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 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

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

The S100A10 Subunit of the Annexin A2 Heterotetramer Facilitates L2-Mediated Human Papillomavirus Infection

<div><p>Mucosotropic, high-risk human papillomaviruses (HPV) are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t) contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI), or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108–120) specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.</p> </div

shRNA knockdown of A2t reduces internalization of HPV16 L1L2 VLP and infectivity of HPV16 PsV.

<p>(A) HPV16 L1 VLP were fluorescently labeled with CFDA-SE, and the percent of infected cells was measured as the percent that were CFDA-SE positive after exposure to VLP for 1 hour as measured by FACS. To control for free label false positives, cells were treated with VLP pre-incubated with a neutralizing anti-L1 antibody (H16.V5). An identical experiment was performed using HPV16 L1L2 VLP. Both histograms are representative examples of two experiments done in triplicate. (B) HeLa cells were left untreated or transduced with a doxycycline inducible pTRIPZ Tet-On lentiviral vector containing an shRNA against ANXA2. Single cell clones treated with or without doxycycline were incubated with labeled HPV16 VLP for 3 hours at 37° and assessed by FACS. The mean percentage of uptake normalized to the wild type group ± SEM of three independent experiments is presented. (C) Protein was collected from cell populations used in the internalization assay for analysis of ANXA2 and S100A10 via Western blot. GAPDH served as a loading control. (D) mRNA was collected from cell populations in the uptake assay for quantitative RT-PCR analysis of ANXA2 and S100A10 expression. The mRNA expression levels were normalized to GAPDH and the graph is a representative example of an experiment performed in triplicate ± SD. (E) Wildtype HeLa cells or HeLa cells stably transduced with a doxycycline inducible lentiviral vector containing shRNA against ANXA2 or control non-target lentiviral vector were infected with GFP plasmid containing HPV16 pseudovirus. Infection was scored 48 h later by enumeration of GFP-positive cells by flow cytometry. The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the no doxycycline treated groups ± SD are presented. (*<i>P</i><0.05 and **<i>P</i><0.01 as determined by a two-tailed, unpaired t-test, as compared to the no doxycycline-treated groups). Figure is representative of two independent experiments. (F) Protein was collected from cell populations used in the infection assay for analysis of ANXA2 and S100A10 via Western blot. GAPDH served as a loading control.</p

Surface expression of A2t on HaCaT and HeLa human epithelial cell lines. (

<p>A) HaCaT and HeLa cells were incubated with an anti- S100A10 antibody, then incubated with fluorophore-conjugated secondary antibodies, and mounted with DAPI containing media. For control staining, cells were either stained with a mouse or rabbit IgG isotype control followed by secondary antibody staining. Images were acquired using an upright confocal fluorescent microscope. (B) HeLa and HaCaT cells were incubated with PBS supplemented with Ca²⁺ or PBS with increasing concentration of EDTA for 45 min. The supernatants were collected and the presence of ANXA2 and S100A10 were analyzed via Western blot.</p

HPV16 binds to A2t on the cell surface of HeLa cells.

<p>HeLa cells were incubated with HPV16 L1L2 VLP, HPV16 PsV or HPV16 L1 VLP for 1 hour at 37°C. The cells were washed and surface proteins cross-linked. HPV16 VLP and PsV were precipitated out of cell lysates with an anti-L1 antibody (H16.V5) conjugated to magnetic beads. Elutions were analyzed via Western blot for the presence of ANXA2 and S100A10. Band density was determined by Licor Odyssey imaging software. HPV L1 western blot shows equivalent amounts of HPV16 particles were precipitated in lanes 3–5. The table below the figure indicates which components were included in each treatment. Data are representative of at least three independent experiments.</p

Mutations in HPV16 L2_108–111 reduce PsV binding to A2t and PsV infectivity.

<p>(A) ELISA plate wells were coated with 500 ng of A2t prior to overnight incubation with 400 ng HPV16 PsV or HPV16 L1–L2(GGDD) mutant PsV and subsequently incubated with mouse anti-L1 H16.V5 or goat anti-SLPI antibodies. Anti-mouse and anti-goat HRP-conjugated secondary antibodies were added prior to the substrate. In control experiments, no ligands were used. The graph represents the mean absorbance measured at 490 nm ± SD (***<i>P</i><0.001 as determined by a two-tailed, unpaired t-test between WT and mutant PsV). (B) HaCaT cells were infected with wild type (WT) or mutant (L2_108–111 LVEE to GGDD) HPV16 pseudovirions containing a GFP plasmid. Infectivity was scored at 48 h post infection by enumerating GFP-positive cells by flow cytometry. The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the WT PsV group ± SD are presented of two combined independent experiments. Inset shows the L1 band of a coomassie blue stained SDS-PAGE gel loaded with an equivalent amount of WT and mutant PsV used in the infectivity assays. (***<i>P</i><0.001 as determined by a two-tailed, unpaired t-test between WT and mutant PsV group).</p