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 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

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

Analysis of Sp17 expression in ID8 cells.

<p>Sp17 mRNA in the murine ID8 tumor cells and in mouse testis (positive control). The mRNA levels were analyzed by RT-PCR for Sp17 expression. A cellular housekeeping gene, B-actin, was included as a control. A PCR-only control (no RT step) failed to generate a product, indicating that there was no DNA contamination in the samples. In addition to the RT-PCR (see figure <b>1a</b>), Western blot was performed and figure <b>1b</b> shows the expression of Sp17 at the protein level. The ID8 cell line was further characterized by an immunocytochemistry (ICC) and immunofluorescence (IF). This figure shows a cytospin of ID8 cells permeabilized (P) and a positive staining for Sp17 at the cytoplasm level. In addition, Sp17 was confirmed by IF in the cytoplasm. Additionally, in figure 1c, the non permeabilized (NP) cells show clear expression via IF and less by ICC. Panel d shows ID8 characterization for surface expression of Sp17 and MHC-I; isot. ctrl, isotypic control antibody; percentage indicate positive-staining cells. MHC-I expression was evaluated with or without IFN-g stimulation.</p

(a,b) Measurement of cytokines in mice treated with different vaccines.

<p>On day 300, serum was collected and analyzed by E.L.I.S.A. for the measurement of the indicated cytokine levels in prophylactically (<b>a</b>) or therapeutically (<b>b</b>) vaccinated mice. For both regimens, IFN-gamma, TNF-alpha and GM-CSF statistically significant increments were detected only in Sp17+CpG vaccinated mice compared with controls (PBS). No significant differences were evidenced for IL-2, IL-4, IL-5 or IL-10 levels.</p

Cytotoxicity assay in the prophylaxis regimen.

<p>Splenocytes from the three different formulations of prophylactically vaccinated mice and controls were collected and analyzed by ⁵¹Chromium-release assay on days 7 (first vaccine), 97 (third vaccine) and 270 (ninth vaccine), using splenocytes as effector cells and ID8 as target cells. These results were obtained from three independent experiments. X axis indicate effector:target ratios.</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

Analysis of Th-17 and T-reg population frequencies.

<p>Splenocytes from mice that received prophylactic or therapeutic vaccinations or controls (mice with no tumor or tumor-bearing mice without vaccinations) were collected at different time points (no tumor: day 0; ID8 only: day 45; prophylactic and therapeutic vaccines: day 270) analyzed by flow-cytometry for the measurement of a) Th-17 population (CD4/IL-17-double positive) or b)T-reg population (CD4/Foxp3-double positive) frequency.</p

(a, b, c, d) Analysis of ID8 cells growth and dissemination in vivo.

<p><b>a</b> shows on the left side a control mouse (width of mouse 20 mm) not injected with ID8 cells and on the right side a mouse injected with 2×106 ID8 cells after 40 days (width of mouse 44 mm). These mice represent experiments with similar results. <b>b</b> shows the full peritoneal cavity of ascites induced from an i.p injection of 2×106 ID8 in 40 days (left) and an open view of the cavity with several metastatic nodes and tumor masses (center and right, after aspirating 22 mL of ascites). <b>c</b> shows a peritoneum negative for Sp17 from a control mouse not injected with ID8 cells. <b>f</b> shows a positive expression of Sp17 on the peritoneum of a mouse injected with 2×106 ID8 cell after 40 days. Testis is the positive control for Sp17 by ICH (<b>g</b>). Figure <b>d</b> shows PCR for Sp17 DNA (and B-actin control): a tissue panel derived from (1) the organs of a 2×106 ID8 injected mouse was positive for Sp17 and a panel of tissues derived from organs of a healthy mouse revealed no expression of SP17. Positive controls (ID8 and testis) for Sp17 are also shown. Panel <b>h</b> shows in vivo fluorescence pictures of a control (left) and ID8-injected (right) mouse for the localization of GFP-positive ID8 cells.</p