Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, Propidium Iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (15 minutes) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.

Anti-tumor vaccines are a relatively non-toxic alternative to conventional chemotherapeutic strategies to control breast cancer. Immunization with tumor-associated antigens (TAAs) triggers anti-tumor cytotoxic T lymphocytes (CTL), which can limit tumor progression. Here we report on the development and effectiveness of a TAA-based DNA vaccine encoding Mage-b1/2, the mouse homologue of the human MAGE-B1/2. As model system, we used immune competent Balb/c mice with syngeneic non-metastatic (64pT) or metastatic (4TO7cg) breast tumors. First, the presence of Mage-btranscripts in the 64pT and 4TO7cg breast tumors and metastases was demonstrated by RT-PCR, Southern blotting, and DNA sequencing. A DNA-based vaccine was developed from transcripts of one of the 64pT tumors, encoding the complete Mage-b1/2 protein, and subsequently tested for its preventive efficacy in both breast tumor models. Mice were immunized two times intramuscularly with the vaccine (pcDNA3.1-Mage-b1/2-V5), the control vector (pcDNA3.1-V5), or saline. Two weeks after the last immunization, the syngeneic 4TO7cg or 64pT tumor cell lines were injected in a mammary fat pad. Mice were monitored during the next 4 weeks for tumor formation, latency and size, and subsequently sacrificed for analysis. While the Mage-b1/2 vaccine had only a minor effect on the latency and growth of primary tumors, a significant and reproducible reduction in the number of 4TO7cg metastases was observed (vaccine versus control vector, p=0.0329; vaccine versus saline, p=0.0128). The observed protective efficacy of the Mage-b DNA vaccine correlated with high levels of vaccine-induced IFNgamma in spleen and lymph nodes upon re-stimulation in vitro. These results demonstrate the potential of TAA-based DNA vaccines in controlling metastatic disease in breast cancer patients.

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.

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Previous work from our laboratory demonstrated nanopore formation in cell membranes following exposure to nanosecond pulsed electric fields (nsPEF). We observed differences in sensitivity to nsPEF in both acute membrane injury and 24h lethality across multiple cells lines. Based on these data, we hypothesize that the biological response of cells to nsPEF is dependent on the physical properties of the plasma membrane (PM), including regional cholesterol content. Results presented in this paper show that depletion of membrane cholesterol disrupts the PM and increases the permeability of cells to small molecules, including propidium iodide and calcium occurring after fewer nsPEF. Additionally, cholesterol depletion concurrently decreases the "dose" of nsPEF required to induce lethality. In summary, the results of the current study suggest that the PM cholesterol composition is an important determinant in the cellular response to nsPEF.

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