CTLA-4 immunotherapy exposes differences in immune response along with different tumor progression in colorectal cancer

Abstract

Tumor growth is accompanied by a changing tumor microenvironment and mutations that increase the resistance to therapy. Here, we used syngeneic models to evaluate the drug response of tumors of the same type of different sizes. We used the in vivo efficacy and Ki-67 immunohistochemistry (IHC) assay to assess the difference in responses in response to treatment with the same concentration of anti-CTLA-4. Flow cytometry analysis revealed changes in the immune subpopulations changes the spleen, peripheral blood, lymph node, and tumor tissue across different tumor growth phases. For example, naive CD4+T, CD4+TCM, CD8+TEM, T, B, Treg, CD8+TCM exhibited different percentages depending on the specific immune organ. To further expose the changes in the immune microenvironment, the level of expression of PD-1 and CTLA-4 showed statistically significant difference in related subsets for each four immune tissues in different tumor sizes. In addition, the ratios of CD4 + Teff/ CD4 + Treg and CD8 + T/Treg in corresponding immune tissue were also associated with statistically significant differences alongside tumor growth in different animal models. These results reveal the ongoing changes in the immune microenvironment during tumor progression and anti-CTLA-4 antibody immunotherapy effect depends on the expression level of immune factors.

Keywords: CTLA-4; central memory T cell (TCM); effector memory T cell (TEM); tumor-immune microenvironment.

Figure 1

Different tumor sizes depending on the start treatment (100 to 800 mm³) of the subcutaneously inoculated CT26 show the different anti-tumor effects. The cell numbers of inoculation is 3 × 10⁵ into 100 ul for each mouse. Tumor volume trace after administering anti-CTLA-4 Ab to female BALB/c mice inoculated with the CT26 syngeneic model. (A) Volume of tumors from mice inoculated with CT26 subcutaneously. Mean tumor volumes (mm³) ± SE (n = 5 mice per group) are shown. Data are statistically significant for anti-CTLA-4 antibody 10 mg/kg intraperitoneal injection, weekly; ***, P < 0.001, on day 17. (B) Photos of the tumors isolated from mice inoculated with CT26 cells in the study.

Figure 2

Ki-67 immunohistochemistry analysis of the proliferation of the tumor tissue in the difference groups. Ki-67 staining in the vehicle group (A), the 200 mm³ group (B), the 400 mm³ group (C), and the 800 mm³ group (D); n = 5 mice per group. Data are statistically significant for anti-CTLA-4 antibody 10 mg/kg intraperitoneal injection weekly; **, P < 0.01 on day17, Photos of the tumors isolated from mice inoculated with the CT26 cells. For group 2 (start treatment with the tumor size of 100mm³), no IHC staining is shown due to the small tumor volume on the final day.

Figure 3

Percentages of the different subpopulations and expression levels in different tissues of the subcutaneous xenograft model. Distribution of immune cell subpopulations in the tumor, spleen, lymph node, and blood isolated from the same tumor progression animal model (CT26 cells). The gating schema for the study and the percentage of immune cell subpopulations in TILs (A), lymph node (B), spleen (C), and peripheral blood (D).

Figure 4

Immune microenvironment differences accompany the tumor progression changes in the different immune tissues. The percentage of immune cell subpopulations in different immune tissues isolated from both CT26 and Colon26 animal models in Balb/c female mice. (A) Different immune factor expression ratios in TILs. (B) Different immune factor expression ratios in the lymph node isolated from the armpit. (C) Different immune subpopulations expression ratio in the spleen tissue isolated from an immune-competent mouse. (D) Different immune subpopulation expression ratios in peripheral blood isolated from both CT26 and Colone26 animal models.

Figure 5

Expression of PD-1 and CTLA-4 checkpoints in each subpopulation of different immune tissues at different stages of the tumor progression. The different colors represent the different levels of expression. The differential expression of PD-1 and CTLA-4 in immune cell subpopulations in different immune tissues isolated from the CT26 animal model in Balb/c female mice. (A) Different expression levels of PD-1 and CTLA-4 in each subpopulation in TILs. (B) Different expression levels of PD-1 and CTLA-4 in each subpopulation in the lymph nodes. (C) Different expression levels of PD-1 and CTLA-4 in each subpopulation in the spleen (no sufficient data could be gathered for the MFI of CTLA-4 in the different subpopulations when the tumor size was 400 mm³ and 800 mm³). (D) Different expression levels of PD-1 and CTLA-4 in each subpopulation in the peripheral blood samples in the CT26 animal model. The statistical analysis in this figure was obtained by analyzing the raw data. The dendrogram was plotted from the raw data of each subpopulation and biomarker using the R language.

Figure 6

Expression of the PD-1 and CTLA-4 checkpoints in each subpopulation of different immune tissues at each stage of tumor progression. The different colors represent different levels of expression. The differential expression of PD-1 and CTLA-4 in immune cell subpopulations in different immune tissues isolated from the Colon26 animal model. Different expression levels of PD-1 and CTLA-4 in each subpopulation in TILs (A), lymph nodes (B), spleen (C), and peripheral blood samples (D) in the Colon26 animal model. The statistical analysis in this figure was obtained by analyzing the raw data. The dendrogram was plotted from the raw data of each subpopulation and biomarker using the R language.

Figure 7

Ratio of CD4+ Effector T / regulatory T cells (Teff/Treg) and CD8+ T/Treg (T/regulatory T cells) in each immune tissue. The error bar represents the variation of the data in the same group. The different colors represent the different tumor sizes in the corresponding tissues. (A) Differential ratio of Teff/Treg in CD4T and CD8T/Treg in the difference tumor progression phase in the CT26 animal model. (B) Differential ratio of Teff/Treg in CD4T and CD8T/Treg in the difference tumor progression phase in the Colon26 animal model. The statistical analysis in this figure was obtained by analyzing the raw data.