Abstract 5021: Regulatory T-cells and effects of anti-CTLA4 and anti-PD1 therapy in a transgenic murine model of neuroblastoma
Authors: Chan Randall , Shirinbak Soheila , Muthugounder Sakunthala , Hung Long , Gnanachandran Janahan , Hajidaniel Michael , Asgharzadeh Shahab 
Published: May 2015

Background:

The tumor microenvironment is important in the prognosis of Neuroblastoma (NBL), a common childhood cancer of the sympathetic nervous system. However little is know about the role of T regulatory-cells (Treg cells) in NBL. We hypothesize that Treg cells may play an important role in NBL development and immunotherapy targeted to Treg cells may modify the natural course of murine model of neuroblastoma driven by SV40's large T-antigen (NBL-Tag).

Methods:

Treg cells (positive for CD25/FOXP3/CD4/CD3ϵ) along with other common leukocytes were identified by flow cytometry in single-cell suspensions of adrenal tumors, lymph nodes, spleens, bone marrow and blood of NBL-Tag mice at 14-22 weeks of age. Age dependent pattern of T-cell infiltration and expression of 45 inflammation-relate genes (including CD4) were assessed by CD4 immunohistochemical staining (IHC), and RT-PCR (TaqMan-Low-Density-Array:TLDA), respectively.

A luciferase-expressing NBL-Tag cell line (NBTH-Luc) was used to establish a subcutaneous neuroblastoma models in C57Bl/6 mice and tumors were profiled using similar flow cytometry strategies as above. Animals with subcutaneous tumors or transgenic animals were subjected to immunotherapy using anti-CTLA-4, anti-PD-1, or combination of the two (with appropriate isotype controls) in low and high tumor burden scenarios.

Results:

IHC showed presence and increasing number of CD4+ cells in tumors of NBL-Tag mice over time. In comparison, no CD4+ cells were identified in wild-type adrenal glands. TLDA analysis also showed on average a 4-fold increased expression of CD4 cells in tumors compared to wild-type adrenal glands. Flow cytometry data demonstrated that the CD4+CD25+FOXP3+ Treg cells infiltrate NBL-Tag tumors at all ages (mean=2.9% of all CD45+ cells, n=17 mice). Subcutaneous syngeneic tumors were also infiltrated with CD4+CD25+FOXP3+ Treg cells (mean=8.6% of all CD45+ cells, n=7 mice). Combination of anti-CTLA-4 or anti-PD-1 was not effective in well-established tumors in either NBL-Tag transgenic model or subcutaneous model (p=NS). However, combination immunotherapy prevented formation of subcutaneous tumors (minimal residual disease model) compared to control group (p&lt;0.0001, n=5 per group).

Conclusion and Future Directions:

Our results demonstrate presence of Treg cells in NBL-Tag tumors. Treatment with anti-CTLA-4 and anti-PD-1 prevented formation of neuroblastoma tumors in a syngeneic subcutaneous model. Our data provides pre-clinical evidence that Ipilimumab and Nivolumab, recently approved melanoma drugs, may be useful in treating neuroblastoma children with minimal residual disease.

Citation Format: Randall Chan, Soheila Shirinbak, Sakunthala Muthugounder, Long Hung, Janahan Gnanachandran, Michael Hajidaniel, Shahab Asgharzadeh. Regulatory T-cells and effects of anti-CTLA4 and anti-PD1 therapy in a transgenic murine model of neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5021. doi:10.1158/1538-7445.AM2014-5021