Nano-Pulse Stimulation induces immunogenic cell death in human papillomavirus-transformed tumors and initiates an adaptive immune response

<div><p>Nano-Pulse Stimulation (NPS) is a non-thermal pulsed electric field modality that has been shown to have cancer therapeutic effects. Here we applied NPS treatment to the human papillomavirus type 16 (HPV 16)-transformed C3.43 mouse tumor cell model and showed that it is effective at eliminating primary tumors through the induction of immunogenic cell death while subsequently increasing the number of tumor-infiltrating lymphocytes within the tumor microenvironment. <i>In vitro</i> NPS treatment of C3.43 cells resulted in a doubling of activated caspase 3/7 along with the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, indicating programmed cell death activity. Tumor-bearing mice receiving standard NPS treatment showed an initial decrease in tumor volume followed by clearing of tumors in most mice, and a significant increase in overall survival. Intra-tumor analysis of mice that were unable to clear tumors showed an inverse correlation between the number of tumor infiltrating lymphocytes and the size of the tumor. Approximately half of the mice that cleared established tumors were protected against tumor re-challenge on the opposite flank. Selective depletion of CD8⁺ T cells eliminated this protection, suggesting that NPS treatment induces an adaptive immune response generating CD8⁺ T cells that recognize tumor antigen(s) associated with the C3.43 tumor model. This method may be utilized in the future to not only ablate primary tumors, but also to induce an anti-tumor response driven by effector CD8⁺ T cells capable of protecting individuals from disease recurrence.</p></div

NPS treatment of tumors results in a CD8-dependent adaptive immune response.

<p>Shown are the individual tumor growth profiles of primary and rechallenge events in mice receiving NPS with or without selective depletion. Growth curves of primary (black) and re-challenge tumors (red) of NPS-treated mice with or without selective depletion of CD4 or CD8 T cells are displayed. <b>(A)</b> Mice received NPS treatment of primary tumor only (3 pps, 30 kV/cm, 70 A). <b>(B and C)</b> Mice received NPS treatment of primary tumor (3 pps, 30 kV/cm, 70A) followed by selective depletion of CD4 cells <b>(B)</b> or CD8 cells <b>(C)</b> with the administration of either an αCD4 mAb (yellow dots) or αCD8 mAb (green dots. The red arrows indicate the day of tumor re-challenge.</p

NPS treatment of primary tumors results in significant levels of tumor clearance, enhanced survival, and is effective during multiple applications.

<p>Groups of 10 mice were s.c. challenged with C3.43 tumors. 10-days post tumor challenge mice were given NPS (3 pps, 30kV/cm) treatment at the tumor site. Mice with recurring tumors received a second treatment on day 31 <b>(A)</b> Mean tumor volume (±SEM) of untreated and NPS treated mice (*p<0.05, unpaired students t-test at each time point). Volume measurements of untreated group displayed until there was a loss of 3 or more mice within the group due to euthanasia endpoints met <b>(B)</b> 50-day survival curve of groups with no treatment (naïve, median survival 39 days) or NPS treatment (NPS) (p<0.0012, Mantel-Cox Log Rank test). Data are representative of 2 independent experiments.</p

NPS application.

<p><b>(A)</b> Photo of a typical shaved C3.43 tumor prior to treatment. <b>(B)</b> Pinch electrode used to treat these tumors. <b>(C)</b> Pinch electrode sandwiching a tumor as NPS is applied. <b>(D)</b> Oscilloscope trace of voltage (top) and current (bottom) applied to the tumor in each pulse. <b>(E)</b> Photo of the treated tumor in “A” 11 days later.</p

NPS treatment of C3.43 cells results in significant upregulation of caspase 3/7 activity at lower treatment energies.

<p><b>(A)</b> Measured levels of activated caspase 3/7 in cells at 3h post NPS treatment for a range of NPS energy densities. Data shown as the mean of 4 experiments and the error bars represent the standard error of the mean (**p<0.01 ***p<0.001, One-way ANOVA followed by Dunnett’s multiple comparisons test to untreated cells). <b>(B)</b> Mean distribution of treated C3.43 tumor cells in early and late apoptosis at 3 h post-treatment with indicated NPS energy density <b>(C)</b> Data collected 24 h post NPS treatment.</p