Human GM-CSF/IL-3 enhance tumor immune infiltration in humanized HCC patient-derived xenografts

Abstract

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.

Material and methods: NOG (NOD/Shi-scid/IL-2Rγ^null) 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.

Results: Humanized NOG-EXL mice demonstrated earlier and higher levels of human chimerism compared to humanized NOG mice (82.1% vs. 43.8%, p <0.0001) with a greater 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 exhibited greater human immune cell infiltration (57.6% vs. 30.2%, p = 0.04) with higher proportions of 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 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.

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 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.

Keywords: HCC mouse models; humanized mouse; liver cancer; precision medicine; tumor immune microenvironment.

Graphical abstract

Fig. 1

Impact of human GM-CSF and IL-3 on circulating human immune cells in HIS mice. (A) Percentage of peripheral blood cells that were human immune cells (huCD45+) by flow cytometry. (B) Percentage of human immune cells by subtype in peripheral blood at week 15 post-humanization. (C–H) Percentage of peripheral blood cells that were human immune cell subtypes from time of humanization. (I-J) Percentage of liver and spleen cells that were human immune cells (huCD45+). (K–P) Percentage of human immune cells by subtypes in liver and spleen. Blue circle = HIS NOG; Purple square = HIS NOG-EXL. ∗p <0.05; ∗∗p <0.01 (Student’s t test, ANOVA). GM-CSF, granulocyte-macrophage colony-stimulating factor; HIS, humanized immune system; NOG, NOD.Cg-Prkdc^scidIl2rg^tm1Sug; NOG-EXL, NOD.Cg-Prkdc^scidIl2rg^tm1Sug Tg(SV40/HTLV-IL3,CSF2).

Fig. 2

Influence of human GM-CSF and IL-3 on tumor immune cell infiltration in HIS mice. (A) Proportion of mice with tumor engraftment. (B-D) Tumor growth rate by individual tumor, latency, and doubling time. (E-F) Percentage of tumor-infiltrating immune cells that were human immune cells and human T cells by flow cytometry. (G-I) Percentage of tumor-infiltrating human T cells that were CD4+, regulatory T cells, and PD-1⁺. (J-K) Number of human immune cells and human T cells per high-power field by IHC. (L) Percentage of tumor-infiltrating immune cells that are human macrophages by flow cytometry. (M) Number of human macrophages per high-power field by IHC. (N) Percentage of tumor-infiltrating immune cells that are human neutrophils by flow cytometry. Black triangle = NOG control; Blue circle = HIS NOG; Purple square = HIS NOG-EXL. ∗p <0.05; ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.00001 (Student’s t test, ANOVA). GM-CSF, granulocyte-macrophage colony-stimulating factor; HIS, humanized immune system; IHC, immunohistochemistry; IL-3, interleukin-3; NOG, NOD.Cg-Prkdc^scidIl2rg^tm1Sug; NOG-EXL, NOD.Cg-Prkdc^scidIl2rg^tm1Sug Tg(SV40/HTLV-IL3,CSF2).

Fig. 3

Effect of HCC tumor on circulating immune cells and chemokines in HIS mice. (A) Percentage of peripheral blood cells that were human immune cells (huCD45⁺) by flow cytometry in HIS mice with and without HCC PDX tumors. (B-G) Proportion of circulating human immune cells that were each subtype by flow cytometry in HIS mice with and without HCC PDX tumors. (H–R) Concentration of human cytokines and vascular growth factors in peripheral blood of HIS mice with and without HCC PDX tumors. Black circle, large dash = HIS NOG sham; Blue circle = HIS NOG HCC; Black square, small dash = HIS NOG-EXL sham; Purple square = HIS NOG-EXL. ∗p <0.05; ∗∗∗p <0.001 (Student’s t test, ANOVA). HCC, hepatocellular carcinoma; HIS, humanized immune system; NOG, NOD.Cg-Prkdc^scidIl2rg^tm1Sug; NOG-EXL, NOD.Cg-Prkdc^scidIl2rg^tm1Sug Tg(SV40/HTLV-IL3,CSF2); PDX, patient-derived xenograft.

Fig. 4

Immune engraftment in HIS mice depends on source of CD34+ cells. (A-D) Percentage of peripheral blood cells that were human immune cells (huCD45+) by flow cytometry for varying numbers of adult bone marrow-derived CD34+ cells (BM-CD34+ cells) compared to fetal liver-derived CD34+ cells (FL-CD34+ cells). (E-G) Proportion of human immune cells that were each subtype in peripheral blood at week 9 post-humanization based on number and source of CD34+ cells. (H–K) NOG-EXL mice humanized with 500,000 BM-derived CD34+ (HIS-BM) or 300,000 FL-derived CD34+ (HIS-FL) were orthotopically implanted with human HCC PDX tumors 2 weeks after humanization. Tumor growth was monitored by ultrasound to determine tumor engraftment rates at 24 weeks and growth latency compared to tumors implanted in non-humanized NOG-EXL mice. ∗p <0.05; ∗∗p<0.01; ∗∗∗p <0.001 (Student’s t test, ANOVA). BM, bone marrow; FL, fetal liver; HCC, hepatocellular carcinoma; HIS, humanized immune system; NOG, NOD.Cg-Prkdc^scidIl2rg^tm1Sug; NOG-EXL, NOD.Cg-Prkdc^scidIl2rg^tm1Sug Tg(SV40/HTLV-IL3,CSF2); PDX, patient-derived xenograft.