Immune characterization of pre-clinical murine models of neuroblastoma

Emily R Webb,Carol Wareham,Stephen A Beers,Mark S Cragg,Stuart N Dunn,Alistair Easton,Sonya James,Silvia Lanati,Freja M Sadler,Margaret Ashton-Key,Juliet C Gray,Tatyana Inzhelevskaya

doi:10.1038/s41598-020-73695-9

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.

Highlights

Immunotherapy offers a potentially less toxic, more tumor-specific treatment for neuroblastoma than conventional cytotoxic therapies
Transgenic TH-MYCN mice developed large tumor masses in the abdominal cavity, with adhesion to the spine, with one or both kidneys encased in the tumor mass
As previously reported[18,22], TH-MYCN tumors were histological complex (Fig. 1Ei), with islets of multinucleated neuroblastoma cells divided by septae of fibrous tissue (Fig. 1Eii)

Summary

Introduction

Immunotherapy offers a potentially less toxic, more tumor-specific treatment for neuroblastoma than conventional cytotoxic therapies. 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. 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. Anti-GD2 mAbs have been shown to improve outcome in first line treatment for high risk neuroblastoma, and are considered a key component of treatment[9,10]. Survival and histological structure differ between subcutaneous and spontaneous neuroblastoma tumor models. Pre-clinical modeling is essential in order to compare and prioritize different therapies, to understand mechanisms of action and resistance, and to identify potential biomarkers. The number of potential combinational therapies is vast, and careful pre-clinical assessment is vital to rationally guided trial design, given the small potential patient population

Objectives

Methods

Results

Conclusion