Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor

Abstract

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

Fig 1. TdTomato/Luc expression does not alter cell surface markers of LL/2 cells.

Flow cytometry plots of surface markers expressed on LL/2 and LL/2-tdTomato/Luc cells with or without IFN-γ treatment. LL/2 cells (A) and LL/2-tdTomato/Luc cells (B) were cultured with or without IFN-γ (100 ng/ml) for 72 hours. Cells were harvested and stained for FACS analysis as described in the Methods section.

Fig 2. TdTomato/Luc expression by LL/2 cells inhibits its tumorigenicity.

(A) Tumor cell growth in vitro, and (B) tumor growth in vivo. (C) Spleen weight of tumor-bearing mice (n = 3). Mean ± SEM is plotted. Significance of differences was determined by using unpaired student t-tests. Values of p<0.05 were considered significant (*p < 0.05, **p < 0.01, ***p ≤ 0.001).

Fig 3. TdTomato/Luc expression by LL/2 cells increases tumor immunogenicity and inhibits tumor-induced G-CSF and MDSC levels.

(A-C) Tumors from Fig 2B were harvested and analyzed for tumor infiltrating cell counts of (A) CD4+ T cells (CD45+, CD3+, CD4+), (B) CD8+ T cells (CD45+, CD3+, CD8+), and (C) B cells (CD45+, CD19+) are shown. (D-F) MDSC levels in the spleen, tumor and blood samples of LL/2 and LL/2-tdTomato/Luc tumor bearing mice. (G) The levels of common MDSC-inducing factors in the plasma of mice including G-CSF, GM-CSF, TNF-α, IFN-γ, IL-1β, and IL-6. (H) The concentration of G-CSF in the plasma of mice. Mean ± SEM is plotted. Significance of differences was determined by using unpaired student t-test. Values of p<0.05 were considered significant (*p < 0.05, **p < 0.01, ***p ≤ 0.001).

Fig 4. LL/2-tdTomato/Luc tumor growth is inhibited by CD4+ T cells, CD8+ T cells and NK cells in vivo.

(A) Experimental design of in vivo cell depletion study (n = 5). (B) Tumor growth and (C) tumor-free survival. Data were analyzed by unpaired student t-tests on day 30. Mean ± SEM is plotted. Values of p<0.05 were considered significant. *p < 0.05 LL/2-tdTomato/Luc vs. all the other groups. Tumor-free survival was analyzed using Kaplan-Meier analysis. Values of p<0.05 were considered significant. *p < 0.05 LL/2-tdTomato/Luc vs. all the other groups.

Fig 5. ICI blockade does not inhibit LL/2 or LL/2-tdTomato/Luc tumor growth.

(A) Experimental design of the in vivo study (n = 5). (B) Tumor size of mice treated with anti-PD-1 or anti-CTLA-4 mAb. Mean ± SEM is plotted. Significance of differences was determined by using unpaired student t-test. Values of p<0.05 were considered significant.