Method for normalizing abnormal glycolytic metabolism in cancer cells

Abstract

The viability of cancer cells (e.g., glioblastoma cells) can be reduced by administering mannose to the cancer cells and applying an AC electric field to the cancer cells at a frequency between 100 and 500 kHz. Sensitivity to AC electric field treatment can be determined by measuring the uptake of a PKM2 probe (e.g., [18F]DASA) before and after AC electric field treatment. Notably, experiments show that the combination of mannose and AC electric fields produces synergistic anti-glioblastoma results.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 953,704, filed on December 26, 2019, which is incorporated herein by reference in its entirety.

Background Art

[0002] Tumor Treating Fields (TT Fields) is an effective anti - malignant tumor treatment method delivered by the non - invasive application of an alternating electric field of low intensity and intermediate frequency (e.g., 100 - 500 kHz). TT Fields exert a directional force on microtubules and interfere with the normal assembly of the spindle. Such interference with microtubule dynamics results in abnormal spindle formation and subsequent mitotic arrest or delay. Cells can die in mitotic arrest or proceed to cell division leading to the formation of either normal or abnormal aneuploid progeny. The formation of tetraploid cells can occur either by mis - segregated mitotic exit or during inappropriate cell division. Abnormal daughter cells can die during subsequent quiescence, undergo permanent arrest, or proliferate by further mitosis where they are then subjected to further TT Fields attack. Giladi M et al., Sci Rep. 2015;5:18046.

[0003] In an in vivo environment, TT Fields therapy can be delivered using a wearable and portable device (Optune®). The delivery system includes an electric field generator, four adhesive patches (a non - invasive insulated transducer array), a rechargeable battery, and a carrying case. The transducer array is applied to the skin and connected to the device and the battery. The therapy is designed to be worn for as long as possible, day and night.

[0004] In a preclinical setting, TT fields can be applied in vitro, for example, using the Inovitro™ TT Field Laboratory Bench System. Inovitro™ includes a TT field generator and a base plate containing eight ceramic dishes per plate. Cells are placed on a 22 mm round coverslip disposed inside each dish. In each dish, TT fields are applied using two pairs of perpendicular transducer arrays insulated by a high dielectric constant ceramic. The orientation of the TT field in each dish is switched by 90° every second, thus covering various orientation axes of cell division.

[0005] Pyruvate kinase M2 (PKM2) is an important marker of cancer metabolic reprogramming as it catalyzes the final step of glycolysis. 1-((2-Fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine (hereinafter, [18F]DASA-23) is a radiotracer for measuring abnormally expressed PKM2 in glioblastoma (GBM). Approved treatments for tumors such as GBM include surgery, temozolomide (TMZ) chemotherapy, radiotherapy, and TT fields. And there is an important need to early evaluate whether a patient's GBM responds to a given therapy (e.g., TT field therapy).

[0006] Mannose is a monosaccharide that has been shown to inhibit tumor growth in vitro and in vivo. Gonzalez et al., Mannose impairs tumour growth and enhances chemotherapy, Nature, Vol. 563, pp. 719 - 723 (2018). Mannose and glucose share the transporters responsible for cellular uptake. (Ibid.)

Prior Art Documents

Patent Documents

[0007] [Patent Document 1] U.S. Patent No. 7,565,205 [Non-Patent Document]

[0008] [Non-Patent Document 1] Giladi M et al., Sci Rep. 2015;5:18046 [Non-Patent Document 2] Gonzalez et al., Mannose impairs tumour growth and enhances chemotherapy, Nature, Vol. 563, pp. 719 - 723 (2018) [Non-Patent Document 3] Ji et al., Tumor Biology, June 2017:1 - 11 [Non-Patent Document 4] Gao et al., J Cancer Res Clin Oncol. January 2011;137(1):65 - 72 [Non-Patent Document 5] Shin et al., Electrophoresis, Vol. 30, No. 12, June 2009, pp. 2182 - 2192 [Non-Patent Document 6] Shi et al., Cancer Science, Vol. 101, No. 6, June 2010, pp. 1447 - 1453 [Summary of the Invention] [Means for Solving the Problems]

[0009] The present inventors have determined that (1) a PKM2 probe (e.g., [18F]DASA - 23, etc.) can be used to determine the sensitivity of a patient to the treatment of glioblastoma in a TT field, and (2) a synergistic result is provided by treating a tumor (e.g., glioblastoma) with a combination of mannose and a TT field. The present inventors have also determined that labeled mannose can be used as a probe for detecting changes in glioblastoma metabolism and for determining the sensitivity of a patient to the treatment of glioblastoma with a TT field and mannose.

[0010] Aspects described in this specification include the steps of administering a PKM2 probe to a patient having glioblastoma; measuring a first level of PKM2 uptake in cells derived from glioblastoma; exposing the glioblastoma to treatment after the measurement of the first level using an alternating electric field having a frequency between 100 and 500 kHz; measuring a second level of PKM2 uptake in cells derived from glioblastoma after exposing the glioblastoma to the alternating electric field; and determining whether the patient has sensitivity to treatment using the alternating electric field based on whether the first level is at least 5% higher than the second level, thereby providing a method for determining a patient's sensitivity to treatment of glioblastoma with an alternating electric field.

[0011] A further aspect provides a method for reducing the viability of glioblastoma cells by the steps of administering a PKM2 probe to glioblastoma cells of a patient having glioblastoma; measuring a first level of PKM2 expression or uptake of the PKM2 probe in the glioblastoma cells; after measuring the first level, exposing the glioblastoma cells to an alternating electric field having a frequency between 100 and 500 kHz for a first time; measuring a second level of PKM2 expression or uptake of the PKM2 probe in the glioblastoma cells after the first time; and continuing to expose the glioblastoma cells to the alternating electric field when the first level is at least 5% higher than the second level.

[0012] In some examples, methods are provided for determining a patient's susceptibility to treatment of glioblastoma by an alternating electric field. These methods include administering mannose labeled with an imaging probe to cells of a patient having glioblastoma; measuring a first level of uptake of the mannose labeled with the imaging probe in the glioblastoma cells; after measuring the first level, treating the glioblastoma with an alternating electric field having a frequency between 100 and 500 kHz for a first time; measuring a second level of uptake of the mannose labeled with the imaging probe in the glioblastoma cells after the first time; and continuing treatment of the glioblastoma using the alternating electric field if the first level is at least 10% lower than the second level.

[0013] Exemplary methods are provided for reducing the viability of glioblastoma cells. These methods include administering mannose to cells of a patient having glioblastoma and then exposing the glioblastoma cells to an alternating electric field having a frequency between 100 and 500 kHz.

[0014] Aspects described herein provide a method for determining a patient's susceptibility to treatment of cancer with an alternating electric field by administering a PKM2 probe to a patient having cancer; measuring a first level of PKM2 uptake in the patient's cancer cells; after measuring the first level, exposing the cancer cells to treatment with an alternating electric field having a frequency between 100 and 500 kHz; measuring a second level of PKM2 uptake in the cancer cells; and determining whether the patient is susceptible to treatment with the alternating electric field based on whether the first level is at least 5% higher than the second level.

[0015] Exemplary methods are provided for determining a patient's susceptibility to treatment of cancer using an alternating electric field. These methods include administering to the patient mannose labeled with an imaging probe; measuring a first level of uptake of the mannose labeled with the imaging probe in cancer cells derived from the patient; after measuring the first level, treating the cancer cells with an alternating electric field at a frequency between 100 and 500 kHz for a first time; after the first time, measuring a second level of uptake of the mannose labeled with the imaging probe in the cancer cells; and continuing treatment of the cancer cells with the alternating electric field if the first level is at least 10% lower than the second level.

[0016] Aspects described herein provide a method of reducing the viability of cancer cells by administering mannose to a patient having cancer and exposing cancer cells derived from the patient to an alternating electric field at a frequency between 100 and 500 kHz.

[0017] Further aspects provide a method of reducing the viability of cancer cells by administering a PKM2 probe to a patient having cancer; measuring a first level of PKM2 expression or uptake of the PKM2 probe in cancer cells derived from the patient; after measuring the first level, exposing the cancer cells to an alternating electric field at a frequency between 100 and 500 kHz for a first time; after the first time, measuring a second level of PKM2 expression or uptake of the PKM2 probe in the cancer cells; and administering a chemotherapeutic agent to the cancer cells if the first level is at least 5% higher than the second level. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

Figure 1

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Figure 9

Figure 10

Figure 11

Figure 12A

Figure 12B

DETAILED DESCRIPTION OF THE INVENTION

[0019] All references cited herein, including but not limited to patents and patent applications, are hereby incorporated by reference in their entirety.

[0020] [18F]DASA-23 can be used to detect metabolic changes in GBM responsive to TMZ and TT field therapy. In one experiment, human U87 GBM cells were subjected to a 200 kHz TT field, IC 50 of TMZ, or vehicle for 3 or 6 days (n≧3 / condition), and then [18F]DASA-23 uptake was evaluated (e.g., Figures 2 and 6). Immunofluorescence for PKM2 was performed to confirm the [18F]DASA-23 uptake results (e.g., Figure 9). Western blot analysis was performed to determine the effects of TMZ and TT field exposure on PKM2 expression. Two-way ANOVA with multiple comparisons was performed (e.g., Figure 8). Data are reported as mean ± SD.

[0021] The TT field reduces PKM2 expression in GBM and shows a conversion from abnormal glycolysis (i.e., the Warburg effect) to oxidative phosphorylation, as confirmed by radioactive tracer uptake, Western blot, and immunofluorescence assays. PKM2 expression is a biomarker of this conversion (e.g., FIGS. 2, 6-9). The embodiments described herein provide a non-invasive assessment of the glycolytic response of GBM to various therapies using [18F]DASA-23.

[0022] Further embodiments provide a method of inhibiting the growth of GBM cells and providing a synergistic inhibition of GBM cells by administering mannose and a TT field to glioblastoma cells of a patient in need of treatment (e.g., FIGS. 10 and 11).

[0023] An important feature of cancer cells is the metabolic peculiarity known as the Warburg effect, whereby tumor cells prefer fermentation as an energy source over the more efficient mitochondrial pathway of oxidative phosphorylation (OxPhos) even in the presence of oxygen. Normal tissues use only this less efficient pathway in the absence of oxygen. Biochemically, this metabolic reprogramming appears at several stages of the glycolytic pathway. Among them are notable changes in the expression and distribution of membrane-associated glucose transporters and alterations in the activity and expression of the main enzymes involved in the final stages of glycolysis, particularly pyruvate kinase.

[0024] [18F]DASA-23 radiotracer has been developed to measure the expression of PKM2, while [18F]deoxyglucose ([18F]-FDG) is used to monitor the promotion of glucose uptake into cancer cells. Glioblastoma (GBM) is traditionally treated with surgical resection, temozolomide (TMZ) chemotherapy, and / or radiotherapy. Application of tumor treatment electric fields (TT fields), i.e., alternating electric fields to tumors (e.g., 100 - 500 kHz, 1 - 4 V / cm), is the fourth recommended treatment in GBM. There is an important need to early evaluate whether a patient's GBM responds to a given treatment including, but not limited to, TT field therapy.

[0025] [18F]DASA-23 was evaluated for its ability to detect metabolic changes responsive to TMZ and TT field therapy in human GBM cell cultures and orthotopic mouse models (Figure 6). There was a significant interaction between treatment (vehicle, TMZ, or TT field) and treatment duration (3 or 6 days) with respect to PKM2 expression as measured by intracellular uptake of [18F]DASA-23 (p = 0.005, two-way ANOVA) (Figure 6).

[0026] From immunofluorescence of PKM2 in U87-MG cells exposed and not exposed to TT fields, a decrease in cell number and lower intensity of PKM2 staining by TT fields were revealed.

[0027] Mannose and TT fields interact synergistically to reduce the viability of GBM cells

[0028] Mannose is known to occupy the same transporter system as that of glucose. Therefore, it acts as a competitive inhibitor against glucose for glucose transporters. It has been shown to directly affect cell proliferation by inhibiting the glycolytic metabolism of glucose and thus altering cancer metabolism. The inventors conducted a combination intervention with mannose and TT field and showed a significant synergistic interaction between the two interventions regarding the decrease in the number of glioblastoma cells.

[0029] From the mannose + TT field data (Figures 10 and 11), it is suggested that the TT field affects the metabolic pathways involving glucose and mannose uptake. Without being bound by this theory, the TT field is thought to induce the conversion from abnormal glycolysis (the so-called "Warburg effect") to normal oxidative phosphorylation. In one aspect, mannose labeled with an imaging probe could have both diagnostic and therapeutic effects (i.e., theranostic). The mannose + TT field data was generated as follows:

[0030] Proliferation conditions for human glioblastoma cells

[0031] U87-MG and MDA-MB-231 were grown in DMEM (Invitrogen / Life Technologies, Carlsbad, CA, USA / 10% FBS / and 1X Antibiotic-Antimycotic) and 1X antibiotic / antimycotic. GBM2 and GBM39 were grown in a defined serum-free medium of a 1:1 mixture of Neurobasal-A medium (1X) / DMEM / F12 (1X) which also contained HEPES buffer solution (10 mM), MEM sodium pyruvate solution 1 mM, MEM non-essential amino acid solution 10 mM (1X), GlutaMAX-I supplement (1X) and Antibiotic-Antimycotic (1X). These solutions were obtained from Invitrogen / Life Technologies (Carlsbad, CA, USA). The complete functional medium also contained H-EGF (20 ng / mL), H-FGF-basic-154 (20 ng / mL), H-PDGF-AA (10 ng / mL), H-PDGF-BB (10 ng / mL) and heparin solution, 0.2% (2 μg / mL) as growth factors (all from Shenandoah, Warwick, PA, USA), and B-27 (Invitrogen / Life Technologies, Carlsbad, CA, USA) as a supplement.

[0032] Proliferation experiments in the Inovitro(™) system

[0033] In this aspect, 50,000 single cells were suspended in 200 μL of medium and seeded in the center of a 22 mm diameter cover glass. The cover glass was placed in a 6-well plate and incubated in a conventional tissue culture incubator (37 °C, 95% air, 5% CO 2) It was incubated overnight. Once the cells adhered to the cover glass, an additional 2 mL of medium was added to each well. The cells were maintained on the cover glass for 2 - 3 days to acquire a growth phase before being transferred to a ceramic dish of the Inovitro™ system, and then mounted on an Inovitro™ base plate (Novocure Ltd., Haifa, Israel). A TT field set at 1 - 4 V / cm was applied by an Inovitro™ power generator, with the frequency ranging from 50 - 500 kHz. The incubation temperature ranged from 20 - 27 °C at a target temperature of 37 °C for the ceramic dish during the application of the TT field. The cultures were incubated during a 24 - hour control period prior to treatment. The treatment time was continuous for 1 - 6 days, after which the cover glass was removed and the cell number per cover glass was determined. Throughout the experiment, the culture medium was manually changed every 24 hours. Equivalent cover glasses within the ceramic dish were placed in a conventional tissue culture incubator (37 °C, 5% CO 2 ) to conduct corresponding control experiments and grow the cells in parallel with the cover glasses exposed to the TT field. Unless otherwise noted, all experiments per condition were in triplicate samples, with 4 measurements (cell number) per sample.

[0034] The term "[18F]DASA - 23" refers to 1 - ((2 - fluoro - 6 - [18F]fluorophenyl)sulfonyl)-4 - ((4 - methoxyphenyl)sulfonyl)piperazine having the following chemical structure:

[0035]

Chem.

[0036] and its pharmaceutically acceptable salts. [18F]DASA - 23 can be used in combination with a pharmaceutically acceptable carrier for administration to a patient.

[0037] As used herein, the term "reducing the viability of cancer cells" or "reducing the viability of glioblastoma cells" refers to reducing the growth, proliferation, or survival of cancer cells (e.g., GBM cells). In some embodiments, reducing the viability of cancer cells includes reducing the clonogenic survival rate of cancer cells, increasing the cytotoxicity of cancer cells, inducing apoptosis in cancer cells, and reducing the tumor volume of tumors formed from at least a portion of cancer cells.

[0038] Aspects described herein provide a method for determining a patient's susceptibility to treatment of glioblastoma with TT fields (e.g., alternating electric fields), comprising administering a PKM2 probe to a patient having glioblastoma, measuring a first level of PKM2 uptake in cells derived from the glioblastoma, exposing the glioblastoma to TT field treatment (e.g., alternating electric fields), measuring a second level of PKM2 uptake in cells derived from the glioblastoma, and determining whether the patient is susceptible to treatment with TT fields based on whether the first level is at least 5% (e.g., at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90%) higher than the second level. In this aspect, a decrease in the level of PKM2 uptake indicates a metabolic change that renders glioblastoma cells susceptible to treatment with TT fields.

[0039] The PKM2 probe can be administered to the patient by any suitable method known in the art (e.g., injection, oral administration, and ex vivo). In some examples, glioblastoma or tumor cells can be removed from the patient (e.g., by biopsy), and measurement of PKM2 expression or uptake can be performed in cell culture, for example, before and after exposure to TT fields.

[0040] A further aspect provides a method of reducing the viability of glioblastoma cells, comprising administering a PKM2 probe to cells of a patient having glioblastoma, measuring a first level of PKM2 expression or uptake of the PKM2 probe in the glioblastoma cells, exposing the glioblastoma cells to a TT field for a first time, measuring a second level of PKM2 expression or uptake of the PKM2 probe in the glioblastoma cells after the first time, and continuing the exposure of the glioblastoma cells to the TT field when the first level is at least 5% (e.g., at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90%) higher than the second level. In this aspect, a decrease in the level of PKM2 uptake as described herein indicates a metabolic change that renders glioblastoma cells sensitive to treatment with a TT field. In one aspect, the PKM2 probe comprises [18F]DASA-23.

[0041] Yet a further aspect provides a method of determining a patient's sensitivity to treatment of glioblastoma with a TT field, comprising administering to cells of a patient having glioblastoma mannose labeled with an imaging probe, measuring a first level of uptake of the imaging probe-labeled mannose in the glioblastoma cells, exposing the glioblastoma cells to a TT field for a first time, measuring a second level of uptake of the imaging probe-labeled mannose in the glioblastoma cells after the first time, and continuing the exposure of the glioblastoma cells to the TT field and mannose when the first level is at least 5% (e.g., at least 10, 15, 20, 30, 40, 50, 60, 70, 90, or 100%) lower than the second level. In this aspect, an increase in the level of mannose uptake as described herein indicates a metabolic change that renders glioblastoma cells sensitive to treatment with a TT field.

[0042] As shown in Figure 6, the expression of PKM2 in U87 glioblastoma cells over 30 minutes is reduced by at least 10% after treatment with chemotherapy (TMZ - temozolomide) and by at least 10% after exposure to TT fields.

[0043] Figure 7 shows that after exposure to TT fields, (1) the [18F]DASA - 23 uptake decreases by at least 5% over 30 minutes and by at least 10% over 60 minutes in U87 cells, and (2) the [18F]DASA - 23 retention rate decreases by at least 20% over 60 minutes.

[0044] Figure 8 shows that exposure to TT fields reduces the expression of PKM2 in U87 cells by about 50% after 3 days and by about 80% after 6 days as detected by Western blot.

[0045] Figure 9 shows that exposure to TT fields reduces the expression of PKM2 in U87 cells as detected by immunofluorescence with blue (dark) indicating DAPI nuclear staining and green (bright) indicating PKM2. The reduction in PKM2 expression is greater than 50%.

[0046] As shown in Figures 10 and 11, there is an approximately 10 - fold difference in IC50 between treatment with mannose alone and in combination with TT fields. Without being bound by theory, this effect is thought to be reduced by approximately 5 - fold in vivo and, for example, by the use of a mannose PET (positron emission tomography) probe. Therefore, it is thought that an approximately 20% increase in mannose uptake results in a metabolic treatment response to mannose + TT fields.

[0047] As shown in FIGS. 12A-12B, application of the TT field reduces the PKM2 protein level. FIGS. 12A and 12B show the results of an exemplary experiment comparing the levels of PKM2 protein in lysates generated from control OVCAR3 human ovarian adenocarcinoma cells among (1) cells subjected to 72 hours of TT field application, (2) cisplatin at 300 nM, (3) a combination of cisplatin and the TT field, and (4) a control, in a Western blot. As seen in FIG. 12A, the Western blot results were quantified relative to housekeeping gene expression (GAPDH).

[0048] The OVCAR-3 cell line was obtained from ATCC. Cells were cultured in ATCC-formulated RPMI-1640 medium, catalog number 30-2001, supplemented with 0.01 mg / ml bovine insulin, 20% fetal bovine, and antibiotics. 30,000 single cells were suspended in 500 μL of medium and seeded in the center of a 22 mm diameter coverslip.

[0049] For induction of 72 hours of TT field application, the coverslip was placed in a ceramic dish of the Inovitro™ system and incubated overnight in a conventional tissue culture incubator (37° C., 95% air, 5% CO 2 2). Once the cells had adhered to the coverslip, an additional 1.5 mL of medium was added to each well and covered with paraffin (P7793, Sigma Aldrich) to avoid evaporation of the medium. Cisplatin was added at a final concentration of 300 nM. After overnight incubation, the dish was mounted on an Inovitro™ baseplate (Novocure Ltd., Haifa, Israel). A TT field set at 1-6 V / cm was applied by an Inovitro™ power generator, with a frequency of 200 kHz. The incubation temperature was 18° C. at a target temperature of 37° C. for the ceramic dish during application of the TT field. Equivalent coverslips within the ceramic dish were placed in a conventional tissue culture incubator (37° C., 5% CO 2By placing it in ), a corresponding control experiment was conducted, and cells were grown in parallel with the cover glass exposed to the TT field.

[0050] Cell Lysates and Immunoblotting

[0051] After TT field application, cells were transferred to cold PBS plates for washing.

[0052] RIPA lysis buffer (R0278, Sigma-Aldrich) supplemented with a cocktail of protease inhibitors (cOmplete Mini, Roche) and phosphatase inhibitors (Halt #78420, Thermo Scientific) was added to the plates, and cells were scraped with approximately 100 μl of adjusted RIPA buffer for 8 Inovitro™ dishes.

[0053] The extracts were shaken for a period of 30 minutes at 4 °C. The samples were centrifuged (20 minutes, 14,000 rpm, 4 °C). The supernatant was transferred, and the protein concentration was determined using a BCA protein assay kit (BCA Protein Assay Kit, ab102536, Abcam).

[0054] After determining the protein concentration, 30 μg of protein was lysed under reducing conditions (Bolt Sample Reducing Agent, #2060435 and Sample Buffer #2045289, Novex), and the samples were boiled at 100 °C for 5 minutes. The samples were run on SDS polyacrylamide gel electrophoresis (Bolt 8% Bis-Tris based gel NW00080BOX, Thermo-Fischer).

[0055] After electrophoresis, the proteins were transferred to a 0.2 μm polyvinylidene difluoride membrane (Immuno-Blot PVDF #162-0177, Bio-Rad) and probed with appropriate primary antibodies: GAPDH (SC-32233, Santa Cruz) and PKM2 (ab137852, abcam), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit 7074, Cell Signaling and goat anti-mouse 7076, Cell Signaling) and chemiluminescent substrate (WBLUF0100, Sigma-Aldrich). Band quantification was performed using Image J software.

[0056] In another aspect, there is provided a method of reducing the viability of glioblastoma cells, comprising the steps of administering mannose to cells of a patient having glioblastoma and exposing the glioblastoma cells to TT fields.

[0057] Aspects described herein provide a method of reducing the viability of cancer cells (e.g., GBM) by administering mannose to the cancer cells and applying an alternating electric field to the cancer cells, wherein the alternating electric field has a frequency between 100 and 500 kHz. In some aspects, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step.

[0058] A further aspect provides a method for determining a patient's susceptibility to treatment of cancer with an alternating electric field, comprising the steps of administering a PKM2 probe to a patient having cancer, measuring a first level of PKM2 uptake in cancer cells derived from the patient, exposing the cancer cells to treatment with an alternating electric field at a frequency between 100 and 500 kHz, measuring a second level of PKM2 uptake in the cancer cells, and determining whether the patient is susceptible to treatment with the alternating electric field based on whether the first level is at least 5% (e.g., at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 90, or 100%) higher than the second level. In this aspect, a decrease in the level of PKM2 uptake as described herein indicates a metabolic change that renders glioblastoma cells sensitive to treatment with TT fields.

[0059] Another aspect provides a method for reducing the viability of cancer cells, comprising the steps of administering a PKM2 probe to cancer cells derived from a patient having cancer, measuring a first level of PKM2 expression or PKM2 probe uptake in the cancer cells, exposing the cancer cells to an alternating electric field at a frequency between 100 and 500 kHz for a first time, measuring a second level of PKM2 expression or PKM2 probe uptake in the cancer cells after the first time, and continuing to expose the cancer cells to the alternating electric field if the first level is at least 5% (e.g., at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 90, or 100%) higher than the second level. In this aspect, a decrease in the level of PKM2 uptake as described herein indicates a metabolic change that renders glioblastoma cells sensitive to treatment with TT fields.

[0060] In any of the above aspects, the PKM2 probe may optionally comprise [18F]DASA-23 having the following structure:

[0061]

Chemical Structure

[0062] A further aspect provides a method of determining a patient's susceptibility to treatment of cancer with an alternating electric field, comprising the steps of administering mannose labeled with an imaging probe to cells of a patient having cancer; measuring a first level of uptake of the mannose labeled with the imaging probe in the cancer cells; treating the cancer cells with an alternating electric field at a frequency between 100 and 500 kHz for a first time; after the first time, measuring a second level of uptake of the mannose labeled with the imaging probe in the cancer cells; and continuing treatment of the cancer with the alternating electric field when the first level is at least 5% (e.g., at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 90, or 100%) lower than the second level. In this aspect, an increase in the level of mannose uptake as described herein indicates a metabolic change that renders glioblastoma cells sensitive to treatment with TT fields.

[0063] Another aspect provides a method of reducing the viability of cancer cells, comprising the steps of administering mannose to a patient having cancer and exposing the patient's cancer cells to an alternating electric field at a frequency between 100 and 500 kHz.

[0064] Aspects described herein provide a method of delivering mannose to cancer cells (e.g., GBM cells) at a therapeutically effective concentration, wherein the alternating electric field has a field strength of at least 1 V / cm in at least a portion of the cancer cells.

[0065] The term "therapeutically effective concentration" as used herein refers to a concentration of mannose sufficient to achieve the intended purpose (e.g., treatment of cancer, treatment of GBM). In one aspect, the therapeutically effective concentration of mannose is between 1 and 10 mM.

[0066] In another aspect, the step of applying the electric field has a duration of at least 72 hours. The application of the 72-hour electric field may be achieved in a single 72-hour interval. Alternatively, the application of the electric field may be interrupted by short breaks. For example, six sessions each having a duration of 12 hours with a 2-hour break between sessions may be conducted. In another aspect, the step of applying the electric field has a duration of at least 4 hours.

[0067] In yet another aspect, the frequency of the alternating electric field is between 180 and 220 kHz. In another aspect, mannose is delivered to cancer cells at a therapeutically effective concentration, and the alternating electric field has a field strength of at least 1 V / cm in at least a portion of the cancer cells.

[0068] In yet another aspect, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step.

[0069] In a further aspect, mannose is delivered to cancer cells at a therapeutically effective concentration, and the alternating electric field has a field strength of at least 1 V / cm in at least a portion of the cancer cells. Optionally, the applying step has a duration of at least 72 hours, and the frequency of the alternating electric field is between 180 and 220 kHz. Optionally, at least a portion of the applying step may be performed simultaneously with at least a portion of the administering step.

[0070] Aspects described herein include the steps of administering a PKM2 probe to a patient having cancer; measuring a first level of PKM2 expression or PKM2 probe uptake in cancer cells derived from the patient; exposing the cancer cells to an alternating electric field at a frequency between 100 and 500 kHz for a first time; measuring a second level of PKM2 expression or PKM2 probe uptake in the cancer cells after the first time; and, when the first level is at least 5% (e.g., at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 90, or 100%) higher than the second level, administering a chemotherapeutic agent (e.g., tamoxifen, cisplatin, 5-fluorouracil (5-FU), and docetaxel) to the cancer cells to reduce the viability of the cancer cells. In some examples, the chemotherapeutic agent is cisplatin. In some examples, the method further includes the step of continuing to expose the cancer cells to the alternating electric field.

[0071] Without being bound by theory, lower [18F]DASA-23 uptake is thought to correlate with a decrease in PKM2 activity, and indeed, Western blot analysis of protein samples from treated cells revealed lower PKM2 expression after treatment (Figures 8, 12A - 12B). Significantly lower [18F]DASA-23 uptake has been shown following TT field application in glioma cells. Decreasing PKM2 expression is thought to increase the sensitivity of cancer cells to various cytotoxic agents where resistance to treatment has been shown to be associated with increased PKM2 activity. Ji et al., Tumor Biology, June 2017: 1 - 11; Gao et al., J Cancer Res Clin Oncol. January 2011; 137(1): 65 - 72; Shin et al., Electrophoresis, Vol. 30, No. 12, June 2009, 2182 - 2192; Shi et al., Cancer Science, Vol. 101, No. 6, June 2010, 1447 - 1453. For example, after decreasing PKM2 expression, combining TT field treatment with a chemotherapeutic agent may increase the treatment effect and may decrease the therapeutically effective dose of the chemotherapeutic agent.

[0072] The in vitro experiments described herein were conducted using the Novocure Inovitro™ system. In these experiments, the direction of the alternating electric field was switched between two perpendicular directions at intervals of 1 second. However, in alternative embodiments, the direction of the alternating electric field can be switched at a faster rate (e.g., at intervals between 1 and 1000 ms) or at a slower rate (e.g., at intervals between 1 and 100 seconds).

[0073] In the in vitro experiments described herein, the direction of the alternating electric field was switched between two perpendicular directions by applying an alternating voltage to two pairs of electrodes that were arranged 90° apart from each other in a 2D space in an alternating order. However, in alternative embodiments, the direction of the alternating electric field can be switched between two non-perpendicular directions or between three or more directions (assuming additional pairs of electrodes are provided) by repositioning the pairs of electrodes. For example, the direction of the alternating electric field can be switched between three directions, each of which is determined by the arrangement of its own pair of electrodes. Optionally, these three pairs of electrodes can be arranged such that the resulting electric fields are arranged 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 arrangements described in U.S. Patent No. 7,565,205, which is incorporated herein by reference. In other alternative embodiments, the direction of the electric field is constant.

[0074] In the in vitro experiments using the Inovitro™ system described herein, the Inovitro™ system used conductive electrodes disposed on the outer surface of the sidewall of the dish, and since the ceramic material of the sidewall acts as a dielectric, the electric field was capacitively coupled to 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 such that the conductive electrodes are disposed on the inner surface of the sidewall instead of on the outer surface of the sidewall).

[0075] The methods described herein can also be applied in an in vivo environment by applying an alternating electric field to a target region of a living subject's body (e.g., using the Novocure Optune® system). This can be achieved, for example, by placing electrodes on or under the subject's skin such that an alternating electric field is imposed on the target region of the subject's body by the application of an alternating voltage between a selected subset of these electrodes.

[0076] For example, in a situation where the relevant cells are located in the subject's brain, a pair of electrodes could be placed in front of and behind the subject's head, and a second pair of electrodes could be placed on the right and left of the subject's head. In some embodiments, the electrodes are capacitively coupled to the subject's body (e.g., by using electrodes that include a conductive plate and have a dielectric layer disposed 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 could be inserted subcutaneously under the patient's skin. The alternating voltage generator applies an alternating voltage at a selected frequency (e.g., 200 kHz) between the left and right electrodes for a first time period (e.g., 1 second), which induces an alternating electric field in which the most significant component of the lines of force is parallel to the transverse axis of the subject's body.

[0077] Next, the alternating voltage generator applies an alternating voltage between the front and rear electrodes at the same frequency (or a different frequency) for a second time period (e.g., 1 second), which induces an alternating electric field in which the most important component of the electric field lines is parallel to the sagittal axis of the object body. This two-step sequence is then repeated for the duration of the treatment. Optionally, the electrodes may include temperature sensors, and the alternating voltage generator may be set such that the amplitude of the alternating voltage applied to the electrodes decreases if the temperature sensed at the electrodes is too high. In some embodiments, one or more additional pairs of electrodes may be added and included in the sequence. In alternative embodiments, only a single pair of electrodes is used, in which case the direction of the electric field lines cannot be switched. Note that any of the parameters for this in vivo embodiment (e.g., frequency, field strength, duration, direction switching rate, and electrode placement) may vary as described above in relation to the in vitro embodiment. However, in the in vivo environment, care must be taken to ensure that the electric field always remains safe for the subject.

[0078] Note that in the experiments described herein, the TT field was applied for a continuous time period (e.g., 72 hours or 14 days). However, in alternative embodiments, the application of the TT field may be interrupted, preferably by short breaks. For example, a 72-hour period can be met by applying an alternating electric field to six 12-hour blocks with a 2-hour break between each block.

[0079] The present invention has been disclosed with reference to specific embodiments, but numerous modifications, changes, and variations to the described embodiments are possible without departing from the scope and scope of the invention as defined in the appended claims. Accordingly, the present invention is not limited to the described embodiments, but is intended to have the broadest scope defined by the language of the following claims or their equivalents.

[0080] (References) TIFF0007687698000003.tif67170

Claims

1. administering a PKM2 probe to a patient having glioblastoma; measuring a first level of PKM2 uptake in cells derived from glioblastoma; exposing the glioblastoma to treatment after the measurement of the first level using an alternating electric field having a frequency between 100 and 500 kHz; after exposing the glioblastoma to the alternating electric field, measuring a second level of PKM2 uptake in cells derived from glioblastoma; and determining whether the patient is sensitive to treatment using the alternating electric field based on whether the first level is at least 5% higher than the second level A combination of a PKM2 probe and an alternating electric field generator for use in a method of determining a patient's sensitivity to treatment of glioblastoma with an alternating electric field, comprising.

2. The PKM2 probe has the following structure: 【Chemical 1】 The product according to claim 1, comprising [18F]DASA-23 having.

3. The product according to claim 1, wherein the alternating electric field has a frequency between 180 and 220 kHz.

4. administering a PKM2 probe to glioblastoma cells of a patient having glioblastoma; measuring a first level of PKM2 expression or PKM2 probe uptake in glioblastoma cells; after measuring the first level, exposing the glioblastoma cells to an alternating electric field having a frequency between 100 and 500 kHz for a first time; after the first time, measuring a second level of PKM2 expression or PKM2 probe uptake in glioblastoma cells; and continuing to expose the glioblastoma cells to the alternating electric field when the first level is at least 5% higher than the second level A combination of a PKM2 probe and an alternating electric field generator for use in a method of reducing the viability of glioblastoma cells, comprising.

5. The PKM2 probe has the following structure: [Chemical 2] The product according to claim 4, comprising [18F]DASA-23 having.

6. The product according to claim 4, wherein the alternating electric field has a frequency between 180 and 220 kHz.

7. administering mannose labeled with an imaging probe to cells of a patient having glioblastoma; measuring a first level of uptake of mannose labeled with an imaging probe in glioblastoma cells; after measuring the first level, treating the glioblastoma with an alternating electric field having a frequency between 100 and 500 kHz for a first time; Measuring a second level of uptake of imaging probe-labeled mannose in glioblastoma cells after a first time; and continuing treatment of glioblastoma using an alternating electric field when the first level is at least 10% lower than the second level, A combination of an imaging probe-labeled mannose and an alternating electric field generator for use in a method for determining a patient's sensitivity to treatment of glioblastoma by an alternating electric field.

8. The combination according to claim 7, wherein the alternating electric field has a frequency between 180 and 220 kHz.

9. Administering mannose to cells of a patient having glioblastoma, and then exposing the glioblastoma cells to an alternating electric field having a frequency between 100 and 500 kHz, A combination of mannose and an alternating electric field generator for use in a method for reducing the viability of glioblastoma cells.

10. The combination according to claim 9, wherein the alternating electric field has a frequency between 180 and 220 kHz.

11. Administering a PKM2 probe to a patient having cancer; Measuring a first level of PKM2 uptake in the patient's cancer cells; After measuring the first level, exposing the cancer cells to treatment using an alternating electric field having a frequency between 100 and 500 kHz; Measuring a second level of PKM2 uptake in the cancer cells; and Determining whether the patient is sensitive to treatment using an alternating electric field based on whether the first level is at least 5% higher than the second level A combination of a PKM2 probe and an alternating electric field generator for use in a method for determining a patient's sensitivity to treatment of cancer with an alternating electric field.

12. The PKM2 probe has the following structure: [Chemical Formula 3] The combination according to claim 11, comprising [18F]DASA-23 having the structure.

13. Administering a PKM2 probe to a patient having cancer; Measuring a first level of PKM2 expression or uptake of the PKM2 probe in cancer cells derived from the patient; After measuring the first level, exposing the cancer cells to an alternating electric field having a frequency between 100 and 500 kHz for a first time; After the first time, measuring a second level of PKM2 expression or uptake of the PKM2 probe in the cancer cells; and, A step of continuing the exposure of cancer cells to an alternating electric field when the first level is at least 5% higher than the second level A combination of a PKM2 probe and an alternating electric field generator for use in a method of reducing the viability of cancer cells, comprising **Claim 14** The PKM2 probe has the following structure: 【Chemical Formula 4】 The combination according to claim 13, comprising [18F]DASA-23 having **Claim 15** A step of administering mannose labeled with an imaging probe to a patient; A step of measuring a first level of uptake of mannose labeled with an imaging probe in cancer cells derived from the patient; After measuring the first level, a step of treating the cancer cells with an alternating electric field having a frequency between 100 and 500 kHz for a first time; After the first time, a step of measuring a second level of uptake of mannose labeled with an imaging probe in the cancer cells; and A step of continuing the treatment of the cancer cells using an alternating electric field when the first level is at least 10% lower than the second level, comprising A combination of mannose labeled with an imaging probe and an alternating electric field generator for use in a method of determining a patient's sensitivity to the treatment of cancer using an alternating electric field. **Claim 16** A combination of mannose and an alternating electric field generator for use in a method of reducing the viability of cancer cells, comprising a step of administering mannose to a patient having cancer and a step of exposing cancer cells derived from the patient to an alternating electric field having a frequency between 100 and 500 kHz. **Claim 17** A step of administering a PKM2 probe to a patient having cancer; A step of measuring a first level of PKM2 expression or uptake of the PKM2 probe in cancer cells derived from the patient; After measuring the first level, a step of exposing the cancer cells to an alternating electric field having a frequency between 100 and 500 kHz for a first time; After the first time, a step of measuring a second level of PKM2 expression or uptake of the PKM2 probe in the cancer cells; and A step of administering a chemotherapeutic agent to the cancer cells when the first level is at least 5% higher than the second level A combination of a PKM2 probe, an alternating electric field generator, and a chemotherapeutic agent for use in a method of reducing the viability of cancer cells, comprising **Claim 18** The combination according to claim 17, wherein the chemotherapeutic agent is selected from the group consisting of tamoxifen, cisplatin, 5-fluorouracil (5-FU), and docetaxel.

19. The substance according to claim 18, wherein the chemotherapeutic agent is cisplatin.

20. The substance according to claim 17, further comprising the step of continuing to expose the cancer cells to an alternating electric field.

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