Cancer therapy

Abstract

Disclosed herein is a method of promoting drug uptake via TRPC1 channels using a pulsed electromagnetic field (PEMF). Also disclosed herein are methods of treating cancer using PEMF adjuvant therapy and methods of selecting cancer patients for PEMF adjuvant therapy by detecting TRPC1 levels in a cancer sample.

Description

[0001] CANCER THERAPY

[0002] Technical field

[0003] The present invention relates, in general terms, to cancer therapy and more specifically to methods of promoting drug uptake via TRPC1 channels.

[0004] Background

[0005] Anthracyclines such as doxorubicin (DOX) are widely used chemotherapeutic agents due to their broad- spectrum efficacy against numerous cancers. However, anthracycline chemotherapy is associated with undesirable systemic side effects, particularly in tissues exhibiting elevated basal levels of mitochondrial respiration, such as cardiac and central nervous tissues. Some patients also develop resistance to anthracycline chemotherapy.

[0006] Recent efforts to mitigate anthracycline toxicity have focused on developing liposomal delivery systems to provide more targeted delivery to tumour tissues. Although liposomal DOX delivery has been shown to reduce cardiotoxicity, additional dose-limiting side effects such as mucosal and cutaneous toxicity, along with the high cost of liposomal preparations, may limit their wider clinical adoption. There is thus an unmet need for anthracycline therapies with reduced side effects and comparable or improved efficacy.

[0007] It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

[0008] Summary

[0009] Disclosed herein is a method of promoting Transient Receptor Potential Channel 1 (TRPC1) channel-mediated uptake of a drug into a mammalian cell, the method comprising: a) contacting a TRPCl-expressing mammalian cell with a drug; and b) exposing the mammalian cell to a pulsed electromagnetic field (PEMF) to promote TRPC1 channel- mediated uptake of the drug into the mammalian cell. Disclosed herein is a method of treating a subject found likely to have a cancer that expresses TRPC1, the method comprising administering an anti-cancer drug to the subject and exposing the subject to PEMF to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

[0010] Disclosed herein is an anti-cancer drug for treating a subject found likely to have a cancer that expresses TRPC1, wherein the anti-cancer drug is to be administered to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC1 channel- mediated uptake of the drug into a cancer cell in the subject.

[0011] Disclosed herein is the use of an anti-cancer drag in the manufacture of a medicament for treating a subject found likely to have a cancer that expresses TRPC1, wherein the anticancer drug is to be administered to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC 1 channel-mediated uptake of the drug into a cancer cell in the subject.

[0012] Disclosed herein is a method of selecting a subject with cancer for an anti-cancer therapy, the method comprising: (a) detecting the level of TRPC 1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to the anti-cancer therapy; and (b) selecting a subject found likely to respond to the anti-cancer therapy for treatment with the anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anti-cancer drug to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC1 channel- mediated uptake of the drug into a cancer cell in the subject.

[0013] Disclosed herein is a method of treating a subject with cancer, the method comprising: (a) detecting the level of TRPC1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to an anti-cancer therapy; and (b) treating a subject found likely to respond to the anti-cancer therapy with the anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anti-cancer drag to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC 1 channel-mediated uptake of the drag into a cancer cell in the subject. Brief description of the drawings

[0014] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0015] Figure 1. Immunohistochemistry (IHC) staining of breast tumour and neighboring normal breast tissue. (A) Representative IHC DAB images showing TRPC1 and Ki-67 staining (brown) in breast tumour (Grade 1 and 3) and neighboring normal breast tissue. Tissue sections were counterstained with hematoxylin (blue). (B) Semi-quantitative IHC analysis of TRPC1 and Ki-67 staining intensity. Staining intensity was scored as: 0 (no staining), 1 (weak), 2 (moderate) or 3 (strong staining). Data represent mean ± standard error of the mean (SEM). The number of independent samples is reflected within each bar and were analyzed using one-way ANOVA followed by Sidak’s multiple comparison post hoc test Statistical significance is indicated by **, p < 0.01 and ***, p < 0.001.

[0016] Figure 2, Magnetic field exposure increases intracellular DOX concentration in a timedependent manner. (A) Intracellular DOX concentration (expressed as fold change) of 4T1 murine breast cancer cell line incubated in 500 nM DOX for 5 min prior to magnetic exposure for 10 or 30 min as indicated. Control cells were not treated with DOX or magnetic fields. (B) Intracellular DOX concentration (expressed as fold change) of MCF7 human breast cancer cell line incubated in 500 nM DOX for 5 min prior to magnetic exposure for 10 min as indicated. Control cells were not treated with DOX or magnetic fields. The intracellular DOX concentrations were calculated from a standard curve. (C) generated by measuring the intrinsic autofluorescence of DOX at 480 / 560 nm absorbance from serial dilutions in lysis buffer. Data represent mean ± standard error of the mean (SEM) (n=4, with 3 technical replicates) and were analyzed using one-way ANOVA followed by Sidak’s multiple comparison post hoc test. Statistical significance is indicated by *, p < 0.05; **, p < 0.01 and ***,p < 0.001.

[0017] Figure 3. Magnetic fields induced DOX uptake correlates with TRPC1 expression. (A) Bar chart showing the fold change in intracellular DOX concentration of 4T1 cells pre-treated with 50 pM SKF-96365 for 15 min and 500 nM DOX for 5 min prior to 10 min magnetic exposure (n=4). (B) Bar chart showing fold changes in TRPC1 transcript levels as detected by qPCR in 4T1 cells transfected with scrambled or TRPC1 dsiRNA after 24 h. Data represent mean ± standard deviation, (n=3 technical replicates). (C) Bar chart showing fold change in intracellular DOX concentration in 4T1 cells transfected with scramble or TRPC1 dsiRNA. Cells were pre-treated with 500 nM DOX for 5 min prior to 10 min magnetic exposure (n=3). (D) Bar chart showing fold change in TRPC1 transcript level detected by qPCR in MCF7 and MCF7 stable cell line overexpressing TRPC1 (MCF7-TRPC1) cells (n=3). (E) Western Blot of GFP-TRPC1 in MCF7 and MCF7-TRPC1 cells (n=4). (F) Bar chart showing the fold change in intracellular DOX concentration of MCF7 and MCF7- TRPC1 cells pre-treated with 500 nM DOX for 5 min prior to 10 min of magnetic exposure (n=4). Unless otherwise stated, data represent mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA, followed by Sidak’s multiple comparison post hoc test. Significance is indicated by ns (not significant); *, p < 0.05; **, p < 0.01; ***,p < 0.001; and ****, / ? < 0.0001.

[0018] Figure 4. Magnetic exposure enhances DOX mediated cytotoxicity in 4T1 cells. (A) Doseresponse curve of 4T1 cells treated with increasing concentrations of DOX for 24 h, with IC50 values as extrapolated from the red (unexposed, IC50 = 506.7 nM) or blue (magnetically exposed, IC50 = 279.7 nM) symbols as generated using cellular DNA content. (B) Doseresponse curve of 4T1 cells treated with increasing concentrations of DOX for 48 h. IC50 values as extrapolated from the red (uncxposcd) or blue (magnetically exposed) symbols as generated measuring mitochondrial respiration (MTT). (C) Bar chart corresponding to panel A showing % cell viability of 4T1 cells 24 h after treatment with magnetic fields and DOX. (D) Bar chart corresponding to panel B showing % cell viability of 4T1 cells 48 h after treatment with magnetic fields and DOX. Panels A and C were generated using a CyQuant DNA content assay kit. B and D were generated using a MTT assay kit. In all cases, DOX was added 5 min prior to magnetic exposure for 10 min. Data represent mean ± standard error of the mean (SEM) (n=5, with 8 technical replicates for CyQuant; n=2, 8 technical replicates for MTT). Statistical analysis was performed using 2-way ANOVA, followed by Sidak’s multiple comparison post hoc test. Significance is indicated by *, p < 0.05 and ****, p < 0.0001.

[0019] Figure 5. Muscle cells are not susceptible to PEMF-enhanced, DOX-mediated, cytotoxicity. (A) Dose-response curve of C2C12 myoblasts treated with increasing concentrations of DOX for 24 h, with IC50 values as extrapolated from the red (uncxposcd, IC50 = 581.5 nM or blue (magnetically exposed, IC50 = 651.6 nM) symbols as generated using cellular DNA content. (B) Dose-response curve of C2C12 myoblast cells treated with increasing concentrations of DOX for 48 h. IC50 values as extrapolated from the red (unexposed), or blue (magnetically exposed) symbols as generated measuring mitochondrial respiration (MTT). (C) Bar chart corresponding to panel A showing % cell viability of C2C12 myoblasts treated with increasing concentrations of DOX for 24 h after treatment with magnetic fields and DOX. (D) Bar chart corresponding to panel B showing % cell viability of C2C12 myoblasts treated with increasing concentrations of DOX for 48 h after treatment with magnetic fields and DOX. (E) Dose-response curve of C2C12 myotube cells treated with increasing concentrations of DOX for 24 h. (F) Dose-response curve of C2C12 myotube cells treated with increasing concentrations of DOX for 48 h. (G) Bar chart corresponding to panel E showing % cell viability of C2C12 myotubes treated with increasing concentrations of DOX for 24 h after treatment with magnetic fields and DOX. (H) Bar chart corresponding to panel F showing % cell viability of C2C12 myotubes treated with increasing concentrations of DOX for 48 h after treatment with magnetic fields and DOX. Panels A, C, E, and G were generated using a CyQuant DNA content assay kit. Panels B, D, F, and H 'ere generated using a MTT assay kit. In all cases, DOX 'as added 5 min prior to magnetic exposure for 10 min. Data represent mean ± standard error of the mean (SEM) (n=4, with 8 technical replicates for myoblasts, 4 technical rep-licates for myotubes). Statistical analysis was performed using 2-w'ay ANOVA, followed by Sidak’s multiple comparison post hoc test. A similar data set generated with the muscle specific exposure paradigm (1.5 mT for 10 min) is provided in Figure 7.

[0020] Figure 6. Magnetic exposure loads cell-derived vesicles (CD Vs) 'ith DOX that can be then transferred to breast cancer cells for targeted killing. Fold change in the viability of 4T1 murine breast cancer cells after treatment w'ith CDVs that were either magnetically exposed (1.5 mT), or not (0 mT) for 10 min, 'hilc in the absence or presence of 12 pM DOX. Each repetition of a condition was provisioned with the quantity of CDVs generated from approximately 1.5 x 106wild type C2C12 myoblasts. An optimization of the concentration of CDVs to observe the greatest cell response was not attempted here; the only objective was to detect differential cell responses. Cell viability was ascertained by quantifying cellular DNA using CyQuant. Data represent mean ± standard error of the mean (SEM) (n=5, with 6 technical replicates). Statistical analysis was performed using one-way ANOVA, followed by Sidak’s multiple comparison post hoc test. Significance is indicated by ns (not significant); *, p < 0.05; **, p < 0.01. A similar data set generated on MCF-7 breast cancer cells as recipients for the CD Vs is provided in Figure 8.

[0021] Figure 7. Muscle cells are not susceptible to PEMF-enhanced, DOX-mediated, cytotoxicity. (A) Dose-response curve of C2C12 myoblasts treated with increasing concentrations of DOX for 24 h, with IC50 values as extrapolated from the red (unexposed, IC50 = 581.5 nM or blue (muscle signature - magnetically exposed, IC50 = 644.3 nM) symbols as generated using cellular DNA content. (B) Dose-response curve of C2C12 myoblast cells treated with increasing concentrations of DOX for 48 h. IC50 values as extrapolated from the red (unexposed), or blue (muscle signature - magnetically exposed) symbols as generated measuring mitochondrial respiration (MTT). (C) Bar chart corresponding to panel A showing % cell viability of C2C12 myoblasts treated with increasing concentrations of DOX for 24 h after treatment with magnetic fields and DOX. (D) Bar chart corresponding to panel B showing % cell viability of C2C12 myoblasts treated with increasing concentrations of DOX for 48 h after treatment with magnetic fields and DOX. (E) Dose-response curve of C2C12 myotube cells treated with increasing concentrations of DOX for 24 h. (F) Doseresponse curve of C2C12 myotube cells treated with increasing concentrations of DOX for 48 h. (G) Bar chart corresponding to panel E showing % cell viability of C2C12 myotubes treated with increasing concentrations of DOX for 24 h after treatment with magnetic fields and DOX. (H) Bar chart corresponding to panel F showing % cell viability of C2C12 myotubes treated with increasing concentrations of DOX for 48 h after treatment with magnetic fields and DOX. Panels A, C, E, and G were generated using a CyQuant DNA content assay kit. Panels B, D, F, and H were generated using a MTT assay kit. In all cases, DOX was added 5 min prior to magnetic stimulation for 10 min. Data represent mean ± standard error of the mean (SEM) (n=4, with 8 technical replicates for myoblasts, 4 technical replicates for myotubes). Statistical analysis was performed using 2-way ANOVA, followed by Sidak’ s multiple comparison post hoc test.

[0022] Figure 8. Magnetic exposure loads CDVs with DOX that can be then transferred to breast cancer cells for targeted killing. Fold change in the viability of MDA-MB231 human breast cancer cells after treatment with CDVs that were either magnetically exposed (1.5 mT), or not (0 mT) for 10 min, while in the absence or presence of 100 nM DOX. Cell viability was ascertained by quantifying cellular' DNA using CyQuant. Each repetition of a condition was provisioned with the quantity of CDVs generated from approximately 1.5 x 106wild type C2C 12 myoblasts. An optimization of the concentration of CDVs to observe the greatest cell response was not attempted here; the only objective was to detect differential cell responses. Data represent mean ± standard error of the mean (SEM) (n=3, with 6 technical replicates). Statistical analysis was performed using Student’s t-test. Significance is indicated by *, p < 0.05.

[0023] Figure 9 shows that HTRA1 potentiates DOX cytotoxicity in cancer cells (n = 3).

[0024] Figure 10 is a perspective view of a breast coil system for PEMF application to a human subject.

[0025] Figure 11 is a schematic showing PEMF-mediated enhancement of DOX entry into cells through TRPC1 channels.

[0026] Detailed description

[0027] Transient Receptor Potential Canonical 1 (TRPC1) is a channel protein which mediates calcium transport across the cell membrane of mammalian cells. The inventors have discovered that TRPC1 channels can also mediate cellular- uptake of drags including doxorubicin (DOX) and structurally related anthracyclincs. Other drugs which, like DOX, have a net positive charge at physiological pH (such as carboplatin, cisplatin and other platinum-based drugs), may also enter cells through TRPC1.

[0028] TRPC1 is overexpressed in many cancers, including breast, prostate, pancreatic, liver, lung, colorectal, gastric, kidney and thyroid cancers, glioblastoma, and certain lymphomas and sarcomas. Elevated TRPC1 expression in cancer cells is associated with increased invasivcncss and malignancy. It is known that pulsed electromagnetic fields (PEMF) can activate TRPC1 -mediated calcium entry into cells. The inventors found that magnetic field stimulation can also improve DOX uptake through TRPC1 channels in TRPC1 -expressing cancer cells. Cell-derived vesicles (CDVs) enriched in TRPC1 can be loaded with DOX upon magnetic exposure, suggesting that TRPC1 per se is sufficient for DOX entry.

[0029] The sum of these findings suggest that magnetic field exposure can be used as a non-invasive adjuvant therapy for chemotherapy. By promoting selective uptake of anti-cancer drugs into cancer cells with elevated TRPC1 expression, PEMF exposure advantageously allows lower drug dosing, with the concomitant benefit of reduced systemic side effects, it was discovered that healthy tissues (such as muscle tissue), on the other hand, are less susceptible to PEMF- induced DOX uptake. The adjuvant therapy can also be used to enhance drug uptake in drugresistant cancer cells, such as cancer cells overexpressing P-glycoprotein (Pgp), a transporter responsible for the efflux of various anti-cancer drugs including DOX.

[0030] Accordingly, this disclosure provides methods of promoting drug uptake into cells through TRPC1 -channels using pulsed electromagnetic fields (PEMF). Also provided are methods of cancer treatment and patient selection based on detection of TRPC1 levels in patient samples.

[0031] Disclosed herein is a method of promoting TRPC1 channel-mediated uptake of a drug into a mammalian cell, the method comprising: a) contacting a TRPC1 -expressing mammalian cell with a drug; and b) exposing the mammalian cell to a pulsed electromagnetic field (PEMF) to promote TRPC1 channel-mediated uptake of the drug into the mammalian cell.

[0032] General definitions

[0033] A “mammalian cell” herein may be a primary cell or a cell line. A non-limiting list of exemplary cells includes cells from connective, nervous, muscle, bone, cartilage, blood, epithelial, adipose, and vascular tissues. Exemplary cells are cells from breast, prostate, brain, spinal cord, heart, liver, intestine, pancreas, gallbladder, kidney, lung, ovary, thyroid, cartilage, muscle, skin, blood, immune system, as well as stem cells, etc. The cell may be derived from any mammal, including but not limited to rodents (such as mice, rats, guinea pigs and rabbits), primates (including humans and non-human primates), dogs, cats, pigs, cows, oxen, goats, horses, camels, and sheep.

[0034] As used herein, the term “subject” includes any mammal, including but not limited to farm animals (such as, for example, horse, cow, pig), companion animals (such as, for example, dog, cat), laboratory animals (such as, for example, mouse, rat, rabbits), non-human primates (such as, for example, apes and monkeys) and humans. In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. The term “precancerous” refers to a condition or a growth that typically precedes or develops into a cancer. By “non-metastatic” is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, brain cancer, cancer of the head and neck, endometrial cancer, rectal cancer, esophageal cancer and hematological cancers (such as leukemias, lymphomas and myelomas).

[0035] A “cell-derived vesicle (CDV)” herein refers to a membrane-bound structure released by cells. CDVs may be shed naturally by cells during the course of cellular activity or cell culture. Alternatively, cells may be induced to release CDVs using methods known in the ait, such as acoustic or electrical stimulation, mechanical loading, cell stress induction and pharmacological stimulation. CDVs can contain proteins, lipids, nucleic acids, metabolites and organelles. Exemplary CDVs include exosomes, micro vesicles, apoptotic bodies and oncosomes. CDVs may be identified based on size, biochemical composition, cell of origin, or condition under which they are released. Cell-derived vesicles herein are preferably isolated from eukaryotic cells.

[0036] The term “sample” herein is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, including both biological and environmental sources. A “biological sample” includes within its scope a collection of similar fluids, cells, or tissues isolated from a biological source, such as a whole organism or in vitro culture. Samples include but are not limited to tissue biopsies, tissue resections, tissue aspirates, swabs (e.g., buccal swabs), whole blood, plasma, serum, urine, saliva, cerebrospinal fluid, and cell cultures, and may be obtained using any suitable method known in the art. Archival tissues, such as those having treatment or outcome history may also be used for sample extraction. The sample may be pooled from multiple aliquots. Samples include untreated, treated, diluted and concentrated samples. The terms “increased” and “increase” are used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased” and “increase” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or up to and including a 100%> increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0037] The terms “decreased” and “decrease” are used herein to mean a decrease by a statistically significant amount. In some embodiments, the terms “decreased” and “decrease” can mean a decrease of at least 10% as compared to a reference level, for example a decrease of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or up to and including a 100% decrease or any decrease between 10-100% as compared to a reference level.

[0038] The terms “overexpress” and “overexpressed”, in the context of a cell expressing a biomarker, refer to the production of the biomarker at a level that is greater than the amount produced by a corresponding control or reference cell. The control or reference cell may be, for example, a cell that does not express the biomarker or a healthy (i.e., non-diseased) cell. The increase in biomarker production may be determined at the nucleic acid level (e.g., mRNA) or protein level, as measured by any suitable quantitative method. Overexpression encompasses both transient and stable increases in expression, and includes an increase of any magnitude that is statistically significant relative to the control. In certain embodiments, overexpression refers to an increase in expression of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to and including a 100% increase or any increase between 10-100% as compared to a control or reference, or at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a control or reference. As used herein “treatment” or “treating” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve amelioration of, or complete reduction of, one or more symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0039] As used herein a “therapeutically effective dose” or “effective dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations. For purposes of this disclosure, a therapeutically effective dose of a drug or treatment is an amount that is sufficient to palliate, ameliorate, stabilise, reverse, prevent, slow or delay the progression of a cancer. The effective dose may vary depending upon the health and physical condition of the subject to be treated, the taxonomic group of the subject, the formulation of the composition, the assessment of the medical situation, and other relevant factors.

[0040] As used herein, the term “standard dose” refers to a therapeutically effective amount of a drug that is typically administered to a general patient population. The standard dose encompasses a fixed dose, weight-adjusted dose, or a range of doses that is clinically approved or commonly prescribed for the treatment of a given indication in the patient population. This dose may vary depending on prevailing clinical practice and the medical condition of a subject.

[0041] Two or more treatments administered “in combination” means that the two or more treatments arc administered cither simultaneously, or sequentially in any order at different points in time. If not administered simultaneously, then the two or more treatments are administered to a subject sufficiently close in time so that the treatments can act in concert to provide a desired therapeutic effect. Therapeutic agents administered “in combination” may be in a single composition or formulation, or may be in separate compositions or formulations, and may be administered by any suitable route.

[0042] By “adjuvant therapy” is meant any treatment that is given with a primary therapy (such as an anti-cancer drug) to improve disease and / or patient outcomes from the primary therapy. The adjuvant therapy may be administered sequentially (e.g., before or after) or simultaneously with the primary therapy.

[0043] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0044] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0045] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0046] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase “consisting essentially of’, and valuations such as “consists essentially of’ will be understood to indicate that the recited clcmcnt(s) is / arc essential i.c. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0047] Anti-cancer drugs

[0048] Methods herein comprise the use of PEMF as adjuvant therapy to improve uptake of anticancer drugs into cancer cells through TRPC1 channels. The anti-cancer drug may be any therapeutic agent capable of being transported into cells through a TRPC1 channel.

[0049] In some embodiments, the anti-cancer drug is an anthracycline compound. Non-limiting examples of anthracycline compounds include doxorubicin, valrubicin, myocet, pirarubicin, daunorubicin, idarubicin, cpirubicin, and zorubicin. In one embodiment, the anti-cancer drug is doxorubicin (DOX). Suitable therapeutic agents may be structurally and / or electrostatically similar to anthracyclines. Such compounds may include natural derivatives, synthetic analogues, or non-anthracycline scaffolds engineered to mimic the functional structural or electrostatic profile of anthracyclines.

[0050] Structurally similar compounds may contain a molecular scaffold that shares structural features of an anthracy cline, such as a planar polycyclic ring system that enables DNA intercalation (analogous to the tetracyclic anthracycline backbone), an amino sugar substituent capable of interacting with topoisomerase 11 (analogous to the daunosamine moiety in anthracyclines), and / or similar substituent patterns and stereochemistries around key functional groups as anthracyclines. An anthracycline structural analogue may additionally be of comparable molecular dimensions that allow fitting between DNA base pairs.

[0051] Electrostatically similar compounds may exhibit comparable charge distribution patterns, hydrogen bonding capabilities, and / or electronic properties to anthracyclines, even if their core structure may differ from that of anthracyclines. For example, electrostatic analogues may contain ionisablc groups positioned to interact with the negatively charged phosphate backbone of nucleic acids, the side groups of amino acids at a physiological pH, or may be positively charged themselves at physiological pH.

[0052] In some embodiments, the anti-cancer drug is positively charged at physiological pH.

[0053] In some embodiments, the anti-cancer drug is a platinum-based compound, such as a platinum(II) or a platinum(IV) complex. Without being bound by theory, platinum- based drugs are typically administered as neutral complexes, and may be aquated in vivo (i.e., the chloride ions or other leaving ligands in the complex are replaced with water molecules) to form active positively-charged species that can interact with TRPC1 channels. Non-limiting examples of platinum-containing drags include cisplatin, carboplatin, oxaliplatin, lobaplatin, enloplatin, nedoplatin and satraplatin.

[0054] The anti-canccr drug may be provided in a pharmaceutically acceptable carrier for delivery. Such carriers include but are not limited to liposomes, lipid nanoparticles, polymeric micelles, dendrimers, polymeric nanoparticles, albumin nanoparticles, peptide nanoparticles, and extracellular vesicles. In some embodiments, the drug is comprised in a cell-derived vesicle (CDV), such as encapsulated in a CDV. The CDV may be a TRPC1- enriched CDV as described herein below.

[0055] The anti-cancer drug may be combined with other therapies to synergistically enhance drug efficacy. For example, the drug may be administered with a Pgp inhibitor to further improve intracellular drug accumulation. In one embodiment, the drug is administered with HTRA1 to improve drug efficacy, as discussed below.

[0056] Cells, cancers and biomarkers

[0057] Mammalian cells herein may be any mammalian cell expressing or capable of expressing TRPC1.

[0058] In some embodiments, the mammalian cell is a cancer cell. The cancer cell may be a carcinoma, sarcoma, leukemia, lymphoma or myeloma cell. In one embodiment, the cancer cell is a tumour cell. The cancer may be breast, prostate, pancreatic, liver, lung, colorectal, gastric, renal, thyroid, sarcoma or glioblastoma. In one embodiment, the cancer is a breast cancer or prostate cancer cell.

[0059] In some embodiments, the cancer is an early-stage (e.g., Stage I or II) or low to intermediate grade (e.g., Grade 1 or 2) cancer.

[0060] In other embodiments, the cancer is a late-stage (e.g., Stage III or IV) or high-grade (e.g., Grade 3 or 4) cancer. The cancer may be a poorly differentiated or undifferentiated cancer. In one embodiment, the cancer is a locally advanced or invasive cancer. In one embodiment, the cancer is a metastatic cancer. Highly proliferative cancers are most vulnerable to chemotherapeutic agents such as anthracyclines, and this sensitivity may be enhanced through the application of PEMF during chemotherapy. A way to determine how fast cancer cells are proliferating is by measuring the Ki-67 proliferation index. Ki-67 is a nuclear protein that is found only in cells that are actively dividing. The protein may be detected using immunohistochemical (IHC) staining of a cancer sample. The proliferation index is typically expressed as the percentage of positively stained cells relative to the total number of cancer cells evaluated in the sample. A Ki-67 score greater than 10% may be considered high for solid cancers. In one embodiment, the cancer is a highly proliferative cancer characterised by a Ki-67 index of at least 20%.

[0061] Highly proliferative and aggressive cancers include but are not limited to triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), glioblastoma, pancreatic adenocarcinoma, ovarian carcinoma, hepatocellular carcinoma (HCC), oesophageal cancer, and melanoma. In one embodiment, the cancer is triple-negative breast cancer (TNBC), or breast cancer characterised by a Ki-67 index of at least 20%.

[0062] In one embodiment, the cancer is one for which anthracyclines and / or platinum-based drugs arc a standard-of-carc therapy. Anthracyclines, for example, arc typically part of first-line regimens for breast cancer, ovarian cancer, and soft tissue sarcomas. Platinum-based drugs may be used as first-line treatment for breast, ovarian, lung, gastrointestinal, esophageal, pancreatic, head and neck cancer, and mesotheliomas.

[0063] The cell or cancer may express other biomarkers in addition to TRPC1. For example, Ki-67, SBEM, EGFR and STAT3 are known to be co-expressed or co-activated with TRPC1 in certain cancers. The inventors have found that both TRPC1 and Ki-67 arc ovcrcxprcsscd in high-grade breast cancers.

[0064] In some embodiments, the cell or cancer also expresses a biomarker indicative of likely sensitivity to the anti-cancer drug. Exemplary biomarkers that may predict sensitivity to anthracyclines include TOPO2A.

[0065] In some embodiments, the cell or cancer also expresses HTRA1. As discussed herein, the inventors have found that HTRA1 can surprisingly potentiate the cytotoxic effects of certain chemo therapeutics such as anthracyclines, possibly through an effect on TRPC1.

[0066] The level of biomarker expression may be compared to a reference to determine changes in expression (such as overexpression or upregulation). The reference may be the biomarker level in a healthy or non-cancerous cell or tissue in the same subject (such as healthy tissue adjacent a tumour), or the biomarkcr level in a subject of the same species without cancer. Alternatively, the reference may be an average biomarker expression level in a population of subjects of the same species (e.g., of varying ages, ethnic backgrounds and genders) without cancer.

[0067] In some embodiments, the mammalian cell or cancer is characterised by an overexpression of TRPC1. For example, the cell or cancer may express TRPC1 at a level that is at least about 10%, at least about 20%), at least about 30%, at least about 40 ;, at least about 50 ;, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3- fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5- fold greater than a reference. In one embodiment, the mammalian cell or cancer is characterised by an expression of TRPC1 that is at least 1.5-fold greater than the expression level in a reference.

[0068] In some embodiments, the cell or cancer overexpresses TRPC1 and at least one of Ki-67, HTRA1 , SBEM, EGFR, STAT3 or TOPO2A. In one embodiment, the cell or cancer overexpresses TRPC1 and Ki-67. For example, the cell or cancer may be characterised by a Ki-67 index of at least 20%. In one embodiment, the cell or cancer overexpresses TRPC1 and HTRA1. In one embodiment, the cell or cancer overexpresses TRPC1 and SBEM. In one embodiment, the cell or cancer ovcrcxprcsscs TRPC1 and EGFR. In one embodiment, the cell or cancer overexpresses TRPC1 and STAT3. In one embodiment, the cell or cancer overexpresses TRPC1 and TOPO2A.

[0069] In some embodiments, the cell overexpresses TRPC1 and two or more of Ki-67, SBEM, EGFR, STAT3 or TOPO2A. In one embodiment, the cell overexpresses TRPC1, Ki-67 and TOPO2A. In one embodiment, the cell overexpresses TRPC1, Ki-67 and HTRA1. In one embodiment, the cell ovcrcxprcsscs TRPC1, Ki-67, HTRA1 and TOPO2A.

[0070] In some embodiments, the mammalian cell or cancer is resistant to the drug. Drug resistance may be determined, for example, by a reduced response or the absence of a response of the cell or cancer to the drag. In one embodiment, the mammalian cell or cancer expresses the drug efflux pump P-gly coprotein (Pgp). In one embodiment, the cancer is one that is resistant to an anthracycline or a platinum-based drug. In one embodiment, the cancer is anthracycline-resistant breast cancer, including but not limited to anthracycline-resistant TNBC.

[0071] PEMF adi

[0072] Methods herein comprise exposing cells, cell-derived vesicles, tissues, organs or organisms to low-amplitude pulsed electromagnetic fields (PEMF) to stimulate expression and / or activity of TRPC1 channels to improve TRPC1 -mediated drug uptake. In this sense, PEMF treatment can serve as adjuvant therapy for the drug. PEMF exposure may additionally induce cellular' or tissue activity that complements the effects of the drug or further enhance drug uptake. For example, exposure of muscle tissue to PEMF may enrich the muscle secretome with myokines, metabolites or proteins with anti-cancer activity, such as HTRA1 (Tai et al., Cells. 2024 Mar 5; 13(5):460).

[0073] Devices for generating PEMF may be based on classical Helmholtz-coil configurations used in existing clinical MRI scanning apparatus. The device may comprise a field applicator (field generating coil) and a power amplifier. The field generating coil may be suitably configured to generate a spatially homogeneous, time-varying magnetic field within the field applicator volume. The amplifier supports the power requirement for the field applicator to generate specified pulsed electromagnetic fields and may comprise control systems for interrupting treatment in the event of current overflow or excessive field exposure to the subject. The device may be a stand-alone device for application directly to a subject. Alternatively, the device may be accommodated in a standard patient bed to allow for comfortable positioning of the patient during the entire course of the exposure. In some embodiments, the device is configured for local PEMF application to a specific body part of the subject, such as to a breast or limb, or to the head, neck, chest, torso, abdomen or genitals of the subject. Exemplary systems for PEMF application are described in WO2016178631.

[0074] In some embodiments, methods herein comprise exposing a cancerous tissue in the subject, such as a breast or prostate cancer tissue or a tumour, to PEMF.

[0075] In some embodiments, methods herein further comprise exposing a muscle tissue in the subject to PEMF. The inventors have found that muscle tissue is less susceptible to PEMF- stimulated DOX uptake through TRPC1 channels. Exposure of muscle (limbs, arms, etc.) to PEMF can promote muscle health and resilience to DOX as well as increase the production of HTRA1. Without being bound by theory, HTRA1 can enhance DOX uptake into cancer cells via TRPC1, possibly by activating TRPC1. TRPC1 activation is known to be important in muscle development (Yap, J.L.Y. et al., FASEB J. 2019 Nov;33(ll): 12853-12872).

[0076] The muscle tissue treated with PEMF may be muscle that is adjacent to or near a tumour or cancer site. Alternatively, the muscle may be in a different part of the body from the tumour or cancer tissue. In some embodiments, the muscle is in a limb closest to the tumour or cancer site. For example, the muscle may be in the upper leg (e.g., the quadriceps) or the upper limb (e.g., the biceps). The PEMF may be applied to the muscle and the cancerous tissues concurrently or sequentially. Two different and independent sources of PEMF (i.c., PEMF coils) may be used to separately direct PEMF to the cancerous tissue and the muscle tissue. In one embodiment, PEMF of the same amplitude is applied to the muscle and the cancerous tissue.

[0077] The PEMF is preferably applied locally at a target tissue or organ, such as at or near a cancerous tissue or organ, or directly to a limb or muscle.

[0078] Exposure to PEMF may be concurrent with the administration of a drug, or may occur minutes to hours before or after drug administration.

[0079] Multiple exposures to PEMF may be performed. In one embodiment, a first PEMF exposure (preconditioning exposure) is performed before drug administration, followed by a second PEMF exposure concurrent with or after drug administration. The preconditioning exposure may be applied to the cancer tissue, a muscle tissue, or both. This preconditioning may increase TRPC1 expression in target cells and upregulate HTRA1 production in muscle cells to further enhance drug uptake in the tumour.

[0080] In one embodiment, the subject is administered the drag concurrently with PEMF exposure. For example, PEMF may be applied to the subject during infusion of a chemotherapeutic drug. In another embodiment, the subject is administered the drug before PEMF exposure. The PEMF therapy may be initiated within 2 hr after the completion of drug administration, such as within about 30 minutes, within about 1 hr, within about 1.5 hr, or within about 2 hr after completion of drug dosing, hi one embodiment, the PEMF therapy is initiated within about 2 hr after completion of drug administration.

[0081] The duration and amplitude of PEMF exposure may be suitably varied by the skilled person depending on the location and nature of the cell or tissue to be treated and the drug to be administered, to achieve a desired treatment outcome.

[0082] In some embodiments, the PEMF comprises barrages of 20 on and off pulses (each with a duration of 150 ps), repeated at a frequency of 15 Hz.

[0083] In some embodiments, the PEMF is applied at an amplitude of about 1 mT to about 5 nil, such as at an amplitude of about 1 mT, about 1.5 mT, about 2 mT, about 2.5 mT, about 3 mT, about 3.5 mT, about 4 mT, about 4.5 mT, or about 5 mT. In one embodiment, the PEMF is applied at an amplitude of about 3 mT.

[0084] In some embodiments, the PEMF is applied for a duration of at least 5 minutes. Advantageously, the inventors have found that even a short exposure to PEMF was sufficient to stimulate TRPC1 -mediated drug uptake and / or increase TRPC1 expression. Longer and stronger exposures can further enhance drug uptake but can also induce collateral stress on surrounding cells or tissues.

[0085] In some embodiments, the PEMF is applied for a duration of less than about 30 minutes. In some embodiments, the PEMF is applied for a duration of about 10 minutes to about 30 minutes, such as for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes to induce TRPC1 -mediated uptake into cancer cells while reducing systemic uptake.

[0086] In one embodiment, the PEMF is applied at an amplitude of about 1 mT to about 5 mT for a duration of less than about 30 minutes. In one embodiment, the PEMF is applied at an amplitude of about 3 mT for a duration of about 10 minutes to about 30 minutes. In one embodiment, the PEMF is a directional PEMF. In one embodiment, the PEMF is applied in substantially the same direction as gravity, with further enhancement when the downwardly directed fields are additionally applied orthogonally to a longitudinal axis of the cell.

[0087] In one embodiment, the PEMF is applied orthogonally to a longitudinal axis of the cell in substantially the same direction as gravity (i.e., a downward directional PEMF). For application to a subject, the PEMF may be applied in substantially the same direction as gravity to the subject in prone position.

[0088] The PEMF therapy may be administered together with an aminoglycoside. Aminoglycosides arc known to block cation transport through TRPC1 channels (Yap, J.L.Y. ct al, FASEB J. 2019 Sep 13;33(1 1 ): 12853-12872) and may also inhibit TRPC1 -mediated drug transport. Aminoglycoside treatment may hence be used as a protective measure against drug toxicity in collateral healthy tissues such as muscle tissue.

[0089] In some embodiments, the aminoglycoside is provided at a dose effective to reduce TRPC1- mediated uptake of the drug into a healthy cell (such as a muscle cell or a non-cancerous cell) in the subject, while still allowing TRPC1 -mediated uptake of the drug into cancer cells. The aminoglycoside may, for example, be administered intramuscularly or intravenously. In some embodiments, the aminoglycoside is administered with the anti-cancer drug. In some embodiments, the aminoglycoside is administered prior to or concurrently with the PEMF therapy. Exemplary aminoglycosides include but are not limited to gentamicin, streptomycin, kanamycin, spectinomycin, neomycin, paromomycin, tobramycin, amikacin, apramycin, dibekacin, sisomicin, netilmicin, and plazomicin.

[0090] In one embodiment, the aminoglycoside that is administered is gentamicin. In one embodiment, gentamicin is administered intravenously at a dose of 5 mg / kg or less, such as at a dose of about 5 mg / kg, about 4.9 mg / kg, about 4.8 mg / kg, about 4.7 mg / kg, about 4.6 mg / kg, about 4.5 mg / kg, about 4.4 mg / kg, about 4.3 mg / kg, about 4.2 mg / kg, about 4.1 mg / kg, about 4 mg / kg, about 3.9 mg / kg, about 3.8 mg / kg, about 3.7 mg / kg, about 3.6 mg / kg, about 3.5 mg / kg, about 3.4 mg / kg, about 3.3 mg / kg, about 3.2 mg / kg, about 3.1 mg / kg, about 3 mg / kg, about 2.9 mg / kg, about 2.8 mg / kg, about 2.7 mg / kg, about 2.6 mg / kg, about 2.5 mg / kg, about 2.4 mg / kg, about 2.3 mg / kg, about 2.2 mg / kg, about 2.1 mg / kg, about 2 mg / kg, about 1.9 mg / kg, about 1.8 mg / kg, about 1.7 mg / kg, about 1.6 mg / kg, about 1.5 mg / kg, about 1.4 mg / kg, about 1.3 mg / kg, about 1.2 mg / kg, about 1.1 mg / kg, about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, or about 0.1 mg / kg.

[0091] Methods of treatment and patient selection

[0092] In some embodiments of methods herein, TRPC1 -expressing mammalian cells are contacted with a drag (such as an anti-cancer drag) and exposed to PEMF to promote TRPC1 channel- mediated uptake of the drug into the mammalian cells.

[0093] In one embodiment, the contacting occurs in vitro. In vitro contacting can be conducted in any suitable manner. For example, the cell may be treated in adherent culture, or in suspension culture.

[0094] In some embodiments, the mammalian cell is used to screen for drags that may be taken up through TRPC1 channels. In a non-limiting example, TRPC1 -expressing cells and cells which do not express TRPC1 are exposed to a range of concentrations of a drag compound and concurrently or sequentially to a pulsed electromagnetic field (PEMF). Cellular uptake of the drug in the two types of cells is compared, for example, using liquid chromatographymass spectrometry or other types of chromatography methods. The biological effect of the drag (e.g., drag cytotoxicity) on the two types of cells may also be compared. Cellular drug uptake and drag effect(s) on cells may be expected to be more prominent for drugs that are transported through TRPC1 channels. Further validation of TRPC1 -mediated drug transport may be performed by exposing TRPC1 -expressing cells to a TRPC1 inhibitor (such as an aminoglycoside like gentamicin or streptomycin) during drag / PEMF treatment, and monitoring for a decrease in drag uptake or biological effect in the cells.

[0095] In some embodiments, contacting of the cell with the drag occurs in vivo. In vivo contacting may be performed, e.g., by administering the drag to a subject or to a tissue containing the cell.

[0096] Disclosed herein is a method of treating a subject found likely to have a cancer that expresses TRPC1, the method comprising administering an anti-cancer drug to the subject and exposing the subject to PEMF to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

[0097] Disclosed herein is an anti-cancer drug for treating a subject found likely to have a cancer that expresses TRPC1, wherein the anti-cancer drug is to be administered to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC1 channel- mediated uptake of the drug into a cancer cell in the subject.

[0098] Disclosed herein is the use of an anti-cancer ding in the manufacture of a medicament for treating a subject found likely to have a cancer that expresses TRPC1, wherein the anticancer drug is to be administered to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC 1 channel-mediated uptake of the drug into a cancer cell in the subject.

[0099] Disclosed herein is a method of selecting a subject with cancer for an anti-cancer therapy, the method comprising: (a) detecting the level of TRPC 1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to the anti-cancer therapy; and (b) selecting a subject found likely to respond to the anti-cancer therapy for treatment with the anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anti-cancer drug to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC1 channel- mediated uptake of the drug into a cancer cell in the subject

[0100] Disclosed herein is a method of selecting a subject with cancer for pulsed electromagnetic field (PEMF) adjuvant therapy, the method comprising: (a) detecting the level of TRPC 1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to the PEMF adjuvant therapy; and (b) selecting a subject found likely to respond to PEMF adjuvant therapy for an anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anti-cancer drug to the subject in combination with the PEMF adjuvant therapy to promote TRPC1 channel- mediated uptake of the drug.

[0101] Disclosed herein is a method of treating a subject with cancer, the method comprising: (a) detecting the level of TRPC1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to an anti-cancer therapy; and (b) treating a subject found likely to respond to the anti-cancer therapy with the anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anti-cancer drug to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC 1 channel-mediated uptake of the drug into a cancer cell in the subject.

[0102] Disclosed herein is a method of treating a subject with cancer, the method comprising: (a) detecting the level of TRPC1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to a PEMF adjuvant therapy; and (b) treating a subject found likely to respond to the PEMF adjuvant therapy with an anti-cancer therapy, wherein the anti-canccr therapy comprises administering an anti-cancer drug to the subject in combination with the PEMF adjuvant therapy to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

[0103] In some embodiments, the method further comprises detecting the level of Ki-67, HTRA1, SBEM, EGFR, STAT3 and / or TOPO2A in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 and at least one of Ki-67, HTRA1, SBEM, EGFR, STAT3 or TOPO2A.

[0104] In one embodiment, the method further comprises detecting the level of Ki-67 in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 and Ki-67. In one embodiment, the method further comprises detecting the level of HTRA1 in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 and HTRA1. In one embodiment, the method further comprises detecting the level of SBEM in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 and SBEM. In one embodiment, the method further comprises detecting the level of EGFR in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 and EGFR. In one embodiment, the method further comprises detecting the level of STAT3 in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 and STAT3. In one embodiment, the method further comprises detecting the level of TOPO2A in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 and TOPO2A.

[0105] In some embodiments, the method further comprises detecting the level of Ki-67, HTRA1, SBEM, EGFR, STAT3 and / or TOPO2A in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 and at least two of Ki-67, HTRA1, SBEM, EGFR, STAT3 or TOPO2A. In one embodiment, the method further comprises detecting the level of Ki-67 and TOPO2A in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1, Ki-67 and TOPO2A. In one embodiment, the method further comprises detecting the level of Ki-67 and HTRA1 in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to ovcrcxprcss TRPC1, Ki-67 and HTRA1. In one embodiment, the method further comprises detecting the level of Ki-67, HTRA1 and TOPO2A in the cancer sample, wherein the subject is treated or selected for treatment when the cancer is found to overexpress TRPC1 , Ki-67, HTRA1 and TOPO2A.

[0106] Expression of TRPC1 and other biomarkers can be detected at the nucleic acid level or protein level using methods known in the art. Exemplary nucleic acid detection methods include blotting techniques (c.g., Northern blots), probe hybridisation-based methods, nucleic acid amplification-based methods and nucleic acid sequencing. Exemplary protein detection methods include gel electrophoresis, immunoassays, aptamer-based detection assays, protein activity assays and mass spectrometry.

[0107] In some embodiments, TRPC1 and other biomarkers (such as Ki-67, HTRA1, SBEM, EGFR, STAT3 and TOPO2A) are detected by detecting an RNA product of the biomarker gene. For example, a nucleic acid probe that hybridises to a nucleic acid (c.g., an mRNA) encoding the biomarker may be used for detection.

[0108] In other embodiments, TRPC1 and other biomarkers (such as Ki-67, HTRA1 , SBEM, EGFR, STAT3 and TOPO2A) are detected by detecting a polypeptide product of the biomarker gene. For example, an antibody or aptamer that binds to the biomarker may be used for detection. Sequences for genes and expression products herein may be accessed on known databases of genome, transcriptome or proteome information, such as the GenBank or RefSeq databases maintained by the National Center for Biotechnology Information (NCBI), USA, or the Uniprot database maintained by the European Bioinformatics Institute (EMBL-EBI).

[0109] Exemplifications of the human proteins, as identified by their Uniprot accession identifiers, are provided below:

[0110] Human TRPC1: P48995

[0111] Human Ki-67: P46013

[0112] Human HTRA1: Q92743

[0113] Human SBEM: Q96DR8

[0114] Human EGFR: P00533

[0115] Human STAT3: P40763

[0116] Human TOPO2A: Pl 1388

[0117] Disclosed herein is a kit for detecting the responsiveness of a subject to an anti-cancer therapy, the kit comprising reagents for detecting an RNA or polypeptide product of the TRPC1 gene in a sample from the subject. The anti-cancer therapy may comprise the administraton of an anti-cancer drug to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC 1 channel-mediated uptake of the drug into a cancer cell in the subject. In some embodiments, the kit further comprises reagents for detecting the level of Ki-67, HTRA1 , SBEM, EGFR, STAT3 and / or TOPO2 A in the sample.

[0118] In some embodiments, the kit comprises nucleic acids for detecting an mRNA product of the one or more biomarker genes of this disclosure (such as TRPC1, Ki-67, HTRA1, SBEM, EGFR, STAT3 or TOPO2A). The nucleic acids may be, for example, oligonucleotide probes or primers capable of hybridising to an mRNA product of the one or more genes. The design of hybridisation probes and primers is well-kno n in the art.

[0119] In some embodiments, the kit comprises antigen-binding molecules (such as antibodies or aptamers) for detecting a polypeptide product of the one or more biomarker genes of this disclosure (such as TRPC1, Ki-67, HTRA1, SBEM, EGFR, STAT3 or TOPO2A). The level of biomarker expression may be compared to a reference to determine changes in expression (such as overexpression or upregulation). The reference may be the biomarker level in a healthy or non-cancerous cell or tissue in the same subject (such as healthy tissue adjacent a tumour), or the biomarker level in a subject of the same species without cancer. Alternatively, the reference may be an average biomarker expression level in a population of subjects of the same species (e.g., of varying ages, ethnic backgrounds and genders) without cancer.

[0120] An overexpression of a biomarker such as TRPC1 may be an expression that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5-fold greater than a reference. In one embodiment, an overexpression of TRPC1 may be an expression that is at least 1 .5-fold greater than the TRPC1 expression level in a reference.

[0121] The anti-cancer drug, anti-cancer therapy and PEMF adjuvant therapy may be as described herein above.

[0122] In some embodiments, the subject is resistant to the anti-cancer drug. In such cases, the anticancer therapy may comprise administering the same anti-cancer drug, a related anti-cancer drug, or a different anti-cancer drug with PEMF adjuvant therapy to promote drug uptake through TRPC1 channels. For example, a subject who was previously found to be resistant to DOX may be administered daunomycin or a different anthracycline, or may be administered a platinum-based drug together with PEMF.

[0123] As PEMF adjuvant therapy can increase both the quantity and rate of drug uptake into cancer cells, a lower dose of the anti-cancer drug may be administered for a shorter duration compared to standard therapy. For example, the inventors have found that PEMF exposure can reduce the effective IC50 dose of DOX by about 30-60% in cancer cells. Additionally, a 10 min PEMF exposure allows the intracellular accumulation of DOX equivalent to that achieved by 30 min of drug exposure without PEMF. Thus, in some embodiments of the methods herein, the subject is administered the anticancer drug at a dose that is lower than a standard dose of the drug when the cancer is found to express TRPC1. In some embodiments, the dose of the drug is reduced by at least about 30% from a standard dose that would have been administered. In some embodiments, the dose of the drug is reduced by between about 30% to about 70% from a standard dose that would have been administered, such as a reduction of about 30%>, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% from the standard dose.

[0124] By way of non-limiting example, a standard unit dose for DOX may be about 60 mg / m2IV. When administered with PEMF, a dose of less than 60 mg / m2may be used, such as a dose of about 55 mg / m2, about 50 mg / m2, about 45 mg / m2, about 40 mg / m2, about 35 mg / m2, about 30 mg / m2, about 25 mg / m2, or about 20 mg / m2.

[0125] HTRA1 combination therapy

[0126] It was found that HTRA1 can surprisingly enhance the efficacy of anthracyclines, possibly through activation of TRPC1 or TRPC1 -mediated drug uptake. Accordingly, this disclosure also provides anti-cancer combination therapies containing an anthracyclinc and recombinant HTRA1.

[0127] Disclosed herein is a pharmaceutical composition for treating cancer, comprising an anthracy cline compound and a HTRA1 polypeptide.

[0128] Disclosed herein is a pharmaceutical combination for treating cancer, comprising an anthracyclinc compound and a HTRA1 polypeptide.

[0129] In one embodiment, the anthracycline is doxorubicin (DOX).

[0130] The HTRA1 polypeptide may comprise an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 9. In one embodiment, the HTRA1 polypeptide may consists of an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 9.

[0131] Human HTRA1

[0132] MQIPRAALLPLLLLLLAAPASAQLSRAGRSAPLAAGCPDRCEPARCPPQPEHCEGG RARDACGCCEVCGAPEGAACGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLC VCASSEPVCGSDANTYANLCQLRAASRRSERLHRPPVIVLQRGACGQGQEDPNSL RHKYNFIADVVEK1APAVVH1ELFRKLPFSKREVPVASGSGF1VSEDGLIVTNAHVV TNKHRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEF VVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVN LDGEVTGTNTLKVTAGTSFATPSDKTKKFLTESHDRQAKGKATTKKKYIGTRMMSLTS SKAKELKDRHRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVSANDVS DVTKRESTLNMVVRRGNEDTMITVTPEEIDP (SEQ ID NO: 9)

[0133] The composition or combination may contain suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

[0134] The carrier must be pharmaceutically “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0135] Preferred unit dosage compositions arc those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.

[0136] It should be understood that in addition to the active ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents, disintegrating agents, coating agents, preservatives, lubricants and / or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl di stearate.

[0137] Disclosed herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of an anthracycline compound in combination with a therapeutically effective amount of a HTRA1 polypeptide to the subject. Disclosed herein is a HTRA1 polypeptide, for use in treating cancer in a subject, wherein a therapeutically effective amount of the HTRA1 polypeptide is to be administered in combination with a therapeutically effective amount of an anthracycline compound to the subject.

[0138] Disclosed herein is an anthracycline compound, for use in treating cancer in a subject, wherein a therapeutically effective amount of the anthracycline compound is to be administered in combination with a therapeutically effective amount of a HTRA1 polypeptide to the subject.

[0139] Disclosed herein is the use of a HTRA1 polypeptide in the manufacture of a medicament for treating cancer in a subject, wherein a therapeutically effective amount of the HTRA1 polypeptide is to be administered in combination with a therapeutically effective amount of an anthracycline compound to the subject.

[0140] Disclosed herein is the use of an anthracycline compound in the manufacture of a medicament for treating cancer in a subject, wherein a therapeutically effective amount of the HTRA1 polypeptide is to be administered in combination with a therapeutically effective amount of an anthracycline compound to the subject.

[0141] Disclosed herein is the use of a combination of an anthracycline compound and a HTRA1 polypeptide in the manufacture of a medicament or a kit for treating cancer in a subject, wherein a therapeutically effective amount of the HTRA1 polypeptide is to be administered in combination with a therapeutically effective amount of an anthracycline compound to the subject.

[0142] Disclosed herein is a method of treating a subject found likely to have a cancer that expresses TRPC1, the method comprising administering an anthracycline compound in combination with a HTRA1 polypeptide to the subject, and exposing the subject to PEMF to promote TRPC1 channel-mediated uptake of the anthracycline into a cancer cell in the subject.

[0143] Disclosed herein is an anthracycline compound for treating a subject found likely to have a cancer that expresses TRPC1, wherein the anthracycline compound is to be administered to the subject in combination with a HTRA1 polypeptide and a pulsed electromagnetic field (PEMF) to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

[0144] Disclosed herein is the use of an anthracycline compound in the manufacture of a medicament for treating a subject found likely to have a cancer that expresses TRPC1, wherein the anthracycline compound is to be administered to the subject in combination with a HTRA1 polypeptide and a pulsed electromagnetic field (PEMF) to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

[0145] Disclosed herein is a method of selecting a subject with cancer for an anti-cancer therapy, the method comprising: (a) detecting the level of TRPC1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to the anti-cancer therapy; and (b) selecting a subject found likely to respond to the anti-cancer therapy for treatment with the anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anthracycline compound in combination with a HTRA1 polypeptide to the subject, and exposing the subject to PEMF to promote TRPC1 channel-mediated uptake of the anthracycline into a cancer cell in the subject.

[0146] Disclosed herein is a method of treating a subject with cancer, the method comprising: (a) detecting the level of TRPC1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to an anti-cancer therapy; and (b) treating a subject found likely to respond to the anti-cancer therapy with the anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anthracycline compound in combination with a HTRA1 polypeptide to the subject, and exposing the subject to PEMF to promote TRPC1 channel-mediated uptake of the anthracycline into a cancer cell in the subject.

[0147] Disclosed herein is a method of treating a subject with cancer, the method comprising: (a) detecting the level of TRPC1 in a cancer sample from the subject, wherein an increase in the level of TRPC 1 as compared to a reference indicates that the subject is likely to respond to a PEMF adjuvant therapy; and (b) treating a subject found likely to respond to the PEMF adjuvant therapy with an anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anthracycline compound in combination with a HTRA1 polypeptide to the subject, and exposing the subject to PEMF to promote TRPC1 channel-mediated uptake of the anthracycline into a cancer cell in the subject.

[0148] The administration of the combination therapy of the present invention may result not only in a beneficial effect, e.g., an additive or synergistic therapeutic effect, for instance, with regard to alleviating, delaying progression of or inhibiting the symptoms, but also in further surprising beneficial effects. Such other effects may include fewer side effects from the anthracycline, an improved quality of life or a decreased morbidity, compared with a monotherapy applying only the anthracycline used in the combination. A further benefit is that lower doses of the anthracycline can be used. The dosages need not only be smaller but may also be applied less frequently, which may diminish the incidence or severity of side effects.

[0149] The anthracycline compound and HTRA1 polypeptide may be administered simultaneously or sequentially in any order, in jointly therapeutically effective amounts, preferably in synergistically effective amounts. The individual combination partners may be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. Where PEMF adjuvant therapy is used, the HTRA1 polypeptide may be administered simultaneously with PEMF exposure.

[0150] In one embodiment, the anthracycline and HTRA1 polypeptide are both administered simultaneously with PEMF exposure.

[0151] In one embodiment, the subject is administered the anthracycline and HTRA1 polypeptide concurrently, and PEMF exposure is initiated within 2 hr after the completion of drug administration.

[0152] In one embodiment, the subject is administered the anthracycline, and PEMF exposure is initiated concurrently with administration of the HTRA polypeptide within 2 hr after the completion of drug administration.

[0153] Cell-derived vesicles This disclosure also provides methods of loading drugs into cell-derived vesicles (CD Vs) via TRPC1 channel-mediated drug uptake.

[0154] Disclosed herein is a method of preparing drug-loaded cell-derived vesicles (CDVs), the method comprising: a) contacting a population of TRPC1 channel-enriched CDVs with a drug; and b) exposing the population of CDVs to PEMF so as to promote TRPC1 channel- mediated uptake of the drug into the cell-derived vesicles.

[0155] HTRA1 may enhance drug loading by activating TRPC1 channels. Thus, in some embodiments, the CDVs are further contacted with a HTRA1 polypeptide before or during exposure to PEMF to promote TRPC1 -mediated drug loading into the CDVs.

[0156] TRPC1 channel-enriched CDVs may be prepared from any mammalian cell. The mammalian cell may be primary cell (such as a cell obtained from the subject) or a cell line. In one embodiment, the CDVs are prepared from myoblasts, dental pulp stem cells or mesenchymal stem cells. In one embodiment, the CDVs are prepared from myoblasts. In one embodiment, the myoblasts are proliferating myoblasts or myoblasts that are at an early expansion stage. Myoblasts express elevated levels of TRPC1 during early expansion, thus CDVs obtained from proliferating myoblasts may be particularly enriched in TRPC1.

[0157] The CDVs may be isolated from cell cultures or tissue samples using any method known by those in the art. Non-limiting examples include differential centrifugation by ultracentrifugation, sucrose gradient purification and combination filtration / concentration. After isolation, the CDVs can be concentrated to provide a purified population of CDVs. Any appropriate method can be used to concentrate the cell-derived vesicles. Non-limiting examples of such include centrifugation, ultrafiltration, filtration, differential centrifugation and column filtration. Further subpopulations can be isolated using antibodies or other agents that are specific for a specific marker expressed by the desired cellular vesicle population.

[0158] The CDVs can have a heterogeneous size distribution (from <100 nm up to 1 pm). Methods herein may involve a step of selecting CDVs of particular sizes, e.g. <100 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm or about 1 pm. In some embodiments, methods herein comprise exposing a mammalian cell to PEMF to induce TRPC1 protein expression or activity in the cell, and isolating TRPC1 channel- enriched CDVs from the PEMF-exposed cell.

[0159] Exposure of myoblasts and muscle progenitors to PEMF can upregulate the expression of anti-cancer myokines, metabolites or factors such as HTRA1 and KLOTHO, thus producing CDVs that are enriched in these anti-cancer factors.

[0160] In some embodiments, methods herein further comprise contacting the PEMF-exposed cell with an actin cytoskeletal disruptor under mixing conditions to promote release of CDVs.

[0161] The actin cytoskeletal disruptor may be any actin cytoskeletal disruptor that can be used to disrupt the cytoskeleton structure within a cell, including but not limited to cytochalasin B, cytochalasin D, latrunculin A, latrunculin B, migrastatin, gelsolin, chivosazole A, chivosazole F, Clostridium perfringens iota toxin, Clostridium botulinum C2 toxin, and desmethoxymajusculamide C. In one embodiment, the actin cytoskeletal disruptor is cytochalasin B.

[0162] The cells may be mixed with the actin cytoskeletal disruptor under shaking or stirring conditions to promote release of CDVs. In some embodiments, the cells are shaken with the actin cytoskeletal disruptor at about 300 rpm. The shaking may be performed for about 15 mins.

[0163] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0164] Those skilled in the ait will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0165] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0166] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0167] EXAMPLES

[0168] Materials and methods

[0169] Patient samples

[0170] Voluntary female breast cancer patients, who were not pregnant and over the age of 21 years old were recruited for this study. Healthy and breast tumour tissue samples were post- opcrativcly collected from breast cancer patients as part of a mastectomy or a wider local excision.

[0171] IHC Staining

[0172] Breast tissues were maintained in a standard tissue culture incubator for 24 h before overnight fixation with 4% PFA. Subsequently, the tissues were processed using a routine histology protocol of gradual ethanol series (50%, 75%, 90% and 100%), clearing using xylcnc-substitutc (Merck, Sigma Aldrich) and embedded in paraffin, whereafter they were sectioned (5 pm) for IHC analysis. After a standard deparaffinization protocol of xylene - substitute and ethanol series, antigen retrieval was done in Tris-EDTA solution (pH 9) at 60°C for 30 min. The samples were blocked with Tris-buffered saline (pH 7.4) with Tween- 20 supplemented with 5% goat serum and 5% BSA (Merck, Sigma Aldrich). The samples were then stained with the VECTASTA1N® Elite® ABC-HRP kit (Vector Laboratories, CA, USA) per manufacturer’s protocol, followed by a Harris Hematoxylin counterstain (Merck, Sigma Aldrich). Primary monoclonal mouse TRPC1 Antibody (E-6) (1:50; catalogue no.: sc-133076; Santa Cruz Bio-technology, CA, USA) and monoclonal rabbit Antigen Kiel 67 (Ki-67) antibody (1:400; catalogue no.: #9129; Cell Signaling, MA, USA) were used. The slides were mounted using VectaMount (Vector Laboratories, CA, USA) and captured under a standard light microscope.

[0173] Cell culture and pharmacological reagents

[0174] C2C12 mouse skeletal myoblasts were obtained from American Type Culture Collection (LGC Standards, Teddington, United Kingdom) and maintained in DMEM (HyClone; Danaher, Washington, D.C., USA) with 10% FBS (Hyclone). Murine 4T1 breast cancer cells were acquired from a lab in the National University of Singapore and adapted to grow in DMEM with 10% FBS. MCF7 cells were acquired from American Type Culture Collection and maintained in RPMI 1640 (Gibco) supplemented with 10% FBS. TRPC1-GFP ovcrcxprcssing MCF7 generated previously was maintained in RPMI 1640 containing 500 pg / ml Geneticin (Invitrogen) and 10%> FBS; and referred to as MCF7-TRPC1 in the manuscript. All cell lines except MCF7-TRPC1 were cultured without antibiotics in a standard tissue culture incubator. The differentiation of myoblasts was induced with a change in medium serum composition from 10% FBS to 2% horse serum (HyClone; Thermo Fisher Scientific) at 24 h after plating at 6000 cells / cm2. Doxorubicin hydrochloride (DOX) (Abeam, ab 120629) was reconstituted in DMSO to make a stock concentration of 25 mM and stored at -20°C. Subsequent dilutions of DOX were made in PBS to keep DMSO concentration below 0.01%.

[0175] DOX standard curve and uptake assay

[0176] A standard concentration curve for DOX in RIPA cell lysis buffer was constructed using DOX at 0 nM, 100 nM, 250 nM, 500 nM, 1000 nM and 2500 nM. Absorbance was read at 480 / 560 nm using Cytation 5 microplate reader (BioTek) and plotted against their respective concentrations. MCF7 and MCF7-TRPC1 breast cancer cells prc-sccdcd in plO culture dishes were incubated in 500 nM DOX for 5 min and exposed to 3 mT PEMFs for 10 min . 4T1 breast cancer cells pre-seeded in aplO culture dish were treated with 50 uM SKF-96365 (Millipore Sigma) for 15 min prior to the addition of 500 nM DOX for 5 min. The plates were then exposed to 10 min or 30 min of magnetic exposure at 3 mT in the downward direction. Immediately after exposure, MCF7, MCF7-TRPC1 or 4T1 cells were washed twice in ice-cold PBS, lysed in ice-cold RIPA buffer for 10 min and the collected lysate analyzed at 480 / 560 nm using Cytation 5 microplatc reader (BioTek, Winooski, VT, USA). Intracellular DOX concentration was normalized to total protein concentration (2 pg / pl) determined by Pierce™ BCA Protein Assay Kits (Ther-mo Fisher Scientific) following manufacturer’s protocol.

[0177] Real-Time qPCR and TRPC1 Silencing

[0178] Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was carried out using the SYBR green-based detection workflow. Briefly, total RNA was harvested from MCF7, MCF7-TRPC1 and 4T1 cells using RNeasy kit (Qiagen) and 0.5 pg of RNA was reverse transcribed to cDNA using iScript cDNA Synthesis kit (Bio-Rad). Quantification of gene transcript expression was performed using SSoAdvanced Universal SYBR Green (BioRad) on the CFX Touch Real-Time PCR Detection System (Bio-Rad). Relative transcript expression was determined using the 2-AACt method, normalized to 0-actin transcript levels for cells of human origin or P-2-microglobulin (B2M) transcript levels for cells of murine origin.

[0179] The qPCR primers used were: hTRPCl

[0180] F: 5’-AAGCTTTTCTTGCTGGCGTG (SEQ ID NO: 1)

[0181] R: 5’-ATCTGCAGACTGACAACCGT (SEQ ID NO: 2) hB-ACTIN

[0182] F: 5’-AGAAGATGACCCAGATCATGTTTGA (SEQ ID NO: 3)

[0183] R: 5’-AGCACAGCCTGGATAGCAAC (SEQ ID NO: 4) mTRP

[0184] F: 5’-TGGGCCCACTGAGATTTCAA (SEQ ID NO: 5) R:5’-AAGATGGCCACGTGCGCTAAGGAG (SEQ ID NO: 6) mB2M

[0185] F: 5’-GATGTCAGATATGTCCTTCAGCA (SEQ ID NO: 7)

[0186] R: 5’-TCACATGTCTCGATCCCAGT (SEQ ID NO: 8)

[0187] For TRPC1 silencing in 4T1 cells, two pre-designed dicer- sub str ate short interfering RNAs (dsiRNA, IDT) were used to knock down the expression of TRPC1. Both dsiRNAs targeted the coding sequence of TRPC1 (NM_011643). Transfection of dsiRNA was performed using Lipofectamine 3000 reagent (Invitrogen) as per manufacturer’s protocol. TRPC1- silenced cells were validated using qPCR 24 h post dsiRNA transfection using primers against TPRC1, as indicated above, relative to cells transfected with scramble dsiRNA. Western Analysis

[0188] Cell lysates were prepared in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris (pH 8.0) supplemented with protease (Nacalai Tesque) and phosphatase inhibitors (PhosphoSTOP, Roche). Cells were lysed for 20 min and centrifuged for 10 min at 13,500 rpm. The protein concentration of the soluble fractions was determined using a BCA reagent (Thermo Fisher Scientific). 25-50 pg of total protein was resolved using 10% or 12% denaturing polyacrylamide gel electrophoresis and transferred to PVDF membrane (Immobilon-P, PVDF). Proteins on PVDF membranes were blocked using 5% Bovine Serum Albumin in TBST containing 0.1 % Tween-20 and incubated with the primary antibody in SuperBlock TBS (Thermo Fisher Scientific) overnight at 4°C. The primary antibodies used were: GFP (1: 1000; Proteintech, 50430-2-AP), GAPDH (1:10,000; Proteintech, 60004- 1-Ig). The membranes were washed in TBST. Anti-rabbit (31460) or anti-mouse (31430) antibody conjugated to horseradish peroxidase (HRP) were diluted (1:3000, Thermo Fisher Scientific) in 5% Bovine Serum Albumin in TBST and were incubated with the membranes for 1 h at room temperature. The membranes were incubated in SuperSignal West Pico or West Femto chemiluminescent substrate (Thermo Fisher Scientific), detected and analyzed using LI-COR Image Studio.

[0189] Cell viability assay using CyQuant Cell Proliferation Assay Kit

[0190] Cell viability was assessed by measuring DNA content (CyQuant Cell Proliferation Assay kit, Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s protocol. Briefly, 4T1 cancer cells or C2C12 myoblasts were seeded in a 96-well format with 8 technical replicates per condition. C2C12 myotubes were used in a 24- well format with 4 technical replicates per condition. The pre-seeded cells were similarly treated with DOX for 5 min prior to magnetic stimulation for 10 min (3 mT) and analyzed 24 h later using CyQuant at 480 / 520 nm on a Cytation 5 microplate reader (BioTek, Winooski, VT, USA).

[0191] Cell viability assay using MTT Cell Proliferation Kit

[0192] Cell viability was also assessed by measuring metabolic activity (MTT Cell Prolif-eration Kit, Roche) according to manufacturer’s protocol. Briefly, 4T1 cancer cells or C2C12 myoblasts were seeded in a 96-well format with 8 technical replicates per condition. C2C12 myotubes were used in a 24-well format with 4 technical replicates per condition. The pre- seeded cells were similarly treated with DOX for 5 min prior to magnetic stimulation for 10 min (3 mT) and analyzed 48 h later using MTT labelling agent and solubilization solution at 600 nm on a Cytation 5 microplate reader (BioTek, Winooski, VT, USA).

[0193] Cell-derived vesicles preparation

[0194] Cell-derived vesicles (CD Vs) were generated as previously described. CD Vs are derived from lipid raft domains originating from the muscle cell surface after enzymatic treatment followed by mechanical dissociation. Briefly, C2C12 myoblasts were grown in a 75 cm2flask for 24 h to about 60% confluence. Cells were detached and incubated in 2 ml of RPMI 1640 with 10 pM cytochalasin B (Sigma, Switzerland) per T75 flask. The flasks were subjected to orbital shaking at 280 rpm for 15 min at 37°C to promote the formation of CDVs. To aid the detachment of vesicles from cells, the flasks were gently tapped, and the supernatant was collected, ft was first centrifuged at 700 g for 5 min at 4°C to remove cells and debris, and then centrifuged again at 10,000 g for 30 min at 4°C to enrich for CDVs. The pelleted CDVs were resuspended in 50 pl PBS with or without 12 pM DOX (final concentration). The CDVs were immediately ex-posed to PEMFs at 1.5 mT for 10 min. Following magnetic exposure, the CDVs were spun down at 10,000 g for 30 min at 4°C, reconstituted in warm PBS and then provided to pre-plated recipient cancer cells for viability assessment.

[0195] Statistical analysis

[0196] All statistics were carried out using GraphPad Prism (Version 10) software. Unless otherwise stated, statistical analyses were performed using One-Way analysis of vari-ance (ANOVA) to compare the values between two or more groups followed by Sidak’s multiple comparison post hoc test. For the comparison between two independent sam-ples, the Student’s t-test was performed.

[0197] Example 1: PEMF device

[0198] PEMF was generated using a device that produces spatially homogeneous, time-varying magnetic fields, consisting of barrages of 20 x 150 ps on and off pulses for 6 ms repeated at a frequency of 15 Hz. The magnetic flux density rose to predetermined maximal level within ~50 ps (~17 T / s) when driving field amplitudes to 3 mT. All PEMF-treated samples were compared with time-matched control samples (0 mT). For PEMF delivery to breast tissue, a breast coil was constructed based on a classical Helmholtz-coil configuration optimised for field uniformity within the dimensions of 120 mm height and 75 mm of radius (Figure 10). The coil dimensions were derived from existing clinical MRI breast scanning coils for human patients. The coil is accommodated in a standard patient bed to allow for comfortable positioning of patients during the entire course of the exposure.

[0199] The breast coil system consists of a field applicator module (field generating coil) and a power amplifier module. A proprietary coil configuration was unitised for optimal signal generation within the field applicator. A precision wire winding process ensured the generation of a uniform electromagnetic signal within the field applicator volume.

[0200] The amplifier module supports the power requirement for the field applicator to generate specified pulsed electromagnetic using a firmware fine-tuned with minimal heat dissipation, ensuring that the various signal output specifications are within defined tolerances. A power and current consumption safety monitoring module is designed to monitor current consumption of the field applicator in real-time with a feedback mechanism to the microcontroller. The system allows active interruption of treatment in the event of current overflow or excessive field exposure to the subject using the device.

[0201] Example 2: TRPC1 is overexpressed in human breast cancer

[0202] Breast tumour and adjacent normal tissues were obtained from post-operative specimens following mastectomy or wide local excision. The formalin-fixed, paraffin-embedded tissue samples were processed for immunohistochemistry (IHC) to assess the protein expression of TRPC1 and Ki-67. Normal breast tissue displayed preserved adipose and epithelial architecture, characterized by minimal immunoreactivity for TRPC1 (Figure 1A, normal breast tissue). By contrast, grade 1 and grade 3 breast cancer showed disrupted cytoarchitecture, displaying enlarged and irregular nuclei size (Figure 1A, grade 1 and 3 breast cancer). TRPC1 channel expression was predominantly membrane -bound and cytoplasmic in these pathological tissues. Quantification revealed that TRPC1 expression was highest in grade 3 tumours and lowest in normal tissue, showing a clear trend of increasing TRPC1 expression with advancing cancer grades (Figure IB). Moreover, TRPC1 expression showed correlation with Ki-67 expression (Figure 1A and IB), a marker for cell proliferation, and cancer staging in breast cancers. This association suggests a potential role for TRPC1 in promoting cancer cell proliferation and progression.

[0203] Example 3: PEMF exposure enhances DOX uptake into cancer cells via TRPC1

[0204] DOX entry into cells has been proposed to occur via its passive diffusion across the membrane despite saturation kinetics being reported. Here the possibility that TRPC1 channels are involved in the uptake mechanism of DOX into cancer cells and moreover, could be stimulated with targeted magnetic exposure, is explored. To this end, 4T1 murine breast cancer cells were pretreated with 500 nM DOX for 5 min followed by exposure to PEMFs (3 mT) for cither 10 or 30 min. Uncxposcd 4T1 cells (red bars) exhibited a timedependent accumulation of intracellular DOX from 10 min to 30 min (Figure 2 A). Magnetic field exposure (blue bars) further augmented the intracellular accumulation of DOX by twofold relative to time-matched unstimulated controls. Notably, 30 min of magnetic exposure was capable of accumulating DOX within the cancer cell to approximately 40% of its extracellular levels as calculated in accordance with a standard curve (Figure 2C). Similarly, exposure of MCF7 human breast cancer cells to 3 mT PEMFs for 10 min resulted in a 70% increase in accumulation of intracellular DOX (Figure 2B). Given that a 10 min PEMF exposure significantly enhanced DOX uptake in both human and mouse cell lines, this exposure paradigm was selected to maximize therapeutic efficacy while minimizing potential uptake into healthy cells.

[0205] A pharmacological strategy was next undertaken to elucidate the potential contribution of TRPC1 in PEMF-induced DOX entry. SKF-96365 is an accepted inhibitor of TRPC channels. 4T1 cells were pre-treated with the TRPC channel inhibitor SKF-96365 (50 pM) for 15 min prior to 5 min incubation with DOX (500 nM) and subsequently exposed to PEMFs (3 mT) for 10 min. Pre-treatment of 4T 1 cells with SKF-96365 completely abolished the uptake of DOX in response to PEMF exposure (Figure 3 A) while non-SKF-96365 treated cells remained unperturbed in their accumulation of DOX. To further validate the role of TRPC1 in the magnetic induction of cellular DOX entry, transient knockdown of TRPC1 was performed in 4T1 cells using two independent dsiRNAs, achieving approximately 50% reduction in TRPC1 transcript levels (Figure 3B). DOX uptake in TRPC1 knockcd-down cells revealed a comparable level of inhibition of DOX uptake (Figure 3C), similar to that observed with pharmacological inhibition. Finally, an established MCF7 cell line stably overexpressing GFP-TRPC1 (MCF7-TRPC1) was employed to confirm the necessity of TPRC1 for DOX uptake. This cell line exhibited elevated TRPC1 expression at both mRNA (Figure 3D) and protein levels (Figure 3E). Notably, the MCF7-TPRC1 cell line demonstrated enhanced DOX uptake upon magnetic exposure (Figure 3F), with a 2-fold increase in PEMF-induced DOX uptake compared to control MCF7 cells. Taken together, these findings demonstrate that TRPC1 is essential for PEMF-induced DOX uptake, highlighting its value as a biomarker with which to determine the potential efficacy of magnetic field therapy for certain cancers.

[0206] Cellular DNA content and metabolic activity were evaluated in 4T1 cells to assess the therapeutic potential of PEMF-induccd DOX uptake (Figure 4A and 4B). The IC50 (half maximal inhibitory concentration) for DOX in 4T1 cells at 24 and 48 h were 506.7 nM and 228.4 nM, respectively. Remarkably, a 10-min magnetic exposure (3 mT) in the presence of DOX significantly enhanced the efficacy of DOX, reducing the IC50 to 279.9 nM (24 h, CyQuant) and 124.8 nM (48 h, MTT). Notably, the greatest degree of synergism was observed in the lowest concentration range of DOX (50 nM) (Figure 4C and 4D), where magnetic exposure significantly reduced cell viability by an additional 30% (MTT) and 20% (CyQuant) compared to unstimulatcd controls receiving the same DOX dose.

[0207] The feasibility of PEMF intervention as part of clinical workflow was also evaluated in metastatic breast cancer patients undergoing surgical resection or chemotherapy. Preliminary investigations showed that PEMF exposure of 30 mins did not cause any wound healing complications for surgical patients. It was also found that PEMF exposure did not pose any side effects or complicate clinical care for patients undergoing their fourth cycle of chemotherapy.

[0208] Example 4: Muscle cells are less responsive to PEMF-mediated DOX uptake

[0209] The potential for collateral toxicity is of important consideration, particularly as TRPC 1 is ubiquitously expressed across tissues. The consequences of the magnetic induction of DOX uptake into a murine muscle cell line (C2C12), pre- and post-myogenic differentiation, was evaluated as an indication of potential collateral cytotoxicity. Employing the same experimental protocol as conducted in cancer cells (Figure 4), no significant difference in viability between stimulated and unstimulated myoblasts was detected (Figure 5A-D). Similarly, no significant difference in viability was detected between stimulated and unstimulated differentiated myotubes (Figure 5E-H). Furthermore, 1.5 mT magnetic exposure, which coincides with the optimal magnetic strength for myogenic enhancement did not provide any enhancement in DOX-mediated cytotoxicity across varying DOX concentrations (Figure 7). Finally, a slight increase in IC50 values in myoblasts was apparent with magnetic stimulation (0 mT = 581.5 nM, 1.5 mT = 644.3 nM, 3mT = 651.6 nM), suggestive of a slight protective effect of magnetic field stimulation against DOX cytotoxicity.

[0210] Example 5: HTRA1 promotes DOX-mediated cytotoxicity

[0211] It was previously shown that magnetically stimulated muscle exposed to 10 min of 1.5 mT PEMF upregulated expression of HTRA1 and produced a conditioned medium with anticancer properties. To investigate if HTRA1 has an effect on DOX efficacy, MCF-7 cells were treated with recombinant HTRA1 or DOX alone, or a combination of both. It was found that HTRA1 potentiates the effect of DOX in MCF-7 cells (Figure 9). Treatment with HTRA1 or DOX alone resulted in reduced cell viability, but a further -10% reduction in viable cancer cells was observed when both HTRA1 and DOX were administered in combination, indicating a synergistic interaction.

[0212] Example 6: Biomarker screening in breast cancer patients

[0213] To determine if a breast cancer patient is likely to respond to DOX and PEMF adjuvant therapy, a sample from a biopsy or surgical resection can be fixed and embedded for immunohistochemical (IHC) staining using antibodies specific for TRPC1, Ki-67, HTRA1, EGFR, STAT3 or SBEM. Staining intensity and the proportion of positive tumor cells are evaluated, and an H- score may be calculated for each marker. Patients with an IHC score of 2+ or higher for TRPC1 and optionally a Ki-67 index of 20% or greater may be selected for treatment with DOX and PEMF.

[0214] Biomarker expression may also be evaluated using ELISA on cell lysates prepared from cancer samples. The results from ELISA may be compared to a reference profile of biomarker expression levels established from healthy tissue samples from the same patient (such as non-cancerous tissue adjacent the breast tumor). The cancer is determined to be TRPC1 -positive if the expression levels of TRPC1 is increased by 1.5 fold or greater compared to the median expression level in the control tissue. Patients with TRPC1 -positive breast cancer may then be selected for treatment with DOX and PEMF.

[0215] For breast cancer patients who are refractory to a standard-of-care anthracycline chemotherapy paradigm (e.g., DOX), PEMF may be administered in conjunction with another variation of an anthracycline-based regimen.

[0216] Example 7: Loading of TRPCl-enriched cell-derived vesicles (CDVs) with DOX via magnetic field exposure

[0217] A method was developed for the generation of cell-derived vesicles (CDVs) that are enriched in TRPC1 expression and retain magnetosensitivity. Brief exposure to low energy magnetic fields could load these CDVs with Ca2+, corroborating membrane integrity. Moreover, the fusogenic attributes of these CDVs, conferred by the enrichment of the outer leaflet of their phospholipid bilayer membranes with phosphatidylserine, enabled their use as a versatile delivery system. These CDVs could be loaded with DOX upon magnetic exposure (1.5 mT, 10 min), and upon delivery to breast cancer cells were capable of inducing DOX-dcpcndcnt cytotoxicity (Figure 6). This result would suggest that TRPC1 and magnetic exposure was sufficient for the loading of DOX into a greatly minimized vesicular system (mean diameter of 300 nm) largely devoid of cellular reticular structure. Neither magnetic exposure of CDVs alone (-DOX, 1.5 mT; 1st blue), nor incubation of CDVs with DOX alone (+DOX, 0 mT; 2nd red), produced CDVs capable of compromising cancer cell viability. That is, the simultaneity of magnetic and DOX exposures was necessary for effective CDV-mediated DOX delivery. Analogous results were obtained with the human breast cancer cells (MDA- MB231) treated with DOX-PEMF-loaded CDVs (Figure 8). The presence of TRPC1 per se thus appears to be sufficient to support DOX entry upon magnetic exposure.

[0218] Example 8: Discussion

[0219] Several TRP channel classes have been implicated as oncogenic drivers in a variety of cancers. The mammalian TRP channel superfamily consists of 28 cation channel subunits that are subdivided into six subfamilies based on sequence homology that include the founding canonical TRP (TRPC), vanilloid TRP (TRPV), melastatin-related TRP (TRPM), ankyrin TRP (TRPA), mucolipin TRP (TRPML) and polycystic TRP (TRPP) subfamilies. Functional TRP channel complexes are generally formed as tetramers of individual TRP channel subunits of familial homomultimeric, or heteromultimeric, stoichiometry. TRP channels commonly receive, integrate and transduce biophysical and chemical stimuli into developmental responses that include cell growth, proliferation, and survival.

[0220] Although TRP channels are not inherently oncogenic per se, certain TRP channels have been found to be overexpressed or deregulated in cancer, thereby contributing to oncogenic transformation and tumour progression. For instance, the elevated expression of TRPM2 is correlated with breast, lung and prostate cancer cell survival, whereas TRPV6 ovcrcxprcssion has been reported in prostate cancer as well as in breast cancer metastasis. Critically, TRPC1 has been shown to contribute to the aberrant Ca2+signaling that confers upon healthy cells oncogenic features such as enhanced migra-tion, invasion, proliferation and survival.

[0221] The TRPC subfamily is the most ubiquitously expressed of the TRP superfamily with the TRPC1 subunit being the most predominantly expressed member of all. The TRPC1 subunit is hypothesized to regulate the activity of the other TRP channel subunits within a channel multimer, uniting the unique gating sensitivities of the individual subunits into a single channel complex for the effective integration of diverse biophysical stimuli into a unified developmental response. Accordingly, the dysregulated expression of TRPC1 correlates with breast, pancreas and lung cancers. TRPC1 expression may thus serve as prognostic for breast cancer given its strong correlation to tumour progression, metastasis and EMT. For instance, Figure 1 shows that TRPC1 expression was positively correlated with the grade of breast cancer, indicating correspondence with more aggressive tumour growth and poorer patient outcomes, as well as Ki-67 staining, an indicator for poor breast cancer prognosis. As Ki-67 is a well-established marker of cellular proliferation, its co-expression with TRPC1 underscores the role of TRPC1 in proliferative signaling pathways. Elevated TRPC1- mediated Ca2+entry also has been shown to stimulate known oncogenic processes including MAPK / ERK, P13K / AKT, and Wnt / p-cathenin signaling. These proliferative pathways are routinely hijacked in cancers. By contrast, loss of TRPC1 channel expression has been shown to attenuate proliferation, migration, invasion and sternness in cancer. TRPC1 also appears to regulate the activity of the tumour-associated macrophages (TAMs) within the tumour microenvironment (TME). TAMs can assume either pro-tumourigenic or anti-tumourigenic states. Whereas the tumour cancer cell secretome polarizes TAMs towards the pro-tumoural state, TRPC1 -mediated Ca2+entry into TAM promotes polarization towards the anti-tumoural state. DOX has also been shown to polarize macrophages towards the anti-tumoural state. The ability of PEMFs to activate TRPC1 residing on TAMs may hence be exploited to enhance their loading with DOX to decisively polarize them into the anti-tumour status. Furthermore, localizing the PEMF exposure specifically to the tumour may ultimately allow for the lowering of systemic DOX administration, as local DOX uptake may be enhanced.

[0222] Localized PEMF Exposure of Breasts Tumours May Permit Reduction in DOX Dosing

[0223] It is shown that a brief magnetic field exposure enhances DOX uptake in correlation with TRPC1 expression, rendering breast cancer-specific cytotoxicity. Given the fundamental role of TRPC1 in cell proliferation, TRPC1 represents a promising prognostic marker for breast cancer. Moreover, the positive correlation between TRPC1 expression and cancer progression provides a therapeutic window of opportunity; TRPC1 expression can be leveraged with magnetic field treatment in combination with DOX administration to achieve greater anti-tumour outcomes. Targeting TRPC1, a key cancer supporting factor, may confer enhanced selectivity to this therapeutic approach.

[0224] Magnetic fields can induce a mitohormetic effect whereby a moderate increase in oxidative stress adaptively strengthens the antioxidant defenses of a cell and promotes its survival. However, an excessive amount of oxidative stress can overwhelm the existing antioxidant defenses of the cell and undermine its survival. The ultimate effect of magnctically-induccd ROS on cell fate depends on the underlying inflammatory status of the cell. Cancer cells tend to be more vulnerable to ROS-induced damage than healthy cells due to a reduced ROS buffering capacity.

[0225] In muscle cells, brief exposure to low amplitude magnetic fields (1.5 mT, 10 min) supports adaptive cell growth, whereas in cancer cells higher amplitude exposure for longer periods (3 mT, 1 h) inhibits malignant cell growth, without affecting healthy cells or tissues. Thus, PEMF exposure can be exploited for adaptive (e.g., in muscle) or damaging (e.g., in cancer) mitohormetic responses by varying the amplitude and time of magnetic field exposure.

[0226] Extending the magnetic utility of the anti-cancer paradigm described above, the present study revealed that a brief exposure to moderately high-amplitude PEMFs (3 mT, 10 min) further significantly enhanced DOX-mediated cancer cell killing and identified TRPC1 as a crucial mediator of magnetically induced DOX uptake. DOX is typically administered intravenously at a dose of 60 mg / m2and was estimated to achieve a maximum blood concentration (Cmax) of -5 pg / ml (8.6 pM). On the other hand, mathematical modelling has predicted DOX concentrations in plasma and breast adipose tissue to be on the order of 1 uM and 860 nM, respectively. Adipose DOX concentration was further calculated to drop by 80% (to 172 nM) at 48 h after administration.

[0227] Here it is shown that the in vitro DOX ICso for breast cancer cells at 24 h following administration was reduced by almost half (507 nM to 280 nM) with brief magnetic exposure (3 mT for 10 min) (Figure 4). Based on these findings, the standard clinical dose of 60 mg / m2may theoretically be reduced by half, to 30 mg / m2, to achieve a plasma Cmax of 500 nM and adipose accumulation of 430 nM, which would fall within the determined magnetic efficacy window (Figure 4). Hypothetically, local magnetic therapy applied to the breast can permit a reduction of systemic DOX doses by enhancing local uptake of the drug into breast cancer cells. This can reduce the side effects of DOX chemotherapy experienced by patients.

[0228] In summary, the synergistic combination between PEMF treatment and DOX treatment can reduce the effective ICso dose of DOX by 30-60%. This means that, for example, when DOX is delivered at a concentration of 0.2-0.4 pM together with PEMF exposure to TRPC1- expressing breast cancer cells, it can achieve a similar level of cytotoxicity as 0.5-1.0 pM of DOX delivered alone. Additionally, application of 10 min PEMF (3 mT) can accelerate DOX accumulation to that achieved by 30 min of drug exposure alone.

[0229] Combining Muscle and Breast Tumour Anti-Cancer Magnetic Therapies

[0230] DOX intercalates within mtDNA to trigger mitochondrial dysfunction and oxidative stress. The formation of DOX-Fc complexes also generates semiquinone radicals that further contribute to the production of mtROS. These DOX-Fe complexes also deplete iron that compromises mitochondria efficiency, exacerbating the accumulation of mtROS that further damages mitochondria and result in cell death via ferroptosis. The constitutive uptake of DOX by collateral tissues and the detrimental effect it has over organismal resilience (Figure 5) underscores the clinical need for the development of therapeutic measures to reduce the level of systemic DOX administration. DOX- associated collateral damage preferentially affects organs enriched with mitochondria such as the heart, skeletal muscle and central nervous s stem.

[0231] The muscle secretome is one of the body’s defences against cancer. The muscle secretome is a downstream response limb of mitochondria respiration and as such, may be expected to be compromised by DOX-dependent muscular mitochondrial disruption. However, it is shown here that muscle cells arc less sensitive than breast cancer cells to DOX-mcdiatcd cytotoxicity when administered in conjunction with magnetic exposure (Figure 5), possibly reflecting inherently lower expression levels of TRPC1 in healthy muscle. Muscle-targeted magnetic therapy may thus improve systemic resilience against DOX chemotherapy, by harnessing the anti-cancer potential of the muscle secretome. The deployment of a combinatorial magnetic therapy can exploit the unique anti-cancer magnetic responses of muscle and breast cancer for enhanced chemotherapeutic outcomes.

[0232] Evidence for TRPCl-Mediated DOX Uptake

[0233] A protocol for the creation of cell-derived vesicles (CDVs) was developed that produces CDVs that are selectively enriched for TRPC1 and TRPA1. It is shown that these CDVs can be loaded with Ca2+upon exposure to PEMF. Most notably, these CDVs could restore magnetically induced respiratory capacity to TRPC1 knockdown cells.

[0234] It was previously shown that the fluorescent cationic dye, FM1-43 (~12 A diameter, 611 Da), was capable of being taken up into cells in association with TRPC1 and in correlation with cell proliferation. TRPA1 also has been shown to support the entry of FM1-43 into cells. The pore of TRPA1 has an estimated diameter of -11 A and has been shown to be dynamically regulated to accommodate large fluorescent molecules, such as FM1 -43, upon biophysical activation. TRPA1 has been shown to coimmunoprecipitate with TRPC1, suggesting that TRPC1 and TRPA1 can heteromultimerise to form a functional channel complex of potentially regulatable permeability to larger macromolecules. One possibility, therefore, is that TRPC1 / TRPA1 heteromultimers are capable of accommodating DOX entry into cells upon magnetic stimulation. Accordingly, here it is shown that these same CDVs could be loaded with DOX upon magnetic exposure that was then capable of transmitted DOX-mediated cytotoxicity to breast cancer cells (Figure 6).

[0235] Cancer Specificity of Magnetically Stimulated DOX Uptake

[0236] In healthy tissues, TRPC1 channel expression is developmentally regulated, being most heightened during the proliferative phase and downregulated after terminal differentiation. Breast cancer cells exist in a state of sustained sternness and hence retain aberrantly elevated TRPC1 expression throughout most of the course of the disease. Breast cancer is hence developmentally poised for preferential vulnerability to this magnetic therapeutic strategy that targets TRPC1 expression. Healthy tissues, on the other hand, predominantly restrict the expression of TRPC1 to the early phase of cell proliferation that is transient and short lived. For example, Figures 5 and 7 show that skeletal muscle is no more vulnerable to DOX even under PEMF exposure. In this respect, PEMF therapy may potentially be selective towards cancers characterized by elevated TRPC1 expression.

[0237] . The combination of PEMF exposure can reduce the IC50 of DOX for treating cancer cells by at least 30%. Healthy tissues, on the other hand, arc less susceptible to PEMF-induccd DOX uptake.

[0238] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method of promoting Transient Receptor Potential Channel 1 (TRPC1) channel- mediated uptake of a drug into a mammalian cell, the method comprising: a) contacting a TRPC1 -expressing mammalian cell with a drug; and b) exposing the mammalian cell to a pulsed electromagnetic field (PEMF) to promote TRPC 1 channel-mediated uptake of the drug into the mammalian cell.

2. The method of claim 1, wherein the mammalian cell is a cancer cell.

3. The method of claim 1 or 2, wherein the cancer cell is a breast cancer, prostate cancer, pancreatic cancer, liver cancer, lung cancer, colon cancer, gastric cancer, renal cancer, thyroid cancer, sarcoma, or glioblastoma cell.

4. The method of any one of claims 1 to 3 , wherein the mammalian cell is characterised by an overexpression of TRPC1.

5. The method of any one of claims 1 to 4, wherein the mammalian cell overexpresses TRPC1 and Ki-67.

6. The method of any one of claims 1 to 5, wherein the mammalian cell overexpresses TRPC1 and HTRAl .

7. The method of any one of claims 1 to 6, wherein the mammalian cell overexpresses TRPC1 and TOPO2A.

8. The method of any one of claims 1 to 7, wherein the mammalian cell overexpresses TRPC1 and EGFR.

9. The method of any one of claims 1 to 8, wherein the mammalian cell overexpresses TRPC1 and STAT3.

10. The method of any one of claim 1 to 9, wherein the mammalian cell overexpressesTRPC1 and SBEM.1 1 . The method of any one of claims 1 to 10, wherein the mammalian cell is resistant to the drug.

12. The method of any one of claims 1 to 11, wherein the drug is an anthracycline compound or a platinum-based drug.

13. The method of claim 12, wherein the drug is doxorubicin (DOX).

14. The method of any one of claims 1 to 13, wherein the drug is comprised in a cell-derived vesicle.

15. The method of any one of claims 1 to 14, wherein the method comprises exposing the mammalian cell to about 1 mT to about 5 mT of PEMF.

16. The method of any one of claims 1 to 15, wherein the method comprises exposing the mammalian cell to PEMF for about 10 minutes to about 30 minutes.

17. The method of any one of claims 1 to 16, wherein the PEMF is applied in substantially the same direction as gravity.

18. The method of any one of claims 1 to 17, wherein the PEMF is applied orthogonally to a longitudinal axi of the mammalian cell.

19. The method of any one of claims 1 to 18, wherein step b) is performed before, concurrent with or after step a).

20. The method of claim 19, wherein step b) is performed concurrent with step a).21 . A method of treating a subject found likely to have a cancer that expresses TRPC1 , the method comprising administering an anti-cancer drug to the subject and exposing the subject to PEMF to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

22. The method of claim 21 , wherein the cancer is breast cancer, prostate cancer, colon cancer, liver cancer, gastric cancer, renal cancer, lung cancer, pancreatic cancer, thyroid cancer, glioblastoma or sarcoma.

23. The method of claim 21 or 22, wherein the cancer is characterised by an overexpression of TRPCl.

24. The method of any one of claims 21 to 23, wherein the cancer overexpresses TRPC1 and Ki-67.

25. The method of any one of claims 21 to 24, wherein the cancer overexpresses TRPC1 and HTRAl.

26. The method of any one of claims 21 to 25, wherein the cancer overexpresses TRPC1 and T0P02A.

27. The method of any one of claims 21 to 26, wherein the cancer overexpresses TRPC1 and EGFR.

28. The method of any one of claims 21 to 27, wherein the cancer ovcrcxprcsscs TRPC1 and STAT3.

29. The method of any one of claim 21 to 28, wherein the cancer overexpresses TRPC1 and SBEM.

30. The method of any one of claims 21 to 29, wherein the subject is resistant to the anticancer drug.

31. The method of any one of claims 21 to 30, wherein the method comprises exposing a cancerous tissue in the subject to PEMF.

32. The method of claim 31 , wherein the method further comprises exposing a muscle tissue in the subject to PEMF.

33. The method of any one of claims 21 to 32, wherein the method comprises exposing the subject to about 1 mT to about 5 mT of PEMF.

34. The method of any one of claims 21 to 33, wherein the method comprises exposing the subject to PEMF for about 10 minutes to about 30 minutes.

35. The method of any one of claims 21 to 34, wherein the PEMF is applied in substantially the same direction as gravity to the subject in prone position.

36. The method of any one of claims 21 to 35, wherein the PEMF is applied orthogonally to a longitudinal axis of the subject’s body.

37. The method of any one of claims 21 to 36, wherein the anti-cancer drug is an anthracycline compound or a platinum-based drug.

38. The method of claim 37, wherein the drug is doxorubicin (DOX).

39. The method of any one of claims 21 to 38, wherein the ding is comprised in a cell- derived vesicle.

40. The method of any one of claims 21 to 39, wherein the subject is administered the anticancer drug at a dose that is lower than a standard dose of the drug.

41. The method of any one of claims 21 to 40, wherein the anthracycline compound is administered at a dose of less than about 60 mg / m2.

42. The method of any one of claims 21 to 41, wherein the subject is administered the drug before, concurrent with or after PEMF exposure.

43. The method of claim 42, wherein the subject is exposed to PEMF concurrently with drug administration.

44. The method of claim 42, wherein the subject is exposed to PEMF within 2 hr of drug administration.

45. The method of any one of claims 21 to 44, further comprising administering an aminoglycoside to the subject at a dose effective to reduce TRPC1 -mediated uptake of the drug into a healthy cell in the subject.

46. The method of claim 45, wherein the aminoglycoside is gentamicin.

47. The method of claim 45 or 46, wherein the healthy cell is a muscle cell.

48. An anti-cancer drug for treating a subject found likely to have a cancer that expresses TRPC1, wherein the anti-cancer drug is to be administered to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

49. Use of an anti-cancer drug in the manufacture of a medicament for treating a subject found likely to have a cancer that expresses TRPC1 , wherein the anti-cancer drug is to be administered to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

50. A method of selecting a subject with cancer for an anti-cancer therapy, the method comprising: (a) detecting the level of TRPC1 in a cancer sample from the subject, wherein an increase in the level of TRPC1 as compared to a reference indicates that the subject is likely to respond to the anti-cancer therapy; and (b) selecting a subject found likely to respond to the anti-cancer therapy for treatment with the anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anti-cancer drug to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC1 channel-mediated uptake of the drug into a cancer cell in the subject.

51. The method of claim 50, further comprising detecting the level of Ki-67 in the cancer sample, wherein the subject is selected for treatment when the cancer is found to overexpress TRPC1 and Ki-67.

52. The method of claim 50 or 51 , further comprising detecting the level of HTRA 1 in the cancer sample, wherein the subject is selected for treatment when the cancer is found to overexpress TRPC1 and HTRA1.

53. The method of any one of claims 50 to 52, further comprising detecting the level of TOPO2A in the cancer sample, wherein the subject is selected for treatment when the cancer is found to overexpress TRPC1 and TOPO2A.

54. The method of any one of claims 50 to 53, further comprising detecting the level of EGFR in the cancer sample, wherein the subject is selected for treatment when the cancer is found to ovcrcxprcss TRPC1 and EGFR.

55. The method of any one of claims 50 to 54, further comprising detecting the level of STAT3 in the cancer sample, wherein the subject is selected for treatment when the cancer is found to overexpress TRPC1 and STAT3.

56. The method of any one of claims 50 to 55, further comprising detecting the level of SBEM in the cancer sample, wherein the subject is selected for treatment when the cancer is found to overexpress TRPC1 and SBEM.

57. A method of treating a subject with cancer, the method comprising: (a) detecting the level of TRPC1 in a cancer sample from the subject, wherein an increase in the level of TRPC 1 as compared to a reference indicates that the subject is likely to respond to an anti-cancer therapy; and (b) treating a subject found likely to respond to the anti-cancer therapy with the anti-cancer therapy, wherein the anti-cancer therapy comprises administering an anti-cancer ding to the subject in combination with a pulsed electromagnetic field (PEMF) to promote TRPC1 channel- mediated uptake of the drug into a cancer cell in the subject.

58. The method of claim 57, further comprising detecting the level of Ki-67 in the cancer sample, wherein the subject is treated when the cancer is found to overexpress TRPC1 and Ki-67.

59. The method of claim 57 or 58, further comprising detecting the level of HTRA1 in the cancer sample, wherein the subject is treated when the cancer is found to overexpress TRPC1 and HTRA1.

60. The method of any one of claims 57 to 59, further comprising detecting the level of TOPO2A in the cancer sample, wherein the subject is treated when the cancer is found to overexpress TRPC1 and TOPO2A.

61. The method of any one of claims 57 to 60, further comprising detecting the level of EGFR in the cancer sample, wherein the subject is admtreated when the cancer is found to ovcrcxprcss TRPC1 and EGFR.

62. The method of any one of claims 57 to 61, further comprising detecting the level of STAT3 in the cancer sample, wherein the subject is treated when the cancer is found to overexpress TRPC1 and STAT3.

63. The method of any one of claims 57 to 62, further comprising detecting the level of SBEM in the cancer sample, wherein the subject is treated when the cancer is found to overexpress TRPC1 and SBEM.

64. The method of any one of claims 50 to 63, wherein the subject is administered the anticancer drug at a dose that is lower than a standard dose of the drug.

65. The method of any one of claims 50 to 64, wherein the subject is resistant to the anticancer drag.

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