肝细胞癌（HCC）是最常见的原发性肝癌，也是全球所有癌症相关死亡的第四大原因。肝移植、手术和局部消融是早期 HCC 的治疗方法。然而，治疗后结果可能因组织病理学阶段而异。与治疗后高分化 HCC 相比，低分化 HCC 与较高的肿瘤进展率和较低的总生存率相关。在这项研究中，我们的目的是在体外环境中表征组织病理学证明的分化良好和分化不良的 HCC 的癌症干细胞 (CSC) 谱。我们根据每种类型 HCC 的表面标志物和对 NK 细胞介导的细胞毒性的敏感性来表征其干细胞样特征。流式细胞术用于量化良好和不良之间 MHC-I 类、CD54 和 CD44 的差异表达。分化型 HCC。未处理的原代 NK 细胞、IL-2 刺激的原代 NK 细胞以及超荷 (sNK) 细胞介导的细胞毒性针对高分化和低分化 HCC 进行了评估。来自各个 NK 细胞实验组的 IFN-γ 上清液也用于诱导 HCC 的分化。最后，我们使用成像和阻抗的实时定量分析（eSight 研究）来表征颞 NK 效应细胞的细胞毒性。低分化 HCC 表现出 MHC-I 类和 CD54 的低表面表达，以及 CD44 的高表达。治疗分泌 IFN-γ 的 NK 细胞或诱导 HCC 分化的 IFN-γ 细胞因子。与分化良好的 HCC 相比，低分化的 HCC 对原代 NK 细胞、IL-2 刺激的原代 NK 细胞和 sNK 细胞中 NK 细胞介导的细胞毒性更敏感。与未经处理或 IL-2 刺激的原代 NK 细胞相比，sNK 细胞对分化良好的 HCC 具有显着更高的细胞毒性。这些发现通过实时定量成像分析进行了概括。低分化 HCC 被发现具有 CSC 的表面标记模式，使其对基于 NK 细胞的免疫疗法高度敏感。基于 NK 细胞的疗法有可能用作低分化 HCC 的新辅助或辅助疗法。增压的 NK 细胞可以快速扩增至治疗水平，具有独特的溶解低分化和高分化 HCC 的能力。这一发现表明，sNK 细胞不仅表现出针对 NK 细胞靶标的增强功能，而且能够激活 T 细胞，诱导针对高表达 MHC I 类的分化良好的 HCC 的细胞毒性。版权所有 © 2023 Jiang, Chen, Pham, Nguyen 、考尔、拉曼和朱厄特。

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is the fourth-leading cause of all cancer-related deaths around the world. Liver transplantation, surgery, and local ablation are curative therapies for early-stage HCC. However, post-treatment outcomes can vary based on histopathologic stage. Poorly-differentiated HCC are associated with higher rates of tumor progression and lower overall survival compared to well-differentiated HCC after therapy. In this study, we aimed to characterize the cancer stem cell (CSC) profile of histopathologically-proven well and poorly-differentiated HCCs in an in-vitro environment. We characterized the stem-like profile of each type of HCC based on their surface markers and susceptibility to NK cell-mediated cytotoxicity.Flow cytometry was used to quantify differential expression of MHC-class I, CD54, and CD44 between well- and poorly-differentiated HCCs. Primary untreated NK cells, IL-2 stimulated primary NK cells, and supercharged (sNK) cell-mediated cytotoxicity was assessed against well- and poorly-differentiated HCCs. IFN-γ supernatant from each respective NK cell experimental arm was also used to induce differentiation of HCCs. Finally, we characterized the temporal NK effector cell cytotoxicity using real-time quantitative analysis of imaging and impedance (eSight study).Poorly-differentiated HCCs demonstrated low surface expression of MHC-class I and CD54, and high expression of CD44. Treatment of NK cells secreted IFN-γ or IFN-γ cytokine induced differentiation in HCCs. Poorly-differentiated HCCs in comparison to well-differentiated HCC were more susceptible to NK cell-mediated cytotoxicity in primary NK cells, IL-2 stimulated primary NK cells, and sNK cells. sNK cells induced significantly higher cytotoxicity against well-differentiated HCCs in comparison to untreated or IL-2-stimulated primary NK cells. These findings were recapitulated with real-time quantitative imaging analysis.Poorly-differentiated HCCs were found to have surface marker patterns of CSCs, making them highly susceptible to NK cell-based immunotherapy. NK-cell based therapy can potentially be leveraged as a neoadjuvant or adjuvant therapy in poorly-differentiated HCCs. Supercharged NK cells, which can be rapidly expanded to therapeutic levels, are uniquely capable of lysing both poorly- and well-differentiated HCCs. This finding suggests that sNK cells not only exhibit enhanced features against NK cells' targets but also are capable of activating T cells to induce cytotoxicity against well-differentiated HCCs with high expression of MHC class I.Copyright © 2023 Chiang, Chen, Pham, Nguyen, Kaur, Raman and Jewett.