP-α）、β-半乳糖苷结合蛋白9（ Galectin-9）及其受体T细胞免疫球蛋白黏蛋白分子3（T cell immunoglobulin domain and mucin domain-3，TIM-3）等。表达在肿瘤细胞表面的PD-L1通过与T细胞表面PD-1结合抑制T细胞的正常功能；CD47是调节巨噬细胞吞噬功能的最有前景的靶点之一，位于肿瘤细胞表面的CD47与巨噬细胞表面的SIRPα相互作用时能够抑制巨噬细胞的吞噬活性。近年来一系列的临床研究表明，靶向PD-1/PD-L1和CD47的抗体类免疫检查点抑制剂（Immune checkpoint inhibitors，ICIs）能够显著提高病人的生存质量、延长生存期，但也存在治疗响应率低、价格昂贵、不能进入肿瘤内部等局限性。与抗体类药物相比，小分子药物在生产工艺、剂型设计和给药方式等方面都具有天然优势，研发能阻断PD-1/PD-L1、CD47/SIRPα之间相互作用的天然小分子化合物是下一代肿瘤免疫疗法的新方向。本研究中，我们对金丝桃苷下调非小细胞肺癌 （NSCLC）细胞中免疫检查点PD-L1的表达发挥抗肿瘤作用，以及染料木苷诱导结肠癌（CRC）细胞中c-Myc降解进而下调PD-L1和CD47水平发挥抗肿瘤作用进行了探究。

方法  第一部分研究中，以NSCLC 细胞系H1975、HCC827为实验对象，通过Western blot、流式细胞术及PD-1-PD-L1蛋白结合实验检测金丝桃苷对H1975、HCC827细胞膜表面和细胞中PD-L1蛋白表达水平的影响；通过MTT方法检测金丝桃苷对NSCLC细胞增殖的影响；通过实时定量荧光PCR（qRT-PCR）的方法检测金丝桃苷对PD-L1 mRNA表达水平的影响；细胞阻抗检测传感实验、结晶紫染色实验用来验证与NSCLC细胞共培养细胞的T细胞的杀伤活性；在C57BL/6J小鼠Lewis肺癌移植瘤模型中检测金丝桃苷体内抑瘤活性。

第二部分研究中，以CRC细胞SW480、HCT116为实验对象，通过Western Blot检测化合物染料木苷对c-Myc、PD-L1和CD47的蛋白表达的影响；流式细胞术检测染料木苷对膜表面PD-L1表达的影响；qRT-PCR分析染料木苷对c-Myc下游靶基因的影响；细胞阻抗检测传感实验、结晶紫染色实验检测共培养细胞的T细胞和PBMC细胞对HCT116细胞的杀伤活性；在C57BL/6J小鼠皮下MC38移植瘤模型中检测染料木苷体内抑瘤活性；同时利用多色流式细胞仪分析在染料木苷作用下肿瘤组织中浸润性CD4⁺、CD8⁺T细胞、调节性T细胞（Regulatory T-lymphocytes，Tregs）数量的影响和巨噬细胞（Macrophages） M2型向M1型极化的比例，以及γ-干扰素（Interferon-γ，IFN-γ）和颗粒酶B（Granzyme B，GzmB）含量的变化；在BALB/C裸鼠体内探究染料木苷对皮下移植瘤的体内抑瘤效果；将染料木苷与鼠源PD-1单抗联用，探究其对C57BL/6J小鼠中MC38移植瘤的协同抑瘤效果。

结果  金丝桃苷能分别以浓度依赖性和时间依赖性方式下调NSCLC细胞中以及细胞膜表面PD-L1的水平，这与其抑制NSCLC细胞中PD-L1 mRNA的表达水平有关。同时金丝桃苷可降低NSCLC细胞中转录因子c-Myc的蛋白表达量，从而在转录水平下调PD-L1的表达。此外，金丝桃苷可促进共培养的T细胞对肿瘤细胞的杀伤作用。小鼠荷瘤实验证实，金丝桃苷可显著抑制C57BL/6小鼠中Lewis移植瘤的生长。以上结果说明，金丝桃苷通过降低NSCLC细胞中c-Myc的蛋白表达量，在转录水平抑制PD-L1的表达，从而发挥抗肿瘤作用。

此外，染料木苷能够以时间和剂量依赖性方式诱导CRC细胞中c-Myc蛋白的降解并抑制其下游靶基因Pd-l1、Cd47、Cdk4和Glut1的表达。Western blot和流式细胞分析结果表明，染料木苷可下调肿瘤细胞中和细胞表面PD-L1和CD47的蛋白水平。PD-1/PD-L1结合实验结果证实，染料木苷可减少CRC细胞表面PD-L1与重组PD-1蛋白的结合并抑制T细胞中TCR信号的激活。细胞阻抗检测传感实验、结晶紫染色实验结果显示，染料木苷在增强共培养的T细胞对肿瘤细胞的杀伤活性的同时，还可促进巨噬细胞对CRC细胞的吞噬活性。MC38小鼠皮下荷瘤实验证实，染料木苷可显著抑制移植瘤的生长，同时增加CD8⁺T细胞的浸润与激活（IFN-γ、GzmB分泌增加），促进巨噬细胞发生M1极化和NK细胞的浸润，并减少Treg的数量，说明染料木苷通过重塑肿瘤免疫微环境发挥其抗肿瘤作用。最后，染料木苷与抗PD-1单抗联合用药后，可使MC38皮下移植瘤的瘤体积进一步减少，证实二者联用后可发挥协同抗肿瘤效果。

结论 我们的研究证实，金丝桃苷可以通过降低NSCLC细胞中PD-L1的表达水平，阻断PD-1/PD-L1相互作用，从而促进T细胞对NSCLC细胞的杀伤活性，发挥其抗肿瘤免疫作用。染料木苷能够诱导CRC细胞中c-Myc蛋白的降解并下调PD-L1和CD47的水平，阻断PD-1/PD-L1和CD47/SIRPα之间的相互作用，通过激活CD8⁺T细胞和诱导巨噬细胞发生M1极化重塑肿瘤免疫微环境，达到抑制肿瘤生长的效果。本研究为将金丝桃苷和染料木苷发展成为新的肿瘤免疫治疗药物提供一定的数据基础与科学依据。

Backgrounds: In recent years, tumor immunotherapy has attracted extensive attention from researchers. It has become the fourth type of cancer therapy after surgery, chemoradiotherapy and targeted therapy. Tumor immunotherapy is considered to be the most promising method to cure cancer.  Among them, immune checkpoint blockage therapy plays a crucial role in immunotherapy. Common immune checkpoints include PD-L1, and its receptor PD-1, CD47 and its signal-regulatory protein α SIRP-α, galectin-9 and its receptor TIM-3.When PD-L1 binding to PD-1,it can inhibit the normal function of T cells. CD47 is one of the most promising targets for the regulation of macrophage function. CD47 located on tumor cell surface,it can inhibit the phagocytic activity of macrophages when interacting with SIRPα on macrophages. The development of natural small molecule compounds that block the immune checkpoint PD-1/PD-L1 interaction and CD47/SIRPα interaction is a new direction for the next generation of tumor immunotherapy. In this study, we investigated the antitumor effects of hyperoside in down-regulating the expression of immune checkpoint PD-L1 in non-small cell lung cancer (NSCLC) cells, and the antitumor effects of genistin in inducing c-Myc degradation in colorectal cancer (CRC) cells, then reduce PD-L1 and CD47 levels.

Methods: In the first part, we used H1975 and HCC827 cells for experiments. Western blot, flow cytometry and PD-1-PD-L1 protein binding assay were used to detect the effects of hyperoside on cell membrane surface and total PD-L1 protein expression level of H1975 and HCC827. MTT assay was used to detect the effects of hyperoside on cell proliferation and apoptosis.The effect of hyperoside on PD-L1 mRNA expression was detected by qRT-PCR. Cell RTCA and crystal violet assay were used to verify the cytotoxic activity of T cells against NSCLC cells. The antitumor activity of hyperoside was detected in C57BL/6J mouse Lewis lung cancer transplanted tumor model.

In the second part, colon cancer cells SW480 and HCT116 were used for experiments.  The effect of genistin on the protein expression of c-Myc, PD-L1 and CD47 were detected by Western-blot assay. Flow cytometry was used to detect the expression of PD-L1 on the membrane surface. Cell RTCA and crystal violet assay were used to detect the cytotoxic activity of PBMC cells against tumor cells. The antitumor activity of genistin was detected in C57BL/6J mice carried MC38 colon cancer transplanted tumor.Meanwhile, the influence of genistin on the number of infiltrating CD4⁺, CD8⁺T cells，regulatory T-cells (Tregs),the proportion of macrophages M2 type to M1 type polarization,Interferon-γ (IFN-γ) and Granzyme B (GzmB) contents were detected by multi-color flow cytometry. The tumor suppressive effect of genistin on immunodeficient mice was investigated in BALB/C nude mice. The combination of genistin and mouse anti-PD-1 protein was administered to C57BL/6J mice carried MC38 colon cancer transplanted tumor.

Results: Our results in the first part showed that hyperoside down-regulated the total and cell membrane surface PD-L1 levels in a concentration-dependent and time-dependent manner, which is related to the inhibition of PD-L1 mRNA expression level in NSCLC cells. At the same time, hyperoside reduced the protein expression of transcription factor c-Myc in NSCLC cells, thus down-regulating the expression of PD-L1 at the transcriptional level. In addition, hyperoside promoted the killing effect of T cells against tumor cells. Animal experiments in C57BL/6 mice confirmed that hyperoside can significantly inhibited the growth of Lewis transplanted tumor in C57BL/6 mice. These results suggested that hyperoside can inhibit the expression of PD-L1 at the transcriptional level by reducing the protein expression level of c-Myc in NSCLC cells, thus resulting in an anti-tumor role.

Results in the second part showed that genistin induced the degradation of c-Myc protein and inhibited its targets gene expression including Pd-l1、Cd47、Cdk4 and Glut1, down-regulated the expression of PD-L1 and CD47 in a time-dependent and dose-dependent manner in HCT116,SW480 and MC38 CRC cells. Flow cytometry results confirm that genistin could significantly down-regulated the expression of PD-L1 on CRC membrane surface. PD-1/PD-L1 binding assay showed that genistin reduced the binding of PD-L1 in tumor cells with PD-1 recombinant protein. Cell RTCA and crystal violet staining showed that genistin enhanced the cytotoxic activity of co-cultured PBMC cells against tumor cells. Animal experiments confirmed that genistin suppressed tumor growth in C57BL/6 mice colon transplanted tumor model, without affecting the mouse body weight. Meanwhile, genistin increased the infiltration of CD8⁺T cells and the secrection of IFN-γ, GzmB, the infiltration of M1-type macrophages, and inhibited the number of Treg. There was no tumor suppressive effect in immunodeficient nude mice, indicating that the tumor suppressive effect of genistin is through the activation of the T cells in tumor immune microenvironment. The combined application of genistin and anti-PD-1 antibody showed while genistin or anti-PD-1 antibody alone both reduced mouse tumor burden from control, combination treatments of genistin and anti-PD-1 antibody further decreased xenograft tumor volume, suggesting that combination treatments of genistin and anti-PD-1 have synergistic anti-tumor effects.

Conclusions: Our findings demonstrate that hyperoside can reduce total and cell membrane surface PD-L1 of NSCLC, reduce the expression of c-Myc, promote the killing activity of co-cultured T cells, and have tumor suppressive effect in normal immune system mouse. In addition, our results also indicate that genistin induces c-Myc-dependent transcriptional inhibition of PD-L1 and CD47 in CRC cells and thus ameliorates the immunosuppressive tumor microenvironment and improves antitumor T-cell immunity stimulate Our study thus provides an important material basis and scientific basis for the development of hyperoside and genistin as new small molecule drugs for anti-tumor immunotherapy.