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Abstract：
Background: Immunotherapy has demonstrated its potential to improve the prognosis of patients with hepatocellular carcinoma (HCC); however, patients\' responses to immunotherapy vary a lot. A comparative analysis of the tumor microenvironment (TME) in responders and non-responders is expected to unveil the mechanisms responsible for the immunotherapy resistance and provide potential treatment targets. Methods: We performed sequencing analyses using 10x Genomics technology on six HCC patients who responded to anti-PD-1 therapy and one HCC patient who did not respond. Additionally, we obtained single cell data from untreated, responsive, and nonresponsive HCC patients from public databases, and used part of the datasets as a validation cohort. These data were integrated using algorithms such as Harmony. An independent validation cohort was established. Furthermore, we performed spatial transcriptomic sequencing on the tumor adjacent tissues of three HCC responsive patients using 10x Genomics spatial transcriptomic technology. Additionally, we analyzed data about three HCC patients obtained from public databases. Finally, we validated our conclusions using immunofluorescence, flow cytometry, and in vivo experiments. Results: Our findings confirmed the presence of \"immune barrier\" partially accounting for the limited efficacy of immunotherapy. Our analysis revealed a significant increase in TREM2+ Macrophages among non-responsive patients expressing multiple immunosuppressive signals. anti-Csf1r monoclonal antibodies effectively eliminated these macrophages and augmented the therapeutic effects of anti-PD-1 therapy. TCR+ Macrophages possessed direct tumor-killing capabilities. IL1B+ cDC2 was the primary functional subtype of cDC2 cells. Absence of THEMIShi CD8+ T subtypes might diminish immunotherapeutic effects. Furthermore, CD8+ T cells entered a state of stress after anti-PD-1 treatment, which might be associated with CD8+ T cell exhaustion and senescence. Conclusions: The profiles of immune TMEs showed differences in HCC patients responsive, non-responsive and untreated. These differences might explain the discounted efficacy of immunotherapy in some HCC patients. The cells and molecules, which we found to carry unique capabilities, may be targeted to enhance immunotherapeutic outcomes in patients with HCC.
摘要：
背景：免疫治疗已证明其改善肝细胞癌（HCC）患者预后的潜力;然而，患者对免疫疗法的反应差异很大。对应答者和非应答者中的肿瘤微环境(TME)的比较分析预期揭示负责免疫疗法抗性的机制并提供潜在的治疗靶标。方法：我们使用10x基因组学技术对6名对抗PD-1治疗有反应的HCC患者和1名没有反应的HCC患者进行测序分析。此外,我们从未经处理的单细胞数据中获得，响应，和来自公共数据库的无反应HCC患者，并将部分数据集用作验证队列。这些数据使用Harmony等算法进行整合。建立了独立的验证队列。此外，我们使用10倍基因组学空间转录组学技术对3例HCC反应患者的肿瘤旁组织进行了空间转录组测序。此外,我们分析了从公共数据库获得的3例HCC患者的数据.最后,我们用免疫荧光验证了我们的结论，流式细胞术,和体内实验。结果：我们的发现证实了“免疫屏障”的存在，部分原因是免疫治疗的疗效有限。我们的分析揭示了表达多种免疫抑制信号的无反应患者中TREM2+巨噬细胞的显著增加。抗Csf1r单克隆抗体可有效消除这些巨噬细胞并增强抗PD-1治疗的疗效.TCR+巨噬细胞具有直接的肿瘤杀伤能力。IL1B+cDC2是cDC2细胞的主要功能亚型。缺乏THEMIShiCD8T亚型可能会降低免疫治疗作用。此外，CD8+T细胞在抗PD-1治疗后进入应激状态，这可能与CD8+T细胞衰竭和衰老有关。结论：免疫TMEs的概况显示HCC患者反应不同，无反应和未经处理。这些差异可能解释了一些HCC患者免疫疗法的疗效下降。细胞和分子，我们发现它具有独特的能力，可能有针对性地提高肝癌患者的免疫治疗结果。
