Pulse Biosciences, 849 Mitten Rd., Ste 104, Burlingame, CA 94010 USA.
J Immunother Cancer. 2017 Apr 18;5:32. doi: 10.1186/s40425-017-0234-5. eCollection 2017.
We have been developing a non-thermal, drug-free tumor therapy called Nano-Pulse Stimulation (NPS) that delivers ultrashort electric pulses to tumor cells which eliminates the tumor and inhibits secondary tumor growth. We hypothesized that the mechanism for inhibiting secondary tumor growth involves stimulating an adaptive immune response via an immunogenic form of apoptosis, commonly known as immunogenic cell death (ICD). ICD is characterized by the emission of danger-associated molecular patterns (DAMPs) that serve to recruit immune cells to the site of the tumor. Here we present evidence that NPS stimulates both caspase 3/7 activation indicative of apoptosis, as well as the emission of three critical DAMPs: ecto-calreticulin (CRT), ATP and HMGB1.
After treating three separate cancer cell lines (MCA205, McA-RH7777, Jurkat E6-1) with NPS, cells were incubated at 37 °C. Cell-culture supernatants were collected after three-hours to measure for activated caspases 3/7 and after 24 h to measure CRT, ATP and HMGB1 levels. We measured the changes in caspase-3 activation with Caspase-Glo® by Promega, ecto-CRT with anti-CRT antibody and flow cytometry, ATP by luciferase light generation and HMGB1 by ELISA.
The initiation of apoptosis in cultured cells is greatest at 15 kV/cm and requires 50 A/cm. Reducing this current inhibits cell death. Activated caspase-3 increases 8-fold in Jurkat E6-1 cells and 40% in rat hepatocellular carcinoma and mouse fibrosarcoma cells by 3 h post treatment. This increase is non-linear and peaks at 15-20 J/mL for all field strengths. 10 and 30 kV/cm fields exhibited the lowest response and the 12 and 15 kV/cm fields stimulated the largest amount of caspase activation. We measured the three DAMPs 24 h after treatment. The expression of cell surface CRT increased in an energy-dependent manner in the NPS treated samples. Expression levels reached or exceeded the expression levels in the majority of the anthracycline-treated samples at energies between 25 and 50 J/mL. Similar to the caspase response at 3 h, secreted ATP peaked at 15 J/mL and then rapidly declined at 25 J/mL. HMGB1 release increased as treatment energy increased and reached levels comparable to the anthracycline-treated groups between 10 and 25 J/mL.
Nano-Pulse Stimulation treatment at specific energies was able to trigger the emission of three key DAMPs at levels comparable to Doxorubicin and Mitoxantrone, two known inducers of immunogenic cell death (ICD). Therefore NPS is a physical modality that can trigger immunogenic cell death in tumor cells.
我们一直在开发一种非热、无药物的肿瘤治疗方法，称为纳秒脉冲刺激（NPS），它向肿瘤细胞输送超短电脉冲，从而消除肿瘤并抑制继发性肿瘤生长。我们假设抑制继发性肿瘤生长的机制涉及通过免疫原性细胞凋亡的形式刺激适应性免疫反应，通常称为免疫原性细胞死亡（ICD）。ICD 的特征是发出危险相关分子模式（DAMP），这些模式有助于将免疫细胞募集到肿瘤部位。在这里，我们提供的证据表明，NPS 刺激 caspase 3/7 的激活，表明细胞凋亡，以及三种关键 DAMP 的释放：细胞外钙网蛋白（CRT）、ATP 和 HMGB1。
用 NPS 处理三种不同的癌细胞系（MCA205、McA-RH7777、Jurkat E6-1）后，将细胞在 37°C 下孵育。收集细胞培养上清液，在 3 小时后测量激活的 caspase 3/7，在 24 小时后测量 CRT、ATP 和 HMGB1 水平。我们使用 Promega 的 Caspase-Glo®测量 caspase-3 激活的变化，使用抗 CRT 抗体和流式细胞术测量细胞外 CRT，使用荧光素酶发光生成测量 ATP，使用 ELISA 测量 HMGB1。
培养细胞中凋亡的起始在 15 kV/cm 时最大，需要 50 A/cm。降低此电流会抑制细胞死亡。Jurkat E6-1 细胞中激活的 caspase-3 在治疗后 3 小时增加 8 倍，大鼠肝癌和小鼠纤维肉瘤细胞中增加 40%。这种增加是非线性的，在所有场强下都在 15-20 J/mL 时达到峰值。10 和 30 kV/cm 场表现出最低的反应，而 12 和 15 kV/cm 场刺激了最大量的 caspase 激活。我们在治疗后 24 小时测量了这三种 DAMP。NPS 处理样品中细胞表面 CRT 的表达呈能量依赖性增加。在 25 至 50 J/mL 的能量范围内，表达水平达到或超过大多数蒽环类药物处理样品中的表达水平。与 3 小时时的 caspase 反应类似，分泌的 ATP 在 15 J/mL 时达到峰值，然后在 25 J/mL 时迅速下降。HMGB1 释放随着治疗能量的增加而增加，在 10 至 25 J/mL 之间达到与蒽环类药物处理组相当的水平。
在特定能量下的纳秒脉冲刺激治疗能够触发三种关键 DAMP 的释放，其水平可与阿霉素和米托蒽醌相媲美，阿霉素和米托蒽醌是两种已知的免疫原性细胞死亡（ICD）诱导剂。因此，NPS 是一种物理模式，可以在肿瘤细胞中触发免疫原性细胞死亡。
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