Determining frequencies for TTFields therapy based on the electrical properties of targeted cancer cells

Abstract

To provide a method of testing the efficacy of a cancer treatment in a subject having cancerous tissue.SOLUTION: The present invention provides a method of testing the efficacy of a cancer treatment in a subject having cancerous tissue, the method comprising: determining an electrical property of at least one cancer cell removed from a sample of cancerous tissue obtained from the subject; and if the determined electrical property matches a known electrical property possessed by a reference cancer cell, testing the efficacy by comparison with a criterion that a frequency of an alternating electric field effective to treat a reference cancer cell is effective as a frequency of an alternating electric field applied to the subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 810,823, filed February 26, 2019, which is incorporated by reference herein in its entirety. [Background technology]

[0002] Tumor Treating Electric Fields (TT Fields) are an effective anti-neoplastic treatment modality delivered by application of low intensity (e.g., 1-5 V / cm), intermediate frequency (e.g., 100-300 kHz) alternating electric fields. TT Fields therapy has received FDA approval for the treatment of glioblastoma multiforme brain tumors and appears to be highly promising for a number of other tumor types. TT Fields therapy is delivered using a wearable, handheld device (Optune®). The delivery system includes four adhesive, non-invasive, insulating "transducer arrays," an electric field generator, multiple rechargeable batteries, and a carrying bag. The transducer arrays are affixed to the skin near the tumor and connected to the electric field generator.

[0003] In a preclinical setting, TT fields can be applied in vitro using the Inovitro™ system with vertical electrode pairs insulated with high dielectric constant ceramic. The Inovitro™ (TT field lab bench system) consists of a TT field generator and a base plate containing eight ceramic dishes per plate. Summary of the Invention [Means for solving the problem]

[0004] One aspect of the present invention is directed to a first method of treating cancer in a subject having cancerous tissue, the first method including obtaining from the subject a sample of cancerous tissue having at least one cancer cell, determining an electrical property of the at least one cancer cell, determining a frequency of an alternating electric field to apply to the subject to treat the cancer based on the determined electrical property, and treating the cancer by applying an alternating electric field to the subject at the determined frequency of the alternating electric field.

[0005] In some examples of the first method, the step of determining the electrical properties includes measuring the dielectrophoretic force of at least one cancer cell at each of a plurality of frequencies less than 35 kHz. In some examples of the first method, the step of determining the frequency includes selecting a frequency that is effective in treating a reference cancer cell having known electrical properties that match the determined electrical properties. In some examples of the first method, the step of determining the electrical properties includes determining a cell membrane capacitance.

[0006] Another aspect of the invention is directed to a second method of treating cancer in a subject having cancerous tissue, the second method comprising obtaining from the subject a sample of cancerous tissue having at least one cancer cell, measuring at least one physical parameter of the at least one cancer cell that can determine an electrical characteristic of the at least one cancer cell, determining a frequency of an alternating electric field to apply to the subject to treat the cancer based on the measured at least one physical parameter, and treating the cancer by applying an alternating electric field to the subject at the determined frequency of the alternating electric field.

[0007] In some examples of the second method, the cell membrane capacitance of the at least one cancer cell may be determined from the at least one physical parameter.

[0008] Another aspect of the invention is directed to a third method of treating cancer in a subject having cancerous tissue, the third method including obtaining from the subject a sample of cancerous tissue having at least one cancer cell, determining an electrical characteristic of the at least one cancer cell, predicting whether application of an alternating electric field to the subject will be effective to treat the cancer based on the determined electrical characteristic, and if the prediction indicates that application of an alternating electric field to the subject will be effective to treat the cancer, treating the cancer by applying an alternating electric field to the subject.

[0009] In some examples of the third method, the step of determining the electrical properties comprises measuring the dielectrophoretic force of the at least one cancer cell at each of a plurality of frequencies less than 35 kHz. In some examples of the third method, the step of predicting is based on whether a reference cancer cell having an electrical property matching the determined electrical property is susceptible to treatment using an alternating electric field. In some examples of the third method, the step of determining the electrical properties comprises determining a cell membrane capacitance.

[0010] Another aspect of the present invention is directed to a fourth method of treating cancer in a subject having cancerous tissue, the fourth method comprising the steps of obtaining from the subject a sample of cancerous tissue having at least one cancer cell, measuring at least one physical parameter of the at least one cancer cell, which can determine an electrical characteristic of the at least one cancer cell, predicting whether application of an alternating electric field to the subject will be effective to treat the cancer based on the measured at least one physical parameter, and treating the cancer by application of an alternating electric field to the subject if the prediction indicates that application of an alternating electric field to the subject will be effective to treat the cancer.

[0011] In some examples of the fourth method, the cell membrane capacitance of the at least one cancer cell can be determined from the at least one physical parameter.

[0012] Another aspect of the invention is directed to a fifth method of selecting a frequency of an alternating electric field to be used to treat cancer in a subject having cancerous tissue, the fifth method including determining an electrical property of at least one cancer cell isolated from a sample of cancerous tissue obtained from the subject, and determining a frequency of an alternating electric field to apply to the subject to treat the cancer based on the determined electrical property.

[0013] In some examples of the fifth method, determining the electrical properties includes measuring a dielectrophoretic force of at least one cancer cell at each of a plurality of frequencies less than 35 kHz. In some examples of the fifth method, determining the frequency includes selecting a frequency effective for treating a reference cancer cell having known electrical properties matching the determined electrical properties. In some examples of the fifth method, determining the electrical properties includes determining a cell membrane capacitance.

[0014] Another aspect of the invention is directed to a sixth method of selecting a frequency of an alternating electric field to be used to treat cancer in a subject having cancerous tissue, the sixth method including measuring at least one physical parameter of at least one cancer cell isolated from a sample of cancerous tissue obtained from the subject, the physical parameter capable of determining an electrical characteristic of the at least one cancer cell, and determining a frequency of an alternating electric field to apply to the subject to treat the cancer based on the at least one measured physical parameter.

[0015] In some examples of the sixth method, the cell membrane capacitance of the at least one cancer cell can be determined from the at least one physical parameter. [Brief description of the drawings]

[0016] [Figure 1] FIG. 13 shows the change in dielectrophoretic force between two groups of cell lines with optimal TT field frequencies of 150 kHz and 200 kHz, respectively. [Diagram 2]FIG. 1 illustrates a process for selecting frequencies for TT Field therapy based on dielectrophoretic force measurements. [Diagram 3] FIG. 13 illustrates a process for selecting a frequency for TT Field therapy based on optical measurements when it is possible to determine electrical properties (e.g., dielectrophoretic force or cell membrane capacitance) from the optical measurements. [Figure 4] FIG. 1 shows the change in dielectrophoretic force between two groups of cell lines, one sensitive to TT-Fields and one not sensitive to TT-Fields. [Diagram 5] FIG. 1 illustrates a process for determining whether to treat a particular subject with TT Fields based on dielectrophoretic force measurements. [Figure 6] FIG. 13 illustrates a process for determining whether to treat a particular subject with TT Fields based on optical measurements, where it is possible to determine electrical properties (e.g., dielectrophoretic force or cell membrane capacitance) from the optical measurements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Various embodiments are described in detail below with reference to the accompanying drawings, in which like reference numbers refer to like elements, and in which:

[0018] In the past, when it was decided to use TT fields to treat a particular subject, the frequency of the TT fields applied to the subject was based on the particular type of tumor being treated.For example, in the treatment of GBM, 200kHz is the recommended frequency of the TT fields, and in the treatment of gastric cancer, 150kHz is the recommended frequency of the TT fields.However, using only one frequency for all subjects will not provide the best results for each and every one of these subjects.More specifically, while 200kHz may be the best frequency for most subjects with GBM, some individuals with GBM may respond better to different frequencies (e.g., 175kHz or 225kHz).To date, there is no predictive marker that determines the optimal frequency to be used for any particular individual subject.

[0019] Some of the embodiments described herein may improve outcomes for many subjects by customizing the frequency of the TT Fields applied for each individual subject. The determination of the frequency to use for any particular individual subject is based on at least one electrical property of the cancer cells excised from the individual subject. In another embodiment, the determination of the frequency to use for any particular individual subject may be based on a physical parameter capable of determining the electrical property of the excised cancer cells. The ability to predict in advance the best frequency of the TT Fields to apply for each individual subject may advantageously improve the efficacy of TT Fields therapy.

[0020] Furthermore, previously, there have been no predictive markers that determine whether TT Fields will be effective for any particular individual. In some of the embodiments described herein, it is possible to predict whether TT Fields will be effective for a particular individual. The prediction is based on at least one electrical property of cancer cells excised from the individual. In another embodiment, the prediction may be based on a physical parameter that can determine the electrical property of the excised cancer cells. The ability to predict in advance the effectiveness of TT Fields treatment for any particular subject can advantageously improve outcomes (e.g., by selecting the most effective treatment for each individual subject).

[0021] Examples of electrical properties that can be used in these predictions include, but are not limited to, dielectrophoretic forces, cell membrane capacitance, cell membrane resistance, cytoplasmic conductivity, and other measures such as the permittivity, conductivity, capacitance of various cellular structures. The electrical properties used to make the predictions may be measured directly. Alternatively, the electrical properties may be measured indirectly (e.g., by measuring other physical properties, which may be either different electrical properties or non-electrical properties such as light intensity, and determining the electrical properties from the other physical properties). In situations where the electrical properties can be determined from other physical properties, it may be possible to map directly from the other physical properties to a desired TT Field frequency (or efficacy prediction) without intermediate calculation of the relevant electrical properties, as described in more detail below.

[0022] In some embodiments, the electric property used to make the prediction is the dielectrophoretic force on the cancer cells. Any of a variety of commercially available systems can be used to measure dielectrophoretic force, including but not limited to the 3DEP™ 3D dielectrophoretic cell analysis system (dielectrophoresis is a physical action that generates a force on a polarizable particle that experiences a non-uniform electric field, and can therefore be used as a technique to analyze how cells move in electric fields of various frequencies). It should be noted that instead of determining the electric property by dielectrophoretic force measurement on the cancer cells (as in the 3DEP™ system), various alternative methods for determining the electric property of the cancer cells can also be used, as will be apparent to those skilled in the art.

[0023] First set of embodiments: Determining the frequency of TTFields treatment to be applied based on electrical properties of cancer cells To establish how the measured electrical properties can be used to determine the frequency of TT Fields that should be applied to a subject to treat their cancer, the baseline electrical properties (dielectric constant and conductivity) of 18 cell lines derived from a variety of tumor types were determined using the 3DEP™ Cell Analysis System. Among these 18 cell lines, it was first established that 10 of these cell lines (HepG2, A549, H1299, MDA231, LLC-1, C3A, AGS, KATO III, H2052, and RN5) were most susceptible to treatment using TT Fields at a frequency of 150 kHz, and 8 of these cell lines (A172, A2780, U87, A375, LN18, LN229, DKMG, and U251) were most susceptible to treatment using TT Fields at a frequency of 200 kHz. The optimal TTFields frequency for all 18 cell lines was determined by testing the cytotoxic effects of TTFields at different frequencies using the Inovitro™ system.

[0024] The electrical properties of each of the cell lines were then compared to the optimal TT field frequency and sensitivity for each cell line. The results of this comparison are shown in Figure 1, which shows the difference in the cellular dielectrophoretic force versus frequency curves in the lower frequency range (3-35 kHz) between a first group of 10 cell lines (optimal TT field frequency of 150 kHz) and a second group of 8 cell lines (optimal TT field frequency of 200 kHz).

[0025] The curves were analyzed using a two-way analysis of variance. A comparison of the dielectrophoretic force of the first cell line group with the dielectrophoretic force of the second cell line group shows that there is a significant difference between the dielectrophoretic force of these two groups in the dielectrophoretic force vs. frequency curves at the lower frequency range. More specifically, the results show a significant difference (p<0.001) between the dielectrophoretic force vs. frequency curves at the lower frequency range, such as between the first cell line group and the second cell line group. Based on the difference in the curves at this lower frequency range, the inventors have determined that the electrical properties of the dielectrophoretic force at this lower frequency range are a good predictor of the optimal frequency for TT Fields therapy.

[0026] Figure 2 shows a process that takes advantage of this difference. In step S22, tumor cells are removed from a subject (e.g., by resection / biopsy / taking from circulating tumor cells). Then, in step S24, the electrical properties of the cancer cells are determined. One means of doing this is to dissociate the removed tumor cells (using a method such as a tumor dissociation kit) into a single cell suspension. The electrical properties of the cells in the single cell suspension can be examined directly using an instrument that measures electrical properties (e.g., using a 3DEP™ instrument that measures dielectrophoretic forces), or can be further purified to create a more homogenous cell population by sorting from other invasive cells using a method such as fluorescence activated cell sorting (FACS) or using cell specific beads, and then the electrical properties are measured by an instrument such as 3DEP™.

[0027] After determining the electrical properties of at least one cancer cell from the subject, the frequency of the TT field to be applied to the subject to treat the cancer of the subject can be determined based on the electrical properties determined in step S26. For example, in a situation where the electrical property being analyzed is dielectric force, if the measured dielectric force of the tumor cells from the subject more closely matches the dielectric force of the first cell line group, then treatment for the subject should proceed using a TT field at a frequency of 150 kHz. On the other hand, if the measured dielectric force of the tumor cells from the subject more closely matches the dielectric force of the second cell line group, then treatment for the subject should proceed using a TT field at a frequency of 200 kHz.

[0028] After determining the frequency for the TT field treatment, in step S28, the cancer is treated by applying a TT field to the subject at the determined frequency.

[0029] From dielectrophoretic force vs. frequency data (such as that shown in FIG. 1), the cell membrane capacitance can be determined, since the dielectrophoretic force vs. frequency curve in the lower frequency range corresponds to the membrane capacitance of the cell. Thus, the inventors have determined that the electrical properties of the cell membrane capacitance are also good predictors of the optimal frequency for TT Fields therapy. Using the cell membrane capacitance as a predictor of the optimal frequency for TT Fields therapy in a situation where the cell membrane capacitance is calculated based on dielectrophoretic force measurements is an example of indirectly determining a second electrical property (i.e., the cell membrane capacitance) based on a first electrical property (i.e., the dielectrophoretic force) and then using the second electrical property as a predictor of the optimal frequency for TT Fields therapy.

[0030] The process shown in FIG. 2 also applies to situations where cell membrane capacitance is used as the electrical property. In this situation, in step S22, tumor cells are removed from the subject (e.g., as described above). Then, in step S24, the cell membrane capacitance of the cancer cells is determined. This can be done, for example, by using a 3DEP™ device to measure the dielectrophoretic force and then determining the cell membrane capacitance based on the measured dielectrophoretic force. Then, in step S26, a mapping between cell membrane capacitance and optimal TT field frequency is performed. For example, if the cell membrane capacitance of tumor cells from the subject more closely matches the cell membrane capacitance of the first cell line group, then treatment for the subject should proceed using a TT field with a frequency of 150 kHz. On the other hand, if the cell membrane capacitance of tumor cells from the subject more closely matches the cell membrane capacitance of the second cell line group, then treatment for the subject should proceed using a TT field with a frequency of 200 kHz. Then, in step S28, the cancer is treated by applying a TT field to the subject at the determined frequency.

[0031] As mentioned above, the dielectrophoretic force data can be used to predict the optimal frequency for TT Fields therapy. One means of measuring the dielectrophoretic force (used in the 3DEP™ system) is to first perform optical measurements to determine the distance the excised cancer cells move in an electric field, then convert these optical measurements to dielectrophoretic force data (which is an electrical property), and then map the dielectrophoretic force data to an optimal frequency. Thus, it is possible to omit the conversion step and perform a direct mapping from the underlying optical measurements to the optimal frequency for TT Fields therapy. This is an example of measuring the physical parameters (i.e., optical parameters) of the excised cancer cells, when the electrical properties (i.e., dielectrophoretic force) of the cancer cells can be determined from the physical parameters, and then determining the TT Field frequency based on the physical parameters (even though the dielectrophoretic force value is never actually determined).

[0032] The process shown in FIG. 3 applies in this context. Here, in step S32, tumor cells are removed from the subject (e.g., as described above with respect to step S22). Then, in step S34, the physical properties of the cancer cells (e.g., their movement in the wells in the 3DEP™ system determined by measuring the light intensity) are determined. Then, in step S36, a mapping between the measured light intensity and the optimal TT field frequency is performed. For example, if the light intensity measured for the tumor cells from the subject more closely matches the light intensity measured for the first cell line group, then treatment for the subject should proceed using a TT field at a frequency of 150 kHz. On the other hand, if the light intensity measured for the tumor cells from the subject more closely matches the light intensity measured for the second cell line group, then treatment for the subject should proceed using a TT field at a frequency of 200 kHz. Then, in step S38, the cancer is treated by applying a TT field to the subject at the frequency determined.

[0033] Second set of embodiments: Predicting cancer cell response to TTFields treatment based on electrical properties of cancer cells In other embodiments, a prediction can be made as to whether TTFields therapy will be effective for any particular subject based on the electrical properties of cancer cells within the body of the particular subject, which can be determined using any of the techniques described above with respect to the frequency-selective embodiments (e.g., 3DEP™).

[0034] To establish this prediction method, 3DEP™ measurements were performed on a first group of seven different cell lines known to experience high cytotoxic effects when subjected to TTFields (LN18, LN229, A375, A2780, MDA231, LLC-1, and AGS) and on a second group of six different cell lines known to experience low cytotoxic effects when subjected to TTFields (MCF7, U251, DKMG, KATO III, CT26, and RN5). The cytotoxic effects of TTFields on the various cell lines were determined using the Inovitro™ System.

[0035] The electrical properties of these two cell line groups were then compared. The results of this comparison are shown in Figure 4, which shows the difference in the cellular dielectrophoretic force versus frequency curves in the lower frequency range (3-35 kHz) between the first and second cell line groups. As also mentioned above, these curves in this lower range correspond to the membrane capacitance.

[0036] This difference can be used to aid in determining whether a particular subject should or should not be treated with TTFields, or whether they should be treated using TTFields in combination with another modality.

[0037] These results show significant differences (2-way ANOVA, p<0.001) between the dielectrophoretic force versus frequency curves (corresponding to the membrane capacitance of the cells) at the lower frequency range, such as between the first and second cell line groups. Based on the differences in the curves at this low frequency range, the inventors have determined that the electrical properties of the dielectrophoretic force at this low frequency range are good predictors of whether a particular group of cancer cells will respond well to TTFields treatment.

[0038] Figure 5 shows a process that takes advantage of this difference. In step S52, tumor cells are removed from a subject (e.g., by resection / biopsy / harvesting from circulating tumor cells). Then, in step S54, the electrical properties of the cancer cells are determined (e.g., as described above with respect to step S24).

[0039] After determining the electrical properties of at least one cancer cell from the subject, in step S56, a prediction is made as to whether application of a TT field to the subject will be effective to treat the cancer based on the determined electrical properties. For example, in a situation where the electrical property being analyzed is dielectric force, if the dielectric force measurements of the tumor cells from the subject more closely match a first cell line population (known to experience high cytotoxicity when TT fields are applied), then treatment for the subject using TT fields has a high probability of effectiveness. On the other hand, if the dielectric force measurements of the tumor cells from the subject more closely match a second cell line population, then the probability of effectiveness using TT fields is low and treatment for the subject should proceed using an alternative approach.

[0040] Finally, if the results of the prediction in step S56 indicate that treatment using TT fields has a high probability of effectiveness, then in step S58, the cancer is treated by appropriately applying TT fields to the subject at an appropriate frequency.

[0041] From the dielectrophoretic force vs. frequency data (such as that shown in FIG. 4), the cell membrane capacitance can be determined, since the dielectrophoretic force vs. frequency curve in the lower frequency range corresponds to the membrane capacitance of the cell. Thus, the inventors have determined that the electrical properties of the cell membrane capacitance are also good predictors of the efficacy of TT Fields therapy. Using the cell membrane capacitance as a predictor of the efficacy of TT Fields therapy in the context of calculating the cell membrane capacitance based on dielectrophoretic force measurements is an example of indirectly determining a second electrical property (i.e., the cell membrane capacitance) based on a first electrical property (i.e., the dielectrophoretic force) and then using the second electrical property as a predictor of the efficacy of TT Fields therapy.

[0042] The process illustrated in FIG. 5 also applies to the situation where cell membrane capacitance is used as the electrical property. In this situation, in step S52, tumor cells are removed from a subject (e.g., as described above with respect to step S22). Then, in step S54, the cell membrane capacitance of the cancer cells is determined (e.g., as described above with respect to step S24). Then, in step S56, a prediction is made as to whether TT Fields treatment will be effective based on the determined cell membrane capacitance. For example, if the cell membrane capacitance of tumor cells from the subject more closely matches the cell membrane capacitance of a first cell line population (known to experience high cytotoxicity upon application of TT Fields), then treatment for the subject using TT Fields has a high probability of effectiveness. On the other hand, if the cell membrane capacitance of tumor cells from the subject more closely matches the cell membrane capacitance of a second cell line population, then the probability of effectiveness using TT Fields is low and treatment for the subject should proceed using an alternative approach. Finally, if the results of the prediction in step S56 indicate that treatment using TT fields has a high probability of effectiveness, then in step S58, the cancer is treated by applying a TT field to the subject at an appropriate frequency.

[0043] Because (a) dielectrophoretic force data can be used to predict whether TT Fields treatment will be effective (as described above with respect to Figures 4-5), and (b) optical measurements can be converted to dielectrophoretic force data (as described above with respect to Figure 3), it is possible to predict directly from the underlying optical measurements whether treatment with TT Fields will be effective for a particular subject, without any intermediate step of determining dielectrophoretic force data. This is an example of measuring the physical parameters (i.e., optical parameters) of excised cancer cells, where the electrical properties (i.e., dielectrophoretic force) of the cancer cells can be determined from the physical parameters, and then predicting whether application of a TT Field will be effective to treat the cancer in a subject based on the physical parameters (even though the dielectrophoretic force value has never actually been determined).

[0044] The process depicted in FIG. 6 applies in this context. Here, in step S62, tumor cells are removed from the subject (e.g., as described above with respect to step S22). Then, in step S64, physical properties of the cancer cells are determined (e.g., movement within a well in a 3DEP™ system determined by measuring light intensity). Then, in step S66, a mapping is made between the measured light intensity and a prediction of whether TT Fields treatment will be effective. For example, if the measured light intensity for tumor cells from the subject more closely matches a first group of cell lines (known to experience high cytotoxicity when TT Fields are applied), then treatment for the subject using TT Fields has a high probability of effectiveness. On the other hand, if the light intensity for tumor cells from the subject more closely matches a second group of cell lines, then the probability of effectiveness using TT Fields is low and treatment for the subject should proceed using an alternative approach. Finally, if the prediction results in step S66 reveal that treatment using TT Fields has a high probability of effectiveness, then in step S68, the cancer is treated by applying a TT Field to the subject at an appropriate frequency.

[0045] Although the present invention has been disclosed with reference to particular embodiments, numerous modifications, variations, and variations to the described embodiments are possible without departing from the scope and spirit of the invention as defined in the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but rather encompass the full scope defined by the language of the claims, and equivalents thereof.

Claims

1. 1. A method for testing the efficacy of cancer treatment by application of TTFields in a subject having cancerous tissue, comprising: determining the dielectric force of at least one cancer cell excised from a sample of cancerous tissue obtained from the subject; and The effectiveness of cancer treatment was evaluated based on the following criteria: if the measured dielectric force of tumor cells from the subject more closely matches a first set of cell lines known to exhibit high cytotoxicity upon application of TTFields, then treatment for the subject using TTFields is likely to be effective; - if the measured dielectric forces of tumor cells derived from the subject more closely match a second set of cell lines known to exhibit low cytotoxicity upon application of TTFields, then the use of TTFields is less likely to be effective; A process for testing based on A method comprising:

2. The method of claim 1, wherein the step of determining the dielectric force includes measuring the dielectrophoretic force of at least one cancer cell at each of a plurality of frequencies less than 35 kHz.

3. The method of claim 1, wherein the step of determining the dielectric force includes determining a cell membrane capacitance.

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