Method for restoring sensitivity to TTFields in TTFields-resistant cancer cells by a PTGER3 inhibitor

Abstract

To provide a method of reducing viability of TTFields-resistant cancer cells in a subject by recommending administering a Prostaglandin E Receptor 3 (PTGER3) inhibitor to the subject and applying an alternating electric field to the cancer cells of the subject, where the alternating electric field has a frequency between 100 and 500 kHz.SOLUTION: Provided herein are methods of reducing the viability of cancer cells, preventing cancer cells of a subject from developing resistance to TTFields, and restoring sensitivity of cancer cells to TTFields by recommending or prescribing a PTGER3 inhibitor to a subject and applying an alternating electric field to the cancer cells. In some instances, sensitivity of cancer cells to TTFields can be restored with one or more PTGER3 inhibitors (e.g., NSAIDs, cox2 inhibitors).SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application Nos. 62 / 826,078, filed Mar. 29, 2019, and 62 / 849,535, filed May 17, 2019, each of which is incorporated herein by reference in its entirety. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Background Art

[0002] Tumor Treating Fields (TTFields) is an effective anti - tumor drug treatment modality delivered by non - invasively applying an alternating electric field of low intensity and intermediate frequency (e.g., 100 - 500 kHz). TTFields 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 arrest or delay of mitosis. Cells may die while undergoing mitotic arrest or progression into cell division, which results in the formation of normal or abnormal aneuploid progeny. The formation of tetraploid cells can occur by mitotic exit due to translational slippage or during inappropriate cell division. Abnormal daughter cells may die during subsequent interphase, undergo permanent arrest, or proliferate by additional mitoses in which they are subject to further TTFields attack. Giladi M et al. Sci Rep. 2015;5:18046.

[0003] In an in vivo setting, TTFields therapy can be delivered using a wearable 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 placed on the skin and connected to the device and the battery. The treatment is designed to be worn for as much time as possible throughout the day and night.

[0004] In pre - clinical settings, TTFields can be applied in vitro, for example, using the Inovitro™ TTFields Laboratory Bench System. Inovitro™ includes a TTFields generator and a base plate containing eight ceramic dishes per plate. Cells are placed on cover glasses positioned inside each dish. TTFields are applied using two orthogonal pairs of transducer arrays insulated by the high - dielectric - constant ceramic of each dish. The orientation of TTFields in each dish switches by 90° every second, thus covering different axes of cell division.

[0005] Glioblastoma (GBM) is the most common and lethal malignant brain cancer in adults, despite surgery and aggressive chemoradiotherapy. Tumor Treating Fields (TTFields) are approved for newly diagnosed GBM in combination with adjuvant temozolomide chemotherapy. Application of TTFields has resulted in a significant improvement in overall survival. TTFields are low - intensity alternating electric fields thought to disrupt the mitotic macromolecular assembly, leading to disrupted chromosome segregation and cell death. However, treatment resistance occurs in many TTFields responders.

[0006] Several human GBM cell lines have been developed that demonstrate relative resistance to the cytotoxic effects of TTFields compared to parental cells.

Prior Art Documents

Non - Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0008] It is recommended to administer a prostaglandin E receptor 3 (PTGER3) inhibitor, and by applying an alternating electric field to the target cancer cells, a method for reducing the survival rate of TTFields-resistant cancer cells in a subject is provided. The alternating electric field has a frequency between 100 and 500 kHz.

[0009] In some examples, it is recommended to administer a PTGER3 inhibitor, and by applying an alternating electric field to the target cancer cells, a method for preventing the development of resistance to the alternating electric field in the target cancer cells is provided. The alternating electric field has a frequency between 100 and 500 kHz.

[0010] In some examples, a method for restoring the sensitivity of TTFields-resistant cancer cells in a subject to TTFields is provided by recommending administration of a PTGER3 inhibitor to the subject. In this aspect, the sensitivity of the TTFields-resistant cancer cells in the subject to TTFields is substantially restored.

[0011] In some examples, a method for preventing the development of resistance to an alternating electric field in a subject's cancer cells is provided by formulating a PTGER3 inhibitor for the subject and applying an alternating electric field to the cancer cells. The alternating electric field has a frequency between 100 and 500 kHz.

[0012] In some examples, a method of restoring sensitivity to TTFields in a subject's TTFields-resistant cancer cells by prescribing a PTGER3 inhibitor. In this aspect, the sensitivity of the subject's TTFields-resistant cancer cells to TTFields is restored after the PTGER3 inhibitor is administered to the subject.

[0013] In some examples, it is recommended to administer to a subject an inhibitor of a target in a resistance pathway controlled by EP-3, and a method is provided for reducing the survival rate of TTFields-resistant cancer cells in a subject by applying an alternating electric field to the subject's cancer cells. The alternating electric field has a frequency between 100 and 500 kHz.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0015] TTFields is an effective anti-tumor drug treatment modality delivered by non-invasively applying an alternating electric field of low intensity and intermediate frequency (e.g., 100 - 500 kHz). However, under certain circumstances, tumor cells can develop resistance to TTFields treatment, which may lead to a decrease in effectiveness under these circumstances. In some embodiments, resistance to TTFields is associated with an increase in "stemness" or a phenotype similar to stem cells. Stemness can be measured by the expression of stem cell markers such as CD44, as well as the ability to grow in serum-free medium as 3D spheres and the ability to form brain tumors when implanted into the brains of immunosuppressed mice.

[0016] Importantly, TTFields-induced chromosomal instability, such as the formation of micronuclei in the cytoplasm, is preserved in resistant cells compared to sensitive counterparts, indicating that resistance to TTFields is mediated by non-biophysical mechanisms. Indeed, the TTFields-induced inflammatory response is significantly suppressed in resistant cells, supporting the process by which resistance to TTFields is conferred by the selective loss of harmful effects induced by biophysical assault. This acquired TTFields-resistant phenotype is associated with a transition to a stem-like state, as determined by the standard neurosphere assay.

[0017] Recently, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING, encoded by TMEM173) pathway, an immune-sensing molecule, has been identified as an important component of cytoplasmic DNA sensing and plays a crucial role in mediating the intracellular immune response. Ghaffari et al., British Journal of Cancer, Vol. 119, pp. 440 - 449 (2018); see, for example, Figure 3. Activation of the STING pathway mediates the immune response in response to intracellular abnormalities (e.g., the presence of cytoplasmic double-stranded DNA (dsDNA)).

[0018] Prostaglandin E receptor 3 (PTGER3) is a G protein-coupled receptor and is one of the four receptors for prostaglandin E2. PTGER3 is related to biological systems and diseases associated with inflammation, cancer, digestion, the nervous system, kidney function, blood pressure, and uterine contractions.

[0019] PTGER3 inhibitors include NSAIDs (e.g., aspirin, ibuprofen), cox2 inhibitors (e.g., celecoxib, valdecoxib, rofecoxib), L798,106, and DG041. NSAIDs are common commercially available drugs used for pain relief and inflammation reduction.

[0020] The embodiments described herein used a systems approach assisted by a series of innovative computational platforms to understand the emergence of "stemness" in resistant cells and identify key regulators of the resistance mechanism. It was found that three networks, including nervous system development regulation, inflammatory response, and cell-cell adhesion, all of which play roles in GBM stem-like cells, collapsed.

[0021] Using a unique key regulator ranking system, prostaglandin E receptor 3 (PTGER3) was identified as an important key regulator at the top of these pathways and a factor in the TTFields resistance phenotype. PTGER3 is rapidly upregulated in GBM cells when exposed to TTFields, leading treated cells away from beneficial inflammatory pathways that TTFields also activates in parallel.

[0022] The PTGER3 signaling pathway is upregulated by interaction with prostaglandin E2 (PGE2). The combination of TTFields with aspirin or other traditional NSAIDs (e.g., cox2 inhibitors) can prevent PGE2 biosynthesis and thus the activation of PTGER3 signaling. Alternatively, PTGER3 receptor antagonists (e.g., F798,106,106, ONO-AE3-240, and DG-041) can be used together with or in combination with other PTGER3 antagonists and inhibitors. Such combinations can be used to restore sensitivity to TTFields in cells that have developed resistance.

[0023] Furthermore, GBM cells treated with a PTGER3 inhibitor while being exposed to TTFields can prevent the cells from developing resistance to TTFields, for example, from about 3 weeks to more than 5 weeks later.

[0024] There is provided a method of reducing the survival rate of TTFields-resistant cancer cells in a subject by administering a prostaglandin E receptor 3 (PTGER3) inhibitor to the subject and applying an alternating electric field to the cancer cells of the subject. The alternating electric field may have a frequency between 100 and 500 kHz.

[0025] The term "reducing the survival rate" as used herein refers to reducing proliferation, inducing apoptosis, or killing cancer cells. The term "TTFields-resistant cancer cells" as used herein refers to cancer cells that show a 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% reduction in sensitivity to TTFields treatment compared to TTFields-sensitive cancer cells. The term "sensitivity" as used herein refers to the responsiveness to TTFields treatment measured, for example, by a reduction in the number of cells after treatment with TTFields.

[0026] The term "recommend" refers to a suggestion or instruction from a healthcare professional, such as a physician, physician assistant, nurse, or nurse practitioner, to a subject such as a patient.

[0027] In one example, the PTGER3 inhibitor is selected from the group consisting of one or more of an NSAID (e.g., aspirin, ibuprofen), a cox2 inhibitor (e.g., celecoxib, valdecoxib, rofecoxib), L798,106, and DG041. In one aspect, the PTGER3 inhibitor is aspirin.

[0028] In one example, the recommended concentration of the PTGER3 inhibitor in a subject is about 1 to 500 nanomolar for L798,106, about 0.1 to 2 millimolar for aspirin, about 0.5 to 50 nanomolar for DG041, or about 1 to 500 nanomolar for celecoxib. The term "recommended concentration" can refer to a recommended dosage sufficient to result in an intermittent or sustained level of the PTGER3 inhibitor in the subject. In one example, the recommended concentration of the PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks. In one example, the cancer cells are selected from the cells of glioblastoma, lung cancer, pancreatic cancer, mesothelioma, ovarian cancer, and breast cancer.

[0029] A further aspect provides a method of recommending administering a prostaglandin E receptor 3 inhibitor to a subject and applying an alternating electric field to the subject's cancer cells to prevent the subject's cancer cells from developing resistance to the alternating electric field. The alternating electric field has a frequency between 100 and 500 kHz. In one example, the alternating electric field has a frequency between 100 and 300 kHz.

[0030] In one example, the PTGER3 inhibitor is selected from the group consisting of one or more of an NSAID (e.g., aspirin, ibuprofen), a cox2 inhibitor (e.g., celecoxib, valdecoxib, rofecoxib), L798,106, and DG041. In one aspect, the PTGER3 inhibitor is aspirin.

[0031] In one example, the recommended concentration of a PTGER3 inhibitor in a subject is about 1 to 500 nanomolar for L798,106, about 0.1 to 2 millimolar for aspirin, about 0.5 to 50 nanomolar for DG041, or about 1 to 500 nanomolar for celecoxib. The term "recommended concentration" can refer to a recommended dosage sufficient to provide an intermittent or sustained level of the PTGER3 inhibitor in the subject. In one example, the recommended concentration of the PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks. In one example, the cancer cells are selected from cells of glioblastoma, lung cancer, pancreatic cancer, mesothelioma, ovarian cancer, and breast cancer.

[0032] A further aspect provides a method of restoring sensitivity to TTFields in TTFields-resistant cancer cells of a subject by recommending administration of a PTGER3 inhibitor to the subject, wherein the sensitivity to TTFields is substantially restored in the TTFields-resistant cancer cells of the subject.

[0033] The term "restoring sensitivity" refers to re - establishing the responsiveness of TTFields - resistant cells relative to the responsiveness of TTFields - sensitive cells. In this aspect, "responsiveness" is measured by counting the number of cells before and after exposure to TTFields. The term "substantially restored" refers to increasing the responsiveness of TTFields - resistant cells. In one example, the responsiveness of TTFields - resistant cells is restored by at least 10%. In one example, the responsiveness of TTFields - resistant cells is restored by at least 25%. In one example, the responsiveness of TTFields - resistant cells is restored by at least 50%.

[0034] In one example, the PTGER3 inhibitor is selected from the group consisting of one or more of an NSAID (e.g., aspirin, ibuprofen), a cox2 inhibitor (e.g., celecoxib, valdecoxib, rofecoxib), F798,106, and DG041. In one aspect, the PTGER3 inhibitor is aspirin.

[0035] In one example, the recommended concentration of a PTGER3 inhibitor in a subject is about 1 to 500 nanomolar for L798,106, about 0.1 to 2 millimolar for aspirin, about 0.5 to 50 nanomolar for DG041, or about 1 to 500 nanomolar for celecoxib. The term "recommended concentration" can refer to a recommended dosage sufficient to result in an intermittent or sustained level of the PTGER3 inhibitor in the subject. In one example, the recommended concentration of the PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks. In one example, the cancer cells are glioblastoma cells.

[0036] Yet another aspect provides a method of reducing the viability of TTFields-resistant cancer cells in a subject by prescribing a PTGER3 inhibitor to the subject and applying an alternating electric field to the cancer cells. The alternating electric field may have a frequency between 100 and 500 kHz.

[0037] The term "prescribing", as used herein, refers to a healthcare provider authorized to write prescriptions providing a prescription for a drug to a subject or transmitting the prescription to a pharmacy or other clinic.

[0038] A further aspect provides a method of preventing the development of resistance to an alternating electric field in cancer cells of a subject by prescribing a PTGER3 inhibitor to the subject and applying an alternating electric field to the cancer cells. The alternating electric field may have a frequency between 100 and 500 kHz.

[0039] Yet another aspect is a method of restoring sensitivity to TTFields in TTFields-resistant cancer cells of a subject by prescribing a PTGER3 inhibitor to the subject, wherein the sensitivity of the subject's TTFields-resistant cancer cells to TTFields is restored after administering the PTGER3 inhibitor to the subject.

[0040] In one example, it is recommended to administer an inhibitor of a target in a resistance pathway controlled by EP3, and a method is provided for reducing the survival rate of TTFields-resistant cancer cells in a subject by applying an alternating electric field to the cancer cells of the subject. The alternating electric field has a frequency between 100 and 500 kHz.

[0041] In one example, the targets in the resistance pathway controlled by EP3 are selected from the group consisting of ZNF488 and PRDM8. Examples of inhibitors of PRDM8 include, but are not limited to, azacitidine and decitabine.

[0042] As shown in Figure 1A, human LN827 glioblastoma cells were treated with TTFields at 200 kHz for 24 hours and then fixed with 4% PFA for 20 minutes. The cells were stained with DAPI (4',6-diamidino-2-phenylindole) at a dilution rate of 1:5000 and incubated at room temperature for 5 minutes to stain the nuclei and micronuclei. Micronuclei can be shown in cells treated with TTFields (lower panel) (arrows). The bar graph shows an approximately 15% increase in the micronuclear structure.

[0043] Figure 1B shows the induction of the inflammatory STING and pyroptosis pathways by dsDNA (double-stranded DNA). dsDNA can be generated from micronuclei induced by abnormal mitosis. Abnormal mitosis can be induced, for example, by TTFields. TTFields can also reduce the integrity of the nuclear envelope, as shown by the disruption of the lamin B1 structure, and lead to the induction of dsDNA in the cytoplasm and the STING pathway as shown.

[0044] In the experiments summarized in Figure 2, TTFields-resistant human GBM cell lines were generated by seeding LN827 cells at a density of 10,000 cells / ml and treated with TTFields repeatedly at a frequency of 200 kHz in a one-week cycle. In each cycle, the cells were counted on days 2, 4, and 7. At the end of each cycle, the cells were reseeded at the same density as on day 0 and collected and processed for RNAseq and lyophilization for further analysis. Resistant cells emerged at least 4 weeks after TTFields treatment. As shown in Figure 2, the cell number was significantly higher in the cells treated with TTFields after the 5th week compared to the previous week, demonstrating the generation of cells resistant to the cell number reduction effect of TTFields in non-resistant cells.

[0045] Human GBM cell lines were treated with or without TTFields at a frequency of 200 kHz for one week (TTF = sensitive cells; R-TTF = resistant cells) (Figure 3). Cells were analyzed for size (flow cytometry), micronucleus structure (immunofluorescence), and type I interferon response genes (qPCR). As shown in the left panel, U87C (sensitive) and U87R (resistant) did not show a change in cell size. However, the right panel (top) shows that micronuclei were still formed (comparing TTF and R-TTF). Interferon-stimulated gene 15 (ISG15), an important type I interferon response gene, was no longer produced in the U87R and LN827R resistant cell lines.

[0046] Figure 4 shows that the inflammation-promoting and resistance pathways induced by TTFields are independent. The STING / AIM2 pathway was depleted in "knockdown" (KD) TTFields-resistant cell lines (U87R, LN428R, LN827R) using shRNA (short hairpin RNA). Cells were treated as shown for 3 days and cell numbers were determined by a cell counter (Bio-Rad TC10). As shown in Figure 4, resistance to TTFields is maintained even when the STING / AIM2 pathway is inhibited by shRNA (for example, compare the bar for TTFields in double KD+TTFields).

[0047] Figure 5 shows the overall gene expression changes in resistant cells in control cells, as well as exemplary changes 1 week and 5 weeks after TTFields exposure in resistant cells (LN428, LN827, and U87).

[0048] Figure 6 shows the changes in the overall gene network in resistant GBM cells using an algorithm and the association of the identified genes with phenotypes related to stemness (e.g., ERG, FOXF1, NFKBIZ, LIF, BCL3, EHF, ZNF488, SLC2A4RG, ETV4, PTGER3) and immune response (e.g., CEBPD, RCOR2, TRIM22, SLC1A3, PLSCR1, FLIl).

[0049] Figure 7A shows the nSCORE ranks for the top major regulators of resistance to TTFields identified using NETZEN (Figure 7A) over four different time points (0 hours, 6 hours, 24 hours, 1 week, 5 weeks) in three different GBM cell lines. Figure 7B shows that the expression of PTGER3 determined by RNAseq and western blotting correlates with the PTGER3 nSCORE rank. Overexpression of EP3 in the 293T cell line serves as a positive control, and B-actin serves as a loading control.

[0050] Figure 8 provides a two-dimensional view of the gene subnetworks controlled by PTGER3, 24 hours after exposure to TTFields. Figure 9 shows how the gene subnetworks controlled by PTGER3 become dominant as resistant cells overtake the cell culture (week 1 + week 5).

[0051] Figure 10 shows that upregulation by PTGER3 correlates, among three, with STING and pyroptosis activation induced by TTFields after exposure to TTFields. Quantitative RT-PCR was utilized to detect the transcriptional levels of PTGER3, IL6, and ISG15 (markers for STING and pyroptosis activation).

[0052] Figure 8 shows that upregulation of PTGER3 correlates in vivo with STING inflammatory cytokines induced by TTFields in a rat GBM model (established by Novocure). TTFields treatment was initiated 1 week after injection and continued for 1 week in an F98 rat orthotopic GBM model (Novocure). The animals were then euthanized and tumors were harvested for RNA. Quantitative RT-PCR was performed to detect the transcriptional levels of PTGER3, IL6, and ISG15.

[0053] Figure 12 exemplifies the PTGER3 pathway, including portions of the signaling pathways affected by aspirin and PTGER3 inhibitors (L798,106 and DG041), based on examples from January 1, 2017, in Markovic et al., Structural features of subtype-selective EP receptor modulators. Drug Discovery Today, Volume 22(1).

[0054] Aspirin significantly reduces the resistance to TTFields in TTFields-resistant cells. Figure 13 shows that aspirin reduces the resistance to TTFields in TTFields-resistant GBM cells (U87R, L428R, and LN827R) (comparing vehicle + TTFields with aspirin + TTFields). In the experiments summarized in Figure 13, resistant human GBM cells were treated for 3 days with either vehicle control or aspirin, with or without TTFields. The drug was replenished daily. The number of viable cells was quantified using a cell counter at the endpoint.

[0055] Therefore, patients who have developed resistance to TTFields treatment (e.g., over the course of long-term use) can reduce their resistance to TTFields and make TTFields treatment more effective over a longer period by taking aspirin (e.g., daily). Without being bound by theory, this approach is thought to be able to improve the effectiveness of TTFields in patients who develop resistance.

[0056] For example, in U87R-resistant cells, TTFields reduced the number of viable cells from 150k / dish to 125k / dish. When aspirin was applied to the cells, the number of viable cells decreased from 150k / dish to 100k / dish. In LN428R-resistant cells, TTFields reduced the number of viable cells from 85k / dish to 70k / dish. When aspirin was applied to the cells, the number of viable cells decreased from 85k / dish to 40k / dish. In LN827R cells, TTFields slightly increased the number of viable cells. When aspirin was applied to the cells, the number of viable cells decreased from 125k / dish to 90k / dish.

[0057] The PTGER3 inhibitor can restore sensitivity to TTFields. Resistant human GBM cells were treated separately with either vehicle control or the EP3 (PTGER3) inhibitor (L798,106 (left panel) or DG041 (right panel)) with or without TTFields at 200 kHz for 3 days (Figure 14). DG041 was obtained from US Biological and L798,106 was obtained from Tocris. In the experiments summarized in Figure 14, the drugs were replenished daily. The number of viable cells was quantified at the endpoint using a cell counter. As shown in Figure 14, the PTGER3 inhibitor restores sensitivity to TTFields (compare the bars for TTFields to L798,106 + TTFields and DG041 + TTFields).

[0058] As shown in Figure 14 (left panel), TTFields reduced the number of viable cells from approximately 200k / dish to 125k / dish for U87 GBM-resistant cells. When the PTGER3 inhibitor L798,106 (0.5 μM) was given to the cells, the number of viable cells was reduced from approximately 200k / dish to 25k / dish. As shown in Figure 14 (right panel), TTFields reduced the number of viable cells from approximately 160k / dish to 100k / dish for U87 GBM-resistant cells. When the PTGER3 inhibitor DG041 (50 nM) was given to the cells, the number of viable cells was reduced from approximately 160k / dish to approximately 40k / dish. This data demonstrates that the PTGER3 inhibitor can restore sensitivity to TTFields even after resistance to TTFields has occurred.

[0059] Enhanced expression of PTGER3 in TTFields-sensitive GBM cells confers resistance to TTFields. As shown in Figure 15A, human GBM cells were transduced with an empty vector control (EV) or a lentivirus expressing PTGER3 and then treated with TTFields at 200 kHz for 3 days. Viable cell numbers were quantified using a cell counter. The efficacy of EP3 overexpression was determined by Western blot (Figure 15B). Resistance was conferred only in cells overexpressing EP3 (U87 and LN428) and not in cells that were unable to overexpress EP3.

[0060] The resistance cell line generation experiment using the original human GBM cell lines from Figure 2 was repeated in more than two groups containing the PTGER3 inhibitors (L798,106 and L798,106 + TTFields) (Figure 16). The L798,106 PTGER3 inhibitor blocked the development of resistance to TTFields. Cells were seeded and counted at the same cell density and time points. Each cycle lasted for 7 days, for a total of within 5 cycles.

[0061] The results shown in Figure 17 demonstrate the presence of EP3 in the nucleus upon exposure to TTFields. These results indicate the presence or translocation of the seven-transmembrane cell surface receptor EP3 to the nucleus, where EP3 acts as a major regulator of resistance to TTFields, which have direct interactions with hundreds of genes. The majority of major regulators are transcription factors located in the nucleus.

[0062] Without being bound by theory, it is thought that EP3 is upregulated and either present in the nucleus or translocates to the nucleus upon exposure to TTFields, providing a mechanism by which EP3 can directly or indirectly regulate other genes by other transcription factors such as the neural stem factor ZNF488. Thus, EP3 is thought to regulate resistance to TTFields by promoting the generation and enrichment of GBM stem cells that are resistant to many treatment modalities (e.g., TTFields) by virtue of their slow recycling rates and other survival and anti-apoptotic pathways.

[0063] As shown in FIGS. 18A - 18B, TTFields - resistant GBM cells are enriched in a "stem - cell - like" phenotype (e.g., tumor - sphere formation and CD44 surface marker). FIG. 18A shows tumor - sphere formation in resistant cells treated with TTFields for one week, while TTFields - sensitive cells treated with TTFields for one week did not show tumor - sphere formation.

[0064] FIG. 18B shows an increase in the CD44 surface marker in TTFields - resistant cells compared to TTFields - sensitive cells. Human GBM cell lines were treated as shown, then seeded into 96 - well plates at a density of 100 cells / well in FBS - free stem - cell culture medium, cultured for 4 weeks, and stained with Calcein AM dye for 30 minutes at room temperature. Images of each well were taken in a plate reader (SpectraMax® i3x) at a wavelength of ex / em = 456 / 541 nm. The expression of CD44 was measured by FACS.

[0065] As shown in FIG. 19, TTFields - resistant cells are enriched in GBM stem cells that lead to increased tumor growth and death. TTFields - resistant cells and TTFields - sensitive cells were implanted into the brains of mice. Equal numbers of cells under each treatment condition were implanted orthotopically into the brains of NSG mice, and survival rates were measured as a proxy for tumor growth.

[0066] 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 range of the present invention as defined in the appended claims. Accordingly, the present invention is not intended to be limited to the described embodiments, but rather is intended to have the full scope defined by the language of the following claims and their equivalents.

Claims

1. A pharmaceutical composition for use in a method of reducing the survival rate of TTFields-resistant cancer cells in a subject, the composition comprising an inhibitor of a target in a resistance pathway controlled by EP3, the method comprising the steps of prescribing to the subject an inhibitor of a target in a resistance pathway controlled by EP3, and applying an alternating electric field having a frequency between 100 and 500 kHz to the cancer cells of the subject, wherein the target in the resistance pathway controlled by EP3 is selected from the group consisting of ZNF488 and PRDM8, and the inhibitor is selected from the group consisting of aspirin, L798,106 and DG041.

2. The composition according to claim 1, wherein the alternating electric field has a frequency between 100 and 300 kHz.

3. The composition according to claim 1, wherein the TTFields-resistant cancer cells are selected from the group consisting of cells of glioblastoma, lung cancer, pancreatic cancer, mesothelioma, ovarian cancer, and breast cancer.

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