Introduction: 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/cm2, 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-3-bearing 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 PC-3 (p=0.0061, 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 CD3-positive 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.
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