Abstract
BackgroundWe 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.MethodsAfter 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.ResultsThe initiation of apoptosis in cultured cells is greatest at 15 kV/cm and requires 50 A/cm2. 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.ConclusionNano-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.
Highlights
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 provide evidence that NPS treatment initiates all of the classic Danger associated molecular pattern (DAMP) indicative of immunogenic cell death (ICD) and at levels that are comparable with two anthracycline chemotherapeutics that are known ICD inducers, doxorubicin and mitoxantrone [10]
We demonstrate that NPS is a physical modality capable of inducing an immunogenic form of cell death comprised of features of apoptosis when delivered at specific energies
Summary
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 have been developing a non-thermal, drug-free tumor treatment therapy that applies ultrashort electric pulses in the nanosecond range called Nano-Pulse Stimulation These pulses differ from those in the microsecond range typically used for electroporation in that NPS is 1000 times shorter and much larger in amplitude. The main characteristic that sets nanosecond pulsed fields apart from the microsecond domain is their ability to electroporate internal membranes This unique feature first observed by Schoenbach’s group [7] allows nanosecond pulses to be used to tease new responses out of cells such as the initiation of apoptosis and tumor ablation. One form of cell death known to elicit an immune response through ER-stress and ROS production is an immunogenic form of apoptosis [10]
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