Abstract A140: Check point inhibitor modulation of tumor microenvironment at orthotopic and metastatic sites using bioluminescent syngeneic cell line models in immune competent mice
Authors: McKenzie Andrew , Papadopoulou Nektaria , Jiang Simon , Wrigley Jane , Jones Kelly , Garland Russell , Williams Neil , Kumari Rajendra 
Published: November 2016

Introduction: Syngeneic models are widely used to model the impact of cancer immunotherapy on tumor growth and tumor invading leucocytes (TILs), and the majority of such work is typically carried out in the subcutaneous setting. However, orthotopic models are known to better model cancer in patients as they form a single focal disease area as in the patient situation, facilitate metastatic spread via intra- and extra-thoracic lymph nodes and, in the case of syngeneic models, strain-specific tumor microenvironment interactions (immune and stromal components). Bioluminescent imaging (BLI) increases the usefulness of such models, as it allows for non-invasive longitudinal monitoring of tumor burden, allowing for optimal randomisation and reduction of false positives. It also allows continuous feedback allowing one to optimise treatment regimen mid-study. Herein we describe the generation of several bioluminescent variants of syngeneic cell lines commonly used for immunotherapy studies and assess the impact of orthotopic growth on response to immune check point therapy.

Experimental Procedures: Bioluminescent cell line variants of syngeneic cell lines were established by lentiviral transduction for: 4T1 (breast), B16-F10 (melanoma), MBT-2 (bladder) H22 (Hepatoma), LL/2 (lung), Pan02 (Pancreatic) and RM-1 (prostate). Following establishment of stable cultures, DNA profiling and in vitro cytotoxicity assays was carried out to ensure there was no significant changes in DNA, cell doubling time or response to SoC agents following transduction. Subcutaneous growth of wild-type and bioluminescent variants was compared to assess any impact of luciferase expression on tumor growth, inflammation and TILs. Orthotopic models were established for most cell lines, and a metastatic model for B16-F10, BLI was carried out to assess real-time tumor growth and tumor burden at end stage (Spectrum CT; PerkinElmer). Immune checkpoint therapy was also assessed and TIL infiltration by FACs analysis and IHC.

Results: Stable transduced bioluminescent cell lines were established; cell doubling time, morphology and growth in vitro was found to be consistent with their wild-type counterparts. Bioluminescent 4T1 cells exhibited growth consistent to that previously reported and readily metastasised to the lungs from both the orthotopic and subcutaneous sites; B16-F10 cells readily metastasise to bone following intracardiac administration. Subcutaneous response to immunotherapy did not appear to be affected. Modulation of immune cell infiltration will be reported and correlated to response.

Conclusions: The growth and response to immunotherapy does not appear to be significantly impacted in the bioluminescent cell line models tested; as such they are a useful tool for further assessing the impact of complex orthotopic, spontaneous and experimental metastasis modelling in immune competent mice.

Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Kelly Jones, Russell Garland, Neil Williams, Rajendra Kumari. Check point inhibitor modulation of tumor microenvironment at orthotopic and metastatic sites using bioluminescent syngeneic cell line models in immune competent mice [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A140.