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| Mechanism of action of triple therapy with radiotherapy and Gemcitabine combined with immune checkpoint inhibitors on the microenvironment of triple-negative breast cancer |
| WU Miaomiao1 FANG Kun2 |
1.Tumor Epigenetics Lab, West Branch of the First Affiliated Hospital of University of Science and Technology of China Anhui Provincial Cancer Hospital West Branch of Anhui Provincial Hospital, Anhui Province, Hefei 230031, China;
2.Department of Biomedical Engineering, the First Affiliated Hospital of University of Science and Technology of China Anhui Provincial Hospital, Anhui Province, Hefei 230001, China |
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Abstract Objective To investigate the effect of triple therapy of radiotherapy, Gemcitabine (Gem) combined with immune checkpoint inhibitors on the microenvironment of triple-negative breast cancer, and to reveal its possible mechanism. Methods A total of 29 SPF BALB/c mice aged five weeks and weighing 18-20 g were selected. On the first day of the experiment, about 1×106 4T1 cells were injected subcutaneously into the left abdomen to establish triple-negative breast cancer transplant tumor model. When the tumor volume increased to 100 mm3, the mice were divided into control group (five mice), anti-programmed death receptor ligand 1 (aPD-L1) group (six mice), radiotherapy (RT) +aPD-L1 group (six mice), Gem +aPD-L1 group (six mice), and triple group (six mice) by random number method. In addition to the control group, the other four groups were injected with drugs (Gem 5 mg/kg, aPD-L1 3 mg/kg) in the tumor at the 6th, 8th, and 10th days of the experiment. Twenty-four hours after each injection, RT (2 Gy) was administered to the RT+aPD-L1 group and the triple group. On the 30th day of the experiment, the animals were killed, the tumor weight of each group was weighed, and the main organs were stained with hematoxylin-eosin. The absolute percentage of CD3+CD8+ T cells in peripheral blood and tumor was detected by flow cytometry. The expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in peripheral blood and tumor were detected by enzyme-linked immunosorbent assay. The expressions of Ki-67, microvessel density(MVD)-CD31, and γH2AX were detected by immunohistochemistry, and apoptosis was evaluated by TUNEL detection. The expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 α (HIF-1α) were detected by Western blot analysis. Results There was no significant difference in body weight among all groups (P＞0.05). At the end of the experiment, the tumor weight of RT+aPD-L1 group and triple group were lower than those of aPD-L1 group, and those of triple group were lower than those of RT+aPD-L1 group (P＜0.05). Hematoxylin-eosin staining showed no obvious damage to the heart, liver, spleen, and kidney in each group. The absolute percentage of CD3+CD8+ T cells , TNF-α, and IL-6 in peripheral blood of aPD-L1 group were higher than those of control group. Peripheral blood IL-6 in RT+aPD-L1 group was higher than that in aPD-L1 group, and absolute percentage of CD3+CD8+ T cells , IFN-γ, and IL-6 of peripheral blood in triple group were higher than those in aPD-L1 group. The absolute percentage of CD3+CD8+ T cells of peripheral blood in triple group was higher than that of RT+aPD-L1 group and Gem+aPD-L1 group, and IL-6 was higher than that of Gem+aPD-L1 group (P＜0.05). The absolute percentage of CD3+CD8+ T cells , IFN-γ, TNF-α, and IL-6 of tumor in aPD-L1 group were higher than those in control group. The absolute percentages of CD3+CD8+ T cells of tumor in RT+aPD-L1 group, Gem+aPD-L1 group, and triple group were higher than those in aPD-L1 group, and TNF-α and IL-6 of tumor in triple group were higher than those in aPD-L1 group. The absolute percentage of CD3+CD8+ T cells and IL-6 of tumor in triple group were higher than those in RT+aPD-L1 group and Gem+aPD-L1 group, and IFN-γ was higher than those in RT+aPD-L1 group (P＜0.05). Immunohistochemical staining and TUNEL detection showed that the expression of Ki-67 decreased and the apoptotic cells increased in the tumor tissues of triple group. MVD-CD31 in triple group was lower than that in aPD-L1 group, γH2AX was higher than that in aPD-L1 group and Gem+aPD-L1 group (P＜0.05). VEGF in aPD-L1 group was lower than that in control group, VEGF in RT+aPD-L1 group, Gem+aPD-L1 group, and triple group was lower than that in aPD-L1 group, and HIF-1α in triple group was lower than that in aPD-L1 group and RT+aPD-L1 group (P＜0.05). Conclusion The triple therapy of radiotherapy, Gem combined with immune checkpoint inhibitors can delay tumor growth, increase the absolute percentage of CD3+CD8+ T cells in peripheral blood and tumor, promote the release of inflammatory factors in serum and tumor, improve intratumor hypoxia and inhibit tumor microvascular growth in mouse triple-negative breast cancer transplant tumor model.
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