Immune Response to Bioluminescence Imaging Reporters in Murine Tumor Models

The in vitro and in vivo growth effects of two stably expressed bioluminescence reporter genes, a red-shifted firefly luciferase and a click beetle green luciferase, were evaluated in four different cancer cell lines. Differences in parental and reporter-expressing cancer cell immune cell composition, activation, and secreted cytokine levels were evaluated using flow cytometry, cytokine arrays and ELISAs.

The data revealed no significant differences in in vitro cell proliferation between parental and reporter cancer cell lines. In vivo subcutaneous tumor growth was not observed in tumor cells stably expressing the red-shifted firefly luciferase. Cells labeled with click beetle green luciferase demonstrated no significant differences in in vivo subcutaneous tumor growth compared to parental cells. Tumor cells expressing red-shifted firefly luciferase induced an increase in activated and cytotoxic T cells compared to parental and click beetle green luciferase, suggesting enhanced immunogenicity. Furthermore, the tumor-immune composition and cytokine production were similar between parental and click beetle green luciferase-labeled tumor cells.

These findings demonstrate that the stable expression of click beetle green luciferase in cancer cells, in contrast to red-shifted firefly luciferase, has minimal immunogenicity and does not alter tumor development in immunocompetent mice. We report detailed characterization studies of bioluminescence reporter cells, providing essential considerations for their use in investigating tumor-immune interactions in syngeneic murine tumor models.