Anti-PD-L1 Antibody Enhances T Cell Immune Responses and Reduces Resistance of Breast Cancer Cells to Radiotherapy

Abstract

Immune escape is a frequent occurrence, which limits the duration of antitumor immune responses to radiotherapy. Here, we aimed to ascertain the roles and underlying mechanisms of programmed death ligand 1 (PD-L1) in tolerance of breast cancer (BC) to radiotherapy. We first quantified microRNA-21 (miR-21) and PD-L1 expression in BC tissues and cells, followed by identification of the interactions between miR-21, PD-L1, and programmed cell death protein 4 (PDCD4). miR-21 knock-in mice were used to construct tumor-bearing models, which were then treated with anti-PD-L1 antibody and irradiation, followed by measurement of tumor growth and tumor immune escape. Finally, we evaluated the synergistic effects of radiotherapy and anti-PD-L1 antibody in vivo. The results showed increased miR-21 expression in BC tissues and cells, which was positively correlated with PD-L1 expression. The treatment with radiotherapy or anti-PD-L1 antibody in the miR-21 knock-in mice diminished tumor weight and volume, along with decreased CD3⁺CD8⁺ positive cells, serum IL-2 and IFN-γ levels, and lower PD-L1 expression, but augmented apoptosis of T and BC cells. Moreover, miR-21 significantly augmented PD-L1 expression via PI3K/Akt pathway activation by targeting PDCD4 in BC cells. Thus, radiotherapy and anti-PD-L1 antibody synergistically accelerated the therapeutic effect against BC in mice, thereby implicating a close interplay between radiotherapy, T cells, and the miR-21/PDCD4/PI3K/Akt/PD-L1 axis.

Figure 1

miR-21 expression is increased and positively correlates to PD-L1 expression in BC tissues of patients. (a) Volcano map of differentially expressed miRNAs in 50 BC tissue samples and in 3 adjacent normal tissue samples in the GSE44124 dataset (The x-axis represents the difference log10 p value and the ordinate represents logFoldChange. Each point in the graph represents a gene, the red point represents the upregulated gene with logFoldChange greater than 1 and the green point represents the downregulated gene with logFoldChange less than -1). (b) Heat map of differentially expressed miRNAs in 50 BC tissue samples and in 3 adjacent normal tissue samples in the GSE44124 dataset. (c) miR-21 differential expression in BC tissue samples and normal breast tissue samples using the TCGA database. (d) Expression of miR-21 in BC and adjacent normal tissues determined by RT-qPCR (n = 50). (e) Correlation between PD-L1 and miR-21 expression in BC tissue samples. (f) PD-L1 protein level in BC tissues measured by immunohistochemistry (n = 50, scale bar = 50 μm; the nucleus was stained blue, and the positive cells were stained yellow-brown). (g) PD-L1 protein level in BC tissues measured by Western blot analysis (n = 50). ^∗p < 0.05 compared with adjacent normal tissues. Data (mean ± standard deviation) from two groups were compared using paired t-test or independent sample t-test. Each sample was evaluated three times independently.

Figure 2

miR-21 knock-in increases tolerance of BC cells to radiotherapy and promotes tumor immune escape in mice. WT or miR-21^+/+ mice were either treated or not treated with radiotherapy and anti-PD-L1 antibody. (a) Tumor volume curve of the mice. (b) Tumor weight of the mice. (c) The number of CD3⁺CD8⁺ cells in peripheral bloods and tumor tissues of mice detected by flow cytometry. (d) T lymphocyte apoptosis in peripheral blood and tumor tissues of mice measured using Annexin V-FITC/PI. (e) Level of IL-2 in peripheral blood and tumor tissues of mice measured using ELISA. (f) Level of IFN-γ in peripheral blood and tumor tissues of mice measured using ELISA. ^∗p < 0.05 compared with WT mice; ^#p < 0.05 compared with WT mice treated with radiotherapy. Each sample was evaluated three times independently. Data (mean ± standard deviation) from two groups were compared using independent sample t-test while data for tumor volume were compared using repeated measures ANOVA. There were 10 mice in each group.

Figure 3

miR-21 potentiates PD-L1 expression via activation of the PI3K/Akt pathway by targeting PDCD4 in BC cells. (a) Venn diagram depicting the intersection of target genes of miR-21 predicted with PicTar, miRDB, TargetScan, and starBase databases. (b) Protein-protein interaction network of the miR-21 downstream target genes. (c) Correlation between PDCD4 and miR-21 expression in BC tissue samples from the TCGA database. (d) Binding site between miR-21 and PDCD4 predicted using the TargetScan database. (e) The binding of miR-21 to PDCD4 confirmed by dual luciferase reporter gene assay. (f) Expression of miR-21, PDCD4, and PD-L1 after alteration of miR-21 and overexpression of PDCD4 determined by RT-qPCR in BC cells. ^∗p < 0.05 compared with cells treated with mimic NC. ^#p < 0.05 compared with cells treated with inhibitor NC and p < 0.05 compared with cells treated with miR-21 mimic. (g) The silencing efficiency of si-PDCD4-1 and si-PDCD4-2 in BC cells determined by RT-qPCR. ^∗p < 0.05 compared with cells treated with si-NC. (h) Western blot analysis of PDCD4, PI3K, Akt, and PD-L1 proteins, and the extent of PI3K and Akt phosphorylation in BC cells. ^∗p < 0.05 compared with cells treated with oe-NC. ^#p < 0.05 compared with cells treated with si-NC and p < 0.05 compared with cells treated with si-PDCD4. Data (mean ± standard deviation) from two groups were compared using independent sample t-test while those from multiple groups were compared using one-way ANOVA. All cell experiments were repeated 3 times independently.

Figure 4

Anti-PD-L1 antibody combined with radiotherapy represses BC cell growth and promotes apoptosis in vivo. miR-21^+/+ mice were either treated or not treated with radiotherapy or radiotherapy + anti-PD-L1 antibody. (a) Tumor volume curve of miR-21^+/+ mice following different treatment protocols. (b) Tumor weight of miR-21^+/+ mice following different treatment protocols. (c) The number of PD-L1 positive cells in tumor tissues of miR-21^+/+ mice following different treatment protocols, detected by immunofluorescence staining. (d) The positive rate of Ki67 protein expression in tumor tissues of miR-21^+/+ mice following different treatment protocols, detected by immunohistochemistry (10 mice in each group, 3 sections for each mouse). (e) Cell apoptosis in tumor tissues of miR-21^+/+ mice following different treatment protocols, measured using TUNEL staining. (f) Protein levels of apoptosis-related factors in tumor tissues of miR-21^+/+ mice following different treatment protocols, determined by Western blot analysis. ^∗p < 0.05, compared with miR-21^+/+ mice without treatment; ^#p < 0.05, compared with miR-21^+/+ mice treated with radiotherapy or anti-PD-L1 antibody. Each sample was evaluated three times independently. Data (mean ± standard deviation) from two groups were compared using independent sample t-test. There were 10 mice in each group.

Figure 5

Anti-PD-L1 antibody combined with radiotherapy facilitates T cell immune response in miR-21^+/+ mice in vivo. miR-21^+/+ mice were either treated or not treated with radiotherapy or radiotherapy+anti-PD-L1 antibody. (a) The number of CD3⁺CD8⁺ cells in the peripheral blood of miR-21^+/+ mice following different treatment protocols detected by flow cytometry. (b) T lymphocyte apoptosis rate in peripheral blood of miR-21^+/+ mice following different treatment protocols measured using Annexin V-FITC/PI. (c) Levels of IL-2 in the peripheral blood of miR-21^+/+ mice following different treatment protocols measured using ELISA. (d) Levels of IFN-γ in peripheral blood of miR-21^+/+ mice following different treatment protocols measured using ELISA. ^∗p < 0.05 compared with miR-21^+/+ mice without treatment; ^#p < 0.05 compared with miR-21^+/+ mice treated with radiotherapy or anti-PD-L1 antibody. Each sample was evaluated three times independently. Data (mean ± standard deviation) from two groups were compared using independent sample t-test. The experiments were repeated 3 times independently.

Figure 6

Mechanistic investigations indicated that anti-PD-L1 antibody augmented T cell immune response and alleviated the tolerance of BC to radiotherapy by regulating the miR-21/PDCD4/PI3K/Akt/PD-L1 axis.