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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.

背景

超声（US）主要用于诊断目的，但可通过适合该目的的超声强度或频率用于癌症治疗。前列腺癌（PC）在泌尿外科领域的患病率最高，但PC的免疫检查点抑制剂（ICI）适应证仅限于极少数病例。在本研究中，我们在体外和体内比较了单独超声照射与超声和ICI联合使用的抗肿瘤效果。

方法

我们的实验使用了PC细胞系TRAMP-C2细胞。用脉冲重复频率（PRF）为1、10和100Hz的超声照射TRAMP-C2细胞。通过MTS试验评估细胞增殖，并使用流式细胞术分析凋亡细胞。为了验证超声照射对体内PC的抗肿瘤作用，我们使用小鼠进行了动物实验。用PRF为10和100Hz的超声照射荷TRAMP-C2小鼠。超声照射开始3周后，给小鼠注射抗PD-1抗体。最后，处死小鼠并收集肿瘤。对肿瘤细胞提取物中的裂解型半胱天冬酶-3和CD3进行免疫组织化学（IHC）分析。

结果

细胞增殖试验表明，1Hz和10Hz的超声显著抑制细胞存活（p<0.0001）。此外，超声照射在1、10和100Hz时均诱导凋亡（分别为p=0.0129、p=0.0150和p=0.0017）。在动物实验中，在10Hz和100Hz以及100Hz+ICI时观察到显著的肿瘤生长抑制作用（分别为p<0.05）。苏木精-伊红（H-E）染色显示，在100Hz和100Hz+ICI时肿瘤坏死面积显著增加（分别为p<0.05）。此外，在IHC染色下，100Hz时裂解型半胱天冬酶-3的表达水平和CD3阳性细胞数量增加（分别为p<0.05）。