Weinfurtner Kelley, Tischfield David, McClung George, Crainic Jennifer, Gordan John, Jiao Jing, Furth Emma E, Li Wuyan, Supan Erena Tuzneen, Nadolski Gregory J, Hunt Stephen J, Kaplan David E, Gade Terence P F
Penn Image-Guided Interventions Laboratory, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
Division of Gastroenterology and Hepatology, University of Pennsylvania, Philadelphia, PA, United States.
JHEP Rep. 2024 Nov 8;7(3):101264. doi: 10.1016/j.jhepr.2024.101264. eCollection 2025 Mar.
BACKGROUND & AIMS: Response to immunotherapy in hepatocellular carcinoma (HCC) is suboptimal with no biomarkers to guide patient selection. "Humanized" mice represent promising models to address this deficiency but are limited by variable chimerism and underdeveloped myeloid compartments. We hypothesized that expression of human GM-CSF and IL-3 increases tumor immune cell infiltration, especially myeloid-derived cells, in humanized HCC patient-derived xenografts.
NOG (NOD/Shi-/IL-2Rγ) and NOG-EXL (huGM-CSF/huIL-3 NOG) mice conditioned with busulfan underwent i.v. injection of human CD34+ cells. HCC patient-derived xenograft tumors were then implanted subcutaneously or orthotopically. Following serial blood sampling, mice were euthanized at defined tumor sizes. Tumor, blood, liver, and spleen were analyzed by flow cytometry and immunohistochemistry.
Humanized NOG-EXL mice demonstrated earlier and higher levels of human chimerism compared to humanized NOG mice (82.1% 43.8%, 0.0001) with a greater proportion of human monocytes (3.2% 1.1%, 0.001) and neutrophils (0.8% 0.3%, 0.02) in circulation. HCC tumors in humanized NOG-EXL mice exhibited greater human immune cell infiltration (57.6% 30.2%, 0.04) with higher proportions of regulatory T cells (14.6% 6.8%, 0.04), CD4+ PD-1 expression (84.7% 32.0%, 0.01), macrophages (1.2% 0.6%, 0.02), and neutrophils (0.5% 0.1%, 0.0001). No differences were observed in tumor engraftment or growth latency in subcutaneous tumors, but orthotopic tumors required implantation at 2 rather than 4 weeks post-humanization for successful engraftment. Finally, utilizing adult bone marrow instead of fetal livers enabled partial HLA-matching to HCC tumors but required more CD34+ cells.
Human GM-CSF and IL-3 expression in humanized mice resulted in features more closely approximating the immune microenvironment of human disease, providing a promising model for investigating critical questions in immunotherapy for HCC.
This study introduces a unique mouse model at a critical point in the evolution of treatment paradigms for patients with hepatocellular carcinoma (HCC). Immunotherapies have become the first-line treatment for advanced HCC; however, response rates remain low with no clear predictors of response to guide patient selection. In this context, animal models that recapitulate human disease are greatly needed. Leveraging xenograft tumors derived from patients with unresectable HCCs and a commercially available immunodeficient mouse strain that expresses human GM-CSF and IL-3, we demonstrate a novel but accessible approach for modeling the HCC tumor microenvironment.
肝细胞癌（HCC）对免疫疗法的反应欠佳，且缺乏指导患者选择的生物标志物。“人源化”小鼠是解决这一缺陷的有前景的模型，但受嵌合率可变和髓系区室发育不全的限制。我们假设人GM-CSF和IL-3的表达会增加人源化HCC患者来源异种移植瘤中的肿瘤免疫细胞浸润，尤其是髓系来源的细胞。
用白消安预处理的NOG（NOD/Shi-/IL-2Rγ）和NOG-EXL（huGM-CSF/huIL-3 NOG）小鼠经静脉注射人CD34+细胞。然后将HCC患者来源的异种移植瘤皮下或原位植入。在连续采血后，在肿瘤达到规定大小时对小鼠实施安乐死。通过流式细胞术和免疫组织化学分析肿瘤、血液、肝脏和脾脏。
与人源化NOG小鼠相比，人源化NOG-EXL小鼠表现出更早且更高水平的人嵌合率（82.1%对43.8%，P<0.0001），循环中人类单核细胞（3.2%对1.1%，P<0.001）和中性粒细胞（0.8%对0.3%，P<0.02）的比例更高。人源化NOG-EXL小鼠的HCC肿瘤表现出更大程度的人类免疫细胞浸润（57.6%对30.2%，P<0.04），调节性T细胞（14.6%对6.8%，P<0.04）、CD4+ PD-1表达（84.7%对32.0%，P<0.01）、巨噬细胞（1.2%对0.6%，P<0.02）和中性粒细胞（0.5%对0.1%，P<0.0001）的比例更高。皮下肿瘤在肿瘤植入或生长潜伏期方面未观察到差异，但原位肿瘤在人源化后2周而非4周进行植入才能成功植入。最后，使用成年骨髓而非胎肝可实现与HCC肿瘤的部分HLA匹配，但需要更多的CD34+细胞。
人源化小鼠中人类GM-CSF和IL-3的表达产生了更接近人类疾病免疫微环境的特征，为研究HCC免疫治疗中的关键问题提供了一个有前景的模型。
本研究在肝细胞癌（HCC）患者治疗模式演变的关键节点引入了一种独特的小鼠模型。免疫疗法已成为晚期HCC的一线治疗方法；然而，反应率仍然很低，且没有明确的反应预测指标来指导患者选择。在此背景下，迫切需要能够重现人类疾病的动物模型。利用来自不可切除HCC患者的异种移植瘤和一种表达人GM-CSF和IL-3的市售免疫缺陷小鼠品系，我们展示了一种新颖但可行的方法来模拟HCC肿瘤微环境。
