肝細胞癌(hepatocellular carcinoma, HCC)是全球好發的惡性腫瘤，而腫瘤相關巨噬細胞(tumor-associated macrophages, TAMs)在其腫瘤微環境中扮演關鍵角色。TAMs具有促進腫瘤發展的特性，且與HCC不良預後密切相關，然而，目前針對HCC腫瘤細胞是否會直接活化巨噬細胞轉變為促腫瘤表型的相關研究仍然有限。本研究旨在探討HCC如何驅動巨噬細胞活化及其對腫瘤進程的影響。實驗上，以佛波醇12-十四烷醯-13-乙酸酯（phorbol 12-myristate 13-acetate）誘導THP-1單核細胞分化為巨噬細胞，並將其培養在HCC條件培養基（conditioned medium, CM）。我們的研究結果顯示，HCC組織中巨噬細胞浸潤高於正常肝組織，且HCC CM處理的巨噬細胞能顯著促進HCC細胞增殖。RNA定序資料顯示，HCC CM刺激後的巨噬細胞呈現促腫瘤轉錄重編程，其中程序性死亡配體-1 (programmed cell death ligand 1, PD-L1)表現及趨化因子訊號相關路徑顯著富集。此外，HCC活化的巨噬細胞培養上清中，白介素-1β (Interleukin-1β, IL-1β)、IL-6、IL-10及腫瘤壞死因子α (tumor necrosis factor α, TNF-α)濃度顯著上升。免疫轉漬與流式細胞儀分析確認HCC CM可誘導巨噬細胞PD-L1表現，且此現象經干擾素-γ (interferon-γ)共同刺激後更加顯著；但以初代人類肝細胞CM處理則未觀察到此現象。相反地，巨噬細胞CM並未誘導HCC細胞表現PD-L1。進一步分析發現，HCC CM刺激後，巨噬細胞中CXCL9、CXCL10、CCL1及CCL22等趨化因子mRNA表現顯著提升。綜合以上結果，HCC細胞可直接誘導巨噬細胞變成促腫瘤的表型，表現細胞激素與趨化因子分泌增加及PD-L1上升。HCC對巨噬細胞的直接活化，可能會藉由促進免疫抑制進一步推動HCC的進展。

Hepatocellular carcinoma (HCC) is a leading global malignancy, with tumor-associated macrophages (TAMs) playing a critical role in the tumor microenvironment (TME). TAMs are pro-tumorigenic and linked to poor prognosis in HCC; nevertheless, few studies have investigated whether HCC tumor cells directly activate macrophages into a pro-tumorigenic phenotype. This study aims to explore how HCC drives macrophage activation and its impact on HCC progression. THP-1 monocytes were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA). Following differentiation, these macrophages were incubated with HCC conditioned medium (CM). We demonstrated that HCC tissue showed higher macrophage infiltration than normal liver. HCC CM-treated macrophages promoted HCC cell proliferation. The RNA sequencing (RNA-seq) data showed a reprogrammed pro-tumorigenic transcriptome in HCC CM-treated macrophages, with pathways of programmed cell death ligand 1 (PD-L1) expression and chemokine signaling differentially enriched. Furthermore, interleukin (IL)-1β, IL-6, IL-10, and TNF-α were significantly elevated in the supernatants of HCC-activated macrophages. Immunoblotting and flow cytometry analysis showed that HCC CM induced macrophage PD-L1 expression, which was amplified by interferon-γ (IFN-γ). However, this phenomenon was not observed in macrophages treated with primary human hepatocyte CM. Conversely, macrophage CM did not induce PD-L1 in HCC cells. Moreover, chemokine mRNAs of CXCL9, CXCL10, CCL1, and CCL22 were profoundly enhanced in macrophages after HCC CM stimulation. These findings demonstrate that HCC cells can directly induce a pro-tumorigenic phenotype in macrophages, characterized by increased cytokine and chemokine production and elevated PD-L1 expression. This direct activation of macrophages by HCC may exert pro-tumor effects by linking immunosuppression to HCC progression.

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