Targeting the splicing factor SRSF10 in mouse HCC tumors shapes tumor immunity and immunotherapy. Targeting the splicing factor SRSF10 in mouse HCC tumors shapes tumor immunity and immunotherapy

The efficacy of immune checkpoint blockade (ICB) therapy in hepatocellular carcinoma (HCC) patients remains poor. This article aims to uncover the role and mechanism of SRSF10 in HCC immunotherapy. SRSF10 is upregulated in a variety of tumors and is associated with poor prognosis. SRSF10 binds to the 3’UTR region of MYB and enhances the stability of MYB RNA levels. This activation leads to increased transcriptional expression of key enzymes associated with glycolysis, such as GLUT1, HK1, and LDHA, resulting in higher lactate content in tumor cells. The lactate in tumor cells inside can increase histone lactylation so as to upregulated the expression of SRSF10. Besides, the lactate produced by tumors promotes lactylation of the histone H3K18la site upon transport into macrophages, which could activate transcription of M2 macrophage genes and increase protumour macrophage activity, so as to creating a suppressive immune microenvironment. Clinically, SRSF10 can be utilized as a biomarker to assess the resistance to immunotherapy in different types of solid tumors. The pharmacological targeting of SRSF10 with 1C8 has been shown to be effective in both murine and human preclinical models. In conclusion, we prove that the SRSF10/MYB/glycolysis/lactate axis is critical for triggering immune evasion and anti-PD-1 resistance. Overall design: Tumors from WT or shSrsf10 mice were extracted and digested with collagenase type IV (1 mg/ml) and DNase type I (30 U/ml) for 30 min at 37 °C. The cells were filtered through the 70 μm cell strainers, washed with phosphate-buffered saline (PBS), lysed in red blood cell buffer, and resuspended in PBS. Cells were sorted by FACSAria Fusion Flow Cytometer (BD Biosciences). The cell suspension (300-600 living cells per microliter) was loaded onto the Chromium single cell controller (10 x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. Libraries were subsequently prepared sing Single Cell 5’Library and Gel Bead Kit and sequenced on an Illumina NovaSeq 6000 instrument with pair-end 100 bp (PE100) reading strategy.

肝细胞癌（hepatocellular carcinoma, HCC）患者接受免疫检查点阻断（immune checkpoint blockade, ICB）治疗的疗效仍欠佳。本研究旨在阐明SRSF10在肝细胞癌免疫治疗中的作用及机制。
SRSF10在多种肿瘤中呈高表达，且与不良预后相关。SRSF10可结合至MYB的3’非翻译区（3’untranslated region, 3’UTR），并增强MYB RNA的稳定性。该激活过程可上调糖酵解关键酶的转录表达，如葡萄糖转运蛋白1（GLUT1）、己糖激酶1（HK1）及乳酸脱氢酶A（LDHA），进而导致肿瘤细胞内乳酸含量升高。
肿瘤细胞内的乳酸可增强组蛋白乳酸化修饰，从而上调SRSF10的表达。此外，肿瘤分泌的乳酸经转运进入巨噬细胞后，可促进巨噬细胞组蛋白H3K18la位点的乳酸化修饰，激活M2型巨噬细胞相关基因的转录，增强促肿瘤巨噬细胞活性，进而形成免疫抑制性肿瘤微环境。
临床层面，SRSF10可作为生物标志物，用于评估不同实体瘤患者对免疫治疗的耐药性。使用1C8靶向抑制SRSF10的药理学手段，已在小鼠及人源临床前模型中被证实具有治疗效果。
综上，本研究证实SRSF10/MYB/糖酵解/乳酸轴在触发肿瘤免疫逃逸及抗PD-1治疗耐药中发挥关键作用。
实验整体设计：提取野生型（wild type, WT）或shSrsf10敲低小鼠的肿瘤组织，使用IV型胶原酶（1 mg/ml）与DNase I（30 U/ml）于37 ℃消化30分钟。将细胞通过70 μm细胞筛过滤，经磷酸盐缓冲液（phosphate-buffered saline, PBS）洗涤后，使用红细胞裂解液裂解，并重悬于PBS中。通过FACSAria Fusion流式细胞仪（BD Biosciences）对细胞进行分选。将细胞悬液（每微升300~600个活细胞）加载至Chromium单细胞控制器（10 x Genomics），依照制造商操作规程制备乳液内单细胞凝胶磁珠。随后使用单细胞5’文库及凝胶磁珠试剂盒构建文库，并采用Illumina NovaSeq 6000测序仪以双端100 bp（PE100）测序策略进行测序。