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.

Fc-null anti-PD-1 monoclonal antibodies deliver optimal checkpoint blockade in diverse immune environments

“…Mouse neuroblastoma 9464D cells were cultured as previously described. 20 Antibodies Parental anti-mouse PD-1 (EW1-9) rIgG1 was raised using conventional hybridoma technology. 28 Mouse and human anti-PD-1 EW1-9 mIgG1, mIgG2a, mIgG1-N297A, hIgG4 and hIgG4-FALA 26 were constructed as previously described.…”

“…To this end, we compared the immunogenic MC38 model, which bears a high tumor mutational burden (TMB), 19 with the 9464D pediatric neuroblastoma model. 20 21 Pediatric cancers represent a paradigm of immunologically cold tumors with a low mutational load, limited T-cell infiltration, and generally poor responsiveness to anti-PD-1/PD-L1. 22 However, like many adult cancers, there is evidence of PD-1/PD-L1 expression in pediatric tumors, 23 24 supporting the use of preclinical models to better understand how to target PD-1.…”

Anti-GD2 antibody therapy alters the neuroblastoma tumor microenvironment and extends survival in TH-MYCN mice

“…Similar results using TH-MYCN +/mice were recently shown, along with a synergistic effect of adding chemotherapy to GD2-directed antibody, analogous to the findings in children with relapsed neuroblastoma in the COG ANBL1221 chemoimmunotherapy trial. [8,17] Moreover, we found a reduced proportion of neuroblasts from 14G2a-treated mice expressed GD2 with decreased MFI when compared with PBS-treated tumors at both the midpoint and terminal time points, suggesting a selective pressure was exerted by 14G2a treatment. Recent work has suggested that GD2 serves as a macrophage checkpoint or "don't eat me" signal between neuroblasts and TAM.…”

“…[45] The TH-MYCN mouse model offers many advantages for the study of immune:tumor interactions, acknowledging there are certain limitations including the low frequency of macrometastases. [13,16,17,46,47] Anti-GD2 therapies against neuroblastoma have been studied…”

“…The tumor niche is important in influencing cancer progression as evidenced by syngeneic neuroblastoma transplant models in which subcutaneous implantation is 6 associated with reduced tumor growth and fewer infiltrating tumor-suppressive immune cells compared with the same tumor cells injected at orthotopic intra-adrenal sites. [17,18] Despite these advantages, an impediment to adoption of the highly penetrant 129x1/SvJ TH-MYCN model for studies into GD2-directed immunotherapies is the prevailing notion that these tumors lack sufficient surface GD2 to serve as an effective tumor antigen, primarily when investigated for syngeneic tumor transplantation studies. In the C57BL/6 background, preferred by many for immunologic studies, tumor penetrance for the TH-MYCN transgene is too low for efficient autochthonous modeling, and tumor-derived cell lines from this strain are found to express insufficient surface GD2 for therapeutic targeting unless they are manipulated pharmacologically or genetically.…”