Combined Stereotactic Body Radiation Therapy and Immune Checkpoint Inhibition Slows Tumor Growth in a Novel Syngeneic Model of Neuroblastoma

Abstract

The majority of pediatric patients diagnosed with advanced-stage neuroblastoma die from their disease despite intensive multimodality therapy. Combination stereotactic body radiation therapy (SBRT) and immune checkpoint inhibition (ICI) represents a promising therapeutic avenue. We sought to develop a highly reproducible, syngeneic mouse model of advanced-stage neuroblastoma, in order to investigate the effects of combination SBRT/ICI on local control and the tumor immune microenvironment. We prepared an intrarenal tumorgraft model using the 9464D cell line, derived from the TH-MYCN transgenic neuroblastoma mouse. Following tumor implantation, mice were randomized to SBRT versus sham RT, as well as concurrently dosed anti-PD-1 antibody (αPD1) versus isotype control. SBRT fractions were delivered using the Small Animal Radiation Research Platform in 360 degree axial arcs to minimize normal tissue toxicity. Given increased radioresistance of 9464D relative to human neuroblastoma cell lines, three 8-Gy fractions were delivered. Tumor volume was monitored via CT and 3D ultrasound. Quantitative multiplex immunohistochemistry (qmIHC) staining was performed for immune markers including CD3 (T lymphocytes), F4/80 (macrophages) and PD-L1. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Columbia University Medical Center. 100% of 47 mice developed tumors, with 0% surgical mortality. Tumor grafts grew more uniformly than tumors derived from intrarenal injection of 9464D cell suspension (P < 0.05). Tumors were clearly delineated within the kidney following injection of iohexol contrast agent, allowing precise radiation targeting. RT induced intratumoral PD-L1 expression. Combined SBRT/αPD1 was more effective than either SBRT or αPD1 alone in reducing the rate of tumor growth (P < 0.001) as well as tumor weight at 35 days post-implant (P < 0.05). Combination therapy, however, was associated with toxicity, with 40% of mice dying within one week following SBRT/αPD1 administration. Tumors treated with combined SBRT/αPD1 demonstrated dense infiltration by macrophages and T lymphocytes. Our intrarenal syngeneic tumor graft model of advanced-stage neuroblastoma produces uniform tumor growth and is amenable to image-guided radiation therapy. SBRT/ICI promotes tumor infiltration by immune cells and slows tumor growth, and further mechanistic studies are warranted.