Immunohistochemical labeling of tumor thin sections with CD8 (red) antibodies and DAPI (blue) nuclear stain.

<p>A: Two typical sections from an untreated control tumor collected 14 days after injecting tumor cells into the liver; B. Two typical sections of a secondary tumor that was fixed 7 days after it was injected into a liver in which the primary tumor had been nanoelectroablated 3 weeks earlier. This secondary tumor exhibited growth inhibition and a photograph of it can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134364#pone.0134364.g005" target="_blank">Fig 5B</a>; C. Percentage of cells expressing CD8 in histological sections of tumors from four different conditions. Each bar represents the mean of measurements taken from 3–7 sections of 2 separate tumors. The untreated tumors were fixed 14 days after injection; “ns+7d” represents tumors fixed 7days after treating with 400 pulses, 15 kV, 100ns; “T2+7d” represents secondary tumors fixed 7 days after injection. CD8 concentration is significantly different from control tumor levels with **p = 0.002; “T2+7d+CD9 AB” represents secondary tumors fixed 7 days after injection with CD8 antibodies injected IP 24 h prior to the tumor cell injection. The CD8 concentration is significantly different from control tumors with *p = 0.005.</p

Transillumination images of subdermal MCA205 tumors in isogenic B6 albino mice.

<p>Tumors are outlined with dotted lines to aid in visualization. Two top rows show tumors growing from cells injected into mice vaccinated 3 weeks earlier with nsPEF-treated MCA205 cells. The 3^rd row down shows a tumor growing from cells injected into a mouse vaccinated with mitomycin C-treated cells. The fourth row down shows a tumor growing from cells injected into a mouse vaccinated with saline vehicle. The bottom row shows a tumor growing from cells injected into a mouse vaccinated with nsPEF-treated cells one day after injecting anti-CD8 antibodies IP.</p

Immunohistochemical labeling of activated caspase 3 and 9.

<p>A. Cleaved caspase 3 (red) and DAPI (blue) labeling of thin section of 2-week old untreated control tumor. B. Cleaved caspase 3 (red) and DAPI (blue) labeling of tumor tissue removed 2 h after treatment (400p, 15 kV, 100 ns). C. Cleaved caspase 9 (red) and DAPI (blue) labeling of 2 week old untreated control tumor. D. Cleaved caspase 9 (red) and DAPI (blue) labeling of tumor tissue removed 2 h after nsPEF treatment. Note how the tumor nuclei in B and D have shrunk after treatment, indicating pyknosis. E. Summary of the analysis of 3–7 thin sections taken from one control tumor 14 days after injection and 2 treated tumors fixed 2 h after 400 p, 15 kV. Bars indicate SEM. The difference in activated caspase label between pulsed tissue and controls is highly significant with p = 0.005.</p

Mean levels of alanine transaminase and aspartate transaminase from 3 Buffalo rats with nsPEF-treated liver tumors.

<p>The levels of two liver enzymes were measured. The normal range of these enzymes was determined from blood samples taken from 170 Sprague Dawley rats. The red line indicates the mean level minus 2 standard deviations. The green line indicates the mean level plus 2 standard deviations from the mean.</p

Normalized growth rates of second tumor from MCA205 cells injected 3 weeks after the mouse was vaccinated with either vehicle or MCA205 cells that were treated as indicated.

<p>A. Average tumor growth rates in 3 Black C57BL/6 mice vaccinated with saline or mitomycin C-treated cells or nsPEF-treated cells. There is no significant difference between the tumor growth rates in the two controls (p = 0.7). There is a very significant difference between the tumor growth rate in the two controls and that in the mice vaccinated with nsPEF-treated cells (p<0.001). This statistical analysis provided the same conclusion for A-C. B. Average tumor growth rates in 3 B6 albino mice vaccinated with saline or mitomycin C-treated cells or nsPEF-treated cells. C. Average tumor growth rates in 6 B6 albino mice vaccinated with either saline vehicle or nsPEF-treated cells. One of the groups (purple line) was injected with anti-CD8 antibody 24 h prior to injecting the tumor cells. The error bars represent the SEM.</p

Calreticulin labeling on the surface of cells that had been exposed to nsPEF.

<p><b>A</b>. Two typical McA-RH7777 cells labeled with CRT antibody 2h after treating with 25 pulses at 15 kV/cm while plated on an Indium-tin-oxide-coated coverslip that was placed against one electrode in an electroporation cuvette. <b>B.</b> Two murine squamous cell carcinoma cells (SCC VII/SF) labeled with CRT antibody 2 h after treating with 15 pulses (25 kV/cm, 100ns). <b>C.</b> Two BxPC-3 human pancreatic cancer cells labeled with CRT antibody 2 h after treating with 10 pulses (25 kV/cm, 100 ns). <b>D.</b> Percentage of treated cells exhibiting surface CRT 2 h after the indicated number of pulses were applied at 25 kV/cm.</p

Comparison of 1st and 2^nd tumor surface areas in controls (A) and CD8⁺-depleted rats (B).

<p>We took blood samples from each rat and counted the number of both CD4⁺ and CD8⁺ cells. The percentage of CD8⁺ cells in this total is plotted for each tumor pair. We plotted the tumor surface area on a log scale to highlight the dramatic increase in the rate of growth of the second tumor when CD8⁺ cells were depleted. In two of the controls, we could not find the second tumor so the mean tumor size was calculated from the four visible second tumors. A Mann-Whitney Rank Sum Test indicates a statistically significant difference between the secondary tumor sizes in these two groups. (P = 0.03).</p

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