bioRxiv : the preprint server for biology · 2024
Human GM-CSF/IL-3 enhance tumor immune infiltration in humanized HCC patient-derived xenografts.
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Weinfurtner Kelley, Tischfield David, McClung George, Crainic Jennifer, Gordan John, Jiao Jing, Furth Emma E, Li Wuyan, Tuzneen Supan Erena, Nadolski Gregory J, Hunt Stephen J, Kaplan David E, Gade Terence P F
Аннотация
BACKGROUND & AIMS: Responses to immunotherapies in hepatocellular carcinoma (HCC) are 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 (PDXs).
MATERIAL AND METHODS: NOG (NOD/Shi- scid /IL-2R null ) and NOG-EXL (huGM-CSF/huIL-3 NOG) mice conditioned with Busulfan underwent i.v. injection of human CD34+ cells. HCC PDX tumors were then implanted subcutaneously (SQ) or orthotopically (OT). Following serial blood sampling, mice were euthanized at defined tumor sizes. Tumor, blood, liver, and spleen were analyzed by flow cytometry and immunohistochemistry.
RESULTS: Humanized NOG-EXL mice demonstrated earlier and increased human chimerism compared to humanized NOG mice (82.1% vs 43.8%, p<0.0001) with increased proportion of human monocytes (3.2% vs 1.1%, p=0.001) and neutrophils (0.8% vs 0.3%, p=0.02) in circulation. HCC tumors in humanized NOG-EXL mice had increased human immune cell infiltration (57.6% vs 30.2%, p=0.04), noting increased regulatory T cells (14.6% vs 6.8%, p=0.04), CD4+ PD-1 expression (84.7% vs 32.0%, p<0.01), macrophages (1.2% vs 0.6%, p=0.02), and neutrophils (0.5% vs 0.1%, p<0.0001). No differences were observed in tumor engraftment or growth latency in SQ tumors, but OT tumors required implantation at two rather than four 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.
CONCLUSIONS: 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.
IMPACT AND IMPLICATIONS: 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 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 advanced 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.
MATERIAL AND METHODS: NOG (NOD/Shi- scid /IL-2R null ) and NOG-EXL (huGM-CSF/huIL-3 NOG) mice conditioned with Busulfan underwent i.v. injection of human CD34+ cells. HCC PDX tumors were then implanted subcutaneously (SQ) or orthotopically (OT). Following serial blood sampling, mice were euthanized at defined tumor sizes. Tumor, blood, liver, and spleen were analyzed by flow cytometry and immunohistochemistry.
RESULTS: Humanized NOG-EXL mice demonstrated earlier and increased human chimerism compared to humanized NOG mice (82.1% vs 43.8%, p<0.0001) with increased proportion of human monocytes (3.2% vs 1.1%, p=0.001) and neutrophils (0.8% vs 0.3%, p=0.02) in circulation. HCC tumors in humanized NOG-EXL mice had increased human immune cell infiltration (57.6% vs 30.2%, p=0.04), noting increased regulatory T cells (14.6% vs 6.8%, p=0.04), CD4+ PD-1 expression (84.7% vs 32.0%, p<0.01), macrophages (1.2% vs 0.6%, p=0.02), and neutrophils (0.5% vs 0.1%, p<0.0001). No differences were observed in tumor engraftment or growth latency in SQ tumors, but OT tumors required implantation at two rather than four 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.
CONCLUSIONS: 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.
IMPACT AND IMPLICATIONS: 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 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 advanced 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.