PD1 blockade alters cell-cycle distribution and affects 3'-deoxy-3'-[18F]fluorothymidine uptake in a mouse CT26 tumor model

Abstract

Objective: We previously reported that alterations of the tumor microenvironment (TME) by programmed death receptor-1 (PD1) blockade affected tumor glucose metabolism and tumor 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) uptake. In cancer cells, high glycolysis allows cells to sustain rapid proliferation since glycolysis is closely related to the proliferation of cancer cells. Therefore, imaging of cellular proliferation may provide more detail of TME alterations. In this study, we investigated how TME alterations by PD1 blockade affects the uptake of 3'-deoxy-3'-[¹⁸F]fluorothymidine ([¹⁸F]FLT), which is a ¹⁸F-radiolabeled thymidine derivative and is taken up by proliferating cells.

Methods: Mice inoculated with murine colon carcinoma CT26 cells were intraperitoneally administered an anti-PD1 antibody on Day 0, when the tumor volume exceeded 50 mm³, and Day 5. [¹⁸F]FLT-PET imaging was performed pre-treatment (Day 0) and post treatment (Day 7). Tumor infiltrating lymphocytes (TILs) were identified by flow cytometry. [¹⁸F]FLT accumulation and localization in tumor tissue was evaluated by autoradiography and immunohistochemistry. The cell-cycle distribution of tumors and CT26 cells exposed to cytokines (interleukin-2, interferon [INF]-γ, and tumor necrosis factor [TNF]-α) was analyzed by flow cytometry.

Results: PD1 blockade increased CD8⁺ and CD4⁺ T cells in tumor tissue and significantly suppressed tumor proliferation; however, tumor [¹⁸F]FLT uptake remained unchanged. Autoradiography and immunohistochemistry showed that [¹⁸F]FLT was mainly taken up by cancer cells, but not TILs. Flow cytometric analysis demonstrated that the population of cells in G2/M phase increased after PD1 blockade. Moreover, INF-γ and TNF-α significantly increased cells in G2/M phase in vitro.

Conclusion: PD1 blockade-induced alteration of the TME increased CT26 tumor cells in the G2/M phase, which have high thymidine kinase 1 activity. Therefore, [¹⁸F]FLT is taken up by tumor cells even if tumor proliferation is suppressed. This observation may be useful for evaluating the response to immunotherapy.

Keywords: Cell cycle; Cytokine; PD1; PET/CT; [18F]FLT.