Xiang He,1,* Yuhang Tian,1,* Jialin Dong,1 Yanchi Yuan,1 Shijie Zhang,2 Hui Jing1 

1Department of Ultrasound, Harbin Medical University Cancer Hospital, Harbin, People’s Republic of China; 2Department of Radiation Oncology, Harbin Medical University Cancer Hospital, Harbin, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Hui Jing, Department of Ultrasound, Harbin Medical University Cancer Hospital, No. 150, Haping Road, Nangang District, Harbin, 150081, People’s Republic of China, Email jinghuihrb@163.com

Background: Sono-photodynamic therapy (SPDT), the combination of sonodynamic therapy (SDT) and photodynamic therapy (PDT), is a promising tumor treatment method. However, the hypoxic tumor microenvironment greatly compromises the efficacy of SPDT. Pyroptosis, a new type of programmed cell death, is mainly induced by some chemotherapeutic drugs in the current research, and rarely by SPDT. RNA sequencing (RNA-seq) is a high-throughput sequencing technique that comprehensively profiles the transcriptome, revealing the full spectrum of RNA molecules in a cell. Here, we constructed IR780@O2 nanobubbles (NBs) with photoacoustic dual response and hypoxia improvement properties to fight triple negative breast cancer (TNBC), and demonstrated that SPDT could kill TNBC cells through pyroptosis pathway. RNA-seq further revealed potential mechanisms and related differentially expressed genes.
Methods: Thin-film hydration and mechanical vibration method were utilized to synthesize IR780@O2 NBs. Subsequently, we characterized IR780@O2 NBs and examined the cytotoxicity as well as ROS production ability. A series of experiments were conducted to verify that SPDT killed TNBC cells through pyroptosis.
Results: IR780@O2 NBs were successfully prepared and had certain stability. Compared with SDT alone, SPDT increased therapeutic effect by 1.67 times by generating more ROS, and the introduction of NBs and O2 NBs (2.23 times and 2.93 times compared with SDT alone) could further promote this process. Other experiments proved that TNBC cells died by pyroptosis pathway. Moreover, the in-depth mechanism revealed that colony stimulating factor (CSF) and C-X-C motif chemokine ligand (CXCL) could be potential targets for the occurrence of pyroptosis in TNBC cells.
Conclusion: The IR780@O2 NBs prepared in this study increased the degree of TNBC cell pyroptosis through SPDT effect and alleviation of hypoxia, and cellular senescence might be a biological process closely related to pyroptosis in TNBC.

Keywords: triple negative breast cancer, sono-photodynamic therapy, hypoxia relief, pyroptosis, RNA-seq