Background: Ultrasound (US) is mostly used for diagnostic purpose but could be used for cancer treatments with a US intensity or frequency fitted to such a purpose. Prostate cancer (PC) has the highest prevalence in the urological field, but indications for immune checkpoint inhibitors (ICIs) for PC are limited to very few cases. In this study, we compared the antitumor effect of US irradiation alone with the combined use of US and ICIs in vitro and in vivo. Methods: PC cell line TRAMP-C2 cells were used in our experiments. TRAMP-C2 cells were irradiated with US with pulse repeated frequencies (PRF) of 1, 10, and 100 Hz. Cell proliferation was evaluated by MTS assay and apoptotic cells were analyzed using flow cytometry. To verify the antitumor effect of US irradiation on PC in vivo, we conducted animal experiments using mice. TRAMP-C2-bearing mice were irradiated with US with PRF of 10 and 100 Hz. Three weeks after the start of US irradiation, anti-PD-1 antibody was administered to the mice. Finally, mice were sacrificed and tumors were collected. Immunohistochemical (IHC) analyses were assessed for cleaved caspase-3 and CD3 in tumor cell extracts. Results: Cell proliferation assays showed that 1 and 10 Hz US significantly inhibited cell survival (p < 0.0001). In addition, US irradiation induced apoptosis at 1, 10, and 100 Hz (p = 0.0129, p = 0.0150, and p = 0.0017, respectively). In animal experiments, a significant tumor growth inhibitory effect was observed at 10 and 100 Hz, and 100 Hz + ICIs (p < 0.05, respectively). Hematoxylin–eosin (H–E) staining showed a significant increase in the necrotic area of the tumor at 100 Hz and 100 Hz + ICIs (p < 0.05, respectively). In addition, under IHC staining the expression level of cleaved caspase-3 and the number of CD3-positive cells increased at 100 Hz (p < 0.05, respectively). Conclusion: US irradiation induced apoptosis in cells and reduced cell viability. In vivo tumor growth was suppressed by combined treatment with US irradiation and ICIs. Further research on immune system activation will lead to less invasive and more efficient treatments for PC.

Background Ultrasound (US) can induce cell injury, and we have previously reported that adjusting the pulse repetition frequency (PRF) of ultrasound output can induce prostate cancer cell destruction without causing a rise in the temperature of the irradiated area. In this study, we examined the mechanism of nonthermal ultrasound cell destruction, which was not fully clarified in our previous reports. Methods In vitro, we evaluated postirradiation cells immediately after treatment and examined membrane disruption by proliferation assay, LDH assay, and apoptosis assay. In vivo, we injected mice with human LNCaP and PC‐3 prostate cancer cells and evaluated the therapeutic effects of US irradiation by H‐E staining and immunostaining. Results Proliferation assays showed inhibition at 3 h postirradiation independently of PRF and cell line (p < 0.05). Quantitative assessment of apoptosis/necrosis by flow cytometry showed widely varying results depending on cell type. LNCaP showed an increase in late apoptosis at 0 h independent of PRF (p < 0.05), while PC‐3 showed no significant difference at 0 h. The LDH assay showed an increase in LDH independent of PRF in LNCaP (p < 0.05 respectively), but no significant difference in PC‐3. In vivo, tumor volume was compared and a significant reduction was observed at 10 Hz for LNCaP (p < 0.05) and 100 Hz for PC‐3 (p < 0.001) at 3 weeks after the start of irradiation. The excised tumors were evaluated with Ki‐67, Caspase‐3, and CD‐31 and showed a significant treatment effect independent of cell type and PRF (p < 0.001 respectively). Conclusion Examining the mechanism behind the therapeutic effect of US irradiation revealed that the main effect was achieved by apoptosis induction rather than necrosis.

INTRODUCTION AND OBJECTIVE: Ultrasound (US) is mostly used for diagnostic purpose but could be used for cancer treatments if the US intensity or frequency fits to such purpose. Prostate cancer (PC) has the highest prevalence in the urological field, but indications of immune checkpoint inhibitors (ICI) for PC are limited to very few cases. Therefore, we verified the antitumor effect of US irradiation alone and the combined use of US and ICI in vitro and in vivo.METHODS: Human-derived PC cell line LNCaP and PC-3, and mouse-derived PC cell line TRAMP-C2 were used in experiments. US (3.0 W/cm 2 , 3 MHz, irradiation time rate: 20 %) was irradiated with repeated frequency of 1, 10, and 100 Hz. Cell proliferation was evaluated by MTS assay and apoptotic cells were analyzed by flow cytometry. We transplanted LNCaP, PC-3 cells into nude mice, and TRAMP-C2 cells into Balb/c nu/nu mice. US was irradiated on mice at 10 Hz and 100 Hz 3 times a week for 6 weeks. We also administered anti-PD-1 antibody to TRAMP-C2-bearing mice 5 times to verify the effect of combined use of US and ICI. After tumor collection, Immunohistochemical (IHC) stain with Ki-67, caspase-3, CD31 and CD3 were conducted.RESULTS: Cell proliferation assay showed US irradiation suppressed cell growth in LNCaP (at 10 Hz and 100 Hz, p<0.0001, respectively), PC-3 (at 1, 10, and 100 Hz, p[0.0002, p<0.0001, and p[0.0132), and TRAMP-C2 (at 1 Hz and 10 Hz, p<0.0001, respectively). In addition, US irradiation induced apoptosis in LNCaP (at 100 Hz, p[0.0368), PC-3 (at 1 Hz and 10 Hz, p[0.0447, p[0.0137), and TRAMP-C2 (at 1, 10, and 100 Hz, p[0.0129, p[0.0150, p[0.0017). In animal experiments, US irradiation at 100 Hz inhibited tumor growth in both LNCaP and PC-3bearing mice (p[0.0419, p[0.0064). From IHC analysis, the expression level of Ki-67 was decreased at 100Hz in PC-3 (p[0.0467), and caspase-3 was decreased at 10 Hz and 100 Hz in both LNCaP (p[0.0007, p[0.0297) and p[0.0115). In addition, the number of vessels was reduced at 10Hz and 100 Hz in both LNCaP (p[0.0290, p[0.0102) and PC3 (p[0.0026, p[0.0007). In TRAMP-C2 in vivo experiments, an antitumor effect was observed at 10 Hz, 100 Hz, and the combined use of ICI and 100 Hz of US (p[0.0365, p[0.0364, p[0.0393) compared to control. IHC stain revealed the number of CD3positive cells increased at 100 Hz (p[0.0499).CONCLUSIONS: We found that US irradiation induces apoptosis and reduces cell growth. Moreover, tumor growth was suppressed by combined use of US and ICI. Further research on immune system activation will lead to a less invasive and more efficient treatment for PC.