Immune characterization of pre-clinical murine models of neuroblastoma

Abstract

Immunotherapy offers a potentially less toxic, more tumor-specific treatment for neuroblastoma than conventional cytotoxic therapies. Accurate and reproducible immune competent preclinical models are key to understanding mechanisms of action, interactions with other therapies and mechanisms of resistance to immunotherapy. Here we characterized the tumor and splenic microenvironment of two syngeneic subcutaneous (NXS2 and 9464D), and a spontaneous transgenic (TH-MYCN) murine model of neuroblastoma, comparing histological features and immune infiltrates to previously published data on human neuroblastoma. Histological sections of frozen tissues were stained by immunohistochemistry and immunofluorescence for immune cell markers and tumor architecture. Tissues were dissociated by enzymatic digestion, stained with panels of antibodies to detect and quantify cancer cells, along with lymphocytic and myeloid infiltration by flow cytometry. Finally, we tested TH-MYCN mice as a feasible model for immunotherapy, using prior treatment with cyclophosphamide to create a therapeutic window of minimal residual disease to favor host immune development. Immune infiltration differed significantly between all the models. TH-MYCN tumors were found to resemble immune infiltration in human tumors more closely than the subcutaneous models, alongside similar GD2 and MHC class I expression. Finally, TH-MYCN transgenic mice were administered cyclophosphamide alone or in combination with an anti-GD2 or anti-4-1BB monoclonal antibody, which resulted in increase in survival in both combination therapies. The TH-MYCN transgenic mouse is a promising in vivo model for testing immunotherapy compounds and combination therapy in a preclinical setting.

Figure 1

Tumor growth kinetics, survival and histological structure differ between subcutaneous and spontaneous neuroblastoma tumor models. (A) AJ or C57BL/6 mice were inoculated subcutaneously with NXS2 or 9464D (respectively) cells. Comparison of tumor growth kinetics of individual mice of NXS2 (grey) and 9464D (black) tumors, and average tumor growth. Tumor size is shown as mm². (B) Survival of mice bearing either NXS2 (grey), 9464D (black) or TH-MYCN (dashed) tumors. NXS2 and 9464D survival was measured from date of inoculation. TH-MYCN survival is measure from date of birth. n = 5. (C) ex vivo tumors were frozen in OCT and stained by H&E. For NXS2 tumor: (i) homogeneous appearance of the tissue; (ii) collapsed blood vessels; (iii) Mitotic figures; (iv) pseudorosettes; (v) muscle fibers. (D) For 9464D tumor: (i) loosely packed appearance with thick capsule; (ii) Large dilated blood vessel and thick capsule around tumor edge; (iii–v) demonstration of ‘holes’ throughout tumor. (E) For TH-MYCN tumor: (i) transverse section shows high complexity of the tumor microenvironment with visibly enlarged vessels ( →) and tertiary lymphoid structures surrounding the tumor mass (*); (ii) islands of cancer cells divided by fibrous septa; (iii) tertiary lymphoid structure; (iv) border between tumor cells (bottom) and adrenal gland (top) with ganglion cells in it; (v) cross section of a nerve with embedded ganglion cells, surrounded by cancer cells, fat tissue and enlarged arteriole. Scale bars, 100 µm.

Figure 2

GD2 and MHC Class 1 expression in NXS2, 9464D and TH-MYCN mouse neuroblastoma tumors. (A) Ex vivo tumors were disaggregated and stained for flow cytometry analysis. Representative histograms of GD2 and MHC I expression on NXS2, 9464D and TH-MYCN tumors. Expression of MHC I is shown on both GD2+ and non-GD2+ cells. Grey—isotype control; black line—anti-GD2 or MHC I antibody. (B) Quantitation of flow cytometry analysis of GD2 expression in ex vivo tumors by geo MFI. (C) Percentage of GD2+ MHC I+ cells and (D) MHC I expression as geo MFI on GD2+ cells. n = 9 (NXS2), n = 7 (9464D + TH-MYCN). Unpaired t-test, significance was assessed as: **< 0.01, ***< 0.001.

Figure 3

Immunophenotyping by flow cytometry of AJ, C57BL/6 and TH-MYCN tumor or non-tumor bearing spleens. (A) Spleens were harvested from either AJ, C57BL/6 or TH-MYCN (tumor bearing or non-tumor bearing) mice and processed into a single suspension for flow cytometry. Gating shown in Supplementary Figs. S4, S6. NK, B and T cells are shown for each strain. (B) Proportion of CD8⁺ and CD4⁺ T cells are shown for each strain, with (C) demonstrating proportion of Tregs as a percentage of CD4⁺ T cells. (D) Percentage of myeloid cells, macrophages, monocytes and neutrophils are demonstrated for each strain. Proportions of cells are shown as a percentage of total cells (singlets) unless otherwise stated. n = 5 per group (AJ and C57BL/6) or 4 per group (TH-MYCN). Significance calculated using Unpaired t-test between TB and NTB mice within each strain, with *< 0.05, **< 0.01, ***< 0.001.

Figure 4

Distribution of TIL populations within NXS2, 9464D and TH-MYCN tumors. (A) Ex vivo tumors were frozen in OCT. Either IHC or IF was performed to analyze the distribution and location of T cell subsets and B cells within the tumor mass. B + T cell distribution is demonstrated for NXS2 tumors by IHC staining for B220 (left—B cells) or CD3 (right—T cells). Images are shown at either ×4 magnification (top) or ×10 magnification (bottom). Dual staining of CD3 and CD4 (left) or CD8 (right) by IF of frozen NXS2 spleens (top) or tumors (bottom). Red = CD3, green = CD4/CD8, blue = DAPI. Same is shown in (B) for 9464D and (C) for TH-MYCN. Representative images are shown. Scale bars, 100 µm.

Figure 5

Quantification of TILs within tumors shows variability between NXS2, 9464D and TH-MYCN models. (A) Ex vivo tumor and spleens were disaggregated into single cell suspensions for detailed immunophenotyping by flow cytometry as detailed in methods. Gating shown in Supplementary Fig. S6. Proportions of NK, B and T cells are shown, with (B) percentages of CD4⁺ and CD8⁺ T cells within tumors. (C) Proportion of Treg cells as a percentage of CD4⁺ cells. (D) CD8:CD4 ratio (left side) and CD8⁺:FoxP3⁺ ratio (right side) is demonstrated for NXS2, 9464D and TH-MYCN tumors. All proportions are shown as percentage of total cells (singlets) unless otherwise stated. n = 7 (NXS2 and TH-MYCN) and n = 5 (9464D). Significance calculated using Kruskal Wallis test with Dunn’s multiple comparisons, with *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001.

Figure 6

Myeloid cells infiltration in the murine NXS2, 9464D and TH-MYCN neuroblastoma microenvironment. (A) Ex vivo tumors were frozen in OCT. IHC for F4/80 expression was performed to analyze macrophages within the tumor mass of (from left to right) NXS2, 9464D and TH-MYCN tumors. Representative images are shown for each tumor type. (B) Ex vivo tumor and spleens were disaggregated into single cell suspensions for detailed immunophenotyping by flow cytometry as detailed in methods. Gating shown in Supplementary Fig. S4. Proportions of myeloid subsets in tumors from (left to right) NXS2, 9464D and TH-MYCN tumor bearing mice. All proportions are shown as percentage of total cells (singlets). n = 9 (NXS2), n = 7 (TH-MYCN) and n = 5 (9464D). Significance calculated using Kruskal Wallis test with Dunn’s multiple comparisons, with *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001. Scale bars, 100 µm.

Figure 7

FcγR expression on myeloid cell infiltration in murine neuroblastoma microenvironment. (A) TH-MYCN tumors were frozen in OCT. H&E staining and IF for F4/80 and murine FcγRs were performed to demonstrate the distribution and location of macrophages and their receptor expression within the TH-MYCN tumor mass. Blue = DAPI, Red = F4/80, green = (from top to bottom) FcγRI, FcγRII, FcγRIII and FcγRIV. Representative images are shown. (B) Tumor and spleens were disaggregated into single cell suspensions for detailed immunophenotyping by flow cytometry as detailed in methods. Gating shown in Sup Fig. 4. FcγR expression was demonstrated as mean fluorescence intensity (MFI) with corresponding activatory:inhibitory (A:I) ratio for macrophages, (C) monocytes and (D) neutrophils, for (left to right) NXS2, 9464D and TH-MYCN tumors. n = 9 (NXS2) and n = 7 (9464D + TH-MYCN). Significance calculated using Kruskal Wallis test with Dunn’s multiple comparisons, with *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001. Scale bars, 100 µm.

Figure 8

Immunotherapy increases survival of TH-MYCN mice in a MRD setting, compared to immunotherapy alone. (A) Tumor-bearing TH-MYCN transgenic mice were treated i.p. with cyclophosphamide (CPM) alone, or 150 µg anti-GD2 (14G2a) mAb alone, or 150 µg anti-4-1BB (LOB12.3) mAb alone, or combinations thereof. Kaplan–Meier curves were generated. Titration of CPM doses to achieve a model of minimal residual disease (MRD). Time taken for tumors to re-present was used as end point. n = 2 (150 mg/kg), n = 3 (75 mg/kg), n = 5 (40 mg/kg) and n = 1 (20 mg/kg). (B) Combination therapy of 40 mg/kg CPM with LOB12.3 in TH-MYCN tumor bearing mice. (C) Combination therapy of CPM with 14G2a in tumor bearing TH-MYCN mice. Given the spontaneous nature of this model, controls accrue over time, and have been used for comparison to treatment arms in (B) and (C).