Investigation

of Electrostatic Effects on Cancer Cell Cultures

W. C. Hymer1 Andrea Mastro2 Max Fomitchev-Zamilov1
1 2

Quantum Potential, State College, PA 16803

Pennsylvania State University, University Park, 16802

Abstract
Electromagnetic fields continue to find useful applications in human health. For instance, hyperthermia resulting from the application of ultra-high frequency (UHF) fields in combination with electroporation technology can be used fordelivery of chemotherapeutic agents under the influence of DC electric currents. Until recently, such forms of therapy were not recommended for fear of greater risk of increased cancer cell proliferation. Nevertheless, in the past decade, there has been a resurgence of interest in applying electromagnetic fields for cancer treatment. Some results have been encouraging. For example, specific combinations of UHF fields and electrophoresis result in epithelial tumor regression and dissolution [10-14]. Low-power alternating fields, in kilohertz frequency range, are reported to destroy cancer cells during mitosis [5-7]. Finally, electromagnetic fields can be selectively tuned to target cancer cells making them more susceptible to chemotherapeutic agents. In this application we propose a Phase I study to explore the effects of electrostatic fields on human mammary cancer cell cultures with the objective of identifying optimal field parameters that render cancer cells non-viable and/or increase their sensitivity to chemotherapy drugs. The application of electrostatic fields for this use is unique. Our long- term goal is to develop a minimally invasive device that will selectively target and destroy both metastatic and non-metastatic cancer cells in humans. To achieve the goals we propose to use human mammary cell cultures two cancer cell lines MDA-MB-231-GFP, one of which exhibits suppressed metastasis, and an immortalized normal mammary line hTERT-HME1 in the following six experiments: 1) qualitative viability study (trypan blue method) to determine electrostatic field parameters that achieves 50% kill rate in cancer cells; 2) quantitative viability study (XTT method) to accurately determine the range of parameters such as the optimal field strength (V/m), optimal exposure time (days), differential sensitivity of normal vs. cancer cells; 3) viability study with cytostatics to determine how electrostatic fields affect cancer cell susceptibility to chemotherapy agents; 4) post-treatment (no electrostatic field, no drug) viability study to ascertain cell culture recovery; 5) cytoskeleton study to identify cellular morphological response to electrostatic treatment; 6) microarray trial to identify potential changes in genetic expression in the treated cells. The team assembled for the project is highly proficient in the aforementioned cell culture techniques and in the physics and engineering of the proposed electrostatic equipment.

Relevance
In the U.S., breast cancer accounts for 1 in 3 cancers diagnosed in women. Breast cancer preferentially metastasizes (i.e. travels) to bone; once this occurs the five-year survival rate drops from >90% to <10%. We propose a new, minimally invasive method for eliminating cancer cells by making them more susceptible to chemotherapeutic agents.

Specific Aims
1) To establish the electrostatic field hardware in our cell culture laboratory and validate its use in preliminary cell viability tests using human mammary cell lines 2) To optimize, identify and select the field strength(s) that will have maximum impact on the malignant cells and minimum impact on the normal cells 3) To use optimized field strength(s), in combination with cyclophosphamide, to determine if there are changes in sensitivity of the malignant cell lines to the drug 4) To use optimized field strength(s) to evaluate possible changes in actin cytoskeleton 5) To use a single, optimized field strength to evaluate possible genetic changes using microarray analysis

Research Strategy
Significance Although the idea that electromagnetic fields, in some form, may be useful in helping fight cancer is not new (see Table 1), it has been under-investigated. There are indications that electromagnetic treatment may reduce the need for surgery by dissolving lip tumors via a combination of UHF- hyperthermia with DC-electrophoresis [10-14]; there are also reports that inoperable brain tumors can be suppressed (although not completely cured) via an application of alternating electric fields that disrupts dividing cancer cells during mitosis [5-8]. However, the need to fight metastasized cancer has not been successfully addressed. This need represents the chief motivation behind our proposal. While weak alternating electric fields have been shown to enhance cancer cell susceptibility to chemotherapeutic agents [7] the physical effects (such as hyperthermia) caused by alternating currents may promote metastasis. Therefore, in our study we will choose static electric fields. Our main hypothesis is that prolonged exposure to a strong electrostatic field will promote destruction of both metastatic and non- metastatic cancer cells and/or increase cancer cell susceptibility to chemotherapeutic agents.
Table 1. Summary of cancer therapy involving electric fields. Important references that use electric fields for cancer therapy.

Years 1987- 1994 1988- 1994

Author(s) (Country) Nordenstrm (Sweden) Molokanov & Shargorodski (USSR)

Field Type Med-voltage polarization High-power UHF + low- power DC

Therapy Medium-voltage (400-600V) DC electric field was used to polarize tumors into regression [9] UHF Hyperthermia of lip (epithelial) tumors combined with electrophoresis allowed eliminating surgery, reducing radiation therapy dosage by 50% and cytostatic dosage by 90% (successful preclinical trials on rats and successful clinical trial on 60 patients) [10-14]

2004- 2010

Kirson & Palti (Israel)

Low-power HF Low-power alternating electric fields inhibit tumor AC proliferation due to killing effect on dividing tumor cells [5-8]

The driving force behind this proposal stems from the following: 1) The pioneering work of Molokanov, who for many years has used combinations of alternating current (AC) and hyperthermia to significantly reduce lip cancer growth in humans [10-14]; 2) The fact that Molokanov is currently employed by Quantum Potential, and 3) A team of biological/medical scientists at Penn State University, with the combined expertise that will be essential for project success. This team is committed to this project and will effectively synergize (see the Team section below). For Quantum Potential, we define success in terms of our ultimate commercialization goal, viz. development of a wearable device in breast cancer patients that renders neoplastic cells more sensitive to chemotherapeutic agents than surrounding tissue cells. Ultimately, it will be important to understand how electrostatic fields exert their killing action. To begin this aspect of our effort we propose to initiate studies in two classes of sub-celluar changes, viz. cytoskeletal and gene expression. Strong electrostatic fields induce intracellular polarization changes that interfere with ion pumps [9, 15], affect cellular growth and morphology [16], and manifest changes in the actin cytoskeleton and gene expression. These processes are magnified in actively dividing cells, ultimately leading to loss of physiological control mechanisms which can lead to cell destruction.

The specific hypotheses under test are: That exposure of malignant human mammary cancer cell lines to defined electrostatic fields in vitro will kill cancer cells, while at the same time have only marginal effects on immortalized normal human mammary epithelial cell lines; That metastatic mammary cancer cell lines will be more sensitive to electrostatic fields than their non-metastatic counterparts; Those electrostatic fields will increase the killing sensitivity of the cells to chemotherapeutic agents.

Innovation
To our knowledge, our experimental designs test, for the first time, the killing efficiency of strong electrostatic fields on malignant cells in vitro carried out under non-hyperthermic conditions (37C). They also tests the idea that non-malignant cells are only marginally affected by strong electrostatic fields and, finally, that malignant cells are rendered more sensitive to chemotherapeutic agents in the electrostatic influence.

Approach
Rows of 96 well tissue culture plates will be placed in between two metal Teflon-insulated capacitor plates energized by a high-voltage DC power supply. The plates are positioned in a wedge shape so that different electric field strengths, at various positions within the capacitor, vary according to the distance between the plates. The field strength is given as E=U/d where U is the applied voltage. As seen in Fig. 1, the location of each 96 multi-well plate will define the field strength. When the capacitor is charged to maximum voltage (5kV) and the distance between the capacitor plates is set to 20 cm (max) and 2 cm (min) 10 dish locations will be exposed to 10 different electric field strengths (250 -2,500 V/cm at ~ 250 V/cm intervals). The experiments flow diagram with the decision point taking into account alternate approach is shown on Fig. 2.

Fig. 1. Experimental setup showing cell cultures in multi-compartment Petri dishes (small boxes) positioned in between the capacitor plates (large box) energized via high-voltage DC power supply.

Cell Lines-Rationale for Use We propose to use two human mammary cancer cells lines: MDA-MB-231-GFP cells [3], derived from a pleural effusion and MDA-MB-231BRMS-GFP cells [4], a human variant with suppressed metastasis to bone and other organs. For normal cells purpose we intend to use an immortalized human mammary cell line hTERT-HME1 from ATTC (Cat # CRL 4010).

Prepare the cell cultures: hTERT-HME1

MDA-MB-231-GFP (met; nonmet)

Trial #1: Qualitative Viability Study Trypan blue assay

Modified trials

Trial #2: Quantitative Viability Study XTT assay

Decision Point: Differential killing achieved?

No

Introduce modified hardware with AC generator (50-100,000 Hz)

Yes Trial #3: Viability Study with Cytostatics Cyclophosphamide assay

Trial #4: Post-Treatment Viability Study (Recovery) No fields

Trial #5: Cytoskeleton Study Confocal microscopy

Trial #6: Microarray Experiment Statistical analysis

Project Completed Write report

Fig. 2. Flow diagram of experiments illustrating the decision point that may call to an alternative approach of introducing an alternating (AC) low-frequency (under 100,000 Hz) electric fields in the trials.

Trial #1: Qualitative Viability Study Rationale: It will first be necessary to establish cell viability exposed to 10 different electrostatic field strengths (250-2,500 V/cm) vs. viability of cells not exposed to the field. Rather than plunging directly into a colorimetric method to quantify cell viability (such as the one we propose to use in Trial #2), we suggest there are advantages to trypsinization, trypan blue staining, and hemocytometer counting because simple microscopic visualization reveals the state of general cell morphology and health. Methods: Cells from the 3 different lines will be seeded (3x103 /well) in 96 well plates under conditions in which confluency is reached in 3-4 days (DMEM, 10% neonatal FBS, in an anti-biotic free environment at 37C for 3 passages prior to use). A total of 3 wells, 1 well for each cell line, will then be established for each of the 10 field strengths (250-2,500 V/cm) in 10 individual multi well plates (Fig. 1). Every 24 hrs, for a total of 5 days, a plate will be removed from the incubator for analysis. Thus the number of wells/processed each day will be 30. An identical set of plates will be set up, but not subjected to the field, and processed each day as a control. Processing will involve trypan blue exclusion testing followed by a subsequent plating efficiency test done in the absence of the field to determine efficiency of recovery. Questions addressed: 1. 2. 3. 4. 5. Is there a range of electrostatic field strengths that kill the cells? What is the 50% kill range for each cell line and is there differential sensitivity between them? Does the killing depend upon days in culture? Will only nominal killing be obtained in the case of the normal cells? Will the treated cells recover in a plating efficiency test?

Estimated time to establish hardware and complete Trial #1: One month. Trial #2: Quantitative Viability Study Rationale: The results from Trial #1 are expected to yield only qualitative answers to the questions being asked. In Trial #2 we propose replication of Trial #1 using the XTT cell proliferation assay to obtain information, with statistical confidence, that repeatable field conditions have been identified for the cancer cell lines. To do this, trial 2 will be repeated 3 times. This assay is based is based on the ability of metabolically active cells to reduce the tetrazolium salt (XTT) to orange colored compounds of formazan. The dye formed is water-soluble and the dye intensity is read in an ELISA plate reader. The healthier the activity of the mitochondrial enzymes, the higher the concentration of dye formed. The assay is a one-step process and can yield data in 2-5 hrs. Questions addressed: These are identical to those given in Trial #1. Estimated time to complete Trial #2: 6 weeks. Decision Point: At this point we will have sufficient information to determine if electrostatic fields meet our goals. If, however, both normal and neoplastic cells are killed to the same extent, OR if the killing effects are insignificant or absent; we will revise our hardware by incorporating a variable high-voltage (0- 10kV) AC power generator to study the effect of low-frequency (50-100,000 Hz) oscillating electric fields on cancer cell cultures (see Fig. 2). This possible hardware change will not affect the character of the planned experiments. Trial #3: Viability Study with Cytostatics Rationale: Assuming positive results are indeed obtained, we will be able to select field strength, as well as the time required to be in that field, to reproducibly kill 50% of the mammary cancer cells. Assuming that there is a difference in kill rate between the metastatic and non-metastatic lines, the choice of the 50% kill point will be with the line having more living cells. Using these refined field strength conditions we will incorporate cyclophosphamide, a DNA alkylating agent, into the culture medium of one half of the experimental culture wells to determine if the electrostatically-treated cells have increased sensitivity to chemotherapeutic agents.

Methods: Based on the study of Kirson et al [7] (which used MDA-MB-231 cells in low intensity (1-2 V/cm) and variable/alternating electric fields) we will add a high (8 mM) and low (0.1 mM) concentration of cyclophosphamide for 3 days to half the cell culture wells. Diluents will be added to the non-treated half. Appropriate controls, viz. cells incubated for 3 days in the absence of the field, but in the presence-or absence- of cyclophosphamide, will be required to establish the 0 V/cm points on the kill curves. XTT assay methodology will be used and will require 3 experiments for statistical reliability. Questions addressed: Will a defined field strength change the sensitivity of the cancer cells to chemotherapy agent? Will metastatic cells be more sensitive than their non-metastatic counterparts? Will normal mammary cells be only marginally affected? Estimated time to complete trial #3: 6weeks. Trial #4: Post-Treatment Viability Study (Recovery) Rationale: The time course of recovery from a 24 hr exposure to an electrostatic field (+/- cyclophosphamide) will be of importance to determine if, and how well, the cancer cells treated with either electrostatic fields or drug alone, or in combination, recover in the subsequent 48-culture period in the absence of any of these treatments. If an electrostatic treatment of mammary cancer in the clinic is eventually realized, the information from this type of experiment will be important. Methods: This trial will be done according to the design described in Trial #3, except that the cells will be cultured in a field-free environment for 48 hrs (explained above). Questions addressed: In addition to those identified in Trial #3, this trial will help determine recovery of cell viability after experimental treatment. Estimated time to complete trial #4: 6 weeks. Trial #5: Cytoskeleton Study Rationale: The purpose of this trial is to begin to identify major changes that are likely to occur in the cell lines exposed to electric fields. As suggested by [16], we believe that cells, exposed to polarizing electric fields, will have altered distribution patterns of their actin cytoskeleton. The mammary cancer cell lines we will use are already tagged with green fluorescent protein (GFP). Fluorescence microscopy will be used not only to evaluate general cell morphology, but also specific fluorescence of the cytoskeleton (using, for example, a red fluorescent dye such as Alexa Fluor 594, Molecular Probes, Inc.). We will test the idea that defined field strengths (optimized from previous trials), or variable fields, or fields using AC will reveal altered patterns of cytoskeletal distributions. Such patterns, if similar to those in apoptotic cells, would suggest one potential mechanism of killing. Methods: In this trial, 96 well culture plates with flat bottoms will be used. Prior to cell seeding, commercially available cell culture cover slips will be placed in individual wells. Following the procedures identified in preceding trials, we will select field and drug conditions that yield predictable kill kinetics. These cover slips, containing attached cells, will be processed and examined by either fluorescence microscopy or, in selected cases by confocal microscopy. These techniques have been used previously in both the Mastro and Hymer laboratories. Question addressed: Does the field change the distribution patterns of the actin cytoskeleton in the cytoplasm of the mammary cancer cell? Estimated time to complete trial #5: 3-4 weeks. Trial #6: Microarray Experiment Rationale: It seems certain that many genetic changes will take place as the cells are treated with electric fields. The question is: can we identify changes in major gene classes that will offer hints as to other mechanisms of field action? Toward the end of the 6 month Phase I effort we would like to try a single microarray trial as we believe positive results will be useful for a follow on Phase II effort. If even

feasible, this trial is limited because there are many types of experimental samples to analyze (9 are estimated); time is clearly limited (we propose to do this trial concurrently with the cytoskeleton trial); and budget constraints do not permit multi-time point sampling. We will seek statistical advice, available through Penn States Microarray Facility, on design of this single experiment. It seems probable that the single time point selected will be prior to the 50% kill point. Method: Cells will be cultured in 35 mm dishes to obtain sufficient RNA. An RNeasy Kit (Qiagen) will be used to isolate RNA. Its purity will be judged by A260 /A280 ratio. Samples will be processed at Penn States Core Genomics Facility (see letter).

Team
A synergistic team has been assembled to work on the project: Dr. Mastro is an expert in mammary cancer cell cultures; Dr. Hymer is a cell biologist/biochemist/physiologist with long-standing experience; Dr. Lumadue is a practicing cancer physician/scientist lending us clinical advice on cytostatics and therapy, and Dr. Fomitchev-Zamilov is a physicist and an expert in electrostatic hardware used for experimentation. This team will work together to meet Phase I goals. Dr. Fomitchev-Zamilov will implement hardware in the laboratory. Kevin Heister, who is currently employed in Dr. Mastros laboratory and uses the cells and the culture methodologies we propose to use, will establish these same procedures in our lab. We have 2 certified cell culture hoods and Mr. Heister is fully trained in the proper handling of the cultures. Data analysis will be using statistical methods Dr. Mastro has described in several recent publications (see her biosketch). Data interpretation will be achieved by input from the entire team; these data will then serve as a foundation for a phase II proposal.
Andrea Mastro Professor of Microbiol. & Cell Biol. Mammary Cancer Scientist Fig 3. Team structure and data flow. Quantum Potential Laboratory Kevin Heister Lab Technician Wes Hymer Emerit. Professor of Biochemistry Project Science Director Jeanne Lumadue Clinical Investigator Consultant Max Fomitchev-Zamilov Physics/computers/management Hardware design & implementation Quantum Potential Max Fomitchev-Zamilov, Director Wes Hymer, Project Director Nikolay Molokanov, Research Associate Phase I Final Report

Data

References
1. van der Zee J. Heating the patient: A promising approach? Annals of Oncology 2002, 13, pp.11731184 2. Wust P, Hildebrandt B, Sreenivasa G, Rau B, Gellermann J, Riess H, Felix R, Schlag PM, Hyperthermia in combined treatment of cancer. The Lancet Oncology 2002, 3, pp.487497 3. Cailleau R, Young R, Olive M, Reeves Jr. WJ. Breast tumor cell lines from pleural effusions. Journal of National Cancer Institute 1974, 53, pp.661-674 4. Samant RS, Debies MT, Hurst DR, Moore BP, Shevde LA, Welch DR. Suppression of murine mammary carcinoma metastasis by the murine ortholog of breast cancer metastasis suppressor 1 (Brms1). Cancer Lett 2006, 235(2), pp.260-265 5. Kirson ED, Gurvich Z, Schneiderman R, Dekel E, Itzhaki A, Wasserman Y, Schatzberger R, Palti Y, Disruption of cancer cell replication by alternating electric fields, Cancer Res. 2004, 64(9), pp. 3288-3295 6. Salzberg M, Kirson E, Palti Y, Rochlitz C, A pilot study with very low-intensity, intermediate-frequency electric fields in patients with locally advanced and/or metastatic solid tumors, Onkologie. 2008, 31(7), pp.362-365 7. Kirson ED, Schneiderman RS, Dbal V, Tovarys F, Vymazal J, Itzhaki A, Mordechovich D, Gurvich Z, Shmueli E, Goldsher D, Wasserman Y, Palti Y, Chemotherapeutic treatment efficacy and sensitivity are increased by adjuvant alternating electric fields (TTFields), BMC Med Phys. 2009, 9(1) 8. www.novocuretrial.com 9. Nordenstrm BEW, Electrostatic Field Interference with Cellular and Tissue Function, Leading to Dissolution of Metastases that Enhances the Effect of Chemotherapy, Eur J Surg. 1994, Suppl 574,pp.121- 135 10. Molokanov NJ, MatychenkovaZP, MoiseyenkovaSD,On the possibility of application of UHF hyperthermia treatment of malignant head and neck tumors, Medical and Biological consequences of hyperthermia1989, Smolensk, pp.72-74 11. Molokanov NJ, NikiforovAI,Hyperthermia device, USSR Patent 1685473, 1991 12. Shargorodski AG, Molokanov NJ, DedenkovAN,Application of local UHF hyperthermia in treatment and prophylaxis of the lower lip cancer, Aktualnyje Voprosy Stomatologii1986, 16, pp.110-113 13. Shargorodski AG, Molokanov NJ, Immediate results of Lip Tumor Treatment by Thermal Method, Symposium on Head and Neck Tumors, Tallinn, 1991, pp.31-32 14. Molokanov NJ,Prophylaxis of post-radiation treatment complications of lower lip cancer with thermal and chemotherapeutic methods, Dr. Sci. Dissertation,Moscow, 1994 15. Murr LE, Plant growth response in a simulated electric field environment, Nature 1963, 200, pp.490- 491 16. Kalinina IM, Krstic V, Tolic-Norrelykke IM, Cell Polarity: Which Way to Grow in an Electric Field?, Current Biology 2010, 20(8), pp.356-356

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Facilities
Quantum Potential will have a well equipped cell culture/biochemistry laboratory (with suitable office space included in the total lab area (~500 ft2 ) available to carry out these Phase I studies. This lab space, located on the Penn State campus in the Central Biological Lab building, will be rented (see Dr. Kennetts letter. To conduct the microarray experiment we shall contract Penn State Genomics Core facility (see the attached letter). Other Quantum Potential facilities include an office, computer lab and a tool shop.

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Budget Justification
To perform the work outlined in the proposal, we request funds to purchase a larger 6.5 ft3 VWR Incubator ($6,400) as our existing incubator is not large enough to house the electrostatic equipment. Its age is also a matter of concern. We budget $5,000, payable to Penn States Genomics Core facility, for microarray analysis.

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