Radiation-Based Multi-Modal Therapy Combining with Immunotherapy to Develop a Vaccine-Like Effective Treatment for Triple-Negative Breast Cancer

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive malignancy with high metastasis and recurrence rates. Current treatments like chemotherapy and immunotherapy face challenges due to chemotherapy side effects, limited immunotherapy applicability, and TNBC's immunosuppressive microenvironment.

Purpose: To achieve a more effective treatment for TNBC, a novel therapeutic strategy has been developed, which uses X-ray excited photodynamic therapy (X-PDT) to activate the tumor immune microenvironment following with the immunotherapy of Anti-CTLA4.

Methods: Base on the 4T1 tumor mouse model, this study initially investigated the regulatory effects of X-PDT on the tumor immune microenvironment. Subsequently, the therapeutic efficacy of combining X-PDT with Anti-CTLA4 was evaluated for its inhibitory effects on primary, metastatic, and recurrent tumors. The underlying mechanisms were further elucidated through comprehensive techniques including flow cytometry, ELISA, and immunofluorescence assays.

Results: The synergistic strategy can effectively ablate the primary tumor while inhibiting metastasis and preventing recurrence like a vaccine. It enhances intratumoural dendritic cells (DCs) maturation (from 25.7% to 58.3%, P < 0.05) and immune T cell infiltration activating a strong anti-tumor immune response. The anti-tumor efficacy of synergistic therapy is enhanced by 2.5 times comparing with immunotherapy alone, while the tumor metastasis has been inhibited significantly. The maturation level of mature dendritic cells was increased from 26.7% to 86.3% (P < 0.01). The intratumoural CD8⁺/CD4⁺ T cells were increased from 0.51% and 1.54% to 15.4% and 23.1% (P < 0.0001), respectively. The synergistic therapy exerts a powerful vaccine-like long-term immune memory function to prevent tumor recurrence with the elevated level of effector memory T (Tem) cells (from 12.8% to 33.3%, P < 0.05).

Conclusion: Based on the 4T1 mouse model, developed an effective vaccine-like therapeutic strategy combining X-PDT with Anti-CTLA4, which can effectively ablate tumors, inhibit metastasis, and prevent tumor recurrence. This work may provide a novel effective therapeutic modality for the clinical treatment of TNBC.

Keywords: X-ray excited photodynamic therapy; cancer vaccine; combination therapies; immunotherapy; nanodrugs.

Figure 1

X-PDT’s immune-stimulation abilities. (a and b) Schematic of X-PDT treatment; (c and e) Quantification of maturation levels of intratumoral dendritic cells (n=3; *, P < 0.05); (d) Immunofluorescence images of CD8 expression in the tumor (DAPI, blue; CD8⁺ T cells, green; scale bar: 50 μm); (f) Semi-quantitative analysis of immunofluorescence results from c (n=3-5; *, P < 0.05; **, P < 0.01). All data is reported as the means ± SEM.

Figure 2

Anti-tumor effects of X-PDT combining with Anti-CTLA4 treatment in Balb/c mice-bearing subcutaneous 4T1 tumors. (a) Schematic of X-PDT plus Anti-CTLA4 combination therapy; (b) Tumor growth curve (*, P < 0.05; **, P < 0.01); (c) Tumor weight (*, P < 0.05; **, P < 0.01; ****, P < 0.0001); (d) Tumor H&E, TUNEL and Ki-67 staining images (scale bar: 100 μm); (e and f) TUNEL and Ki-67 staining statistical analysis (*, P < 0.05; **, P < 0.01; ****, P < 0.0001). The data are reported as the means ± SEM of n=8-10 (a–c) and n=3 (e and f) independent experiments.

Figure 3

Inhibitory effect on lung metastasis. (a) Schematic diagram of lung metastasis evaluation; (b) The statistic results of metastatic foci in the lungs (*, P < 0.05; **, P < 0.01); (c) Representative white light images of the lungs and H&E staining of lungs (The circled sections indicate the locations of metastatic lesions). Data were reported as means ± SEM of n=8-10 (a) and n=3 (b and c) independent experiments.

Figure 4

The mechanism study. (a and c) Flowcytometry quantification of DCs maturation in tumor-draining lymph nodes (*, P < 0.05; **, P < 0.01); (b, d and e) Flowcytometry quantification of intratumoral CD8⁺/CD4⁺ T cell infiltration (*, P < 0.05; **, P < 0.01; ****, P < 0.0001); (f and g) Monitoring of cytokine levels in serum (*, P < 0.05; **, P < 0.01); (h) Immunofluorescence images of intratumoral Treg cells expression (Scale bar: 100 μm). Data were reported as means ± SEM of n=3 (a, b, f and g) independent experiments.

Figure 5

Vaccine-like immune memory effect. (a) Schematic diagram of long-term immune memory effect evaluation; (b) Recurrent tumor growth curves (*, P < 0.05; ****, P < 0.0001); (c) Photographs of mice and excised tumor after different treatments; (d and e) Expression of mouse splenic Tem cells quantified by flowcytometry as well as statistical analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Data were reported as means ± SEM of n=5 (a–c) and n=3 (d and e) independent experiments.

Figure 6

Schematic diagram of X-PDT combined with immune checkpoint blockade to induce anti-tumor immune response.