目的：微血管侵犯（microvascular invasion, MVI）是指门静脉、肝静脉和肿瘤包膜血管内存在肿瘤细胞巢，是公认的肝细胞癌（hepatocellular carcinoma, HCC）早期复发和预后不良的重要危险因素。前期课题组在探究肝细胞癌对多靶点抗肿瘤药物甲苯磺酸索拉非尼的耐药机制中，发现醇脱氢酶1A（Alcohol dehydrogenase 1A，ADH1A）在索拉非尼治疗应答组的蛋白表达水平显著升高，其高表达与患者的生存获益呈现正相关关系，而其表达的下调则可能导致氧化损伤积累、诱导自噬激活，从而促使肿瘤细胞存活和药物抗性。本研究旨在探讨ADH1A在HCC中的表达变化及其与MVI、预后和肿瘤免疫微环境的关系，并揭示其可能的分子机制。

方法：基于公开发表在Nature上101例包括MVI情况的中国肝细胞癌患者蛋白质组数据，比较其肿瘤组织和癌旁组织的ADH1A表达差异。根据ADH1A蛋白表达量将101例患者分为ADH1A高低表达组，绘制其生存曲线，并分析ADH1A蛋白表达与临床病理特征的关系。通过GO （Gene Ontology）生物过程富集分析，分析与ADH1A表达相关的正负基因集主要富集的通路及其显著性，并提取其中共同富集关系最显著的蛋白，检验其在ADH1A高低表达组间的丰度差异。应用CIBERSORT算法对蛋白组数据推测的表达矩阵进行免疫浸润分析，比较ADH1A高低表达组间各免疫细胞亚群的丰度差异。进一步筛选出其中受ADH1A表达影响显著的免疫细胞类型，对这些免疫细胞的丰度与ADH1A表达之间进行Pearson相关性分析。从公开发表Science Advances上的空间转录组数据集中选取2个分别为MVI阳性和MVI阴性的肝细胞癌样本，并分析ADH1A的空间表达特征。纳入2013年2月至2014年8月于中国医学科学院肿瘤医院肝胆外科收治的经病理确诊的6例HCC患者癌组织及配对癌旁组织标本（其中MVI阳性者3例和MVI阴性者3例）进行Western Blot检测，评估肿瘤组织和癌旁组织中ADH1A蛋白表达水平差异。纳入2024年11月在中国医学科学院肿瘤医院肝胆外科收治的经病理确诊的2例HCC患者术后病理标本（其中MVI阳性和MVI阴性各1例）进行免疫组化染色，评估肿瘤组织和肝组织中ADH1A表达特征。  

结果：研究发现ADH1A在HCC肿瘤组织中的表达显著低于癌旁组织（P < 0.001），ADH1A的表达水平与患者微血管侵犯（MVI）显著相关（P = 0.001），且ADH1A的低表达与患者生存时间缩短显著相关。GO分析显示，与ADH1A表达呈正相关的基因主要参与有机酸代谢、脂肪酸代谢等代谢相关过程，而与ADH1A表达呈负相关的基因主要参与细胞-基质黏附、细胞铺展、黏附依赖性细胞运动等过程。进一步的，针对负相关GO条目中显著富集的“细胞黏附”类生物过程，进一步提取其中共同富集的关键黏附相关蛋白（如MDK、MYADM、PTK2、SRC、PARVG、PREX1、ROCK2），分析其在ADH1A高低表达患者组中的蛋白丰度差异，结果显示上述蛋白在ADH1A低表达组中均显著上调，其上调调伴随着MDK、SRC、PTK2等多种促迁移分子的下调，提示ADH1A可能通过抑制黏附相关信号通路限制肿瘤扩散与血管浸润能力。免疫微环境分析结果显示，ADH1A低表达组中免疫抑制型细胞如活化型肥大细胞显著升高，而抗肿瘤免疫细胞如CD4 naive T细胞明显减少，这意味着ADH1A低表达肿瘤可能更倾向于形成免疫抑制性微环境，最后导致肿瘤逃避免疫监视。空间转录组分析显示，在MVI阴性样本中，ADH1A在肿瘤区域的表达较癌旁区域略有下降，但整体表达水平仍维持在较高水平，空间分布相对均匀；而在MVI阳性样本中，ADH1A在肿瘤区域表达明显下调，且空间表达信号较为稀疏。Western blot验证和免疫组化染色结果也同样显示出MVI阳性患者的肿瘤组织中ADH1A表达水平显著低于MVI阴性者。

结论：ADH1A在肝细胞癌中表达下调，与微血管侵犯及不良预后显著相关。ADH1A可能通过调控细胞黏附通路和免疫微环境发挥抗肿瘤作用，具有作为预后生物标志物和治疗靶点的潜力。

Objective: Microvascular invasion (MVI) refers to the presence of tumor cell nests in the portal vein, hepatic vein, and tumor capsule blood vessels, and is a recognized significant risk factor for early recurrence and poor prognosis in hepatocellular carcinoma (HCC). In previous studies, our team found that Alcohol dehydrogenase 1A (ADH1A) expression was significantly elevated in the sorafenib treatment response group, and its high expression was positively correlated with survival benefits for patients. Conversely, downregulation of ADH1A may lead to the accumulation of oxidative damage and induce autophagy activation, promoting tumor cell survival and drug resistance. This study aims to investigate the expression changes of ADH1A in HCC and its relationship with MVI, prognosis, and the tumor immune microenvironment, as well as to explore its potential molecular mechanisms.

Methods: Based on the publicly available proteomic data of 101 Chinese hepatocellular carcinoma (HCC) patients, including microvascular invasion (MVI) status, published in Nature, the expression difference of ADH1A between tumor tissues and adjacent non-tumor tissues was compared. According to the expression level of ADH1A protein, the 101 patients were divided into high and low expression groups. Survival curves were plotted, and the relationship between ADH1A protein expression and clinicopathological features was analyzed. Gene Ontology (GO) biological process enrichment analysis was used to identify the pathways enriched with genes positively and negatively correlated with ADH1A expression and their significance. Key proteins most significantly enriched in these pathways were extracted and tested for abundance differences between high and low ADH1A expression groups. The CIBERSORT algorithm was applied to analyze immune cell infiltration using expression matrices predicted from proteomic data, comparing the differences in the abundance of immune cell subpopulations between the high and low ADH1A expression groups. Furthermore, immune cell types significantly affected by ADH1A expression were selected, and Pearson correlation analysis was performed between the abundance of these immune cells and ADH1A expression. Two HCC samples, one MVI-positive and one MVI-negative, were selected from the publicly available spatial transcriptomics dataset published in Science Advances, and the spatial expression characteristics of ADH1A were analyzed. Six HCC patients, diagnosed by pathology between February 2013 and August 2014 at the Department of Hepatobiliary Surgery, Cancer Hospital of Chinese Academy of Medical Sciences, were included, with matched tumor and adjacent tissue samples (3 MVI-positive and 3 MVI-negative). Western blot analysis was performed to assess the difference in ADH1A protein expression between tumor and adjacent tissues, and the results were compared with spatial transcriptomic data. Include postoperative pathological specimens from 2 HCC patients (1 MVI-positive and 1 MVI-negative) who were pathologically confirmed and treated at the Department of Hepatobiliary Surgery, Cancer Hospital of Chinese Academy of Medical Sciences in November 2024 for immunohistochemical staining to assess the expression characteristics of ADH1A in tumor tissues and liver tissues.

Results: The study found that ADH1A expression in HCC tumor tissues was significantly lower than that in adjacent tissues (P < 0.001). The expression level of ADH1A was significantly associated with microvascular invasion (P = 0.001), and low expression of ADH1A was significantly correlated with shorter survival time in patients. GO analysis indicated that genes positively correlated with ADH1A expression were mainly involved in metabolic processes such as organic acid and fatty acid metabolism, while genes negatively correlated with ADH1A expression were involved in processes such as cell-matrix adhesion, cell spreading, and adhesion-dependent cell migration. Further analysis of the negatively correlated GO terms related to "cell adhesion" revealed key adhesion-related proteins (e.g., MDK, MYADM, PTK2, SRC, PARVG, PREX1, ROCK2), and significant differences in protein abundance between high and low ADH1A expression groups were observed. These proteins were significantly upregulated in the low ADH1A expression group, with an accompanying downregulation of migration-promoting molecules such as MDK, SRC, and PTK2, suggesting that ADH1A may limit tumor spread and vascular infiltration by inhibiting adhesion-related signaling pathways. Immune microenvironment analysis showed that in the low ADH1A expression group, immune-suppressive cells such as activated mast cells were significantly increased, while anti-tumor immune cells like CD4 naive T cells were notably decreased, indicating that tumors with low ADH1A expression may tend to form an immune-suppressive microenvironment, leading to immune escape. Spatial transcriptomic analysis revealed that in MVI-negative samples, ADH1A expression was slightly reduced in tumor regions compared to adjacent areas but remained relatively high with a fairly uniform spatial distribution. In MVI-positive samples, ADH1A expression was significantly downregulated in tumor regions, with sparse spatial expression signals. Western blot validation and immunohistochemical staining also revealed that ADH1A expression levels in tumor tissues were significantly lower in MVI-positive patients than in MVI-negative patients.

Conclusion: ADH1A is downregulated in HCC and is significantly associated with microvascular invasion and poor prognosis. ADH1A may exert its anti-tumor effects by regulating cell adhesion pathways and the immune microenvironment, making it a potential prognostic biomarker and therapeutic target.