肿瘤浸润的免疫细胞及其与肿瘤微环境中的癌细胞的相互作用在塑造肿瘤进展和对疗法的响应中起着关键作用。我们利用人类原代单核细胞构建了三维（3D）肝癌球体，研究了肿瘤相关巨噬细胞（TAMs）与肝细胞癌（HCC）细胞（HepG2和PLC/PRF5）之间的相互作用。通过使用多重基因表达面板，我们鉴定了在使原代人类单核细胞转变为TAMs表型过程中涉及的关键途径。我们使用特定途径的抑制剂来探索该途径在TAMs极化中的作用。在人类HCC细胞系的共培养球体中，浸润的单核细胞呈现了致瘤的M2型巨噬细胞表型。浸润的单核细胞的基因表达面板显示，上调的基因富集于胆固醇代谢途径。胆固醇代谢相关的基因与核受体PPARG和LXR一起上调。当抑制溶酶体酸类脂酶（LAL），这是对脂蛋白水解所必需的关键酶，浸润的单核细胞在3D球体共培养中表现出明显减少的M2标志物和脂负载受体表达和增加的细胞内脂质含量，这表明胆固醇代谢对于塑造TAMs至关重要。此外，LAL抑制剂还减少了HCC细胞系的球体生长和侵袭能力。通过siRNA介导的单核细胞LAL沉默在共培养球体中产生了类似的结果。这些数据表明，肝癌细胞和浸润的单核细胞通过胆固醇代谢进行相互作用，以使单核细胞向TAMs转化，从而有利于肿瘤的生长和生存，从而促进肝癌的进展。© 2023作者。牛津大学出版社代表白血病学会发表。保留所有权利。如需授权，请发送电子邮件至：journals.permissions@oup.com。

Tumor-infiltrating immune cells and their crosstalk with cancer cells in the tumor microenvironment (TME) play a crucial role in shaping tumor progression and response to therapy. We utilized three-dimension (3D) liver cancer spheroids incorporating human primary monocytes to investigate the crosstalk between tumor-associated macrophages (TAMs) and Hepatocellular carcinoma (HCC) cells, HepG2 and PLC/PRF5. Using multiplexed gene expression panels, the critical pathways involved in shaping primary human monocytes to adopt TAMs phenotypes were identified. Specific inhibitor for identified pathway was used to explore its involvement in polarization of TAMs. In the cocultured spheroids comprised of the human HCC cell lines, the infiltrating monocytes resembled protumor M2-like macrophage phenotypes. Gene expression panels of the infiltrating monocytes demonstrated that the upregulated genes were enriched in the cholesterol metabolism pathway. Cholesterol metabolism-related genes were upregulated together with the nuclear receptors, PPARG and LXR. When lysosomal acid lipase (LAL), the key enzyme necessary for the hydrolysis of lipoprotein, was inhibited, infiltrating monocytes in 3D spheroid coculture showed significantly decreased M2 marker and lipid uptake receptor expression as well as increased cellular lipid content, which indicated that cholesterol metabolism was important for conditioning the TAMs. Moreover, LAL inhibition reduced the spheroid growth and invasiveness of HCC cell lines. siRNA-mediated LAL silencing in monocytes yielded similar results upon spheroid coculture. These data indicated that liver cancer cells and infiltrating monocytes participate in crosstalk via cholesterol metabolism to condition monocytes toward TAMs, which favors tumor growth and survival, thereby promoting liver cancer progression.© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.