Use of alternating electric field to increase cell membrane permeability

Abstract

To allow a substance to pass through a cell membrane by increasing the permeability of the cell membrane, since a certain substance (e.g., a large molecule) that cannot normally traverse a cell membrane can be introduced into a cell by applying an alternating electric field to the cell for a certain period of time and the frequency of the alternating electric field is selected so that the application of the alternating electric field increases the permeability of the cell membrane, this approach being particularly beneficial in association with cancer cells (e.g., glioblastoma).SOLUTION: The first method comprises the steps of: applying an alternating electric field to a cell for a certain period of time, in which the application of the alternating electric field increases the permeability of a cell membrane; and introducing a substance near the cell, the increased permeability of the cell membrane allowing the substance to pass through the cell membrane.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Applications Nos. 62 / 693,811 (filed July 3, 2018), 62 / 728,255 (filed September 7, 2018), and 62 / 795,136 (filed January 22, 2019), each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] Treatment of glioblastoma (GBM) using alternating current electric fields is a new and validated therapy that is becoming an additional modality for anticancer treatment (after surgery, chemoradiotherapy, and chemotherapy). Intermediate frequency alternating current electric fields (100-500 kHz) have been studied in detail. Recently, TT-Fields have been shown to prolong (by 5 months) the median survival time of glioblastoma patients undergoing maintenance temozolomide chemotherapy. In the context of tumor treatment, alternating current electric fields of these frequencies are often referred to as "tumor therapy fields" or "TT-Fields".

[0003] While many hypotheses exist regarding the mechanism of TT Fields, the most widely proposed ("canonical") mechanism of TT Fields anticancer action centers on the property that tubulin subunits have an intrinsic dipole moment. Imposing an exogenous 200 kHz TT Field disrupts the function of actively dividing cells through interference with the cytoskeleton that supports the mitotic spindle by aligning microtubule structures along an alternating electric field system. Such stress ultimately promotes impaired cell proliferation. Proof-of-concept experiments and associated technology development have occurred over the past decade, culminating in the approval by the Food and Drug Administration (FDA) of a commercially available clinical TT Field device (Optune®, Novocure, Inc.) for the treatment of recurrent, newly diagnosed glioblastoma.

[0004] Over the past few years, additional details about the mechanism of action have been reported. For example, TTFields have been shown to disrupt the localization of septins (intracellular proteins responsible for anchoring the mitotic spindle during cell division), thereby disrupting mitosis. Some teams have reported extended DNA damage from chemotherapy or radiation therapy together with TTFields, while others have shown effects on mitochondrial function through swelling of the mitochondrial matrix. Other teams have investigated the combination of TTFields with chemotherapy (e.g., temozolomide) in GBM patients. Such studies of combination interventions have revealed other promising effects on glioblastoma. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 7,565,205 [Patent Document 2] U.S. Patent No. 6,868,289 Summary of the Invention [Means for solving the problem]

[0006] One aspect of the present invention relates to a first method for delivering a substance across a cell membrane of a cell, the first method comprising the steps of applying an alternating electric field to the cell for a period of time, where application of the alternating electric field increases the permeability of the cell membrane; and introducing a substance into the vicinity of the cell, where the increased permeability of the cell membrane allows the substance to pass through the cell membrane.

[0007] In some cases of the first method, the cells are cancer cells. In some cases of the first method, the cells are glioblastoma cells. In some cases of the first method, the AC electric field is applied at a frequency of about 200 kHz. In some cases of the first method, the AC electric field is applied at a frequency of 50-190 kHz. In some cases of the first method, the AC electric field is applied at a frequency of 210-400 kHz. In some cases of the first method, the AC electric field has a field strength of at least 1 V / cm.

[0008] In some cases of the first method, the cells are placed in a body of a living subject, an alternating electric field is applied to the cells by applying an electric field to the body of the subject, and the introducing step includes administering the substance to the subject. In these cases, the cells may be cancer cells. In these cases, the cells may be glioblastoma cells. In these cases, the alternating electric field may have a frequency of 50-190 kHz. In these cases, the alternating electric field may have a frequency of 210-400 kHz. In these cases, the alternating electric field may have a field strength of at least 1 V / cm RMS. In these cases, the alternating electric field may have a field strength of 1-4 V / cm RMS. In these cases, the introducing step may begin at a given time, and the applying the alternating electric field may end at least 12 hours after the given time. In these cases, the applying the alternating electric field may begin at least 1 hour before the given time. In these cases, the substance may have a molecular weight of at least 1.2 kDa. In these cases, the substance can have a molecular weight of at least 4 kDa. In these cases, the substance can have a molecular weight of at least 20 kDa. In these cases, the substance can have at least one characteristic that normally prevents the substance from passing through the cell membrane. In these cases, the cell can be a cancer cell that is naturally resistant to treatment with the substance. In these cases, the cell can include bacteria and the substance includes an antibiotic.

[0009] Another aspect of the invention relates to a second method of attacking cancer cells, the second method comprising the steps of: applying a first alternating electric field of a first frequency to the cancer cells for a first period of time, where application of the first alternating electric field of the first frequency to the cancer cells for the first period of time increases the permeability of a cell membrane of the cancer cells; introducing a substance into the cancer cells, where the increased permeability of the cell membrane allows the substance to pass through the cell membrane; and applying a second alternating electric field of a second frequency to the cancer cells for a second period of time, where the second frequency is different from the first frequency, where the second alternating electric field of the second frequency reduces the viability of the cancer cells.

[0010] In some cases of the second method, the cancer cells include glioblastoma cells, the first frequency is 250 kHz to 350 kHz, and the second frequency is 150 kHz to 250 kHz. In some cases of the second method, the cancer cells include uterine sarcoma cells, the first frequency is 125 kHz to 175 kHz, and the second frequency is 75 kHz to 125 kHz. In some cases of the second method, the cancer cells include breast adenocarcinoma cells, the first frequency is 75 kHz to 175 kHz, and the second frequency is 100 kHz to 300 kHz. In some cases of the second method, the step of introducing the substance begins at a given time, and the step of applying the first AC electric field ends at least 12 hours after the given time. In some cases of the second method, the step of applying the first AC electric field begins at least 1 hour before the given time. In some cases of the second method, the second time period includes a plurality of discrete time intervals during which the second alternating electric field at the second frequency is applied to the cancer cells, where the plurality of discrete time intervals totals at least one week.

[0011] In some cases of the second method, the cancer cells are placed in a living subject's body, a first alternating electric field is applied to the cancer cells by applying the first alternating electric field to the subject's body, and a second alternating electric field is applied to the cancer cells by applying a second alternating electric field to the subject's body, and the introducing step includes administering the substance to the subject. In some cases of the second method, the first alternating electric field has a field strength of at least 1 V / cm RMS. In some cases of the second method, the substance has a molecular weight of at least 1.2 kDa. In some cases of the second method, the substance has a molecular weight of at least 4 kDa. In some cases of the second method, the substance has a molecular weight of at least 20 kDa.

[0012] Another aspect of the present invention relates to a third method of treating a tumor in a subject's body and delivering a substance across a cell membrane in the subject's body, the third method comprising the steps of: applying a first alternating electric field of a first frequency to the subject's body for a first period of time, where application of the first alternating electric field of the first frequency to the subject's body for the first period of time increases the permeability of a cell membrane in the subject's body; administering a substance to the subject, where the increase in the permeability of the cell membrane allows the substance to pass through the cell membrane; and applying a second alternating electric field of a second frequency to the subject's body for a second period of time that is at least one week long, where the second frequency is different from the first frequency, and where the second alternating electric field of the second frequency inhibits tumor growth.

[0013] In some cases of the third method, the tumor comprises a glioblastoma in the brain of the subject, the first frequency is between 250 kHz and 350 kHz, and the second frequency is between 150 kHz and 250 kHz. In some cases of the third method, the second time period comprises a plurality of discrete time intervals during which the second alternating electric field of the second frequency is applied to the body of the subject, where the plurality of discrete time intervals total at least one week. In some cases of the third method, the step of administering the substance begins at a given time, and the step of applying the first alternating electric field ends at least 12 hours after the given time. In some cases of the third method, the step of applying the first alternating electric field begins at least one hour before the given time.

[0014] In some cases of the third method, the substance has a molecular weight of at least 1.2 kDa. In some cases of the third method, the substance has a molecular weight of at least 4 kDa. In some cases of the third method, the substance has a molecular weight of at least 20 kDa.

[0015] Another aspect of the invention relates to a first apparatus for treating a tumor in a subject's body and facilitating delivery of a substance across a cell membrane in a subject's body. The first apparatus includes an AC voltage generator operable at a first frequency between 50-500 kHz and a second frequency between 50-500 kHz, where the second frequency is different from the first frequency. The AC voltage generator has a control input, where the AC voltage generator is configured to output the first frequency when the control input is in a first state and to output the second frequency when the control input is in a second state. The first apparatus also includes a controller programmed to: (a) place the control input in a second state such that the AC voltage generator outputs the second frequency; (b) receive a request to switch to the first frequency; (c) upon receiving the request, place the control input in the first state such that the AC voltage generator outputs the first frequency for a time interval; and (d) place the control input in the second state such that the AC voltage generator outputs the second frequency after the time interval has elapsed.

[0016] Some embodiments of the first device further include a set of electrodes configured for attachment to a body of a subject; and wiring connecting the output of the AC voltage generator to the set of electrodes.

[0017] In some embodiments of the first device, the first frequency is between 250 kHz and 350 kHz, and the second frequency is between 150 kHz and 250 kHz. In some embodiments of the first device, the first frequency is between 125 kHz and 175 kHz, and the second frequency is between 75 kHz and 125 kHz. In some embodiments of the first device, the first frequency is between 75 kHz and 175 kHz, and the second frequency is between 100 kHz and 300 kHz. In some embodiments of the first device, the time interval is at least 12 hours. In some embodiments of the first device, the time interval is between 12 and 72 hours. In some embodiments of the first device, the controller is further programmed to, upon receipt of the request, cycle the control input between the first state and the second state.

[0018] In some embodiments of the first apparatus, the AC voltage generator is capable of operating at at least one additional frequency between 50 and 500 kHz, the AC voltage generator is configured to output the at least one additional frequency when the control input is in the at least one additional state, and the controller is programmed to cause the control input to periodically cycle through the second state and the at least one additional state prior to receipt of the request, and to cause the control input to periodically cycle through the second state and the at least one additional state after a time interval has elapsed.

[0019] Some embodiments of the first device further include a user interface, and the request is received through the user interface. In some embodiments of the first device, the request is received through RF. [Brief description of the drawings]

[0020] [Figure 1] Schematic diagram showing alternative effects of TTFields on modulating cell membrane integrity and therefore permeability. [Figure 2A]FIG. 13 depicts an exemplary effect of TT Fields on bioluminescence of U87-MG / eGFP-fLuc cells from bioluminescence imaging scans as a function of time in TT Field vs. no TT Field conditions. [Figure 2B] FIG. 13 depicts an exemplary effect of TT Fields on eGFP fluorescence of U87-MG / eGFP-fLuc cells as a function of time in TT Field vs. no TT Field conditions. [Figure 2C] FIG. 13 depicts the effect of TT Fields on the ratio of fLuc bioluminescence (fLuc-BLI) to eGFP fluorescence (eGFP-FL) for U87-MG / eGFP-fLuc cells as a function of length of TT Field exposure. [Figure 2D] FIG. 13 depicts the effect of TT field exposure vs. no exposure on the fLuc-BLI / eGFP-FL ratio as a function of TT field exposure time. [Figure 3A] FIG. 13 depicts an exemplary effect of TT fields on the time-dependent uptake of ethidium D in U87-MG / eGFP-fLuc cells during no TT field and a TT field (200 kHz). [Figure 3B] FIG. 13 depicts an exemplary effect of TTfield vs. no TTfield conditions on the time course of dextran-FITC uptake for 4 kDa dextran-FITC. [Figure 3C] FIG. 13 depicts an exemplary effect of TT field versus no TT field conditions on the time course of dextran-FITC uptake for 20 kDa dextran-FITC. [Figure 3D] FIG. 13 depicts an exemplary effect of TTfield vs. no TTfield conditions on the time course of dextran-FITC uptake for 50 kDa dextran-FITC. [Figure 4A]FIG. 1 depicts an exemplary effect of TTFields (200 kHz) on 5-aminolevulinic acid (5-ALA) uptake as shown by representative protoporphyrin IX (PpIX) fluorescence for U87-MG cells exposed to TTFields versus no TTFields after 6 and 24 hours. [Figure 4B] FIG. 13 depicts how PpIX fluorescence changed over time in glioblastoma cells versus fibroblasts in a co-culture platform exposed to TTFields. [Diagram 5] FIG. 13 provides quantification of pore number and size from SEM comparison of plasma membrane pores in U87-MG / eGFP-fLuc cells exposed and not exposed to TTFields for 3 days. [Figure 6] FIG. 13 provides quantification of pore number and size from SEM comparison of plasma membrane pores in normal human PCS-201 cells exposed and not exposed to TTFields for 3 days. [Figure 7A] FIG. 13 shows the results of an experiment demonstrating how an AC electric field reversibly increases the uptake of D-luciferin, 5-ALA, and dextran-FITC (4 kDa) in U87-MG cells. [Figure 7B] FIG. 13 shows the results of an experiment demonstrating how an AC electric field reversibly increases the uptake of D-luciferin, 5-ALA, and dextran-FITC (4 kDa) in U87-MG cells. [Figure 7C] FIG. 13 shows the results of an experiment demonstrating how an AC electric field reversibly increases the uptake of D-luciferin, 5-ALA, and dextran-FITC (4 kDa) in U87-MG cells. [Figure 7D] FIG. 1 represents the results of an experiment showing the timing characteristics of permeability induced by application of TTFields to U87-MG cells. [Figure 8A] FIG. 1 depicts the results of an experiment showing how an AC electric field affects the permeability of MDA-MB-435 cell membranes to 7-AAD. [Figure 8B]FIG. 1 depicts the results of experiments showing how an AC electric field affects the permeability of MDA-MB-435 and MDA-MB-435 doxycycline-resistant cell membranes to doxorubicin. [Figure 8C] FIG. 1 depicts the results of an experiment showing how an AC electric field affects the permeability of MCF-7 and MCF-7 mitoxantrone-resistant cell membranes to mitoxantrone. [Figure 9A] FIG. 13 depicts the effect of TTFields on sensitivity to seven different combinations of substances and corresponding cell types. [Figure 9B] FIG. 13 depicts the effect of TTFields on sensitivity to seven different combinations of substances and corresponding cell types. [Figure 9C] FIG. 13 depicts the effect of TTFields on sensitivity to seven different combinations of substances and corresponding cell types. [Figure 9D] FIG. 13 depicts the effect of TTFields on sensitivity to seven different combinations of substances and corresponding cell types. [Figure 9E] FIG. 13 depicts the effect of TTFields on sensitivity to seven different combinations of substances and corresponding cell types. [Figure 9F] FIG. 13 depicts the effect of TTFields on sensitivity to seven different combinations of substances and corresponding cell types. [Figure 9G] FIG. 13 depicts the effect of TTFields on sensitivity to seven different combinations of substances and corresponding cell types. [Figure 10A] FIG. 1 illustrates a preferred timing relationship between application of an AC electric field and introduction of a substance in the vicinity of cancer cells. [Figure 10B] FIG. 1 illustrates a preferred timing relationship between application of an AC electric field and introduction of a substance in the vicinity of cancer cells. [Figure 11A] FIG. 1 depicts the results of an experiment to determine the frequency that provides the highest level of cytotoxicity to U-87 MG cells. [Figure 11B] FIG. 1 depicts the results of an experiment to determine the frequency that provides the greatest increase in plasma membrane permeability of U-87 MG cells. [Figure 12A] FIG. 1 depicts the results of an experiment to determine the frequency that provides the highest level of cytotoxicity to MES-SA cells. [Figure 12B] FIG. 1 depicts the results of an experiment to determine the frequency that provides the greatest increase in plasma membrane permeability of MES-SA cells. [Figure 12C] FIG. 13 depicts the results of an experiment to determine how a 150 kHz alternating electric field affects the permeability of the plasma membrane of MES-SA cells to doxorubicin. [Figure 13A] FIG. 1 depicts the results of an experiment to determine the frequency that provides the highest level of cytotoxicity to MCF-7 cells. [Figure 13B] FIG. 1 depicts the results of an experiment to determine the frequency that provides the greatest increase in cell membrane permeability of MCF-7 cells. [Figure 14] FIG. 1 depicts the results of an experiment to determine the frequency that provides the greatest increase in plasma membrane permeability of GBM39 / Luc cells. [Figure 15] FIG. 1 is a block diagram of a dual frequency device that generates a first frequency for inducing cell permeability and a second frequency for inducing cytotoxicity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Various embodiments are described in detail below with reference to the accompanying drawings, in which like reference numerals represent like elements.

[0022] As used herein, the term "reducing the viability" of a cell refers to reducing the growth, proliferation or survival of the cell, or increasing the cytotoxicity of the cell.

[0023] This application describes a novel approach to temporarily increase the permeability of the plasma membrane of cancer cells using an alternating electric field, so that substances that are normally blocked by the cell membrane can pass through the cell membrane, or substances that are normally prevented by the cell membrane can pass through the cell membrane more easily. In some of the examples described herein, this approach is used to temporarily increase the permeability of glioblastoma plasma membranes using an alternating electric field, so that substances that are normally prevented by the glioblastoma cell membrane can pass through the glioblastoma cell membrane more easily.

[0024] The inventors demonstrated that TTFields treatment synergistically inhibited the proliferation of human glioblastoma cells together with the novel anticancer compound Withaferin A. The inventors hypothesized that such synergistic effects were due to increased accessibility of Withaferin A to glioblastoma cells through the ability of TTFields to transiently increase tumor cell membrane permeability, as depicted diagrammatically in Figure 1. In this figure, 5-ALA = 5-aminolevulinic acid; Ethidium D = ethidium bromide; FITC = fluorescein isothiocyanate.

[0025] Studies were then carried out to test this hypothesis. Specifically, in human glioblastoma cells engineered to express luciferase (Renilla and Firefly), evidence was found to show that TTFields exposure induced greater bioluminescence, and that this induction was due to increased permeation of substrates (D-luciferin and coelenterazine, respectively) across the plasma membrane. The increase in membrane permeability caused by TTFields exposure was also demonstrated with other membrane penetrating agents such as dextran-FITC and ethidium D.

[0026] Increasing membrane permeability in glioblastoma cells using TTFields has also been shown using 5-aminolevulinic acid (5-ALA). 5-ALA is a hemoglobin precursor that is converted to fluorescent protoporphyrin IX (PpIX) in all mammalian cells. However, many malignant cells, including high-grade gliomas, have elevated hemoglobin biosynthesis, reflected in enhanced accumulation of PpIX (compared to non-cancerous cells) in transformed cells and tissues. This property has prompted many medical investigations to use 5-ALA uptake (and consequently its enzymatic conversion to PpIX) as a fluorescent biomarker of tumor cells. However, at the current level of technology, it may be difficult to identify the exact cellular limits between tumor and non-tumor tissues during surgery. The experiments described herein show that TTFields significantly increase the tumor-to-normal cell ratio for PpIX fluorescence (resulting from 5-ALA exposure and uptake) and thus can be used to more precisely indicate tumor limits in an intraoperative setting.

[0027] Follow-up experiments using scanning electron microscopy (SEM) data demonstrate an increase in the number and size of pores in the glioblastoma cell membrane caused by TT Fields exposure, and that the morphology of the glioblastoma cell membrane is destabilized when TT Fields are applied. Across all modalities studied (bioluminescence, fluorescence, and SEM), the effect of TT Fields on GBM cell membrane permeability was found to be reversible following cessation of TT Fields exposure.

[0028] result Induction of TTFields increases bioluminescence (BLI) in glioblastomas expressing luciferase. U87-MG / eGFP-fLuc cells were seeded on top of Thermanox coverslips, allowed to settle and grow, and then placed under TT fields or no TT fields. In this experiment, the use of TT fields (4 V / cm, 200 kHz, 0.5–24 h) significantly increased the bioluminescence intensity (BLI) of U87-MG / eGFP-fLuc cells compared to non-exposed conditions. This increase in BLI occurred as early as 30 min after the initiation of the TT fields and continued up to 24 h of TT field exposure. When ROI quantification was performed, the time course of BLI intensity for TT field-exposed samples was significantly elevated compared to TT field-unexposed samples (p<0.0001, two-way ANOVA, TT field vs. no TT field). Data representing the temporal quantification of the BLI results from these experiments are summarized in Figure 2A. Without being bound by this theory, it is believed that the increase in bioluminescence was not due to a direct effect of TT Fields on firefly luciferase activity because exposure of purified firefly luciferase to 200 kHz TT Fields led to a >1000-fold decrease in enzyme activity 60 min after initiation of the TT Field.

[0029] FIG. 2B depicts the effect of TT-fields on eGFP fluorescence in U87-MG / eGFP-fLuc cells as observed from a time course of representative images (not shown) for TT-field-exposed vs. non-TT-field-exposed U87-MG / eGFP-fLuc. The presence of TT-fields did not significantly increase eGFP fluorescence (eGFP-FL) over the course of the experiment. When the ratio of BLI to eGFP-FL was compared between TT-field and no TT-field samples, there was a significantly increased ratio with respect to TT-field incubation time for TT-field samples, as depicted in FIG. 2C, FIG. 2D (p<0.0001, two-way ANOVA, TT-field vs. no TT-field). More specifically, Figure 2C depicts the effect of TT-fields on the ratio of fLuc bioluminescence (fLuc-BLI) to eGFP fluorescence (eGFP-FL) for U87-MG / eGFP-fLuc cells as a function of length of TT-field exposure, and Figure 2D depicts the effect of TT-field exposure vs. no exposure on the fLuc-BLI / eGFP-FL ratio as a function of TT-field exposure time (hours). TT-fields significantly reduced the activity of purified firefly luciferase compared to no TT-field (p<0.01, two-way ANOVA, TT-field vs. no TT-field).

[0030] Time-dependent application of TTfields to another patient-derived glioblastoma cell line, GBM2 / GFP-fLuc, also induced a time-dependent increase in bioluminescence in TTfield-exposed GBM2 / GFP-fLuc cells when compared to no TTfield controls (p<0.0001, two-way ANOVA, TTfield vs. no TTfield). This same effect was observed in a murine astrocytoma cell line (KR158B) genetically modified to express Renilla luciferase-red fluorescent protein fusion protein (p<0.0001, two-way ANOVA, TTfield vs. no TTfield). Renilla luciferase activity is not dependent on ATP and magnesium (in contrast to firefly luciferase). Thus, it is believed that the induction of bioluminescence by TTfields was not due to changes in the endogenous pool of ATP.

[0031] Effect of TTFields on the uptake of membrane-bound reagents. To test whether imposition of TT-fields affects cell membrane properties and therefore membrane permeability, we determined the effect of TT-fields on the behavior of fluorescently tagged reagents that bind to cell membranes. First, we measured the effect of TT-fields on the binding of Annexin-V-APC to the membrane of U87-MG / eGFP-fLuc cells. Annexin-V-APC binding is a sign of early apoptosis characterized by membrane ruffling. To investigate the visibility of Annexin-V-APC binding to U87-MG / eGFP-fLuc cells, we used a positive control for apoptosis (addition of 21 μM Withaferin A to U87-MG / eGFP-fLuc cells) and showed that such binding could be visualized through fluorescence microscopy in non-TT-field-exposed samples. However, when TT-fields were applied to U87-MG / eGFP-fLuc cells, Annexin-V-APC binding was not observed at any time point of exposure to TT-fields. Thus, TTFields do not appear to induce any significant degree of apoptosis in U87-MG cells.

[0032] Notably, as depicted in FIG. 3A, ethidium D uptake was significantly increased when U87-MG / eGFP-fLuc cells were exposed to 200 kHz TT fields (p<0.0001, two-way ANOVA, TT fields vs. no TT fields). Ethidium D penetrates the plasma membrane and nuclear membrane to interrupt genomic DNA. Thus, these findings suggest that TT fields can have an effect on the permeability of the plasma membrane of U87-MG / eGFP-fLuc cells.

[0033] Another consequence of the enhancement of membrane permeability by TT fields is the alteration of dextran-FITC binding to the cell membrane. Dextran-FITC is known to bind and interrupt the plasma membrane. When U87-MG cells were exposed to 200 kHz TT fields for 1 hour, there was a significant uptake of dextran-FITC with molecular weights of 4 kDa and 20 kDa compared to no TT field exposure, as shown in Figures 3B and 3C. However, there was no significant difference in uptake for 50 kDa Dextran-FITC, as shown in Figure 3D. More specifically, dextran-FITC binding was examined over a time window of 0.5-24 hours exposure in the presence of TTfield, and a significant increase in uptake of 4 kDa dextran-FITC was found (p<0.0001, two-way ANOVA, TTfield vs. no TTfield) compared to non-TTfield exposed samples, a significant increase in uptake of 20 kDa dextran-FITC under TTfield exposure (p<0.01, TTfield vs. no TTfield), and a lack of significant difference in uptake of 50 kDa dextran-FITC under TTfield exposure (p=0.26, non-significant, TTfield vs. no TTfield). These data suggest that the maximum size of dextran-FITC bound and integrated into the plasma membrane under TTfield exposure in this experiment was approximately 20-50 kDa. For all statistical comparisons described in this paragraph, each data point is representative of n=3 experiments. In Figures 3A-3D, APC = allophycocyanin; Ethidium D = ethidium bromide; and FITC = fluorescein isothiocyanate.

[0034] Effect of TTfields on 5-aminolevulinic (5-ALA) acid uptake: single U87-MG cultures. Experiments were performed to determine the effect of TTfields on 5-ALA uptake in glioblastoma cells (measured by PpIX accumulation and resulting fluorescence). Because it is difficult to distinguish the limits between tumor and normal cells using the present 5-ALA bioassay, measurements of PpIX fluorescence were used to address this issue. Studies were performed to determine whether 5-ALA penetration through the cell membrane and into glioblastoma cells could be increased by TTfields exposure. U87-MG cells were exposed or not to TTfields for 6 to 24 hours, respectively. The results, summarized in Figure 4A, were as follows: TTfields exposure resulted in a significant increase in the uptake of 5-ALA into U87-MG / eGFP-fLuc cells as early as 6 hours of TTfield exposure (p=0.047, Student's t-test, TTfield vs. no TTfield), and this increase was maintained by prolonged TTfield exposure of 24 hours (p=0.011).

[0035] To yield the data depicted in Figure 4A, protoporphyrin IX (PpIX) fluorescence panels were obtained for TT-field non-exposed vs. TT-field exposed U87-MG cells after 6 and 24 hours of exposure. Quantification of those images showed a significant increase in PpIX signal in TT-field exposed cells compared to no TT-field at both the 6 hour (p=0.047) and 24 hour (p=0.01) time points. All monovariant statistical comparisons between no TT-field vs. TT-field samples were performed by Student's t-test for n=3 experiments at each time point.

[0036] Effect of TTFields on 5-aminolevulinic acid uptake: Coculture of U87-MG GBM and PCS-201 fibroblasts. During glioblastoma resection in patients, 5-ALA is used to help neurosurgeons pinpoint the gap between the tumor and the surrounding normal brain tissue. Similarly, to identify differences in 5-ALA uptake between glioblastoma and normal cells, co-cultures were developed in which U87-MG cells were seeded in the center of a bed of PCS-201 fibroblasts and exposed or not to TT-fields. In the co-culture setup, fluorescent and bright-field photomicrographs confirmed the presence of distinct glioblastoma versus fibroblast regions. When the co-cultures were stained with hematoxylin-eosin (H&E), photomicrographs revealed a reduced number of GBM cells infiltrating around the fibroblasts for the TT-field-exposed samples.

[0037] In particular, without TTfields exposure, GBM cells formed numerous pockets of adherent neurospheres as previously reported. Fluorescence images showed an increase in PpIX fluorescence in glioblastoma cells relative to fibroblasts in co-culture platforms exposed to TTfields for 6 h. The results, summarized in Figure 4B, were as follows: PpIX fluorescence accumulated over time, but the rate of increase in fluorescence intensity was significantly enhanced in TTfield-exposed co-cultures compared to non-TTfield-exposed co-cultures (p<0.001, two-way ANOVA, TTfields vs. no TTfields). To yield the data depicted in Figure 4B, a fluorescence panel of 5-ALA uptake (and subsequent PpIX fluorescence, Ex=558 nm, Em=583 nm) for no TTfields and TTfields was obtained. Exposure times were 2, 6 and 24 h. Quantification of the time course of PpIX accumulation (and thus the accumulation of fluorescence flux expressed as photons / s) in glioblastoma-fibroblast co-culture platforms under TT-fields exposed vs. non-exposed conditions (p<0.001). Statistical analysis consisted of a two-way ANOVA of no TT-field vs. TT-field conditions and n=3 experiments at each time point.

[0038] In a separate set of experiments, 24 h of TT field application significantly increased the ratio of PpIX fluorescence intensity in U87-MG glioblastoma cells to surrounding PCS-201 fibroblasts compared with the fluorescence intensity ratio of co-cultured cells under no TT field conditions (p=0.043, two-way ANOVA, TT field vs. no TT field).

[0039] Scanning electron micrographs (SEM) show that TTFields alter the membrane morphology of U87-MG / eGFP-fLuc cells. SEM images of low density (5,000 cells / coverslip) U87-MG / eGFP-fLuc cells that were either not exposed to TTfields or exposed to TTfields for 3 days were obtained at magnifications of 2000×, 20,000×, and 60,000×. Data obtained by inspecting these SEM images are summarized in FIG. 5. There were 100 cells in the ROI of TTfields-exposed cells (53.5±19.1) compared to non-TTfields-exposed cells (23.9±11.0), with 100 cells measuring 51.8 nm in size. 2 (9 pixels at 60,000x magnification 2 There was a significantly increased number of larger pores (equivalent to TTFields) in TTFields-unexposed cells (129.8±31.9 nm) (p=0.0002, univariate Mann-Whitney test). 2 ) compared with TTFields-exposed cells (240.6 ± 91.7 nm 2 ), the average size of pores within the ROI was also significantly larger (p=0.0005 (univariate Mann-Whitney test)). To obtain the data depicted in Figure 5, quantification and comparison of pore number and size was performed between TTfields unexposed and exposed cells within a circular region of interest of 500 nm radius. The minimum pore size cutoff was based on the 3.3 and 5.0 nm Stokes radii of 20 kDa and 50 kDa dextran-FITC, respectively. Coverslips from three experiments were used for each condition, and at least five cells per coverglass were analyzed for pore number and size in a double-blind manner.

[0040] The effect of 24 h exposure to TTFields on the plasma membrane of densely seeded U87-MG cells was also visually observed. For TTField-free samples, the cell surface appeared to be encompassed by densely matted, elongated, flattened membrane extensions that resembled wavy membranes and were continuous with the plasma membrane. In contrast, after 24 h exposure to TTFields, the densely matted, elongated structures were replaced by short, puffy, bubble-like structures.

[0041] For comparison, SEM images of normal human PCS-201 cells were also obtained and analyzed. PCS-201 cells were seeded at low density (5,000 cells per 13 mm coverslip). Cells were grown under standard tissue culture conditions (37°C, 95% O2, 5% CO2). Non-TTFields-exposed cells remained under these conditions for the duration of the study. Other cells were exposed to TTFields for 72 hours. After 72 hours, SEM images were obtained at 2000x, 20,000x, and 60,000x magnification. Within a circular region of interest of 500 nm radius between non-TTFields-exposed and exposed cells, areas of ≥ 51.8 nm2 were obtained. 2 of hole (4 nm radius circle, or 9 pixels in an image at 60,000× magnification) 2 Quantification and comparison of the number and size of pores (equivalent to pore size). The minimum pore size cutoff was based on 3.3 nm and 5.0 nm Stokes radii for 20 kDa and 50 kDa dextran-FITC, respectively. The results, depicted in Figure 6, were as follows: There was no significant difference in the number or size of pores between non-exposed and exposed normal human PCS-201 cells with TTFields (Wilcoxon rank sum analysis). Coverslips from three experiments per condition were used and at least five cells per coverglass were analyzed for pore number and size in a double-blind fashion.

[0042] The effect of 24 h exposure to TTFields on the plasma membrane of PCS-201 cells was also observed visually. Unlike the situation described above for U87-MG cells, TTFields did not appear to alter the membrane morphology of PCS-201 cells.

[0043] The effect of TTFields on membrane permeability is reversible. To investigate the reversibility of the effect of TTFields on cancer cells, U87-MG / eGFP-fLuc cells were subjected to three conditions: (1) no TTFields exposure, standard cell culture conditions (37°C, 95% O2, 5% CO2), (2) 24 hours of TTFields exposure, and (3) 24 hours of TTFields exposure followed by 24 hours of no TTFields exposure. BLI, PpIX fluorescence (5-ALA product) and dextran-FITC (4kDa) fluorescence readouts were obtained. All experimental conditions were performed in triplicate. FIG. 7A summarizes the BLI data: the presence of TT-fields (middle bars) for 24 h significantly increased BLI flux compared to no TT-field exposure (left bars) (p<0.0005, two-way ANOVA, TT-field vs. no TT-field), but this increase was significantly attenuated when cells were reintroduced to TT-field conditions for 24 h (right bars) (two-way ANOVA, p<0.005, TT-field for 24 h vs. TT-field for 24 h followed by no TT-field for 24 h). FIG. 7B shows that a similar pattern of reversible readout occurred with PpIX fluorescence (p<0.0005, two-way ANOVA, TT-field (middle bars) vs. no TT-field (left bars), and p<0.0004, TT-field vs. TT-field followed by no TT-field (right bars)). FIG. 7C shows that a similar pattern of reversible readout occurred with 4 kDa dextran-FITC fluorescence (p<0.05, two-way ANOVA, TT field (middle bar) vs. no TT field (left bar); and p<0.05, TT field vs. TT field followed by no TT field (right bar)). In each set of experiments, eGFP fluorescence did not change significantly. SEM examination revealed that the significant increases by TT field in both pore number (p=0.007, two-way ANOVA, TT field vs. no TT field) and pore size (p=0.0007, two-way ANOVA, TT field vs. no TT field) were also reversible after 24 h of no exposure.where NS = non-significant; BLI = bioluminescence imaging; eGFP = enhanced green fluorescent protein; fLuc = firefly luciferase; 5-ALA = 5-aminolevulinic acid; FITC = fluorescein isothiocyanate; PpIX = protoporphyrin IX; and FL = fluorescence.

[0044] In summary, when an AC field was applied, uptake of the relevant compounds was increased (compared to when no AC field was applied). Each of these figures also shows that uptake was substantially reduced after cessation of the AC field for 24 hours. From this, we can infer that the increase in cell membrane permeability induced by the AC field is not a permanent effect, and that permeability will decrease back to normal after cessation of the AC field.

[0045] FIG. 7D shows the results of an experiment to test how quickly the permeability falls back after the cessation of the AC field. More specifically, 7-aminoactinomycin D (7-AAD) is a fluorescent compound with a strong affinity for DNA. 7-AAD is a relatively large molecule (1270.43 g / mol, i.e., 1.27 kDa) that does not normally cross intact cell membranes easily. FIG. 7D shows the timing characteristics of the permeability to 7-AAD induced by the application of TTFields to U87-MG cells. In this experiment, the cells were treated with a 300 kHz AC field with a field strength of 1.62 V / cm RMS for 24 hours at an ambient temperature of 18° C. 7-AAD was introduced into the samples at five different times: 15 min before the cessation of the AC field; immediately after the cessation of the AC field; and 15, 30 and 60 min after the cessation of the AC field. In each case, cells were incubated with 7-AAD for 30 min after introduction of 7-AAD, followed by flow cytometric analysis of the percentage of cells with increased accumulation of fluorescent 7-AAD for each of the different time points. As can be seen in Figure 7D, a significant increase in 7-AAD accumulation was observed only in samples incubated with 7-AAD while exposed to an AC electric field.

[0046] Additional results for different drugs and different types of cancer cells. The methods described herein are not limited to the glioblastoma setting. On the contrary, they are applicable to other types of cancer cells. More specifically, a substance can be delivered across the cell membrane of a cell by (a) applying an alternating electric field to the cell for a period of time, where the application of the alternating electric field increases the permeability of the cell membrane; and (b) introducing the substance into the vicinity of the cell. The increase in the permeability of the cell membrane allows the substance to pass through the cell membrane. Notably, the methods described herein can be used to deliver large molecules (that would not normally pass through the associated cell membrane) through the cell membrane of different types of cells (i.e., cells other than glioblastoma), including but not limited to other types of cancer cells (e.g., MDA-MB-435 and MCF-7 cells).

[0047] FIG. 8A depicts the results of an experiment performed to determine how an AC electric field affects the permeability of the cell membrane of MDA-MB-435 human melanoma cell line cells. In this experiment, MDA-MB-435 cells were treated with a 150 kHz AC electric field with a field strength of 1.62 V / cm for 24 hours at an ambient temperature of 18° C. and a petri dish temperature of 37° C. (The petri dish temperature in this and other examples is higher than the ambient temperature due to heating caused by the AC electric field). After the first 23.75 hours, 7-AAD was added to the culture and incubated for 15 minutes during which the AC electric field was continued (to complete the 24 hours). After this 15 minutes, application of the AC electric field was terminated and the cells were incubated at room temperature for an additional 15 minutes. The percentage of cells with increased accumulation of fluorescent 7-AAD was determined by flow cytometry analysis. Approximately 66% of the cells showed increased 7-AAD accumulation (bar 2 in FIG. 8A), compared to less than 5% of the cells in the control (bar 1) subjected to the same conditions except that no AC electric field was applied. These results show that the AC electric field induces a highly significant increase in the permeability of the cell membrane.

[0048] In a variation of this experiment, MDA-MB-435 human melanoma cell line cells were treated with a 150 kHz AC electric field with a field strength of 1.62 V / cm for 24 hours at an ambient temperature of 18° C. and a petri dish temperature of 37° C. After this 24 hour period, the AC electric field was turned off for 15 minutes, after which 7-AAD was added. After waiting an additional 15 minutes, the percentage of cells with increased accumulation of fluorescent 7-AAD was determined by flow cytometry. This time, only about 20% of the cells showed increased accumulation of 7-AAD (bar 3 in FIG. 8A). These results indicate that the increase in cell membrane permeability induced by the AC electric field is relatively short-lived, and that permeability decreases rapidly and dramatically after cessation of the AC electric field.

[0049] FIG. 8B shows the results of another experiment carried out to determine how an AC electric field affects the permeability of the cell membrane of MDA-MB-435 human melanoma cell line cells to doxorubicin (543.52 g / mol). In this experiment, wild-type and doxorubicin-resistant variants of MDA-MB-435 cells were treated with an AC electric field of 150 kHz with a field strength of 1.62 V / cm for 23 hours. After this 23 hours, doxorubicin was added at a concentration of 10 μM and incubated for 1 hour while the AC electric field was continued. The intracellular accumulation of doxorubicin was then measured. The intracellular accumulation of doxorubicin was increased in both wild-type cells (compare bar 1 with bar 3) and doxorubicin-resistant cells (compare bar 2 with bar 4).

[0050] FIG. 8C shows the results of a similar experiment using MCF-7 human breast adenocarcinoma cell line cells and mitoxantrone (444.481 g / mol). In this experiment, wild-type and mitoxantrone-resistant variants of MCF-7 cells were treated with an alternating electric field of 150 kHz with a field strength of 1.62 V / cm for 23 hours. After this 23 hours, mitoxantrone was added at a concentration of 2 μM and incubated for 1 hour while the alternating electric field was continued. The intracellular accumulation of mitoxantrone was then measured. The intracellular accumulation of mitoxantrone was increased in both wild-type cells (compare bar 1 with bar 3) and mitoxantrone-resistant cells (compare bar 2 with bar 4).

[0051] The above results in conjunction with Figures 8B and 8C indicate that AC electric fields enhance the intracellular accumulation of chemotherapeutic molecules in both wild-type and drug-resistant cells, and that AC electric fields can advantageously restore the intracellular accumulation of chemotherapeutic chemicals in cancer cells after those cells have developed multi-drug resistance to those chemicals.

[0052] Additional experiments were performed to determine whether synergy exists between TT fields and various drugs for various cancer cell lines, and Figures 9A-9G represent the results of some of these experiments. More specifically, Figure 9A shows how applying TT fields for 3 days improves the sensitivity of U87-MG / GFP-Luc cells to various concentrations of lomustine (compared to a control where TT fields were not applied). Figure 9B shows how applying TT fields for 3 days improves the sensitivity of pcGBM2 / GFPLuc cells to various concentrations of lomustine (compared to a control where TT fields were not applied). Figure 9C shows how applying TT fields for 3 days improves the sensitivity of GBM39 cells to various concentrations of lomustine (compared to a control where TT fields were not applied). Figure 9D shows how applying TT fields for 3 days improves the sensitivity of GBM39 cells to various concentrations of temozolomide (compared to a control where TT fields were not applied). Figure 9E shows how application of a TT field for 3 days improves the sensitivity of GBM39 / Luc cells to various concentrations of irinotecan (compared to a control where no TT field was applied). Figure 9F shows how application of a TT field for 3 days improves the sensitivity of MDA-MB-235 cells to various concentrations of doxorubicin (compared to a control where no TT field was applied). Figure 9G shows how application of a TT field for 4 days improves the sensitivity of U87-MG / eGFP-Luc cells to various concentrations of mannose (compared to a control where no TT field was applied).

[0053] To date, synergy has been found with the combination of TT-Fields and Withaferin A for GBM39 / Luc, U87-MG / GFP-Luc, and pcGBM2 / GFP-Luc; synergy has been found with the combination of TT-Fields and Lomustine for GBM39 / Luc, U87-MG / GFP-Luc, and pcGBM2 / GFP-Luc; synergy has been found with the combination of TT-Fields and Irinotecan for GBM39 / Luc; and synergy has been found with the combination of TT-Fields and Mannose for U87-MG / GFP-Luc. Evidence of synergy has also been found with the combination of TT-Fields and Doxorubicin for MDA-MB-235.

[0054] Consideration Previous studies have focused on the effects of TTFields on the nucleus (e.g., microtubules), septins, mitochondria, and autophagy. However, the experiments described herein are believed to be the first to report the effect of TTFields on cancer cell membrane integrity, and demonstrate increased cell membrane permeability for cancer cells (e.g., multiple human GBM cell lines) in the presence of TTFields using a variety of assessment techniques (e.g., bioluminescence imaging, fluorescence imaging, and scanning electron microscopy).

[0055] Observations revealed increased cell membrane permeability for glioblastoma in the presence of TT Fields across multiple human GBM cell lines. Approaches used to test the hypothesis included bioluminescence imaging, fluorescence imaging, and scanning electron microscopy. Observations also revealed increased cell membrane permeability for other types of cancer cells in the presence of TT Fields. Studies of TT Fields in combination with chemotherapy demonstrated both therapeutic additivity and synergy. For this study, we determined that TT Fields mediate increased accessibility to cancer cells. Several experiments demonstrated reversibility of the effect of TT Fields on the membrane, thus demonstrating a causal relationship between TT Fields and increased membrane permeability. Such observations also suggest that TT Fields may be used to modulate drug accessibility to cancer cells.

[0056] Investigation of the cell permeability hypothesis of TT Fields action was initiated in part due to the observation of increased bioluminescence in luciferase-expressing GBM cells by TT Fields. Without being bound by this theory, it is believed that TT Fields induced an increase in permeability in the plasma membrane of GBM cells. The increase in GBM cell permeability to D-luciferin measured by BLI is believed not to be due to an effect of TT Fields on luciferase itself, but rather due to an increased influx of its substrate D-luciferin into cells engineered to express firefly luciferase. Furthermore, this finding held true for both ATP-dependent (FLuc) and ATP-independent luciferases (RLuc). Thus, despite a preliminary report suggesting increased intracellular ATP in CT26 colorectal cancer cells exposed to TT Fields, the observation of increased glioblastoma cell membrane permeability in the setting of TT Fields exposure suggests an independent phenomenon. In these cells, the luciferase enzyme is controlled by the same promoter as eGFP, and since no increase in fluorescent signal was observed in the same cells, increased expression or activation of luciferase upon TT Fields exposure may not explain the increase in BLI signal. However, exposure to TT Fields can affect cellular metabolism, which may be manifested by changes in ATP levels, alterations in membrane morphology, and shifts in oxygen consumption.

[0057] Some key findings supporting the permeability hypothesis came from the dextran-FITC validation experiments described above in connection with Figures 3B-3D. Cell membrane accessibility to small probes in the context of TT Fields was tested with FITC-labeled dextran, which resulted in increased influx of 4 kDa (Stokes radius 1.4 nm) and 20 kDa (Stokes radius 3.3 nm), but not 50 kDa dextran (Stokes radius 5 nm). This suggests that TT Fields render GBM cells more permeable to entities around 20 kDa, but smaller than 50 kDa in size. For reference, luciferin and coelenterazine substrates are small enough in molecular weight to be accessible through the membrane upon TT Field exposure. D-luciferin (a substrate for firefly luciferase) has a molecular weight of 280.3 g / mol (approximately 280 Da), coelenterazine H (a substrate for Renilla luciferase) has a molecular weight of 407.5 g / mol (approximately 408 Da), and 5-ALA has a molecular weight of 167.6 g / mol (169 Da), consistent with the findings for dextran-FITC.

[0058] As described above in relation to FIG. 5, the SEM findings described herein demonstrate that at low seeding density, 3 days of TT Field exposure resulted in an increase in tumor size by 51.8 nm2 in area compared to the no TT Field condition. 2 This pore size cutoff represents a circle with a radius of 4.1 nm, which is the Stokes radius for FITC-dextran molecules of size 20-40 kDa. Thus, the difference in cell membrane disruption visualized by SEM confirms the indirect observations from the FITC-dextran studies described herein.

[0059] Interestingly, exposure of normal human fibroblasts (PCS-201) to TTFields did not cause a significant increase in the number or size of cell membrane pores, thus suggesting that the effect on permeability may have some specificity for cancer cells. Qualitatively, in U87-MG cells, there was a clear development of puffy bubble-like structures for 24 h exposure to TTFields under high seeding density. The appearance of these structures was consistent with increased outer membrane permeability and induction of apoptosis, and there appeared to be little evidence of an apoptotic phenotype with 24 h of TTFields exposure. Furthermore, high-density PCS-201 cells showed no such changes with TTFields exposure (data not shown), thus again suggesting specificity of the effect of TTFields on cancer cells.

[0060] Although cell cycles were not synchronized for the experiments, given that the doubling time of U87-MG cells is approximately 48 h, and that TT fields exert their maximal antiproliferative effect on dividing cells, this may explain the lack of abundant apoptosis observed after 24 h of TT fields exposure. An alternative interpretation may lie in reports that cell bubble formation can confer resistance to cell lysis. Previous reports of asynchronous glioblastoma cells demonstrated that 72 h of TT fields exposure induced cell death, resulting in a significant proportion of Annexin V-positive cells. Using transmission electron microscopy, these reports described signs of autophagy, including autophagosomes, swollen mitochondria, and dilated endoplasmic reticulum. In contrast, the results herein use SEM to better visualize the effects of TT fields, particularly on the plasma membrane.

[0061] The increase in membrane permeability by TT-fields has considerable clinical implications. A co-culture platform of human GBM cells overlaid on normal human fibroblasts was used to study the effect of TT-fields on GBM cell uptake of 5-aminolevulinic acid (5-ALA). TT-fields exposure resulted in a significant increase in 5-ALA uptake in GBM cells compared to fibroblasts. In June 2017, 5-ALA was approved by the Food and Drug Administration for clinical use in the United States to help neurosurgeons pinpoint the tumor-normal brain margin during glioma resection. Thus, pretreatment of glioma patients with TT-fields prior to 5-ALA administration is beneficial to enhance the delineation of invasive tumor margins during tumor resection.

[0062] With regard to detecting and measuring the effects of TTFields on cancer cells, most cell culture-based studies to date have focused on cell number / viability as the primary readout. This is based on the prevailing understanding that TTFields interfere with mitosis of rapidly dividing tumor cells, which results in cancer cell death. Furthermore, computer modeling studies of TTFields in cell culture are currently driven by cell number as the primary outcome of the model.

[0063] Recurrence of GBM is inevitable, with the median time to first recurrence being roughly 7 months despite standard therapy. In clinical application of TTfields to GBM patients, data suggest that increased compliance and duration of TTfield use correlate with improved survival. TTfield compliance (≥75% vs <75%) was an independent predictor of overall survival in a retrospective analysis of the full EF-14 clinical trial dataset, and duration of TTfield use was also found to affect overall survival. Collectively, these data can serve as clinical correlates of the observed effects in the setting of cell culture-based TTfield experiments. That is, a correlation was observed between the length of TTfield exposure and the duration of its effect on cell membrane permeability after cessation of TTfields. At lengths of TTfield exposure between 0.5 and 3 hours, the duration of increased BLI (compared to no TTfield conditions) lasted approximately 5 minutes. However, at 12-25 hours of TTfield exposure, this difference in BLI between TTfield and no TTfield conditions lasted longer than 20 minutes. Similarly, a reanalysis of the data reported by Ram et al. indicates that the percent increase in overall survival (in patients treated with TTFields plus temozolomide versus temozolomide alone) jumped from 32% after 1 year of TTFields exposure to 551% after 5 years of TTFields exposure, respectively.

[0064] The results described herein, i.e., that AC electric fields increase cell membrane permeability, are distinct from previously reported effects of TTFields and should have a significant impact on current surgical and clinical practice in the treatment of glioblastoma as well as other types of cancer.

[0065] In the above in vitro experiments, the frequency of the AC electric field was 200 kHz. However, in alternative embodiments, the frequency of the AC electric field may be another frequency, for example, about 200 kHz, 50-500 kHz, 25 kHz-1 MHz, 50-190 kHz, 25-190 kHz, or 210-400 kHz.

[0066] In the in vitro experiments described above, the field strength of the AC electric field was 1-4 V / cm RMS, however, in alternative embodiments, different field strengths can be used (e.g., 0.1-10 V / cm).

[0067] In the in vitro experiments described above, the AC electric field was applied for a variety of different durations ranging from 0.5 hours to 72 hours. However, in alternative embodiments, different durations can be used (e.g., 0.5 hours to 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied for 2 hours each day.

[0068] In the in vitro experiments using the Inovitro™ system described herein, the direction of the AC electric field was switched between two orthogonal directions at 1 second intervals, however, in alternative embodiments, the direction of the AC electric field can be switched at a faster rate (e.g., at intervals of 1-1000 milliseconds) or at a slower rate (e.g., at intervals of 1-100 seconds).

[0069] In the in vitro experiments using the Inovitro™ system described herein, the direction of the AC electric field was switched between two orthogonal directions by applying an AC voltage to two pairs of electrodes alternately placed 90° apart from each other in 2D space. However, in alternative embodiments, the direction of the AC electric field can be switched between two non-orthogonal directions by rearranging a pair of electrodes, or between three or more directions (assuming additional pairs of electrodes are provided). For example, the direction of the AC electric field can be switched between three directions, each determined by the arrangement of its own pair of electrodes. If necessary, these three pairs of electrodes can be arranged so that the resulting electric fields are placed 90° apart from each other in 3D space. In other alternative embodiments, the electrodes need not be arranged in pairs. See, for example, the electrode arrangement described in U.S. Pat. No. 7,565,205, which is incorporated herein by reference. In other alternative embodiments, the direction of the electric field is constant.

[0070] In the in vitro experiments using the Inovitro™ system described herein, the Inovitro™ system used conductive electrodes placed on the exterior surface of the petri dish sidewalls, with the ceramic material of the sidewalls acting as a dielectric such that the electric field was capacitively coupled into the culture. However, in alternative embodiments, the electric field could be applied directly to the cells without capacitive coupling (e.g., by modifying the Inovitro™ system configuration so that the conductive electrodes were placed on the interior surface of the sidewalls instead of the exterior surface of the sidewalls).

[0071] The methods described herein can also be applied to in vivo situations for glioblastoma cells and other types of cancer cells by applying an AC electric field to a target area of ​​a living subject's body. Applying an electric field to the target area increases the permeability of the cell membrane in the target area, which allows molecules that are normally blocked or prevented by the cell membrane to pass through the cell membrane. This can be accomplished, for example, by placing electrodes on or under the subject's skin such that application of an AC voltage between a selected subset of those electrodes applies an AC electric field to the target area of ​​the subject's body.

[0072] For example, in a situation where the relevant cells are located in the subject's lungs, one pair of electrodes can be placed on the front and back of the subject's chest, and a second pair of electrodes can be placed on the right and left sides of the subject's chest. In some embodiments, the electrodes are capacitively coupled to the subject's body (e.g., by using an electrode that includes a conductive plate and also has a dielectric layer placed between the conductive plate and the subject's body). However, in alternative embodiments, the dielectric layer can be omitted, in which case the conductive plate would be in direct contact with the subject's body. In another embodiment, the electrodes may be inserted subcutaneously into the patient. The AC voltage generator applies an AC voltage of a selected frequency (e.g., 100-200 kHz) between the right and left electrodes for a first period of time (e.g., 1 second), which induces an alternating electric field, but the most significant component of the electric field system is parallel to the transverse axis of the subject's body. The AC voltage generator then applies an AC voltage of the same frequency (or a different frequency) between the front and back electrodes for a second period of time (e.g., 1 second), which induces an alternating electric field, but the most significant component of the electric field system is parallel to the sagittal axis of the subject's body. This two-step procedure is then repeated throughout the treatment. Optionally, a thermal sensor may be included in the electrode, and the AC voltage generator may be configured to reduce the amplitude of the AC voltage applied to the electrode if the temperature sensed at the electrode becomes too high. In some embodiments, one or more additional pairs of electrodes may be added and included in the procedure. In alternative embodiments, only a single pair of electrodes is used, in which case the direction of the electric field system is not switched. It is noted that any of the parameters of this in vivo embodiment (e.g., frequency, field strength, duration, direction switching speed and placement of electrodes) can be modified, as described above in connection with the in vitro embodiment. However, in an in vivo situation, care must be taken to ensure that the electric field is always safe for the subject.

[0073] A wide variety of applications for increasing cell membrane permeability can be easily envisioned in an in vivo context. In one example, local enhancement of drug uptake by tumor cells can be induced by applying an AC electric field to the relevant body part for a period of time (e.g., 12 or 24 hours) before and during administration of chemotherapy or other anti-neoplastic drugs. In another example, drug uptake by multidrug-resistant tumor cells can be restored by applying an AC electric field to the relevant body part for a period of time (e.g., 12 or 24 hours) before and during administration of chemotherapy or other anti-neoplastic drugs. In another example, development of multidrug-resistant metastases can be prevented by applying an AC electric field to a region prone to metastasis for a period of time (e.g., 12 or 24 hours) before and during administration of the appropriate drug (whether or not the subject has a primary tumor that is treated with an AC electric field).

[0074] FIG. 10A illustrates a first preferred timing relationship between application of an AC electric field and introduction of a substance in the vicinity of cancer cells; or between application of an AC electric field and administration of a substance to a living patient in an in vitro context. Based on the data described above in connection with FIGS. 7A-7D and 8A, and assuming that the substance is introduced or administered at a given time t=0, the AC electric field can begin after a given time and continue for a certain period of time (e.g., 12 hours) while the substance is still available in the vicinity of the cells. In this situation, the permeability begins to increase after the AC electric field begins, and this increase in permeability allows the substance to enter the relevant cells. In the context of chemotherapy, this would correspond to administering a chemotherapeutic agent to the patient, followed by application of an AC electric field for a certain period of time (e.g., 12 hours).

[0075] Alternatively, as depicted in FIG. 10B, the AC electric field can begin before a given time (e.g., 1 hour before t=0) and continue for a period of time while the substance is still available in the vicinity of the cells (e.g., up to 12 hours after t=0). In this situation, the permeability of the relevant cells begins to increase before the substance reaches the vicinity of the cells (or before the substance is administered to a living patient). This allows the substance to cross the cell membrane immediately after it reaches the vicinity of the cells. In the context of chemotherapy, this would correspond to starting the application of the AC electric field, followed by administering a chemotherapeutic agent while the AC electric field is still applied, followed by continuously applying the AC electric field for an additional period of time (e.g., up to 12 hours after the time the chemotherapeutic agent was administered).

[0076] It should be noted that the time periods discussed above in connection with Figures 10A and 10B can be uninterrupted or preferably include short interruptions. For example, assuming a time period is 12 hours, it may be filled by a single uninterrupted block of 12 hours. Alternatively, a 12 hour period may be filled by applying an AC electric field for 6 hours, followed by a 1 hour interruption, followed by applying an AC electric field for an additional 6 hours (while the substance is still available near the cells). It is also noted in connection with Figures 10A and 10B that when a substance is administered to a living patient, administration of the substance can be performed using any of a variety of approaches, including, but not limited to, intravenous, oral, subcutaneous, intrathecal, intraventricular, and intraperitoneal.

[0077] The optimal frequency, field strength and switching characteristics can be determined empirically for each combination of a given type of host cell and a given type of substance to be delivered through the cell membrane. In some preferred embodiments, the frequency is less than 190 kHz (e.g., 50-190 kHz or 25-190 kHz). In other preferred embodiments, the frequency is 210-400 kHz.

[0078] One existing approach for treating tumors (e.g., glioblastoma) is by applying an alternating electric field to the tumor at a frequency of 50-500 kHz, preferably 100-300 kHz. For glioblastoma, 200 kHz is the most preferred frequency. Alternating electric fields of these frequencies are called TT fields and are described in U.S. Patent Nos. 6,868,289 and 7,565,205, each of which is fully incorporated herein by reference. Briefly, those two applications describe destroying dividing cells during mitosis. The effectiveness of TT fields is improved when the direction of the electric field is switched periodically, when the field strength in at least a portion of the tumor is at least 1 V / cm, and when the field is applied for extended periods of time (e.g., weeks or months) with as few interruptions as possible.

[0079] Situations may arise where it is desirable to treat tumors with TT Fields and also deliver substances across the cell membrane of tumor cells (e.g., to help a therapeutically effective amount of a chemotherapy drug cross the cell membrane to provide an additional line of attack to the tumor). In some situations, it may be possible to use a single frequency of an alternating electric field to treat the tumor and increase the permeability of the cell membrane. In other situations, it may be desirable to use alternating electric fields at different frequencies: a first frequency selected to provide improved results for increasing the permeability of the cell membrane, and a second frequency selected to provide improved results for the anti-tumor action of the TT Fields.

[0080] 11A and 11B depict the results of two in vitro experiments with U-87 MG glioblastoma cells. More specifically, FIG. 11A depicts the results of a first experiment to determine the frequency that provides the highest level of cytotoxicity to U-87 MG cells; FIG. 11B depicts the results of a second experiment to determine the frequency that provides the greatest increase in cell membrane permeability of U-87 MG cells.

[0081] In the first experiment, U-87 MG cells were subjected to an AC electric field with a field strength of 1.62 V / cm RMS at different frequencies for 72 hours at an ambient temperature of 18° C. After this 72 hour period, the number of cells present in the samples for each of the different frequencies was measured by flow cytometry. As can be seen in FIG. 11A, the lowest cell number (which indicates the highest level of cytotoxicity) was observed in the samples subjected to an AC electric field of 200 kHz.

[0082] In a second experiment, permeability to 7-AAD, a fluorescent chemical with a molecular weight of 1270.43 g / mol that does not normally pass easily through intact cell membranes, was measured. In this experiment, cells were treated with alternating electric fields of different frequencies with a field strength of 1.62 V / cm RMS for a total of 24 hours at an ambient temperature of 18° C. and a petri dish temperature of 37° C. After the first 23.75 hours, 7-AAD was added to the culture and incubated for 15 minutes during which the alternating electric field was continued (to complete the 24 hours). After this 15 minutes, application of the alternating electric field was terminated and the cells were incubated at room temperature for an additional 15 minutes, followed by flow cytometry analysis of the percentage of cells with increased accumulation of fluorescent 7-AAD for each of the different frequencies. As can be seen in FIG. 11B, the highest percentage of cells with increased accumulation of 7-AAD (which indicates the highest level of permeability) was observed in samples subjected to an alternating electric field of 300 kHz.

[0083] 12A and 12B show the results of two in vitro experiments similar to those described above in connection with FIG. 11A / FIG. 11B, except that MES-SA uterine sarcoma cells were used. More specifically, FIG. 12A shows the results of an experiment to determine the frequency that provides the highest level of cytotoxicity to MES-SA cells. The lowest number of MES-SA cells (which indicates the highest level of cytotoxicity) was observed in samples subjected to an AC electric field of 100 kHz. FIG. 12B shows the results of an experiment to determine the frequency that provides the greatest increase in cell membrane permeability of MES-SA cells. The highest percentage of MES-SA cells with increased accumulation of 7-AAD (which indicates the highest level of permeability) was observed in samples subjected to an AC electric field of 150 kHz.

[0084] FIG. 12C shows the results of another experiment performed to determine how a 150 kHz AC electric field affects the permeability of the cell membrane of MES-SA cells to doxorubicin (543.52 g / mol). In this experiment, MES-SA cells were treated with a 150 kHz AC electric field with a field strength of 1.62 V / cm RMS for 24 hours. After the first 23 hours, doxorubicin was added at a concentration of 10 μM and incubated for 1 hour while the AC electric field was continued (to complete the 24 hour period). The intracellular accumulation of doxorubicin was then measured. The intracellular accumulation of doxorubicin was increased by more than 2× in samples treated with a 150 kHz AC electric field.

[0085] Figures 13A and 13B represent the results of two in vitro experiments similar to those described above in connection with Figures 11A / 11B, except that MCF-7 breast adenocarcinoma cells were used. More specifically, Figure 13A represents the results of an experiment to determine the frequency that provides the highest level of cytotoxicity to MCF-7 cells. The lowest number of MCF-7 cells, which indicates the highest level of cytotoxicity, was observed in samples subjected to an AC electric field of 200 kHz. Figure 13B represents the results of an experiment to determine the frequency that provides the greatest increase in cell membrane permeability of MCF-7 cells. The highest percentage of MCF-7 cells with increased accumulation of 7-AAD, which indicates the highest level of permeability, was observed in samples subjected to an AC electric field of 150 kHz.

[0086] The experiments described above in connection with Figures 11-13 reveal that the optimal frequency for inducing cell permeability is different from the optimal frequency for inducing cytotoxicity. More specifically, for glioblastoma, the optimal first frequency (for inducing cell permeability) is 250kHz-350kHz; the optimal second frequency (for inducing cytotoxicity) is 150kHz-250kHz. For uterine sarcoma, the optimal first frequency (for inducing cell permeability) is 125kHz-175kHz; the optimal second frequency (for inducing cytotoxicity) is 75kHz-125kHz. For breast adenocarcinoma, the optimal first frequency (for inducing cell permeability) is 75kHz-175kHz; the optimal second frequency (for inducing cytotoxicity) is 100kHz-300kHz. Sets of frequency ranges for other types of cancer can be experimentally determined.

[0087] When different frequencies are used to induce cell permeability and to induce cytotoxicity, the cytotoxic frequency is preferably applied for the maximum time that the patient can tolerate without inconvenience. Preferably, the cytotoxic frequency is applied for at least one week. More preferably, the cytotoxic frequency is applied for months. If necessary, the time during which the cytotoxic frequency is applied can be divided into multiple discontinuous times separated by interruptions, where the multiple discontinuous times amount to at least one week in total. In contrast, the frequency for inducing permeability is preferably applied such that permeability is high when the relevant material is located near the target cells (e.g., as described above in connection with Figures 10A-10B). The application of these two different frequencies can be achieved using a single AC voltage generator that is controllable to output a first frequency for inducing cell permeability at certain times and a second frequency for inducing cytotoxicity at other times. The same set of transducer arrays (i.e., electrodes) can be used to apply the AC electric field at these two frequencies (depending on which frequency is applied by the AC voltage generator).

[0088] FIG. 15 is a block diagram of an apparatus for generating a first frequency for inducing cell permeability and a second frequency for inducing cytotoxicity. The apparatus includes an AC voltage generator 44, similar to a conventional Optune® electric field generator unit, but with the ability to operate at two different frequencies, each of which is between 50 and 500 kHz. This ability can be implemented, for example, by using a relay to switch a first set of components or a second set of components to a conventional circuit that generates an AC voltage, and adjusting the operating frequency of the oscillator. The AC voltage generator 44 is configured to output the first frequency or the second frequency depending on the state of a control input. When the control input is in the first state, the AC voltage generator 44 outputs the first frequency, and when the control input is in the second state, the AC voltage generator 44 outputs the second frequency. The controller 42 is programmed to place the control input in the second state such that the AC voltage generator 44 outputs the second frequency. The controller 42 is further programmed to receive a request to switch to the first frequency. 15, the request comes through a user interface 40, which may be implemented using any of a variety of conventional approaches, including but not limited to push buttons, touch screen, etc. In alternative embodiments, the request may come through RF (e.g., Bluetooth, WiFi, etc.) from a tablet, smartphone, etc.

[0089] Upon receiving the request, the controller 42 places the control input in a first state such that the AC voltage generator 44 outputs the first frequency for a period of time (e.g., at least 1 hour, at least 12 hours, or at least 24 hours). After this period of time has elapsed, the controller 42 places the control input in a second state such that the AC voltage generator 44 reverts to outputting the second frequency.

[0090] Optionally, depending on the state of the control input, the AC voltage generator 44 can be configured to output one or more additional frequencies (e.g., a third frequency, a fourth frequency, etc.). Preferably, each of these additional frequencies is selected to induce cytotoxicity. In these embodiments, the controller 42 is programmed to cause the control input to periodically cycle through a state that causes the AC voltage generator 44 to output the second frequency and the one or more additional frequencies before a request arrives. The controller 42 is further programmed to receive a request to switch to the first frequency. Upon receiving the request, the controller 42 places the control input in a first state such that the AC voltage generator 44 outputs the first frequency for a period of time (e.g., at least 1 hour, at least 12 hours, or at least 24 hours). After this period of time has elapsed, the controller 42 reverts to causing the control input to periodically cycle through a state that causes the AC voltage generator 44 to output the second frequency and the one or more additional frequencies.

[0091] The system depicted in FIG. 15 is particularly beneficial when an individual has a tumor being treated with a combination therapy including TTFields and chemotherapy. In this situation, the system operates at the second frequency most of the time to provide maximum cytotoxic effect. However, when the individual visits a chemotherapy clinic for administration of chemotherapy, the health care provider (or user) activates the user interface 40 to switch the system to the first frequency, which promotes penetration. In this situation, activation of the user interface may be performed, for example, one hour before the expected start of chemotherapy or a short time after the actual start of chemotherapy.

[0092] Alternatively, upon receiving a request (e.g., from user interface 40), controller 42 can control the control input such that AC voltage generator 44 outputs the first frequency for a period of time (e.g., 1 hour), and then alternates between the second frequency and the first frequency (e.g., switching every hour). Eventually (e.g., when the relevant substance has been depleted from the patient's bloodstream), controller 42 controls the control input such that AC voltage generator 44 reverts to outputting the second frequency.

[0093] A set of electrodes (not shown), similar to conventional electrodes used with Optune®, are connected to the output of AC voltage generator 44.

[0094] Figure 14 shows the results of yet another experiment to determine the frequency that provides the greatest increase in cell membrane permeability of GBM39 / Luc cells. In this experiment, cells were treated with AC electric fields of different frequencies for two days in the presence of 4 kDa dextran-FITC (which does not normally readily pass through intact cell membranes). As can be seen in Figure 14, the highest levels of dextran-FITC fluorescence (which indicates the highest level of permeability) were observed in samples subjected to an AC electric field of 100 kHz.

[0095] The experimental data discussed in connection with Figures 11-14 contain information that is useful for inducing cell permeability to the highest degree possible (to allow more of the relevant material to pass through the cell membrane), regardless of any cytotoxicity that may be occurring as a secondary effect. In these situations, the AC electric field is preferably applied at only a single frequency selected to induce the highest level of cell permeability. In some situations (e.g., GBM39 / Luc, uterine sarcoma, and breast adenocarcinoma), this frequency will be between 50 and 190 kHz; in other situations (e.g., U-87 MG glioblastoma), this frequency will be between 210 and 400 kHz.

[0096] For substances that can normally cross cell membranes to a significant extent, the techniques described herein for increasing cell membrane permeability can be used to increase the amount of substances that enter the cell. This can improve the therapeutic outcome provided by those substances. Examples of substances in this class discussed above include ethidium bromide (size=394 Da), doxorubicin (size=544 Da), mitoxantrone (size=445 Da), etc.

[0097] Of note, the techniques described herein can also be used to allow substances that are normally unable to cross cell membranes to any significant extent to enter cells. Examples of this class of agents discussed above include: (a) compounds that are at least 1.2 kDa (e.g., 7-AAD, which is 1.27 kDa in size); (b) compounds that are at least 4 kDa (e.g., 4 kDa dextran-FITC); and (c) compounds that are at least 20 kDa (e.g., 20 kDa dextran-FITC); (d) genetic material, including but not limited to supercoiled plasmid DNA, siRNA and shRNA constructs; (e) genome editing systems, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and clustered regularly interspersed short palindromic repeats (CRISPR / Cas9); and (f) any form of antibody, including but not limited to IgG, Fab, Fab', F(ab')2, scFv, bi-scFv, sdAb. Such antibodies may be unconjugated or conjugated to cytotoxic agents, toxins, fluorophores, quantum dots and enzymes, (g) charged molecules, and (h) small molecules, therapeutic entities, peptides and proteins that generally do not permeate cell membranes or are destroyed during endocytosis. Providing these substances with the ability to cross cell membranes means that compounds that may have previously been rejected as ineffective in a compound screening process (due to their inability to cross the cell membrane) may suddenly become usable for therapeutic purposes when combined with an alternating electric field that enhances cell permeability.

[0098] The methods described herein may also be beneficial beyond the context of cancer cells. More specifically, the methods described herein may be useful for delivering large molecules (that would not normally pass through the relevant cell membrane) through the cell membrane of certain other non-cancerous cells (e.g., kidney cells, lung cells, liver cells, heart cells, brain cells, muscle cells, bone marrow cells, etc.). The delivery of such drugs can be enhanced by applying an alternating electric field to the relevant body part for a period of time (e.g., 24 hours) before and during the administration of the drugs. Such drug candidates include, but are not limited to, anti-epileptic drugs and psychotropic drugs (e.g., olanzapine, 9-OH risperidone and other variants of risperidone).

[0099] In yet another example, it may be possible to achieve localized enhancement of drug uptake in bacteria by applying an AC electric field to the relevant body part for a period of time (e.g., 24 hours) before and during administration of a suitable antibiotic. In situations where a particular bacterium has become drug-resistant or multi-drug resistant (e.g., based on a mechanism of action involving the cell membrane), application of an AC electric field can increase the permeability of the bacterial cell membrane to the point where resistance can be overcome. A similar approach can be used to enhance drug uptake to combat meningitis, pneumonia, infective endocarditis, etc. It is noted that in an in vivo situation, an AC electric field can be applied to a target area that is free of tumors (e.g., lungs). Alternatively, an AC electric field can be applied to a target area that contains a tumor (e.g., brain containing glioblastoma).

[0100] Although the present invention has been disclosed with reference to certain specific embodiments, numerous modifications, alterations and variations to the described embodiments are possible without departing from the sphere and scope of the invention as defined in the appended claims. Accordingly, the present invention is not limited to the described embodiments, but has its full scope as defined by the language of the following claims and equivalents thereof.

[0101] Materials and Methods Cell Culture Studies: Two patient-derived GBM lines (GBM2, GBM39), a commercially available human GBM cell line (U87-MG from ATCC, Manassas, VA, USA), and a mouse astrocytoma cell line (KR158B; a gift from Dr. Duane Mitchell, Department of Neurosurgery, University of Florida School of Medicine) were used. Human U87-MG, human PCS-201, and mouse KR158B glioblastoma cell lines were grown in DMEM (Invitrogen / Life Technologies, Carlsbad, CA, USA) / 10% FBS / and 1× antibiotic-antimycotic (Invitrogen / Life Technologies, Carlsbad, CA). GBM2 and GBM39 were grown in defined serum-free medium, the composition of which has been previously described.

[0102] Seeding of cells onto coverslips for TT-field experiments: Briefly, cultured cells were trypsinized by standard protocols, and 10,000-50,000 single cells were suspended in 200 or 75 μL of DMEM / 10% FBS / 1× antibiotic-antimycotic and then seeded onto the center of 22 mm or 12 mm diameter glass Thermanox™ coverslips (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Cells were incubated overnight in a humidified 95% air / 5% CO2 incubator set at 37°C. Once the cells had attached to the coverslips, 2 mL or 1 mL of DMEM / 10% FBS / 1× antibiotic-antimycotic was added to each well of a 6-well or 12-well plate, respectively. Unless otherwise noted in the Results section, cells were grown on coverslips for 2-3 days (to ensure cells were in the proliferation phase) before being transferred to ceramic dishes in the inovito™ in vitro TT-Fields device (Novocure, Haifa, Israel). Growth conditions (i.e., the time cells were grown under TT-Fields-exposed vs. non-exposed conditions) are specified in the Results section or corresponding figure legends.

[0103] In vitro tumor treatment electric field setup: Coverslips were transferred to a ceramic dish of the inovito™ system, which in turn was mounted on an inovito™ baseplate (Novocure, Haifa, Israel). A tumor treatment electric field of 200 kHz (1-4 V / cm) was applied through the inovito™ generator. The incubator environmental temperature was 20-27°C, and the target temperature in the ceramic dish after application of TT-fields was 37°C. The time of TT-fields exposure lasted approximately 0.5-72 hours, after which the coverslips were removed and processed for the appropriate bioassay (see below). For reversibility experiments, TT-fields-exposed coverslips were transferred to a regular incubator without TT-fields exposure for 24 hours (off-TT-field period to investigate the reversibility of TT-fields effects on cell membrane permeability) before processing for the appropriate bioassay. To account for evaporation, the medium was manually changed every 24 hours throughout the experiment. Corresponding control experiments (without TT-fields) were performed by placing equivalent coverglasses in 6- or 12-well plates in a conventional humidified tissue culture incubator (37°C, 95% air / 5% CO2) and growing cells in parallel to the TT-field-exposed coverglasses. Unless indicated, all experiments were performed with at least triplicate samples per condition and time point.

[0104] Hemocytometer cell count analysis: Preparation of cells for counting was accomplished through established protocols and visualized with a Zeiss Primo Vert benchtop microscope (Dublin, CA, USA). Unless otherwise stated, cell counts were performed by hemocytometer on trypsinized single cell suspensions and the average of four cell number measurements was calculated and rounded to the nearest integer.

[0105] Bioluminescence imaging: For all bioluminescence studies, we used genetically modified GBM2, GBM39, and U87-MG, whereby glioblastoma cells were transfected with lentiviral vectors expressing firefly luciferase (fLuc for GBM39) or fusion proteins of GFP and firefly luciferase (GFP / fLuc for GBM2 and eGFP-fLuc for U87-MG), or Renilla luciferase-red fluorescent protein fusion (RLuc-RL8 for KR158B). Viral supernatants were used to transduce cells, and luciferase expression was confirmed by measuring cellular luciferase activity (IVIS Spectrum; Perkin Elmer, Waltman, MA) in the presence of D-luciferin (final concentration of 0.3 mg / mL) for fLuc and coelenterazine (1 mg / mL) for rLuc.

[0106] Scanning Electron Microscopy (SEM): 5,000 (low seeding condition) to 50,000 (high seeding condition) U87-MG / eGFP-fLuc cells or PCS-201 fibroblasts were plated on 13 mm coverslips and then prepared for TT-field experiments. Cells were grown under standard tissue culture incubator conditions (37°C, 95% O2, 5% CO2). At the end of the TT-field exposure and TT-field non-exposure experiments (1 day for high seeding condition and 3 days for low seeding condition), coverslips were processed for SEM. All ROI analyses were performed in a blinded manner, where neither the individuals responsible for SEM image acquisition nor those performing data analysis knew the experimental conditions for the samples. A third party had the identity of the samples.

[0107] Chemical Reagents: Unless otherwise stated, all chemicals were purchased from Selleckchem (Houston, TX, USA), Thermo-Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA). Purified firefly luciferin or firefly luciferase (SRE0045-2MG) and Ethidium D Apoptosis Kit (11835246001) were purchased from Sigma Aldrich (St. Louis, MO). Dextran-FITC with molecular weights of 4, 20 and 50 kDa (FD4, FD20 and FD50) were also purchased from Sigma Aldrich. 5-Aminolevulinic acid (5-ALA, AAA16942ME) and Annexin V-APC kit (50712549) were purchased from Thermo-Fisher Scientific (Waltham, MA).

[0108] Statistical analysis: PRISM 7.0 software (Graph Pad Software, La Jolla, CA, USA) was used to determine whether data were normally distributed. Normally distributed data were analyzed with two-way Student's t-test or analysis of variance (ANOVA) comparison of means, and non-normally distributed data were analyzed with non-parametric analysis (e.g., Mann-Whitney U test comparison of medians). The level of statistical significance was set at alpha = 0.05. Bonferroni or Dunnett's post-hoc correction was used to adjust alpha for multiple comparisons. All data are presented as range, mean ± standard deviation, median (interquartile range) or percentage. In all figures, the level of statistical significance is represented by the following: *p<0.05, **p<0.01, and ***p<0.001. [Explanation of symbols]

[0109] 40 User Interface 42 Controller 44 AC Voltage Generator

Claims

1. A composition for use in a method for treating cancer, comprising a substance for delivery across a cell membrane of a cancer cell in the body of a subject, The method further comprising: subjecting the cells to an alternating electric field at a frequency of 50-500 kHz for a period of time, the application of the alternating electric field increasing the permeability of the cell membrane; and introducing a substance into the vicinity of said cells, increasing the permeability of the cell membrane thereby allowing the substance to pass through the cell membrane; A composition comprising:

2. The composition described in claim 1, wherein the frequency is 100 kHz to 300 kHz.

3. The composition of claim 1, wherein the frequency is 200 kHz.

4. The composition of claim 1, wherein the alternating electric field has a field strength of at least 1 V / cm RMS.

5. The composition of claim 1, wherein the alternating electric field has a field strength of 1 to 4 V / cm RMS.

6. The composition described in claim 1, wherein the cancer cells are glioblastoma cells.

7. The composition described in claim 1, wherein the cancer cells are uterine sarcoma cells.

8. The composition of claim 1, wherein the cancer cells are breast adenocarcinoma cells.

9. The composition of claim 1, wherein the substance comprises a chemotherapeutic agent.

10. The composition of claim 9, wherein the chemotherapeutic agent is temozolomide or irinotecan.

11. 2. The composition of claim 1, wherein the time period is at least 12 hours.

12. 2. The composition of claim 1, wherein the time period is from 12 to 72 hours.

13. The composition of claim 1, wherein the time period is uninterrupted.

14. The composition of claim 1, wherein the time periods are interrupted by short interruptions.

15. The composition of claim 1, wherein the substance has a molecular weight of at least 1.2 kDa.

16. The composition of claim 1, wherein the substance has a molecular weight of at least 4 kDa.

17. An apparatus for controlling the permeability of a cell membrane to facilitate delivery of a substance when administered across the cell membrane of a cancer cell, comprising: The apparatus includes an AC voltage generator (44) configured to apply an alternating electric field at a frequency of 50-500 kHz within a subject's body for a period of time.

18. A kit comprising the device of claim 17 and a substance as defined in claim 17.

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