Abstract Background: Subcutaneous syngeneic models are widely used to evaluate the impact of immunotherapy on tumor growth and tumor invading leucocytes (TILs). However, the tumor microenvironment of orthotopic models is more comparable to the patient as they form an organ-specific tumour, facilitate metastatic spread and, in the case of syngeneic models, support immune and stromal component interactions. Bioluminescent imaging (BLI) enables non-invasive longitudinal monitoring of orthotopic/metastatic tumor burden, allowing for optimal randomisation, reduction of false positives and continuous feedback on treatment mid-study. Here we describe the generation of several bioluminescent variants of syngeneic cell lines and assess the impact of orthotopic growth on response to immune checkpoint therapy. Methods: Bioluminescent variants of syngeneic cell lines were established by lentiviral transduction for: 4T1/EMT6 (breast), B16-F10 (skin melanoma), H22 (liver) and Pan02 (Pancreas) murine cancer cell lines. 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 was developed. BLI was carried out to assess real-time tumor burden and at end stage (Spectrum CT; PerkinElmer). Response to immune checkpoint therapy was evaluated and TIL infiltration was assessed by FACs analysis and IHC. Results: No significant differences in growth profiles were exhibited when the bioluminescent variants of B16-F10 and Pan02 (subcutaneous) or EMT6 (mammary fat pad) were compared with the wild-type equivalents. Bioluminescent 4T1 cells readily metastasised to the lungs from both the orthotopic and subcutaneous sites and B16-F10 cells formed metastases. Response to immunotherapy, such as anti-CTLA-4, in the subcutaneous setting between the bioluminescent and WT models was similar. Anti-CTLA-4 therapy resulted in a statistically significant impact on orthotopic Pan02 tumor growth (BLI) and final tumor weight; however, further characterization by IHC and refinement of implantation conditions is required as the tumors exhibited low basal TIL levels, poor stromal and blood capillary infiltration. 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 immunocompetent mice. However, the orthotopic growth and TIL infiltration does vary between subcutaneous and orthotopic/metastatic settings and requires characterisation and validation with immunotherapeutics in order to understand the relevance and utilisation of such models. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Sumanjeet Malhi, Jason King, Kelly Jones, Neil Williams, Rajendra Kumari. Evaluation of checkpoint inhibitors on tumour infiltrating immune cells in the orthotopic and metastatic tumour microenvironment using bioluminescent syngeneic cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1829. doi:10.1158/1538-7445.AM2017-1829