研究目的：
肝细胞癌 (hepatocellular carcinoma, HCC) 是肝癌中最常见的类型，具有独特和复杂的免疫微环境，对肝癌的发生发展起着至关重要的作用，是肝癌研究领域的热点和难点。既往的研究已经发现各种免疫细胞在肿瘤进展中的作用，并且开发了利用免疫细胞的治疗方法，但临床上这些治疗的有效率依然有限或仅对于特定类型的肿瘤具有治疗效果。在肝癌微环境中各种因素的影响下，巨噬细胞通过改变自身的极化状态而影响其他免疫细胞的活化或抑制，从而在肝癌的发生发展中发挥不同的功能。现有研究还表明肝癌中抑制性的免疫微环境不利于CD8+T细胞发挥抗肿瘤功能，因此探索CD8+T细胞失活的机制对肝癌的治疗十分重要。
胰岛素样生长因子2 mRNA结合蛋白3 (Insulin-like growth factor 2 mRNA binding protein 3, IGF2BP3) 是一种RNA结合蛋白，研究表明IGF2BP3在正常成人组织中低表达，在肿瘤的发生发展过程中表达升高。目前在肝癌中，针对IGF2BP3的研究主要集中在其对肿瘤细胞本身的影响及其与疾病恶性进展的相关性。然而，IGF2BP3在肝癌免疫微环境中的作用却鲜有报道。
本研究以肝癌为研究对象，探索IGF2BP3在肝癌中的表达以及对肿瘤微环境中巨噬细胞和CD8+T细胞的影响，并对其中的细胞与分子机制进行探究。同时本研究还在动物水平探讨了敲除Igf2bp3激活抗肿瘤免疫细胞，并联合其他免疫疗法增强治疗效果的可行性，为临床中肝癌的治疗提供了新的思路和理论支持。
研究方法：
第一，利用公共数据库，采用生物信息学的方法分析IGF2BP3在肝癌不同分期的表达以及其与患者预后、肝癌中免疫细胞浸润的关系。利用单细胞测序数据，分析肝癌中表达IGF2BP3的主要细胞类型。利用慢病毒和Crispr-Cas9技术分别构建了稳定过表达及敲除Igf2bp3的Hepa1-6小鼠肝癌细胞系，并利用qPCR, Western blot, Flow cytometry技术进行了验证。使用CCK-8, 划痕实验，Transwell, Flow cytometry分别检测IGF2BP3对肿瘤细胞增殖、迁移和凋亡的影响。
第二，使用小鼠肝癌细胞系Hepa1-6，构建肝癌皮下移植瘤模型。采用流式细胞术分析肿瘤中各种免疫细胞的比例及表型，进一步对比清除巨噬细胞后的小鼠皮下移植瘤模型，验证巨噬细胞在IGF2BP3促进肿瘤中的重要作用。
第三，为了探究IGF2BP3对肿瘤细胞分泌细胞因子的作用，收集肿瘤细胞培养上清，采用细胞因子阵列，Western blot，ELISA，免疫组化染色等方法检测IGF2BP3表达水平的改变对肿瘤细胞所分泌细胞因子的影响。进一步使用CCL5, TGF-β1的中和抗体，分别利用Transwell和Flow cytometry，验证由肿瘤细胞产生的、介导IGF2BP3调控巨噬细胞和CD8+T细胞的细胞因子。
第四，为了探究IGF2BP3对CCL5和TGF-β1的调控机制，采用RIP实验将IGF2BP3结合的RNA片段富集，使用普通PCR和qPCR方法检测目的片段，分析IGF2BP3与目的片段的结合情况。进一步利用TriFc系统验证其结合。最后使用放线菌素D抑制RNA的合成，检测IGF2BP3对CCL5以及TGF-β1 mRNA半衰期的影响。
第五，为了探讨敲除Igf2bp3表达，激活免疫细胞，联合其它免疫治疗增强抗肿瘤疗效的可行性，在小鼠皮下移植瘤模型中，比较敲除Igf2bp3联合anti-CD47的治疗策略与单独敲除Igf2bp3或单独使用anti-CD47的治疗策略对肿瘤发生发展的影响。
实验结果：
1. 生物信息学分析发现，IGF2BP3在肝癌中的高表达与巨噬细胞的高浸润显著相关。并且IGF2BP3高表达，巨噬细胞高浸润与患者的生存时间呈负相关。
2. 肿瘤细胞中的IGF2BP3可以促进巨噬细胞迁移和向M2极化。利用小鼠肝癌皮下移植瘤模型分析发现，敲除肿瘤细胞Igf2bp3，肿瘤组织中巨噬细胞的浸润明显减少，CD8+T细胞浸润比例增加。
3. 细胞机制研究发现肝癌细胞中IGF2BP3的高表达可上调CCL5和TGF-β1的表达和分泌。其中一方面，CCL5的分泌可招募巨噬细胞向肿瘤微环境中浸润；另一方面，IGF2BP3通过促进TGF-β1的分泌，诱导肿瘤微环境中巨噬细胞M2型极化，抑制CD8+T细胞的活化。
4. 分子机制研究发现IGF2BP3可以与CCL5和TGF-β1的mRNA直接结合而发挥调控作用。
5. 在小鼠Hepa1-6皮下移植瘤模型中，敲除Hepa1-6细胞中Igf2bp3并联合CD47抗体的治疗策略，可以显著抑制肿瘤的生长，且较单用其中一种的治疗策略更有效。
结论：
我们研究发现，IGF2BP3主要表达于肝癌细胞。IGF2BP3可以通过上调CCL5的表达与分泌在肿瘤微环境中促进巨噬细胞的浸润；通过上调TGF-β1的表达与分泌诱导巨噬细胞的M2极化，同时抑制CD8+T细胞的活化。在小鼠肝癌模型中敲除Igf2bp3激活抗肿瘤免疫细胞与CD47抗体联合治疗策略具有良好的治疗效果。

Objective

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Its unique and complex immune microenvironment plays a crucial role in the development of HCC and has been a hot topic in the field of HCC research. Previous studies have identified the role of various immune cells in tumor progression and developed therapeutic approaches using immune cells, but the clinical efficacy of these therapies is still limited or only has therapeutic effect on certain types of tumors. Under the influence of various factors in the liver cancer microenvironment, macrophages play different functions in the development of HCC by altering their polarization and influencing the activation or inhibition of other immune cells. Studies have shown that the suppressive immune microenvironment in HCC is not conducive to the anti-tumor function of CD8+T cells. Therefore, it is important to explore the mechanisms of CD8+T cell inactivation for the treatment of tumors.
Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an RNA-binding protein that has been shown to be lowly expressed in normal adult tissues. IGF2BP3 promotes tumor progression by promoting the malignant phenotype of tumor cells. It is up-regulated during the occurrence and development of tumors. Current studies in HCC have focused on the effects of IGF2BP3 on the tumor cells themselves and its relevance to malignant disease progression. However, the role of IGF2BP3 in the immune microenvironment of HCC has rarely been reported.
In this study, we investigated the expression of IGF2BP3 and its effects on macrophages and CD8+T cells in the tumor microenvironment, and explored the cellular and molecular mechanisms involved. Our study also explored the feasibility of knocking out Igf2bp3 to activate anti-tumor immune cells combined with other immunotherapies at animal level to enhance the therapeutic effect, bringing new ideas and theoretical support for the treatment of HCC in clinical settings.

Methods

Firstly, using public databases, a bioinformatics approach was used to analyze the expression of IGF2BP3 in different stages of liver cancer and the relationship with patient prognosis and immune cell infiltration in HCC. Single cell sequencing data were used to analyze the main cell types expressing IGF2BP3 in HCC. Hepa1-6 mouse HCC cell lines stably overexpressing and knocking out Igf2bp3 were constructed using lentivirus and Crispr-Cas9 techniques respectively and validated using qPCR, Western blot, Flow cytometry. The effects of IGF2BP3 on tumor cell proliferation, migration and apoptosis were examined by CCK-8, wound healing assay, Transwell and Flow cytometry, respectively.
Secondly, a mouse subcutaneous transplantation tumor model was constructed by inoculating a mouse HCC cell line, Hepa1-6. The proportion and phenotype of various immune cells in tumors were analyzed by flow cytometry, and the subcutaneous xenograft tumor model after removing macrophages was further compared to verify the important role of macrophages in the promotion of tumors.
Thirdly, to investigate the effect of IGF2BP3 on cytokine secretion by tumor cells, the culture supernatants of tumor cell were collected and the effect of IGF2BP3 expression on cytokine secretion by tumor cells was detected using cytokine arrays, ELISA, Western blot, immunohistochemical staining. Furthermore, the cytokines produced by tumor cells that mediate the regulation of macrophages and CD8+T cells by IGF2BP3 were verified using Transwell and Flow cytometry using neutralizing antibodies to CCL5 and TGF-β1, respectively.
Fourthly, to investigate the regulatory mechanism of IGF2BP3 on CCL5 and TGF-β1, IGF2BP3-bound RNA fragments were enriched using RIP assays and the binding of IGF2BP3 to the target fragments was analyzed using PCR and qPCR methods. The binding was further verified using the TriFc system. Finally, the effect of IGF2BP3 on the half-life of CCL5 and TGF-β1 mRNA was examined using actinomycin D to inhibit RNA synthesis.
Fifth, to investigate the feasibility of knocking out Igf2bp3 expression, activating immune cells and combining with other immunotherapies to enhance anti-tumor efficacy, the effect of knocking out Igf2bp3 combined with anti-CD47 on tumor development was compared with knocking out Igf2bp3 alone or using anti-CD47 alone in a mouse subcutaneous transplantation tumor model.

Results

1. Bioinformatics analysis revealed that high expression of IGF2BP3 in HCC was significantly correlated with high infiltration of macrophages. And high IGF2BP3 expression and high macrophage infiltration were negatively correlated with the survival time of patients.
2. IGF2BP3 in tumor cells can promote macrophage migration and polarization towards M2. Using a mouse HCC subcutaneous transplantation tumor model, it was found that knockout of tumor cells Igf2bp3 resulted in a significant reduction in macrophage infiltration and an increase in the proportion of CD8+T cell infiltration in tumor tissue.
3. Cellular mechanism studies revealed that IGF2BP3 in HCC cells upregulated the expression and secretion of CCL5 and TGF-β1. On the one hand, the secretion of CCL5 could recruit macrophages to infiltrate into the tumor microenvironment; on the other hand, IGF2BP3 induced macrophage M2-type polarization and inhibited CD8+T cell activation by promoting the expression of TGF-β1.
4. Molecular mechanism studies revealed that IGF2BP3 could play a regulatory role by directly binding to the mRNA of CCL5 and TGF-β1.
5. In a mouse subcutaneous transplantation tumor model of HCC, a therapeutic strategy of knockout Igf2bp3 in Hepa1-6 cells in combination with CD47 antibody significantly inhibited tumor growth and was more effective than either of the therapeutic strategies alone.

Conclusion

We found that IGF2BP3 was mainly expressed in HCC cells. IGF2BP3 could promote macrophage infiltration in the tumor microenvironment by upregulating the expression and secretion of CCL5 and induce M2 polarization of macrophages by upregulating the expression and secretion of TGF-β1, while inhibiting the activation of CD8+T cells. Knocking out of Igf2bp3 in a mouse model of HCC activates anti-tumor immune cells in combination with CD47 antibodies has good therapeutic effects.