Gao Yuting, Gong Yi, Song Xiaoyong, Xiong Yajun, Lu Junlan, Yang Yanguang, Gong Yanli, Du Zhimin, Wang Shanshan, Jia Ruilong, Gong Peng, Shi Xinli
Laboratory of Integrated Medicine Tumor Immunology, Shanxi University of Chinese Medicine, Taiyuan, 030000, China.
Laboratory of Integrated Medicine Tumor Immunology, Shanxi University of Chinese Medicine, Taiyuan, 030000, China.
Phytomedicine. 2025 Sep 23;148:157307. doi: 10.1016/j.phymed.2025.157307.
Hepatocellular carcinoma (HCC) is characterized by a 'cold' tumor microenvironment (TME), which limits the efficacy of immune checkpoint inhibitors (ICIs). In a previous study, we demonstrated that dihydroartemisinin (DHA) could disrupt the immunosuppressive TME and delay HCC progression.
This study aimed to explore the precise mechanisms underlying the shift of the TME from an immunosuppressive to an immunostimulatory state.
We constructed HCC subcutaneous allograft mice model to investigate the in vivo antitumor effects of DHA. We further performed single-cell RNA sequencing (scRNA-seq) and multiplex immunohistochemistry (mIHC) to analyze the immune dynamics during DHA-induced TME transition in HCC. Using NCG mice (lacking T, B, and NK cells), BALB/c nude mice (lacking T cells), and CD8 T cell-depleted mice, we assessed whether the antitumor effect of DHA in HCC depends on its immune functions and determined the key role of T cells in antitumor immunity. Additional downstream mechanisms were explored using YAP1 knockdown HCC cells and liver-specific Yap1 knockout mice through techniques such as Western blot, immunofluorescence (IF), metabolic flux analysis, co-immunoprecipitation (Co-IP), chemokine chip, and flow cytometry (FCM).
We observed that DHA transforms the TME of HCC from an immune-cold to an immune-hot state by increasing the abundance and proportion of T cells, NK cells, M1-like TAMs, and DCs. Notably, DHA was found to enhance the proportion of IFN-γ CD8 T cells within the TME of HCC-bearing mice, and its therapeutic effects were dependent on CD8 T cells. Our results suggest that DHA could suppress histone lactylation (Kla) and acetylation (Kac) in HCC cells by inhibiting YAP1. Mechanistically, DHA reduces lactate transport, thereby decreasing lactate accumulation within the TME, lowering P300/CBP catalytic activity, and enhancing the deacetylation effect of HDAC1-3. This finding reveals a new mechanism by which DHA, through metabolic changes and epigenetic regulation, remodels the TME of HCC. Additionally, a combination strategy involving DHA and anti-PD-1 therapy demonstrated the potential to reshape the cold HCC TME via inhibition of the YAP1-lactylation positive feedback loop.
Our study confirms that DHA inhibits histone Kla in HCC cells through YAP1 inhibition, which shifts the TME from an immune-cold to immune-hot state. Moreover, DHA enhance the anti-HCC immune response dependent on CD8 T cells, thereby sensitizing anti-PD-1 therapy. This novel finding positions DHA as a promising strategy for optimizing anti-PD-1 therapy in HCC, warranting further clinical application studies.
肝细胞癌（HCC）的特征是具有“冷”肿瘤微环境（TME），这限制了免疫检查点抑制剂（ICI）的疗效。在先前的一项研究中，我们证明双氢青蒿素（DHA）可以破坏免疫抑制性TME并延缓HCC进展。
本研究旨在探讨TME从免疫抑制状态转变为免疫刺激状态的精确机制。
我们构建了HCC皮下同种异体移植小鼠模型，以研究DHA的体内抗肿瘤作用。我们进一步进行了单细胞RNA测序（scRNA-seq）和多重免疫组化（mIHC），以分析HCC中DHA诱导的TME转变过程中的免疫动态。使用NCG小鼠（缺乏T、B和NK细胞）、BALB/c裸鼠（缺乏T细胞）和CD8 T细胞耗竭小鼠，我们评估了DHA在HCC中的抗肿瘤作用是否依赖于其免疫功能，并确定了T细胞在抗肿瘤免疫中的关键作用。通过蛋白质免疫印迹、免疫荧光（IF）、代谢通量分析、免疫共沉淀（Co-IP）、趋化因子芯片和流式细胞术（FCM）等技术，使用YAP1敲低的HCC细胞和肝脏特异性Yap1基因敲除小鼠探索了其他下游机制。
我们观察到DHA通过增加T细胞、NK细胞、M1样肿瘤相关巨噬细胞（TAM）和树突状细胞（DC）的丰度和比例，将HCC的TME从免疫冷状态转变为免疫热状态。值得注意的是，发现DHA可提高荷HCC小鼠TME中IFN-γ CD8 T细胞的比例，其治疗效果依赖于CD8 T细胞。我们的结果表明，DHA可通过抑制YAP1来抑制HCC细胞中的组蛋白乳酰化（Kla）和乙酰化（Kac）。从机制上讲，DHA减少乳酸转运，从而减少TME内的乳酸积累，降低P300/CBP催化活性，并增强HDAC1-3的去乙酰化作用。这一发现揭示了DHA通过代谢变化和表观遗传调控重塑HCC TME的新机制。此外，涉及DHA和抗PD-1治疗的联合策略显示出通过抑制YAP1-乳酰化正反馈环重塑冷HCC TME的潜力。
我们的研究证实，DHA通过抑制YAP1抑制HCC细胞中的组蛋白Kla，从而使TME从免疫冷状态转变为免疫热状态。此外，DHA增强了依赖于CD8 T细胞的抗HCC免疫反应，从而使抗PD-1治疗敏感化。这一新发现使DHA成为优化HCC抗PD-1治疗的有前景的策略，值得进一步进行临床应用研究。
