Determining the frequency of TTfield treatment based on the electrical properties of targeted cancer cells.

Abstract

Cancer treatment using TTFields (Tumor Therapy Fields) can be tailored to each individual subject. This involves obtaining cancer cells from the subject, determining their electrical properties (e.g., dielectric electrophoretic force, cell membrane capacitance, etc.), determining the frequency for using the TTField based on these properties, and treating the cancer by applying the TTField to the subject at the determined frequency. Furthermore, cancer treatment can be planned for each individual subject by obtaining cancer cells from the subject, determining their electrical properties, predicting whether the TTField will be effective in treating the cancer based on these properties, and treating the subject by applying the TTField if the prediction indicates its effectiveness.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application 62 / 810,823, filed February 26, 2019, which is incorporated herein by reference in its entirety.

[0003] background

[0004] Therapeutic field (TTField) is an effective form of anti-tumor therapy that delivers medication by applying a low-intensity (e.g., 1-5 V / cm), medium-frequency (e.g., 100-300 kHz) alternating electric field. TTField therapy has been FDA-approved for the treatment of glioblastoma multiforme brain tumors and appears very promising for many other types of tumors. Wearable and portable devices (Optune) are also available. ® TTField therapy is delivered. The delivery system comprises four adhesive, non-invasive, insulated transducer arrays, an electric field generator, a rechargeable battery, and a carrying case. The transducer arrays are applied to the skin near the tumor and connected to the field generator.

[0005] In preclinical setup, Inovitro can be used. TM The system applies a TTField externally via a pair of vertical electrodes insulated from high-dielectric-constant ceramic. (Inovitro) TM The (TTField Lab bench System) includes a TTField generator and a substrate, with each board containing eight ceramic disks. Invention Overview

[0007] One aspect of the present invention relates to a first method for treating cancer in a subject with cancerous tissue. The first method includes obtaining a sample of the cancerous tissue from the subject, the sample having at least one cancer cell; determining the electrical properties of the at least one cancer cell; determining, based on the determined electrical properties, the frequency of an alternating electric field applied to the subject to treat the cancer; and treating the cancer by applying the alternating electric field to the subject at the determined frequency of the alternating electric field.

[0008] In some instances of the first method, determining the electrical property includes measuring the dielectric electrophoretic force of the at least one cancer cell at each of a plurality of frequencies below 35 kHz. In some instances of the first method, determining the frequency includes selecting a frequency effective in treating reference cancer cells with known electrical properties, wherein the known electrical properties match the determined electrical properties. In some instances of the first method, determining the electrical property includes determining the cell membrane capacitance.

[0009] Another aspect of the invention relates to a second method for treating cancer in a subject with cancerous tissue. The second method includes obtaining a sample of the cancerous tissue from the subject, the sample having at least one cancer cell; measuring at least one physical parameter of the at least one cancer cell, wherein the electrical properties of the at least one cancer cell can be determined from the at least one physical parameter; determining, based on the measured at least one physical parameter, the frequency of an alternating electric field applied to the subject to treat the cancer; and treating the cancer by applying the alternating electric field to the subject at the determined frequency of the alternating electric field.

[0010] In some cases of the second method, the cell membrane capacitance of the at least one cancer cell can be determined from the at least one physical parameter.

[0011] Another aspect of the invention relates to a third method for treating cancer in a subject with cancerous tissue. The third method includes obtaining a sample of the cancerous tissue from the subject, the sample having at least one cancer cell; determining the electrical properties of the at least one cancer cell; predicting, based on the determined electrical properties, whether applying an alternating electric field to the subject will effectively treat the cancer; and if the prediction indicates that applying an alternating electric field to the subject will effectively treat the cancer, then treating the cancer by applying the alternating electric field to the subject.

[0012] In some instances of the third method, determining the electrical properties includes measuring the dielectric electrophoretic force of the at least one cancer cell at each of a plurality of frequencies below 35 kHz. In some instances of the third method, the prediction is based on whether a reference cancer cell having electrical properties matching the determined electrical properties is sensitive to treatment using an alternating electric field. In some instances of the third method, determining the electrical properties includes determining the cell membrane capacitance.

[0013] Another aspect of the invention relates to a fourth method for treating cancer in a subject with cancerous tissue. The fourth method includes obtaining a sample of the cancerous tissue from the subject, the sample having at least one cancer cell; measuring at least one physical parameter of the at least one cancer cell, wherein the electrical properties of the at least one cancer cell can be determined from the at least one physical parameter; predicting, based on the measured at least one physical parameter, whether applying an alternating electric field to the subject will effectively treat the cancer; and if the prediction indicates that applying an alternating electric field to the subject will effectively treat the cancer, then treating the cancer by applying the alternating electric field to the subject.

[0014] In some cases 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.

[0015] Another aspect of the invention relates to a fifth method for selecting the frequency of an alternating electric field for treating cancer in a subject with cancerous tissue. The fifth method includes determining the electrical properties of at least one cancer cell extracted from a sample of the cancerous tissue obtained from the subject; and determining, based on the determined electrical properties, the frequency of an alternating electric field applied to the subject to treat the cancer.

[0016] In some embodiments of the fifth method, determining the electrical property includes measuring the dielectric electrophoretic force of the at least one cancer cell at each of a plurality of frequencies below 35 kHz. In some embodiments of the fifth method, determining the frequency includes selecting a frequency effective in treating reference cancer cells with known electrical properties, wherein the known electrical properties match the determined electrical properties. In some embodiments of the fifth method, determining the electrical property includes determining the cell membrane capacitance.

[0017] Another aspect of the invention relates to a sixth method for selecting the frequency of an alternating electric field for treating cancer in a subject with cancerous tissue. The sixth method includes measuring at least one physical parameter of at least one cancer cell extracted from a sample of the cancerous tissue obtained from the subject, wherein the electrical properties of the at least one cancer cell can be determined from the at least one physical parameter; and determining the frequency of the alternating electric field applied to the subject to treat the cancer based on the measured at least one physical parameter.

[0018] In some cases 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 attached diagram

[0020] Figure 1 The changes in dielectric electrophoretic forces between the two cell lines were described. The optimal TTField frequencies for the two cell lines were 150 kHz and 200 kHz, respectively.

[0021] Figure 2 This paper describes a method for selecting the frequency of TTField treatment based on dielectric electrophoretic force measurement.

[0022] Figure 3 This describes a method for selecting a frequency for TTField therapy based on optical measurements when electrical properties (e.g., dielectric electrophoretic force or cell membrane capacitance) can be determined from optical measurements.

[0023] Figure 4 Describe the changes in dielectrophoretic forces between two cell lines, one of which is sensitive to TTField and the other is insensitive to TTField.

[0024] Figure 5 This describes a method for determining whether to use a TTField to treat a specific subject based on dielectrophoretic force measurements.

[0025] Figure 6 This describes a method for determining whether to use TTField to treat a particular subject when electrical properties (e.g., dielectric electrophoretic force or cell membrane capacitance) can be determined from optical measurements.

[0026] Various embodiments are described in detail below with reference to the accompanying drawings, wherein similar reference numerals denote similar elements.

[0027] Description of preferred implementation scheme

[0028] Traditionally, once a decision is made to use TTField to treat a given subject, the frequency at which the TTField is applied to the subject is based on the specific type of tumor being treated. For example, the recommended frequency for TTField is 200 kHz when treating GBM, while it is 150 kHz when treating gastric cancer. However, using a single frequency for all subjects may not provide optimal results in every and all of those subjects. More specifically, while 200 kHz may be the optimal frequency for most subjects with GBM, some individuals with GBM may respond better to different frequencies (e.g., 175 kHz or 225 kHz). To date, no predictive marker has been used to determine the optimal frequency that should be used for any given individual subject.

[0029] Some implementations described herein can provide improved outcomes for a large number of subjects by customizing the frequency at which TTField is applied to each individual subject. The decision of which frequency to use for any given individual subject is based on at least one electrical property of the cancer cells extracted from the individual subject. In alternative implementations, the decision of which frequency to use for any given individual subject can be based on physical parameters from which the electrical properties of the extracted cancer cells can be determined. The ability to predict in advance the optimal frequency for applying TTField to each individual subject can advantageously improve the efficacy of TTField treatment.

[0030] Furthermore, traditionally, no predictive marker has been used to determine whether TTField will be effective for any given individual subject. Some embodiments described herein can predict whether TTField will be effective for a given individual subject. The prediction is based on at least one electrical property of cancer cells extracted from the individual subject. In alternative embodiments, the prediction may be based on physical parameters from which the electrical properties of the extracted cancer cells can be determined. The ability to predict in advance the efficacy of TTField treatment for any given subject can advantageously improve outcomes (e.g., by selecting the most effective treatment for each individual subject).

[0031] Examples of electrical properties that can be used for these predictions include, but are not limited to, dielectric electrophoretic force, cell membrane capacitance, cell membrane resistance, cytoplasmic conductivity, and other measures of permittivity, conductivity, capacitance, etc., of various cellular structures. The electrical properties used for prediction can be measured directly. Alternatively, the electrical properties can be measured indirectly (e.g., by measuring other physical properties, which may be different electrical or non-electrical properties, such as light intensity), and the electrical properties can be determined by these other physical properties. Where the electrical properties can be determined from other physical properties, a mapping can be made directly from the other physical properties to the desired TTField frequency (or validity prediction) without intermediate calculations of the associated electrical properties, as explained in more detail below.

[0032] In some implementations, the electrical property used to make the above predictions is the dielectric electrophoretic force on cancer cells. Any of a variety of commercially available systems for measuring dielectric electrophoretic forces can be used, including but not limited to the 3DEP™ 3DDielectrophoresis Cell Analysis System. (Dielectrophoresis is a physical process that generates forces on polarizable particles subjected to a non-uniform electric field, and can therefore be used as a technique to analyze how cells move within electric fields of different frequencies.) Note that instead of determining electrical properties by measuring the dielectric electrophoretic force on cancer cells (as in the 3DEP™ system), various alternative methods for determining the electrical properties of cancer cells, readily apparent to those skilled in the art, can also be used.

[0033] First implementation plan: Determine the frequency at which TTField treatment should be applied based on the electrical properties of cancer cells.

[0034] To establish how to determine the frequency of TTField applied to a subject to treat their cancer using measured electrical properties, the 3DEP™ Cell Analysis System was used to determine baseline electrical properties (capacitance and conductivity) from 18 cell lines of different tumor types. Of these 18 cell lines, 10 were first identified as most susceptible to treatment with a TTField at 150 kHz; and 8 were identified as most susceptible to treatment with a TTField at 200 kHz. The optimal TTField frequency for all 18 cell lines was determined by testing the cytotoxic effects of the TTField at various frequencies using the Inovitro™ system.

[0035] The electrical properties of each cell line were then compared with the optimal TTField frequency and sensitivity for each cell line. The results of this comparison were... Figure 1 The description demonstrates the difference in dielectric electrophoretic force versus frequency curves in the lower frequency range (3–35 kHz) between the first group of 10 cell lines (for which the optimal TTField frequency is 150 kHz) and the second group of 8 cell lines (for which the optimal TTField frequency is 200 kHz).

[0036] The curves were analyzed using two-channel ANOVA. A comparison of the dielectrophoretic forces of the first and second cell lines revealed a significant difference between the two groups in the lower frequency range of the dielectrophoretic force versus frequency curve. More specifically, these results demonstrated a significant difference (p < 0.001) in the lower frequency range of the dielectrophoretic force versus frequency curve between the first and second cell lines. Based on the differences in the curves within this low frequency range, the inventors have concluded that the electrical properties of the dielectrophoretic force within this low frequency range are a good predictor of the optimal frequency for TTField therapy.

[0037] Figure 2 The method for utilizing this difference is described. In step S22, tumor cells are extracted from the subject (e.g., from excised / biopsy / circulating tumor cells). Then, in step S24, the electrical properties of the cancer cells are determined. One way to accomplish this is to dissociate the extracted tumor cells into a single-cell suspension (using, for example, a tumor dissociation kit). The electrical properties of the cells in the single-cell suspension can be measured using a device for measuring electrical properties (e.g., using a 3DEP device).TM The device can be used to measure dielectric electrophoretic force directly, or methods such as fluorescence-activated cell sorting (FACS) or cell-specific beads can be used, followed by measurement via a device (e.g., 3DEP). TM The electrical properties were measured, and other infiltrating cells were further purified by sorting to produce a more homogeneous cell population.

[0038] After determining the electrical properties of at least one cancer cell from the subject, in step S26, the frequency of the TTField to be applied to the subject to treat the subject's cancer can be determined based on the determined electrical properties. For example, if the electrical property being analyzed is dielectric, and the dielectric power measurement of the tumor cells from the subject is closer to matching the dielectric power of a first group of cell lines, then the treatment of the subject should be performed using a TTField at a frequency of 150 kHz. On the other hand, if the dielectric power measurement of the tumor cells from the subject is closer to matching a second group of cell lines, then the treatment of the subject should be performed using a TTField at a frequency of 200 kHz.

[0039] After the frequency for TTField treatment has been determined, in step S28, the cancer is treated by applying TTField to the subject at the determined frequency.

[0040] Because the dielectric electrophoretic force versus frequency curve in the lower frequency range corresponds to the membrane capacitance of the cell, the dielectric electrophoretic force versus frequency data (e.g., in...) can be derived from the data. Figure 1 The data described in [the document] are used to determine the cell membrane capacitance. Therefore, the inventors have concluded that the electrical properties of the cell membrane capacitance are also a good predictor of the optimal frequency for TTField treatment. In the case of calculating the cell membrane capacitance based on dielectric electrophoretic force measurements, using the cell membrane capacitance as a predictor of the optimal frequency for TTField treatment is an example of indirectly determining the second electrical property (i.e., cell membrane capacitance) based on the first electrical property (i.e., dielectric electrophoretic force), and then using the second electrical property as a predictor of the optimal frequency for TTField treatment.

[0041] exist Figure 2 The method described herein is also applicable to cases where cell membrane capacitance is used as an electrical property. In this case, in step S22, tumor cells are extracted 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 achieved, for example, using 3DEP. TMThe device measures the dielectric electrophoretic force, and then determines the cell membrane capacitance based on the measured dielectric electrophoretic force. Then, in step S26, a mapping is performed between the cell membrane capacitance and the optimal TTField frequency. For example, if the cell membrane capacitance of the tumor cells from the subject is closer to that of a matching first cell line, then the treatment of the subject should be performed using a TTField at a frequency of 150 kHz. On the other hand, if the cell membrane capacitance of the tumor cells from the subject is closer to that of a matching second cell line, then the treatment of the subject should be performed using a TTField at a frequency of 200 kHz. Then, in step S28, the cancer is treated by applying the TTField to the subject at the determined frequency.

[0042] As mentioned above, dielectrophoretic force data can be used to predict the optimal frequency for TTField treatment. Measuring dielectrophoretic force (which is used in 3DEP) TM One approach within the system is to first perform optical measurements to determine how far the extracted cancer cells migrate in an electric field, then convert those optical measurements into dielectrophoretic force data (which is an electrical property), and then map the dielectrophoretic force data to an optimal frequency. Therefore, the conversion step can be omitted, and a direct mapping from basic optical measurements to the optimal frequency for TTField treatment can be performed. This is an example of measuring the physical (i.e., optical) parameters of the extracted cancer cells, from which the electrical properties (i.e., dielectrophoretic force) of the cancer cells can be determined, and then the TTField frequency can be determined based on these physical parameters (even if the dielectrophoretic force value is never actually determined).

[0043] Figure 3 The method described herein is applicable to this situation. Here, in step S32, tumor cells are extracted from the subject (e.g., as described above in conjunction with step S22). Then, in step S34, the physical properties of the cancer cells are determined (e.g., by measuring light intensity in 3DEP). TM (Movement within the orifice in the system). Then, in step S36, a mapping is performed between the measured light intensity and the optimal TTField frequency. For example, if the light intensity measured on the tumor cells from the subject is closer to the light intensity measured on the first group of cell lines, then the treatment of the subject should be performed using a TTField at a frequency of 150 kHz. On the other hand, if the light intensity measured on the tumor cells from the subject is closer to the light intensity measured on the second group of cell lines, then the treatment of the subject should be performed using a TTField at a frequency of 200 kHz. Then, in step S38, the cancer is treated by applying the TTField to the subject at the determined frequency.

[0044] The second implementation plan: predicting the response of cancer cells to TTField treatment based on the electrical properties of cancer cells.

[0045] In other implementations, the effectiveness of TTField treatment for any given individual subject can be predicted based on the electrical properties of cancer cells in that individual subject. The electrical properties of cancer cells from a given individual subject can be obtained using any of the methods described above (e.g., 3DEP). TM The frequency selection implementation plan is determined in conjunction with the frequency selection scheme.

[0046] To establish how such predictions can be made, 3DEP was performed on a first group of seven different cell lines (LN18, LN229, A375, A2780, MDA231, LLC-1, and AGS, which are known to experience high cytotoxicity when TTField is applied) and a second group of six different cell lines (MCF7, U251, DKMG, KATO III, CT26, and RN5, which are known to experience low cytotoxicity when TTField is applied). TM Measurement. Using Inovitro TM The system determined the cytotoxic effects of TTField on various cell lines.

[0047] Then the electrical properties of the two cell lines were compared. The results of this comparison were... Figure 4 The description demonstrates the difference in dielectric electrophoretic force versus frequency curves in the lower frequency range (3-35 kHz) between the first and second cell lines. Furthermore, as mentioned above, these curves in this lower frequency range correspond to membrane capacitance.

[0048] This distinction can be used to help determine whether a given subject should be treated with TTField, not treated, or treated with TTField in combination with another form of treatment.

[0049] These results demonstrate a significant difference in dielectric electrophoretic force relative to the frequency curve (which corresponds to the cell membrane capacitance) in the lower frequency range between the first and second cell lines (2-channel ANOVA, p < 0.001). Based on the difference in the curves in this low frequency range, the inventors have concluded that the electrical properties of the dielectric electrophoretic force in this low frequency range are a good predictor of whether a given set of cancer cells will respond well to TTField treatment.

[0050] Figure 5 The method for utilizing this difference is described. In step S52, tumor cells are extracted from the subject (e.g., from excised / biopsy / circulating tumor cells). Then, in step S54, the electrical properties of the cancer cells are determined (e.g., as described above in conjunction with step S24).

[0051] After determining the electrical properties of at least one cancer cell from the subject, in step S56, it is predicted whether applying the TTField to the subject will be effective in treating the cancer based on the determined electrical properties. For example, if the electrical property being analyzed is dielectric, the likelihood of effective treatment of the subject using the TTField is high if the dielectric measurement of the tumor cells from the subject more closely matches a first group of cell lines (known to experience high cytotoxicity when the TTField is applied). On the other hand, if the dielectric measurement of the tumor cells from the subject more closely matches a second group of cell lines, the likelihood of effectiveness using the TTField is low, and alternative methods should be used to treat the subject.

[0052] Finally, if the predicted results in step S56 reveal a high probability of the effectiveness of treatment using TTField, then in step S58 the cancer is treated by applying TTField to the subject at an appropriate frequency.

[0053] Because the dielectric electrophoretic force versus frequency curve in the lower frequency range corresponds to the membrane capacitance of the cell, the dielectric electrophoretic force versus frequency data (e.g., in...) can be derived from the data. Figure 4 The data described in [the document] are used to determine the cell membrane capacitance. Therefore, the inventors have concluded that the electrical properties of cell membrane capacitance are also a good predictor of the effectiveness of TTField treatment. In the case where cell membrane capacitance is calculated based on dielectric electrophoretic force measurements, using cell membrane capacitance as a predictor of the effectiveness of TTField treatment is an example of indirectly determining a second electrical property (i.e., cell membrane capacitance) based on a first electrical property (i.e., dielectric electrophoretic force), and then using the second electrical property as a predictor of the effectiveness of TTField treatment.

[0054] exist Figure 5The method described herein is also applicable to cases where cell membrane capacitance is used as an electrical property. In this case, in step S52, tumor cells are extracted from the subject (e.g., as described above in conjunction with step S22). Then, in step S54, the cell membrane capacitance of the cancer cells is determined (e.g., as described above in conjunction with step S24). Next, in step S56, based on the determined cell membrane capacitance, the effectiveness of TTField treatment is predicted. For example, if the cell membrane capacitance of the tumor cells from the subject is closer to that of a first group of cell lines (known to experience high cytotoxicity when TTField is applied), the likelihood of effectiveness of TTField treatment for the subject is high. On the other hand, if the cell membrane capacitance of the tumor cells from the subject is closer to that of a second group of cell lines, the likelihood of effectiveness of TTField treatment is low, and alternative methods should be used to treat the subject. Finally, if the prediction in step S56 reveals a high likelihood of effectiveness of TTField treatment, the cancer is treated in step S58 by applying TTField to the subject at an appropriate frequency.

[0055] Because (a) dielectric electrophoretic data can be used to predict whether TTField treatment will be effective (as mentioned above) Figure 4-5 (a) as described above; and (b) optical measurements can be converted into dielectric electrophoretic force data (as described above in conjunction with...). Figure 3 As described above, it is possible to directly predict from basic optical measurements whether treatment with TTField will be effective for a particular subject, without the intermediate step of determining dielectric electrophoretic force data. This is an example of measuring the physical (i.e., optical) parameters of extracted cancer cells, where the electrical properties of the cancer cells (i.e., dielectric electrophoretic force) can be determined from the physical parameters, and then predicting based on these physical parameters whether applying TTField will effectively treat the subject's cancer (even if the dielectric electrophoretic force value is never actually determined).

[0056] Figure 6 The method described herein is applicable to this situation. Here, in step S62, tumor cells are extracted from the subject (e.g., as described above in conjunction with step S22). Then, in step S64, the physical properties of the cancer cells are determined (e.g., by measuring light intensity in 3DEP). TM(Movement within the orifice in the system). Then, in step S66, a mapping is made between the measured light intensity and the predicted effectiveness of TTField treatment. For example, if the light intensity measured on the tumor cells from the subject is closer to matching the first group of cell lines (known to experience high cytotoxicity when TTField is applied), the likelihood of effectiveness of TTField treatment on the subject is high. On the other hand, if the light intensity measured on the tumor cells from the subject is closer to matching the second group of cell lines, the likelihood of effectiveness of TTField treatment is low, and alternative methods should be used to treat the subject. Finally, if the prediction in step S66 reveals a high likelihood of effectiveness of TTField treatment, the cancer is treated in step S68 by applying TTField to the subject at an appropriate frequency.

[0057] Although the invention has been disclosed with reference to certain embodiments, many modifications, alterations, and changes can be made to the described embodiments without departing from the field and scope of the invention as defined in the appended claims. Therefore, it is intended that the invention is not limited to the described embodiments, but has the full scope defined by the language of the appended claims and their equivalents.

Claims

1. A method of selecting a frequency of an alternating electric field for treating a cancer in a subject having a cancerous tissue, the method comprising: determining an electrical property of at least one cancer cell extracted from a sample of the 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, wherein determining the electrical property comprises measuring a dielectrophoretic force of the at least one cancer cell at each of a plurality of frequencies below 35 kHz.

2. The method of claim 1, wherein determining the electrical property comprises determining a cell membrane capacitance.

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