Combination of low-intensity pulsed ultrasound irradiating immune organs with immune checkpoint blockade augments systemic anti-tumor immunity on low tumor burden 4T-1 breast cancer.

BACKGROUND: Despite achieving complete remission (CR) through surgery, chemoradiotherapy, targeted therapy, and other treatment modalities, breast cancer, particularly triple-negative breast cancer (TNBC) remains at a high risk of recurrence and metastasis. Enhancing anti-tumor immunity to eliminate residual tumor cells may reduce TNBC relapse. Our previous research indicated low-intensity pulsed ultrasound (LIPUS) can activate the anti‑tumor immunity. Here, we explore the potential of the suitable strategy with LIPUS, and the combination with an immune checkpoint inhibitor, anti-PD-1 antibody (αPD-1) to anti‑tumor immunity in TNBC. METHODS: In the xenografted mouse model, different times (7, 14, 21, and 28d) and organs (spleen, bone marrow, spleen + bone marrow) of LIPUS were received to experimental groups. In 4T-1 low tumor burden model, after LIPUS irradiated different organs for 28d or combined with αPD-1, the tumor volume, lung metastasis, and survival time were observed. Flow cytometry, immunohistochemistry, immunofluorescence, multiplex immunofluorescence, and ELISA were performed to elucidate detailed impacts of LIPUS or combination treatment on anti‑tumor immunity. RNA-seq was used to preliminarily explore the biological mechanism of LIPUS. RESULTS: LIPUS inhibited the growth of 4T-1 xenografted, and the irradiation time was the main factor. In 4T-1 low tumor burden model, further studies had shown that LIPUS of spleen + bone marrow could best active anti‑tumor immunity primarily through CD8(+) cytotoxic T lymphocytes (CD8(+)CTLs) and myeloid-derived suppressor cells (MDSCs), but not reduce the rate of tumor formation. LIPUS combined with αPD-1 not only reduce tumor formation rate but also further enhance anti‑tumor immunity mainly by means of CD8(+)CTLs, as well as led to significant changes in cytokines. The RNA-seq results also suggested the anti-tumor immune biological processes and signaling pathways were generated after LIPUS irradiating immune organs. CONCLUSIONS: Irradiating the spleen and bone marrow with LIPUS for 28 days may be an applicable treatment strategy, as it induces an increase in CD8(+)CTLs and a decrease in MDSCs, as well as an upregulation of mRNA levels in multiple immunomodulatory pathways. LIPUS combined with αPD-1 elicits a superior anti-tumor immune response and inhibits tumor formation in a 4T-1 low tumor burden model. This study provides a novel approach for treating TNBC after achieving CR by combining LIPUS with αPD-1.