Methods and compositions for inhibiting fibroblast activation

Abstract

A method of treating a subject in need of treatment is disclosed, the method comprising applying an alternating current electric field to a target site in the subject in need thereof and administering a cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof. A method of reducing or preventing CAF activation is disclosed, the method comprising applying an alternating current electric field to a cell population comprising one or more fibroblasts and contacting the cell population with a CAF activation inhibitor. A method of reducing CAF signaling is disclosed, the method comprising applying an alternating current electric field to a cell population comprising one or more fibroblasts and contacting the cell population with a cancer-associated fibroblast (CAF) signaling inhibitor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 477,565, filed December 29, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Fibroblasts are responsible for wound healing. They produce extracellular matrix (ECM) proteins, such as collagen, elastin, and other enzymes and cytokines, to contract the wound and repair damaged tissue. Unlike normal wound healing, which ultimately heals, cancer cells affect the tumor stroma and continuously secrete inflammatory cytokines and increased ECM proteins without reaching homeostasis. Cancer-activated fibroblasts (CAFs) are a fibroblast population present in tumor tissue that express and secrete proteins, thus influencing tumor remodeling. CAFs typically express FAP (fibroblast activation protein), alpha-smooth muscle actin (aSMA), platelet-derived growth factor receptor beta (PDGFRb), integrin beta 1 (ITDB1), CD26, and caveolin-1 (CAV1).

[0003] CAFs not only produce and secrete ECM proteins but also actively participate in ECM protein degradation, cross-linking, and ECM protein assembly processes. In such a robust and highly cross-linked tumor stroma, drug penetration is one potential reason for tumor cell evasion from treatment. Additionally, CAF-mediated ECM remodeling is a highly responsive process that receives, processes, and responds to cellular, molecular, and mechanical signals in the tumor microenvironment.

[0004] Alternative strategies to target cancer-activated fibroblasts and treat cancer are needed. Summary of the Invention

[0005] Disclosed herein are studies showing that TT fields increase fibroblast activation in vivo and in vitro. Thus, treatment with TT fields while inhibiting cancer-associated fibroblast activation leads to better patient outcomes.

[0006] Disclosed are methods of treating a subject in need thereof, the methods comprising applying an alternating current electric field to a target site in the subject in need thereof and administering a cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof. In some embodiments, the CAF inhibitor can be a CAF activation inhibitor or a CAF signaling inhibitor.

[0007] A method of treating a subject in need thereof is disclosed, the method comprising applying an alternating current electric field to a target site in the subject in need thereof and administering a CAF activation inhibitor to the subject in need thereof.

[0008] A method of treating a subject in need thereof is disclosed, the method comprising applying an alternating electric field to a target site in the subject in need thereof and administering a CAF signaling inhibitor to the subject in need thereof.

[0009] A method for reducing or preventing CAF activation is disclosed, the method comprising applying an alternating electric field to a cell population comprising one or more fibroblasts and contacting the cell population with a CAF activation inhibitor.

[0010] A method for reducing CAF signaling is disclosed, the method comprising applying an alternating electric field to a cell population comprising one or more fibroblasts and contacting the cell population with a cancer-associated fibroblast (CAF) signaling inhibitor.

[0011] A method for reducing tumor growth in a subject in need thereof is disclosed, the method comprising applying an alternating electric field to a target site in the subject in need thereof and administering a CAF inhibitor to the subject in need thereof.

[0012] A method for reducing tumor invasion in a subject in need thereof is disclosed, the method comprising applying an alternating electric field to a target site in a subject in need thereof and administering a CAF inhibitor to the subject in need thereof.

[0013] A method for reducing tumor angiogenesis in a subject in need thereof is disclosed, the method comprising applying an alternating electric field to a target site in the subject in need thereof and administering a CAF inhibitor to the subject in need thereof.

[0014] A method for reducing tumor metastasis in a subject in need thereof is disclosed, the method comprising applying an alternating electric field to a target site in a subject in need thereof and administering a CAF inhibitor to the subject in need thereof.

[0015] Additional advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed methods and compositions. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. [Brief explanation of the drawings]

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions.

[0017] [Figure 1A] 1 shows upregulation of PDGFRb in response to TT fields. [Figure 1B] 1 shows upregulation of PDGFRb in response to TT fields. [Figure 1C] 1 shows upregulation of PDGFRb in response to TT fields. [Figure 2A]Figure 1 shows upregulation of aSMA in response to TT fields. [Figure 2B] Figure 1 shows upregulation of aSMA in response to TT fields. [Figure 2C] Figure 1 shows upregulation of aSMA in response to TT fields. [Figure 3] The results shown in Figure 2C detail that not only did the amount of cells expressing aSMA increase, but the mean fluorescence intensity (MFI) per cell also increased, indicating that the cells expressed more aSMA. [Figure 4] Figure 4A shows a representative confocal image of aSMA expression in fibroblasts 48 hours after TT field application. Figure 4B shows increased aSMA expression in fibroblasts 48 hours after TT field application compared to untreated fibroblasts. [Figure 5A] Figure 1 shows that aSMA expression is increased in fibroblasts after incubation with conditioned medium (CM) derived from TT field-treated cancer cells. [Figure 5B] Figure 5 shows that aSMA expression increases in fibroblasts after incubation with conditioned medium (CM) from TT field-treated cancer cells. Figure 5A shows a schematic diagram of the experiment. Figure 5B shows a FACS analysis graph and the mean fluorescence of aSMA. [Figure 6] Figure 1 shows the process of isolation and characterization of fibroblasts from cells derived from single cell suspension tumors in the lungs of LLC2-bearing mice treated with TT field application or heat sham as a control. [Figure 7] FIG. 1 shows a scheme of the FACS analysis gating strategy for distinguishing activated fibroblasts in a sample of cells derived from tumor single cell dissociation. [Figure 8] Figure 1 shows that aSMA expression is increased in tumor-derived CD45- / CD31- / PDGFR+CD29+ cells after TT field application. [Figure 9] Figure 1 shows that aSMA expression is increased in tumor-derived CD45- / CD31- / PDGFR+CD29+ cells after TT field application. [Figure 10]Secretion of CAF markers, such as MCP1 and CD26, is shown in TT field-treated cells, indicating differentiation of fibroblasts into CAFs. Conditioned medium from MRC5 fibroblasts after 48 or 72 hours of TT field application at 150 kHz, incubated in a secreted cytokine array assay according to the product manual, revealed increased secretion of MCP1 (monocyte chemoattractant protein 1) secreted by activated fibroblasts and CD26, a CAF marker indicating differentiation of fibroblasts into CAFs. CD26 increased 1.5-fold and 2-fold after 48 and 72 hours of TT field treatment, respectively, and MCP-1 increased 1.5-fold and 1.4-fold after 48 and 72 hours of TT field treatment, respectively. Analysis was performed using HLImage++ QuickSpot software. [Figure 11] Examples of concentrations (pg / ml) of active TGF-β1 and total TGF-β1 measured in serum from mice injected with LL / 2 NSCLC cells and treated for 8 days with TT field or mock heating using ELISA are shown. (N control = 7 TT field = 7) [Figure 12] Examples of concentrations (pg / mg) of active TGF-β1 and total TGF-β1 measured in lung tumors from mice injected with LL / 2 NSCLC cells and treated with TT field or mock heating for 8 days using ELISA are shown. (N control = 7 TT field = 7) [Figure 13] Figure 1 shows the tissue expression of collagen 1 and Lox. A TT field was applied continuously for 10 days at a frequency of 200 kHz to mice bearing ovarian tumors. Treatment with the TT field significantly increased LOX protein expression. [Figure 14] Figure 1 shows the tissue expression of collagen 4 and fibronectin. A TT field was applied continuously for 10 days at a frequency of 200 kHz to mice bearing ovarian tumors. Treatment with the TT field significantly increased collagen 4 protein expression. DETAILED DESCRIPTION OF THE INVENTION

[0018] The disclosed methods and compositions may be more readily understood by reference to the following detailed description of specific embodiments and examples included therein, as well as the figures and accompanying description.

[0019] It is to be understood that the disclosed methods and compositions are not limited to particular synthetic methods, specific analytical techniques, or particular reagents, unless otherwise specified, and that these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0020] Disclosed are materials, compositions, and ingredients that can be used in, can be used in combination with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even though specific reference to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed. Thus, if a class of molecules A, B, and C is disclosed, and further an example of a combined molecule AD with a class of molecules D, E, and F is disclosed, each is considered individually and collectively, even if each is not individually described. Thus, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE, and CF is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C, D, E, and F, and the example combination AD. Likewise, subsets or combinations of these are also specifically contemplated and disclosed. Thus, for example, the subgroups AE, BF, and CE are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C, D, E, and F, as well as example combinations A-D. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed in any particular embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0021] Headings are provided for convenience only and should not be construed as limiting the invention in any way. Embodiments shown under any heading or in any portion of this disclosure may be combined with embodiments shown under the same or other headings or in other portions of this disclosure. A.Definition

[0022] It is understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims.

[0023] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a CAF activation inhibitor" includes a plurality of such CAF activation inhibitors, a reference to "a cell" is a reference to one or more inhibitors and equivalents thereof known to those skilled in the art, and so forth.

[0024] As used herein, a "target site" refers to a specific site or location within or on a subject or patient. For example, a "target site" can refer to, but is not limited to, a cell (e.g., a cancer cell or a cancer-associated fibroblast), a cell population, an organ, a tissue, or a tumor. Thus, the phrase "target cell" can be used to refer to a target site, and a target site is a cell. In some embodiments, a "target cell" can be a cancer cell. In some embodiments, an organ that can be a target site includes, but is not limited to, the brain. In some embodiments, a cell or cell population that can be a target site or target cell includes, but is not limited to, a cancer cell (e.g., a lung cancer cell). In some embodiments, a "target site" can be a tumor target site.

[0025] A "tumor target site" is a site or location within or on a subject or patient that contains one or more cancer cells, is adjacent to cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells. For example, a tumor target site may refer to a site or location within or on a subject or patient that is prone to metastasis. Additionally, a target site or tumor target site may refer to a site or location adjacent to the resection of a primary tumor within or on a subject or patient. Additionally, a target site or tumor target site may refer to a site or location adjacent to the resection of a primary tumor within or on a subject or patient.

[0026] As used herein, the term "alternating electric field" refers to a very low-intensity, directional, mid-frequency alternating electric field delivered to a subject, a sample taken from a subject, or a specific location (e.g., a target site, such as a cell) within a subject or patient. In some embodiments, the alternating electric field can be unidirectional or multidirectional, e.g., alternating directions across the target site. In some embodiments, the alternating electric field can be delivered through two pairs of transducer arrays that generate perpendicular fields within the target site. For example, in the case of the Optune® system (alternating electric field delivery system), one pair of electrodes is placed on the left and right (LR) sides of the target site, and another pair of electrodes is placed on the anterior-posterior (AP) sides of the target site. Cycling the electric field between these two directions (LR and AP) ensures that the widest range of cellular orientations is targeted.

[0027] As used herein, an "alternating electric field" applied to a tumor target site may be referred to as a "tumor treating electric field" or "TT field." TT fields have been established as antimitotic cancer treatments because they disrupt proper microtubule assembly during metaphase, ultimately destroying cells during telophase, cytokinesis, or subsequent interphase. TT fields target solid tumors and are described in U.S. Patent No. 7,565,205, which is incorporated herein by reference in its entirety for its teachings of TT fields.

[0028] In vivo and in vivo studies have shown that the effectiveness of TT field therapy increases with increasing field strength. Therefore, optimizing array placement on a subject to enhance intensity at a subject site or cell is a standard approach for the Optune system. Optimizing array placement may be performed using "rules of thumb" (e.g., placing the array as close as possible to the subject's target site or cell), measurements representing the patient's body shape, the dimensions of the target site, and / or the location of the target site or cell. Measurements used as input may be obtained from image data. Image data includes, for example, single-photon emission computed tomography (SPECT) image data, X-ray computed tomography (X-ray CT) data, magnetic resonance imaging (MRI) data, positron emission tomography (PET) data, and any type of visual data, such as data captured by optical instruments (e.g., photo cameras, charge-coupled device (CCD) cameras, infrared cameras, etc.). In certain embodiments, the image data may include 3D data (e.g., point cloud data) acquired from or generated by a 3D scanner. Optimization can depend on understanding how the electric field is distributed within the target site or target cell as a function of array position, and in some embodiments takes into account variations in the distribution of electrical properties within the heads of different patients.

[0029] The term "subject" refers to a subject of administration, e.g., an animal. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal. For example, the subject can be a human. The term does not denote a particular age or sex. "Subject" can be used interchangeably with "individual" or "patient." For example, the subject to be administered can refer to one that is subjected to an alternating electric field. For example, the subject to be administered can be a patient suffering from cancer, such as ovarian cancer or lung cancer.

[0030] "Treating" means administering or applying a therapeutic agent, such as an alternating electric field and a cancer-associated fibroblast (CAF) inhibitor, to a subject, such as a human or other mammal (e.g., an animal model), having cancer or having an increased susceptibility to developing cancer, to prevent or slow the worsening effects of a disease or infection, or to partially or completely reverse the effects of cancer. For example, treating a subject with lung cancer can include delivering a therapeutic agent to cells within the subject.

[0031] By "prevent" is meant minimizing or reducing the likelihood that a subject will develop cancer.

[0032] As used herein, the terms "administering" and "administration" refer to any method of directly or indirectly providing a CAF inhibitor to a target site in a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, intranasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous, intraarterial, intramuscular, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, the formulation can be administered therapeutically, i.e., to treat cancer. In further various embodiments, the formulation can be administered prophylactically, i.e., to prevent cancer. In one embodiment, a skilled artisan can determine an effective dosage, an effective schedule, or an effective route of administration to treat a subject. In some embodiments, administration includes exposure or application. Thus, in some embodiments, exposing a target site or subject to an alternating electric field or applying an alternating electric field to a target site or subject refers to subjecting the target site or subject to an alternating electric field.

[0033] "Optional" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur, and the description includes both the occurrence or presence of the event, circumstance, or material, and its non-occurrence or absence.

[0034] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such ranges are expressed, ranges from the one particular value and / or to the other particular value are specifically contemplated and considered to be disclosed, unless the context clearly dictates otherwise. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another specifically contemplated embodiment that should be considered disclosed, unless the context clearly dictates otherwise. Furthermore, unless the context clearly dictates otherwise, it is understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint. Finally, it should be understood that all individual values ​​and subranges of values ​​falling within an explicitly disclosed range are also specifically contemplated and should be considered disclosed, unless the context clearly dictates otherwise. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous, intraarterial, intramuscular, and subcutaneous administration.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of the present invention, particularly useful methods, devices, and materials are as described herein. Publications cited herein and the materials for which they are cited are expressly incorporated herein by reference. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The references state what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Although numerous publications are referenced herein, such reference is expressly not an admission that any of these documents constitutes part of the common general knowledge in the art.

[0036] Throughout this description and the claims, the word "comprise" and variations thereof, such as "comprising" and "comprises," mean "including but not limited to" and are not intended to exclude, for example, other additives, ingredients, integers, or steps. In particular, methods described as including one or more steps or operations are specifically contemplated to include each step that is recited (unless that step includes a limiting term such as "consisting of"). In other words, each step is not intended to exclude other additives, ingredients, integers, or steps, etc., not recited in that step. B. AC electric field

[0037] The methods disclosed herein include applying an alternating electric field. In some embodiments, the alternating electric field used in the methods disclosed herein is a tumor-treating electric field. In some embodiments, the alternating electric field may vary depending on the type or condition of the cells to which it is applied. In some embodiments, the alternating electric field may be applied via one or more electrodes placed on the subject's body. In some embodiments, two or more electrode pairs may be present. For example, arrays may be placed on the front / back and sides of the patient and used with the systems and methods disclosed herein. In some embodiments, when two pairs of electrodes are used, the alternating electric field may alternate between the electrode pairs. For example, a first electrode pair may be placed on the front and back of the subject, and a second electrode pair may be placed on either side of the subject, and then the alternating electric field may be applied alternately between the front and back electrodes, and then between the left and right electrodes.

[0038] In some embodiments, the frequency of the AC electric field is 100 kHz to 500 kHz. In some embodiments, the frequency of the AC electric field is 50 kHz to 1 MHz. The frequency of the AC electric field can be, but is not limited to, 50 to 500 kHz, 100 to 500 kHz, 25 kHz to 1 MHz, 50 to 190 kHz, 25 to 190 kHz, 150 to 300 kHz, 180 to 220 kHz, or 210 to 400 kHz. In some embodiments, the frequency of the AC electric field can be 50 kHz, 100 kHz, 150 kHz, 200 kHz, 250 kHz, 300 kHz, 350 kHz, 400 kHz, 450 kHz, 500 kHz, or any frequency therebetween. In some embodiments, the frequency of the AC electric field is about 200 kHz to about 400 kHz, about 250 kHz to about 350 kHz, or may be about 300 kHz.

[0039] In some embodiments, the field strength of the AC electric field can be in the range of 0.5-4 V / cm RMS. In some embodiments, the field strength of the AC electric field can be in the range of 1-4 V / cm RMS. In some embodiments, different field strengths (e.g., 0.1-10 V / cm RMS) can be used. In some embodiments, the field strength can be 1.75 V / cm RMS. In some embodiments, the field strength is at least 1 V / cm RMS. In some embodiments, the field strength can be 0.9 V / cm RMS. In other embodiments, a combination of field strengths is applied, e.g., combining two or more frequencies simultaneously or applying two or more frequencies at different times.

[0040] In some embodiments, the AC electric field can be applied at various intervals ranging from 0.5 hours to 72 hours. In some embodiments, different durations can be used (e.g., 0.5 hours to 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied for 2 hours per day. For example, the AC electric field can be applied for at least 4 hours per day, at least 8 hours per day, at least 12 hours per day, at least 16 hours per day, or at least 20 hours per day. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 2 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 3 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 7 days.

[0041] In some embodiments, the continuous exposure may continue for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or more of continuous exposure.

[0042] In some embodiments, the cumulative exposure may last for at least 42 hours, at least 84 hours, at least 168 hours, at least 250 hours, at least 400 hours, at least 500 hours, at least 750 hours, or more.

[0043] The disclosed method comprises applying one or more alternating current electric fields to cells or subjects.In some embodiments, the alternating current electric field is applied to target site or tumor target site.When applying the alternating current electric field to cells, this can refer to applying the alternating current electric field to the subject that constitutes the cells.Therefore, when applying the alternating current electric field to the target site of the subject, the alternating current electric field is applied to cells. C. Treatment method

[0044] Disclosed are methods of treating a subject in need of treatment, the methods comprising applying an alternating current electric field to a target site in the subject in need thereof and administering a cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof. In some embodiments, the CAF inhibitor can be a CAF activation inhibitor or a CAF signaling inhibitor. Thus, in some embodiments, the CAF inhibitor can prevent or reduce CAF activation or prevent or reduce signaling from CAFs.

[0045] A method of treating a subject in need thereof is disclosed, the method comprising applying an alternating current electric field to a target site in the subject in need thereof and administering a CAF activation inhibitor to the subject in need thereof.

[0046] Fibroblast activation protein (FAP) is a cell membrane-bound serine peptidase that is overexpressed in cancer-associated fibroblasts and activated fibroblasts at sites of wound healing / inflammation.

[0047] Platelet-derived growth factors (PDGFs) and their tyrosine kinase receptors (PDGFRs) are involved in the pathogenesis of many tumor types and play a key role in angiogenesis. Tumor growth can be promoted by PDGFs through autocrine stimulation of malignant cells, by overexpression or overactivation of PDGFRs, or by stimulation of angiogenesis within the tumor.

[0048] Hedgehog pathway inhibitors are being clinically tested to reduce CAF activation: Hedgehog signaling intersects with CAF heterogeneity in pancreatic ductal adenocarcinoma (PDAC). Acute inhibition of this pathway can alter the CAF activation ratio and convert the tumor microenvironment to a more immunosuppressive phenotype.

[0049] Hyaluronic acid, a hyaluronan depletor, is a major component of the ECM that has been shown to bind to tumor cells, contributing to CAF-induced tumor cell migration and invasion. Angiotensin inhibition has also shown promise in attenuating collagen and hyaluronan deposition by CAFs in clinical trials.

[0050] IL-1R inhibition with inhibitors such as anakinra is currently undergoing clinical trials, as pancreatic tumor-secreted IL-1 is known to activate the LIF / JAK / STAT pathway, which in turn activates inflammatory CAFs (iCAFs) in PDAC and promotes tumor growth.

[0051] Hsp90 (heat shock protein 90) is a chaperone protein that helps other proteins fold properly, stabilizes proteins against heat stress, and aids in protein degradation. It also stabilizes many proteins required for tumor growth. In some embodiments, Hsp90 inhibitors can limit the activation of cancer-associated fibroblasts (CAFs).

[0052] Leucine-rich repeat-containing 15 (LLRC15) is a plasma membrane-expressed protein encoded by the LRRC15 gene that is involved in cell-cell and cell-ECM interactions in humans. LRRC15 lacks a distinct intracellular domain. LRRC15 exhibits a highly restricted expression pattern, but is expressed in areas that constitute the innate immune barrier, such as the placenta, skin, activated fibroblasts in wounds, and lymphoid tissues such as the spleen. LRRC15 may play several roles in innate immunity. LRRC15 is aberrantly expressed in cancer. It is highly expressed in CAFs within the stroma of many solid tumors and directly expressed in mesenchymal tumors such as glioblastoma, sarcoma, and melanoma.

[0053] In some embodiments, the CAF activation inhibitor can be, but is not limited to, a fibroblast activation protein (FAP) inhibitor, a selective platelet-derived growth factor receptor beta (PDGFRb) inhibitor, an HSP90 inhibitor, an LLRC15 inhibitor, a hedgehog inhibitor, a hyaluronic acid depletor, or an IL-1R inhibitor.

[0054] In some embodiments, the FAP inhibitor can be, but is not limited to, talabostat or a small molecule inhibitor such as FAP-2286, Simlukafusp alfa (FAP-IL2v, RO6874281 / RG7461), SP-12786, gemigliptin, FAPI-4, FAPI-2, OncoFAP, FAPI-34, TAM558, BR102910, or a neutralizing antibody to a FAP such as TAM558. In some embodiments, the FAP inhibitor can be one of the antibodies provided at www.medchemexpress.com / search.html?q=FAP+inhibitor&ft=&fa=&fp=&fsp=&ftag=&fsc=, which is incorporated herein by reference for its teachings of FAP inhibitors.

[0055] In some embodiments, a selective PDGFRb inhibitor refers to an inhibitor that binds only to PDGFRb and does not bind to other PDGFR ligands. In some embodiments, the selective PDGFRb inhibitor can be, but is not limited to, sPDGFRbIg, crenolanib, orantinib, linifanib, CP-673451, SU16f, tyrphostin AG 1296, tyrphostin AG1433, SU4312, SU6668, AC710, JNJ 10198409, DMPQ, PD 166285, KG5, PDGFR tyrosine kinase inhibitor III, PDGFR-IN-1, ansornithinib, PP58, GZD856, or seraltinib.

[0056] In some embodiments, the Hsp90 inhibitor can be, but is not limited to, XL888, geldanamycin, gedannin, tanspimycin, luminespib, radicicol, 17-DMAG, EC144, herbimycin A, ganetespib, onarespib, NVP-BEP800, SNX-2112, PF-04929113, KW-2478, NMS-E973, zeravespib, pseudolaric acid A, VER-49009, pirimitespib, CH538303, VER-50589, kakuruniacin, HSP990, BIIV021, CCT 018159, 17-AAG. In some embodiments, the Hsp90 inhibitor can be one or more of the antibodies provided at www.medchemexpress.com / search.html?q=hsp90&ft=&fa=&fp=&fsp=&ftag=&fsc=, which is incorporated herein by reference for its teachings of Hsp90 inhibitors.

[0057] In some embodiments, the LLRC15 inhibitor can be, but is not limited to, ABBV-085.

[0058] In some embodiments, the hedgehog inhibitor can be, but is not limited to, IPI-926, vismodegib, KAAD-cyclopamine, cyclopamine, lobotonikinin, 3-epi-vitamin D3, sonidegib (NVP-LDE225), Jervine, HPI 1, dynalestin, TAK-441, TPB15, JK184, cryoblevin A, Hh-Ag1.5, SANT 2, MK-4101, MRT 10, U 18666A, CUR61414, or itraconazole (R51211). In some embodiments, the hedgehog inhibitor can be one or more of those provided at www.medchemexpress.com / search.html?q=hedgehog&ft=&fa=&fp=&fsp=&ftag=&fsc, which is incorporated herein by reference for its teachings of hedgehog inhibitors.

[0059] In some embodiments, the hyaluronic acid depletor can be, but is not limited to, PEGPH20, 4-MU, hyaluronidase, or an angiotensin receptor agent that attenuates hyaluronan secretion, such as losartan, metopivavir, pratosartan, mepicetil, LY285434, azilsartan mopivanil, tasosartan, ZD 7155, BIBS 39, or CGP 48369. In some embodiments, the hyaluronic acid depletor is The antibody may be one or more of the antibodies provided at www.tocris.com / search?keywords=angiotensin+inhibitor or www.medchemexpress.com / search.html?q=angiotensin+II+receptor+antagonist&ft=&fa=&fp=&fsp=&ftag=&fsc=, which are incorporated herein by reference for their teachings of hyaluronic acid depletors.

[0060] In some embodiments, the IL-1R inhibitor can be, but is not limited to, anakinra, AF12198, or diacerein.

[0061] A method of treating a subject in need thereof is disclosed, the method comprising applying an alternating current electric field to a target site in the subject in need thereof and administering a CAF signaling inhibitor to the subject in need thereof.

[0062] In some embodiments, the CAF signaling inhibitor can be, but is not limited to, a Lox (lysyl oxidase) inhibitor or a LoxL (lysyl oxidase-like) 1-4 inhibitor. In some embodiments, the LoxL inhibitor can be, but is not limited to, a monoclonal antibody against lysyl oxidase-like-2 / 3, such as cituzumab, PXS-5120A, PAT-1251, PXS-4787, PXS-6302, an anti-LOXL 1-4 antibody, or a small molecule inhibitor, such as PXS-5153A. In some embodiments, the LOX inhibitor can be, but is not limited to, LOX-IN-3, β-aminopropionitrile (BAPN), CCT365623, PXS-6302, PXS-5505, mIR142-3p, or PXS-4787. In some embodiments, the LOX inhibitor can be one or more of the antibodies provided at www.medchemexpress.com / search.html?q=Lysyl+oxidase&ft=&fa=&fp=&fsp=&ftag=&fsc=&type=inhibitors-and-agonists, which is incorporated herein by reference for its teachings of LOX inhibitors.

[0063] In some embodiments, the CAF signaling inhibitor can be, but is not limited to, a CXCR4 inhibitor or a CXCL12 inhibitor. Because CXCL12 is secreted by CAFs and promotes tumorigenesis, CXCL12-CXCR4 axis antagonism has been tested in clinical trials. CXCR4 inhibitors include, for example, Olaptesed (NOX-A12), BL-8040 CXCR4 antagonist, TC14012, KRH 2955, IT1t, plerixafor (AMD 3100), WZ811, USL311, LY2510924, MSX-127, MSX-130, AMD 3465, CTCE 9908, tannic acid, motixafortide (BL-8040), MSX-122, balixafortide (POL6326), ATI 2341, or CXCR4 antibodies. CXCL12 inhibitors include, for example, UNBS5162, LIT-927, or CXCL12 antibodies. In some embodiments, any of the CAF signaling inhibitors are described in Izumi et al. Int J Cancer. 2016 Mar 1;138(5):1207-19, the entire contents of which are incorporated herein by reference.

[0064] In some embodiments, the target site comprises one or more cancer cells. In some embodiments, the target site comprises one or more mesothelioma cells, ovarian cancer cells, cervical cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, breast cancer cells, hepatocellular carcinoma cells, renal cancer cells, or colon cancer cells. In some embodiments, the target site comprises cancer cells from any type of cancer.

[0065] In some embodiments, the alternating electric field is applied before, after, or simultaneously with administration of a CAF inhibitor (e.g., a CAF activation inhibitor or a CAF signaling inhibitor). In some embodiments, the step of applying the alternating electric field begins at least 1 hour before administration of the CAF inhibitor (e.g., a CAF activation inhibitor or a CAF signaling inhibitor). In some embodiments, the step of applying the alternating electric field begins at least 30 minutes before administration of the CAF inhibitor (e.g., a CAF activation inhibitor or a CAF signaling inhibitor). In some embodiments, simultaneously applying an alternating electric field can mean application within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes before or after administration of the CAF inhibitor (e.g., a CAF activation inhibitor or a CAF signaling inhibitor). In some embodiments, the alternating electric field can be applied and the CAF inhibitor (e.g., a CAF activation inhibitor or a CAF signaling inhibitor) can be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after each other.

[0066] In some embodiments, the CAF inhibitor is administered intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intrathecally, intravascularly, intravenously, intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, topically, via intratumoral injection, or via inhalation.

[0067] In some embodiments, the effect of a CAF activation inhibitor can be determined by counting the number of activated fibroblasts and / or detecting the presence or absence of CAF markers. For example, in some embodiments, the CAF activation inhibitor reduces the number of activated fibroblasts. In some embodiments, the CAF activation inhibitor inhibits or reduces FAP, alpha-smooth muscle actin (aSMA), or PDGFRb expression, hedgehog expression, or hyaluronic acid formation.

[0068] In some embodiments, the effect of a CAF signaling inhibitor can be determined by detecting downstream signaling events, hi some embodiments, the CAF signaling inhibitor can block a ligand or receptor involved in CAF signaling, thereby preventing a signaling event.

[0069] In some embodiments, the CAF inhibitor blocks AC electric field-induced upregulation of FAP, aSMA, PDGFRb, and / or hedgehog expression and / or hyaluronan formation. In some embodiments, the CAF inhibitor blocks AC electric field-induced signaling via FAP, aSMA, PDGFRb, and / or hedgehog.

[0070] In some embodiments, the CAF inhibitor prevents PDGFRb from interacting with or binding to PDGF, e.g., in some embodiments, the CAF inhibitor binds to or interacts with PDGFRb, thus preventing PDGFRb from interacting with or binding to its ligand, PDGF.

[0071] In some embodiments, the CAF activation inhibitor prevents Hsp90 ATPase activity. In some embodiments, Hsp90 ATPase activity upregulates fibroblast activation, and therefore, preventing or inhibiting Hsp90 ATPase activity can inhibit CAF activation.

[0072] In some embodiments, extracellular Hsp90α interacts with ER stress (which can be induced by an AC electric field) to promote fibroblast activation. Thus, in some embodiments, Hsp90 inhibitors can prevent or inhibit CAF activation.

[0073] In some embodiments, the Hedgehog pathway intersects with CAF heterogeneity to promote fibroblast activation. Thus, in some embodiments, Hedgehog inhibitors can prevent or inhibit CAF activation.

[0074] In some embodiments, inhibiting hyaluronic acid formation inhibits the binding of hyaluronic acid to tumor cells, thereby inhibiting the promotion of CAFs that metastasize and invade tumors and promote tumorigenesis.

[0075] In some embodiments, inhibition of angiotensin inhibits hyaluronic acid formation, which inhibits the binding of hyaluronic acid to tumor cells, thereby encouraging CAFs to metastasize and invade tumors and promote tumorigenesis.

[0076] In some embodiments, inhibition of IL-1R inhibits binding to IL-1R and inhibits signaling that leads to activation of CAFs.

[0077] In some embodiments, CAF signaling inhibitors prevent Lox or Loxl from covalently cross-linking collagen and elastin in the extracellular matrix (ECM). The lysyl oxidase (LOX) family of enzymes plays an important role in the formation, maturation, and remodeling of the extracellular matrix (ECM), which supports tumor growth and metastasis. Thus, in some embodiments, inhibiting Lox or Loxl-mediated signaling can treat cancer.

[0078] In some embodiments, inhibition of CXCR4 inhibits the binding of CXCL12 to CXCR4, thus inhibiting the secretion of CXCL12 from CAFs, which promotes tumorigenesis.

[0079] In some embodiments, the frequency of the AC electric field is 100 kHz to 500 kHz. In some embodiments, the frequency of the AC electric field is between 50 kHz and 1 MHz. In some embodiments, the frequency of the AC electric field is about 150 kHz, about 200 kHz, or about 250 kHz. The frequency of the AC electric field can be, but is not limited to, 50 to 500 kHz, 100 to 500 kHz, 25 kHz to 1 MHz, 50 to 190 kHz, 25 to 190 kHz, 150 to 300 kHz, 180 to 220 kHz, or 210 to 400 kHz. In some embodiments, the frequency of the AC electric field can be 50 kHz, 100 kHz, 150 kHz, 200 kHz, 250 kHz, 300 kHz, 350 kHz, 400 kHz, 450 kHz, 500 kHz, or any frequency therebetween. In some embodiments, the frequency of the AC electric field is about 200 kHz to about 400 kHz, about 250 kHz to about 350 kHz, or may be about 300 kHz. In some embodiments, the AC electric field can be in any of the ranges described herein.

[0080] In some embodiments, the AC electric field has a field strength of 0.1-10 V / cm RMS. In some embodiments, the AC electric field has a field strength of 0.5-4 V / cm RMS. In some embodiments, the field strength can be about or at least 1.75 V / cm RMS. In some embodiments, the field strength is about or at least 1 V / cm RMS. In some embodiments, the AC electric field has a field strength of about or at least 0.9 V / cm RMS. In some embodiments, the AC electric field has any of the field strengths described herein. In other embodiments, a combination of field strengths is applied, for example, combining two or more frequencies simultaneously or applying two or more frequencies at different times.

[0081] In some embodiments, the AC electric field can be applied at various intervals ranging from 0.5 hours to 72 hours. In some embodiments, different durations can be used (e.g., 0.5 hours to 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied for 2 hours per day. For example, the AC electric field can be applied for at least 4 hours per day, at least 8 hours per day, at least 12 hours per day, at least 16 hours per day, or at least 20 hours per day. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 2 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 3 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 7 days.

[0082] In some embodiments, the continuous exposure may continue for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or more of continuous exposure.

[0083] In some embodiments, the cumulative exposure may last for at least 42 hours, at least 84 hours, at least 168 hours, at least 250 hours, at least 400 hours, at least 500 hours, at least 750 hours, or more.

[0084] In some embodiments, the disclosed treatment methods may further include administering a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a known cancer therapeutic agent other than an FGF activation inhibitor or a CAF signaling inhibitor. For example, the cancer therapeutic agent can be, but is not limited to, chemotherapy, radiation therapy, immunotherapy, or hormone therapy. In some embodiments, the cancer therapeutic agent can be, but is not limited to, a tyrosine kinase inhibitor, a PI3K inhibitor, an Akt inhibitor, a VEGF inhibitor, or an FGF inhibitor.

[0085] In some embodiments, the alternating electric field is applied before, after, or simultaneously with administration of the cancer therapeutic agent. In some embodiments, the CAF inhibitor (e.g., a CAF activation inhibitor or a CAF signaling inhibitor) is applied before, after, or simultaneously with administration of the cancer therapeutic agent. In some embodiments, the CAF inhibitor (e.g., a CAF activation inhibitor or a CAF signaling inhibitor) and the cancer therapeutic agent are administered simultaneously, and the alternating electric field is applied before or after the CAF activation inhibitor or CAF signaling inhibitor and the cancer therapeutic agent.

[0086] In some embodiments, after applying the AC electric field and before administering the CAF inhibitor, the method can further include detecting an increase in αSMA, FAP, PDGFRβ, ITDB1, CAV1, and / or CD26 expression in the subject. In some embodiments, this detecting step allows confirmation that an increase in αSMA, FAP, PDGFRβ, ITDB1, CAV1, and / or CD26 expression is detected in response to the AC electric field, thus requiring administration of a CAF inhibitor.

[0087] In some embodiments, administration of the CAF inhibitor occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after application of the AC electric field. In some embodiments, the amount of time to wait after application of the AC electric field and before administering the CAF inhibitor can depend on the amount of time it takes for the AC electric field to cause fibroblast activation or increase expression of CAF markers.

[0088] In some embodiments, the target site comprises one or more cancer cells. In some embodiments, the target site comprises one or more mesothelioma cells, ovarian cancer cells, cervical cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, breast cancer cells, hepatocellular carcinoma cells, renal cancer cells, or colon cancer cells. In some embodiments, the target site comprises cancer cells from any type of cancer.

[0089] In some embodiments, the subject in need thereof does not receive chemotherapy concurrently in combination with the alternating electric field. In some embodiments, the subject in need thereof does not receive chemotherapy prior to receiving the alternating electric field. In some embodiments, the subject in need thereof does not receive chemotherapy within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to receiving the alternating electric field.

[0090] In some embodiments, the subject in need thereof is a subject with cancer. D. Methods to Reduce / Prevent CAF Activation

[0091] A method for reducing or preventing CAF activation is disclosed, the method comprising applying an alternating electric field to a cell population comprising one or more fibroblasts and contacting the cell population with a CAF activation inhibitor.

[0092] In some embodiments, reducing or preventing CAF activation can be achieved by inhibiting FAP, PDGFRb, Hsp90, hedgehog, hyaluronic acid formation, IL-1R, and / or LLRC15. Thus, in some embodiments, the CAF activation inhibitor can be, but is not limited to, a fibroblast activation protein (FAP) inhibitor, a selective PDGFRb inhibitor, a selective hedgehog inhibitor, a selective hyaluronic acid depletor, an IL-1R inhibitor, an HSP90 inhibitor, or an LLRC15 inhibitor.

[0093] In some embodiments, the FAP inhibitor can be, but is not limited to, talabostat or a small molecule inhibitor such as FAP-2286, Simlukafusp alfa (FAP-IL2v, RO6874281 / RG7461), SP-12786, gemigliptin, FAPI-4, FAPI-2, OncoFAP, FAPI-34, TAM558, BR102910, or a neutralizing antibody to a FAP such as TAM558. In some embodiments, the FAP inhibitor can be one or more of the provided inhibitors, which are incorporated herein by reference for their teachings of FAP inhibitors.

[0094] In some embodiments, a selective PDGFRb inhibitor refers to an inhibitor that binds only to PDGFRb and does not bind to other PDGFR ligands. In some embodiments, the selective PDGFRb inhibitor can be, but is not limited to, sPDGFRbIg, crenolanib, orantinib, linifanib, CP-673451, SU16f, tyrphostin AG 1296, tyrphostin AG1433, SU4312, SU6668, AC710, JNJ 10198409, DMPQ, PD 166285, KG5, PDGFR tyrosine kinase inhibitor III, PDGFR-IN-1, ansornithinib, PP58, GZD856, or seraltinib.

[0095] In some embodiments, the Hsp90 inhibitor can be, but is not limited to, XL888, geldanamycin, gedannin, tanspimycin, luminespib, radicicol, 17-DMAG, EC144, herbimycin A, ganetespib, onarespib, NVP-BEP800, SNX-2112, PF-04929113, KW-2478, NMS-E973, zeravespib, pseudolaric acid A, VER-49009, pirimitespib, CH538303, VER-50589, kakuruniacin, HSP990, BIIV021, CCT 018159, 17-AAG. In some embodiments, the Hsp90 inhibitor can be one or more of those provided at www.medchemexpress.com / search.html?q=hsp90&ft=&fa=&fp=&fsp=&ftag=&fsc=, which is incorporated herein by reference for its teachings of Hsp90 inhibitors.

[0096] In some embodiments, the LLRC15 inhibitor can be, but is not limited to, ABBV-085.

[0097] In some embodiments, the cell population can be in vitro or in vivo. For example, applying an alternating electric field can include applying the alternating electric field to a culture dish containing the cell population. In some embodiments, applying an alternating electric field can include applying the alternating electric field to a subject, wherein the cell population is within the subject. In some embodiments, applying an alternating electric field to a cell population in a subject includes applying the alternating electric field to a target site in the subject, wherein the target site comprises the cell population. In some embodiments, the target site comprises one or more cancer cells. Thus, in some embodiments, the target site comprises one or more cancer cells. In some embodiments, the target site comprises one or more mesothelioma cells, ovarian cancer cells, cervical cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, breast cancer cells, hepatocellular carcinoma cells, renal cancer cells, or colon cancer cells. In some embodiments, the target site comprises cancer cells from any type of cancer.

[0098] In some embodiments, the AC electric field is applied before, after, or simultaneously with the administration of the CAF activation inhibitor. In some embodiments, the step of applying the AC electric field is initiated at least 1 hour before the addition of the CAF activation inhibitor. In some embodiments, the step of applying the AC electric field is initiated at least 30 minutes before the addition of the CAF activation inhibitor. In some embodiments, applying the AC electric field simultaneously can mean applying the AC electric field within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes before or after the administration of the CAF activation inhibitor. In some embodiments, the AC electric field and the CAF activation inhibitor can be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours apart from each other.

[0099] In some embodiments, the CAF activation inhibitor is administered intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intrathecally, intravascularly, intravenously, intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, topically, via intratumoral injection, or via inhalation.

[0100] In some embodiments, the effect of a CAF activation inhibitor can be determined by counting the number of activated fibroblasts and / or detecting the presence or absence of CAF markers. For example, in some embodiments, the CAF activation inhibitor reduces the number of activated fibroblasts. In some embodiments, the CAF activation inhibitor inhibits or reduces FAP, alpha-smooth muscle actin (aSMA), PDGFRb expression, hedgehog expression, or hyaluronic acid formation.

[0101] In some embodiments, the CAF activation inhibitor blocks the upregulation of FAP, aSMA and / or PDGFRb expression by an AC electric field. In some embodiments, the CAF inhibitor blocks signal transduction via FAP, aSMA and / or PDGFRb by an AC electric field.

[0102] In some embodiments, the CAF activation inhibitor prevents PDGFRb from interacting with or binding to PDGF, e.g., in some embodiments, the CAF inhibitor binds to or interacts with PDGFRb, thus preventing PDGFRb from interacting with or binding to its ligand, PDGF.

[0103] In some embodiments, the CAF inhibitor prevents Hsp90 ATPase activity. In some embodiments, Hsp90 ATPase activity upregulates fibroblast activation, and therefore, preventing or inhibiting Hsp90 ATPase activity can inhibit CAF activation.

[0104] In some embodiments, extracellular Hsp90α interacts with ER stress (which can be induced by an AC electric field) to promote fibroblast activation. Thus, in some embodiments, CAF inhibitors that are Hsp90 inhibitors can prevent or inhibit CAF activation.

[0105] In some embodiments, the frequency of the AC electric field is 100 kHz to 500 kHz. In some embodiments, the frequency of the AC electric field is between 50 kHz and 1 MHz. In some embodiments, the frequency of the AC electric field is about 150 kHz, about 200 kHz, or about 250 kHz. The frequency of the AC electric field can be, but is not limited to, 50 to 500 kHz, 100 to 500 kHz, 25 kHz to 1 MHz, 50 to 190 kHz, 25 to 190 kHz, 150 to 300 kHz, 180 to 220 kHz, or 210 to 400 kHz. In some embodiments, the frequency of the AC electric field can be 50 kHz, 100 kHz, 150 kHz, 200 kHz, 250 kHz, 300 kHz, 350 kHz, 400 kHz, 450 kHz, 500 kHz, or any frequency therebetween. In some embodiments, the frequency of the AC electric field is about 200 kHz to about 400 kHz, about 250 kHz to about 350 kHz, or may be about 300 kHz. In some embodiments, the AC electric field can be in any of the ranges described herein.

[0106] In some embodiments, the AC electric field has a field strength of 0.1-10 V / cm RMS. In some embodiments, the AC electric field has a field strength of 0.5-4 V / cm RMS. In some embodiments, the field strength can be about or at least 1.75 V / cm RMS. In some embodiments, the field strength is about or at least 1 V / cm RMS. In some embodiments, the AC electric field has a field strength of about or at least 0.9 V / cm RMS. In some embodiments, the AC electric field has any of the field strengths described herein. In other embodiments, a combination of field strengths is applied, for example, combining two or more frequencies simultaneously or applying two or more frequencies at different times.

[0107] In some embodiments, the AC electric field can be applied at various intervals ranging from 0.5 hours to 72 hours. In some embodiments, different durations can be used (e.g., 0.5 hours to 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied for 2 hours per day. For example, the AC electric field can be applied for at least 4 hours per day, at least 8 hours per day, at least 12 hours per day, at least 16 hours per day, or at least 20 hours per day. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 2 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 3 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 7 days.

[0108] In some embodiments, the continuous exposure may continue for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or more of continuous exposure.

[0109] In some embodiments, the cumulative exposure may last for at least 42 hours, at least 84 hours, at least 168 hours, at least 250 hours, at least 400 hours, at least 500 hours, at least 750 hours, or more.

[0110] In some embodiments, after applying the AC electric field and before contacting the cells with the CAF activation inhibitor, the method further comprises detecting an increase in αSMA, FAP, PDGFRβ, ITDB1, hedgehog, hyaluronic acid, Hsp90, and / or CD26, and / or CAV1 expression in the cells. In some embodiments, this detecting step allows for confirmation that an increase in αSMA, FAP, PDGFRβ, ITDB1, hedgehog, hyaluronic acid, Hsp90, CD26, and / or CAV1 expression is detected in response to the AC electric field, thus requiring contacting with a CAF inhibitor.

[0111] In some embodiments, administration of the CAF activation inhibitor occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after application of the AC electric field. In some embodiments, the amount of time required to wait after application of the AC electric field and before contact with the CAF activation inhibitor can depend on the amount of time it takes for the AC electric field to cause fibroblast activation or increase expression of CAF markers.

[0112] In some embodiments, when the cell population is in a subject, the subject does not simultaneously receive chemotherapy in combination with the alternating electric field. In some embodiments, when the cell population is in a subject, the subject does not receive chemotherapy prior to receiving the alternating electric field. In some embodiments, when the cell population is in a subject, the subject does not receive chemotherapy within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to receiving the alternating electric field.

[0113] In some embodiments, the subject in need thereof is a subject with cancer. E. Methods for reducing CAF signaling

[0114] A method for reducing CAF signaling is disclosed, the method comprising applying an alternating electric field to a cell population comprising one or more fibroblasts and contacting the cell population with a cancer-associated fibroblast (CAF) signaling inhibitor.

[0115] In some embodiments, the CAF signaling inhibitor can be, but is not limited to, a Lox (lysyl oxidase) inhibitor or a LoxL (lysyl oxidase-like) inhibitor. In some embodiments, the LoxL inhibitor can be, but is not limited to, a monoclonal antibody against lysyl oxidase-like-1 / 2 / 3 / 4, such as cituzumab, PXS-5120A, PAT-1251, PXS-4787, PXS-6302, anti-LOXL 1-4 antibody [LA3], or a small molecule inhibitor, such as PXS-5153A. In some embodiments, the LOX inhibitor can be, but is not limited to, LOX-IN-3, β-aminopropionitrile (BAPN), CCT365623, PXS-6302, PXS-5505, mIR142-3p [LA4], or PXS-4787. In some embodiments, the LOX inhibitor can be, but is not limited to, The antibody may be one or more of the antibodies provided at www.medchemexpress.com / search.html?q=Lysyl+oxidase&ft=&fa=&fp=&fsp=&ftag=&fsc=&type=inhibitors-and-agonists, which is incorporated herein by reference for its teachings of LOX inhibitors.

[0116] In some embodiments, the CAF signaling inhibitor can be, but is not limited to, a CXCR4 inhibitor or a CXCL-12 inhibitor. Because CXCL12 is secreted by CAFs and promotes tumorigenesis, CXCL12-CXCR4 axis antagonism has been tested in clinical trials. CXCR4 inhibitors include, for example, Olaptesed (NOX-A12), BL-8040 CXCR4 antagonist, TC14012, KRH 2955, IT1t, plerixafor (AMD 3100), WZ811, USL311, LY2510924, MSX-127, MSX-130, AMD 3465, CTCE 9908, tannic acid, motixafortide (BL-8040), MSX-122, balixafortide (POL6326), ATI 2341, or CXCR4 antibodies. CXCL12 inhibitors include, for example, UNBS5162, LIT-927, or CXCL12 antibodies.

[0117] For example, applying an alternating electric field can include applying the alternating electric field to a culture dish containing the cell population. In some embodiments, applying an alternating electric field can include applying the alternating electric field to a subject, wherein the cell population is within the subject. In some embodiments, applying an alternating electric field to a cell population in a subject includes applying the alternating electric field to a target site in the subject, wherein the target site comprises the cell population. In some embodiments, the target site comprises one or more cancer cells. Thus, in some embodiments, the cell population is a cancer cell population. In some embodiments, the target site comprises one or more cancer cells. In some embodiments, the target site comprises one or more mesothelioma cells, ovarian cancer cells, cervical cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, breast cancer cells, hepatocellular carcinoma cells, renal cancer cells, or colon cancer cells. In some embodiments, the target site comprises cancer cells from any type of cancer.

[0118] In some embodiments, the AC electric field is applied before, after, or simultaneously with the administration of the CAF signaling inhibitor. In some embodiments, the step of applying the AC electric field begins at least 1 hour before the CAF signaling inhibitor is added. In some embodiments, the step of applying the AC electric field begins at least 30 minutes before the CAF signaling inhibitor is added. In some embodiments, applying the AC electric field simultaneously can mean applying the AC electric field within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes before or after the administration of the CAF signaling inhibitor. In some embodiments, the AC electric field and the CAF signaling inhibitor can be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours apart from each other.

[0119] In some embodiments, the CAF signaling inhibitor is administered intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intrathecally, intravascularly, intravenously, intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, topically, via intratumoral injection, or via inhalation.

[0120] In some embodiments, the effect of a CAF signaling inhibitor can be determined by detecting downstream signaling events, hi some embodiments, the CAF signaling inhibitor can block a ligand or receptor involved in CAF signaling, thereby preventing a signaling event.

[0121] In some embodiments, CAF signaling inhibitors prevent Lox or Loxl from covalently cross-linking collagen and elastin in the extracellular matrix (ECM). The lysyl oxidase (LOX) family of enzymes plays an important role in the formation, maturation, and remodeling of the extracellular matrix (ECM), which supports tumor growth and metastasis. Thus, in some embodiments, inhibiting Lox or Loxl-mediated signaling can treat cancer.

[0122] In some embodiments, because CXCL12 is secreted by CAFs and promotes tumorigenesis, CXCL12-CXCR4 axis antagonism has been tested in clinical trials. CXCR4 inhibitors include, for example, Olaptesed (NOX-A12), BL-8040 CXCR4 antagonist, TC14012, KRH 2955, IT1t, plerixafor (AMD 3100), WZ811, USL311, LY2510924, MSX-127, MSX-130, AMD 3465, CTCE 9908, tannic acid, motixafortide (BL-8040), MSX-122, balixafortide (POL6326), ATI 2341, or CXCR4 antibodies. The CXCL12 inhibitor is, for example, UNBS5162, LIT-927 or a CXCL12 antibody.

[0123] In some embodiments, the frequency of the AC electric field is 100 kHz to 500 kHz. In some embodiments, the frequency of the AC electric field is between 50 kHz and 1 MHz. In some embodiments, the frequency of the AC electric field is about 150 kHz, about 200 kHz, or about 250 kHz. The frequency of the AC electric field can be, but is not limited to, 50 to 500 kHz, 100 to 500 kHz, 25 kHz to 1 MHz, 50 to 190 kHz, 25 to 190 kHz, 150 to 300 kHz, 180 to 220 kHz, or 210 to 400 kHz. In some embodiments, the frequency of the AC electric field can be 50 kHz, 100 kHz, 150 kHz, 200 kHz, 250 kHz, 300 kHz, 350 kHz, 400 kHz, 450 kHz, 500 kHz, or any frequency therebetween. In some embodiments, the frequency of the AC electric field is about 200 kHz to about 400 kHz, about 250 kHz to about 350 kHz, or may be about 300 kHz. In some embodiments, the AC electric field can be in any of the ranges described herein.

[0124] In some embodiments, the AC electric field has a field strength of 0.1-10 V / cm RMS. In some embodiments, the AC electric field has a field strength of 0.5-4 V / cm RMS. In some embodiments, the field strength can be about or at least 1.75 V / cm RMS. In some embodiments, the field strength is about or at least 1 V / cm RMS. In some embodiments, the AC electric field has a field strength of about or at least 0.9 V / cm RMS. In some embodiments, the AC electric field has any of the field strengths described herein. In other embodiments, a combination of field strengths is applied, for example, combining two or more frequencies simultaneously or applying two or more frequencies at different times.

[0125] In some embodiments, the AC electric field can be applied at various intervals ranging from 0.5 hours to 72 hours. In some embodiments, different durations can be used (e.g., 0.5 hours to 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied for 2 hours per day. For example, the AC electric field can be applied for at least 4 hours per day, at least 8 hours per day, at least 12 hours per day, at least 16 hours per day, or at least 20 hours per day. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 2 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 3 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 7 days.

[0126] In some embodiments, the continuous exposure may continue for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or more of continuous exposure.

[0127] In some embodiments, the cumulative exposure may last for at least 42 hours, at least 84 hours, at least 168 hours, at least 250 hours, at least 400 hours, at least 500 hours, at least 750 hours, or more.

[0128] In some embodiments, after applying the AC electric field and before contacting with the CAF activation inhibitor, the method further comprises detecting an increase in αSMA, FAP, PDGFRβ, hedgehog, hyaluronic acid, Hsp90, CD26, and / or CAV1 expression in the cells. In some embodiments, this detecting step allows confirmation that an increase in αSMA, FAP, PDGFRβ, hedgehog, hyaluronic acid, Hsp90, CD26, and / or CAV1 expression is detected in response to the AC electric field, thus requiring administration of a CAF inhibitor.

[0129] In some embodiments, administration of the CAF signaling inhibitor occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after application of the AC electric field. In some embodiments, the amount of time to wait after application of the AC electric field and before administering the CAF signaling inhibitor can depend on the amount of time it takes for the AC electric field to cause fibroblast activation or increase expression of CAF markers.

[0130] In some embodiments, the target site comprises one or more cancer cells. In some embodiments, the target site comprises one or more mesothelioma cells, ovarian cancer cells, cervical cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, breast cancer cells, hepatocellular carcinoma cells, renal cancer cells, or colon cancer cells. In some embodiments, the target site comprises cancer cells from any type of cancer.

[0131] In some embodiments, when the cell population is in a subject, the subject does not simultaneously receive chemotherapy in combination with the alternating electric field. In some embodiments, when the cell population is in a subject, the subject does not receive chemotherapy prior to receiving the alternating electric field. In some embodiments, when the cell population is in a subject, the subject does not receive chemotherapy within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to receiving the alternating electric field. F. Methods of Modifying Tumors

[0132] A method for reducing tumor growth in a subject in need thereof is disclosed, the method comprising applying an alternating electric field to a target site in the subject in need thereof and administering a CAF inhibitor to the subject in need thereof.

[0133] A method for reducing tumor invasion in a subject in need thereof is disclosed, the method comprising applying an alternating electric field to a target site in the subject in need thereof and administering a CAF activity inhibitor to the subject in need thereof.

[0134] A method for reducing tumor angiogenesis in a subject in need thereof is disclosed, the method comprising applying an alternating electric field to a target site in the subject in need thereof and administering a CAF inhibitor to the subject in need thereof.

[0135] Disclosed is a method of reducing tumor metastasis in a subject in need thereof, the method comprising applying an alternating current electric field to a target site in the subject in need thereof, and administering to the subject in need thereof a CAF activation inhibitor, e.g., a FAP inhibitor, a selective platelet-derived growth factor receptor beta (PDGFRb) inhibitor, a hedgehog inhibitor, hyaluronic acid depletion, an Hsp90 inhibitor, an IL-1R inhibitor, or an LLRC15 inhibitor.

[0136] In some embodiments, the target site comprises one or more cancer cells. In some embodiments, the target site comprises one or more mesothelioma cells, ovarian cancer cells, cervical cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, breast cancer cells, hepatocellular carcinoma cells, renal cancer cells, or colon cancer cells. In some embodiments, the target site comprises cancer cells from any type of cancer.

[0137] In some embodiments, the CAF inhibitor can be a CAF activation inhibitor or a CAF signaling inhibitor. Thus, in some embodiments, the CAF inhibitor can prevent or reduce CAF activation or prevent or reduce signaling from CAFs.

[0138] In some embodiments, the CAF activation inhibitor can be, but is not limited to, a fibroblast activation protein (FAP) inhibitor, a selective platelet-derived growth factor receptor beta (PDGFRb) inhibitor, an HSP90 inhibitor, hyaluronic acid depletion, a hedgehog inhibitor, an IL-1R inhibitor, or an LLRC15 inhibitor.

[0139] In some embodiments, the FAP inhibitor can be, but is not limited to, talabostat or a small molecule inhibitor such as FAP-2286, Simlukafusp alfa (FAP-IL2v, RO6874281 / RG7461), SP-12786, gemigliptin, FAPI-4, FAPI-2, OncoFAP, FAPI-34, TAM558, BR102910, or a neutralizing antibody to a FAP such as TAM558. In some embodiments, the FAP inhibitor can be one or more of the provided inhibitors, which are incorporated herein by reference for their teachings of FAP inhibitors.

[0140] In some embodiments, a selective PDGFRb inhibitor refers to an inhibitor that binds only to PDGFRb and does not bind to other PDGFR ligands. In some embodiments, the selective PDGFRb inhibitor can be, but is not limited to, sPDGFRbIg, crenolanib, orantinib, linifanib, CP-673451, SU16f, tyrphostin AG 1296, tyrphostin AG1433, SU4312, SU6668, AC710, JNJ 10198409, DMPQ, PD 166285, KG5, PDGFR tyrosine kinase inhibitor III, PDGFR-IN-1, ansornithinib, PP58, GZD856, or seraltinib.

[0141] In some embodiments, the Hsp90 inhibitor can be, but is not limited to, XL888, geldanamycin, gedannin, tanspimycin, luminespib, radicicol, 17-DMAG, EC144, herbimycin A, ganetespib, onarespib, NVP-BEP800, SNX-2112, PF-04929113, KW-2478, NMS-E973, zeravespib, pseudolaric acid A, VER-49009, pirimitespib, CH538303, VER-50589, kakuruniacin, HSP990, BIIV021, CCT 018159, 17-AAG. In some embodiments, the Hsp90 inhibitor can be one or more of the antibodies provided at www.medchemexpress.com / search.html?q=hsp90&ft=&fa=&fp=&fsp=&ftag=&fsc=, which is incorporated herein by reference for its teachings of Hsp90 inhibitors.

[0142] In some embodiments, the LLRC15 inhibitor can be, but is not limited to, ABBV-085.

[0143] In some embodiments, the hedgehog inhibitor can be, but is not limited to, IPI-926, vismodegib, KAAD-cyclopamine, cyclopamine, lobotonikinin, 3-epi-vitamin D3, sonidegib (NVP-LDE225), Jervine, HPI 1, dynalestin, TAK-441, TPB15, JK184, cryoblevin A, Hh-Ag1.5, SANT 2, MK-4101, MRT 10, U 18666A, CUR61414, or itraconazole (R51211). In some embodiments, the hedgehog inhibitor can be one or more of the inhibitors provided at www.medchemexpress.com / search.html?q=hedgehog&ft=&fa=&fp=&fsp=&ftag=&fsc, which is incorporated herein by reference for its teachings of hedgehog inhibitors.

[0144] In some embodiments, the hyaluronic acid depletor can be, but is not limited to, PEGPH20, 4-MU, hyaluronidase, or an angiotensin receptor agent that attenuates hyaluronan secretion, such as losartan, metopivavir, pratosartan, mepicetil, LY285434, azilsartan mopivanil, tasosartan, ZD 7155, BIBS 39, or CGP 48369 [LA3]°. In some embodiments, the hyaluronic acid depletor can be one or more of the inhibitors provided at www.tocris.com / search?keywords=angiotensin+inhibitor or www.medchemexpress.com / search.html?q=angiotensin+II+receptor+antagonist&ft=&fa=&fp=&fsp=&ftag=&fsc=, which are incorporated herein by reference for their teachings on hyaluronic acid depletion.

[0145] In some embodiments, the IL-1R inhibitor can be, but is not limited to, anakinra, AF12198, or diacerein.

[0146] In some embodiments, the CAF signaling inhibitor can be, but is not limited to, a Lox (lysyl oxidase) inhibitor or a LoxL (lysyl oxidase-like [LA4]) inhibitor. In some embodiments, the LoxL inhibitor can be, but is not limited to, a monoclonal antibody against lysyl oxidase-like-2 / 3, such as cituzumab, PXS-5120A, PAT-1251, PXS-4787, PXS-6302, anti-LOXL 1-4 antibody [LA5], or a small molecule inhibitor, such as PXS-5153A. In some embodiments, the LOX inhibitor can be, but is not limited to, LOX-IN-3, β-aminopropionitrile (BAPN), CCT365623, PXS-6302, PXS-5505, mIR142-3p [LA6], or PXS-4787. In some embodiments, the LOX inhibitor can be one or more of those provided at www.medchemexpress.com / search.html?q=Lysyl+oxidase&ft=&fa=&fp=&fsp=&ftag=&fsc=&type=inhibitors-and-agonists, which is incorporated herein by reference for its teachings of LOX inhibitors [LA7].

[0147] In some embodiments, the CAF signaling inhibitor can be, but is not limited to, a CXCR4 inhibitor or a CXCL12 inhibitor. Because CXCL12 is secreted by CAFs and promotes tumorigenesis, CXCL12-CXCR4 axis antagonism has been tested in clinical trials. CXCR4 inhibitors include, for example, Olaptesed (NOX-A12), BL-8040 CXCR4 antagonist, TC14012, KRH 2955, IT1t, plerixafor (AMD 3100), WZ811, USL311, LY2510924, MSX-127, MSX-130, AMD 3465, CTCE 9908, tannic acid, motixafortide (BL-8040), MSX-122, balixafortide (POL6326), ATI 2341, or CXCR4 antibodies. CXCL12 inhibitors include, for example, UNBS5162, LIT-927, or CXCL12 antibodies.

[0148] In some embodiments, the AC electric field is applied before, after, or simultaneously with the administration of the CAF inhibitor.In some embodiments, the step of applying the AC electric field is started at least 1 hour before the administration of the CAF activity inhibitor or CAF signaling inhibitor.In some embodiments, the step of applying the AC electric field is started at least 30 minutes before the administration of the CAF activity inhibitor or CAF signaling inhibitor.In some embodiments, the AC electric field is applied simultaneously can mean that the AC electric field is applied within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes before or after the administration of the CAF activity inhibitor or CAF signaling inhibitor. In some embodiments, the alternating electric field can be applied and the CAF activity inhibitor or CAF signaling inhibitor can be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours apart from each other.

[0149] In some embodiments, the CAF inhibitor is administered intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intrathecally, intravascularly, intravenously, intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, topically, via intratumoral injection, or via inhalation.

[0150] In some embodiments, the effect of the CAF activation inhibitor can be determined by counting the number of activated fibroblasts and / or detecting the presence or absence of CAF markers. For example, in some embodiments, the CAF activation inhibitor reduces the number of activated fibroblasts. In some embodiments, the CAF activation inhibitor inhibits or reduces FAP, alpha-smooth muscle actin (aSMA), hedgehog, hyaluronic acid, or PDGFRb expression.

[0151] In some embodiments, the effect of a CAF signaling inhibitor can be determined by detecting downstream signaling events, hi some embodiments, the CAF signaling inhibitor can block a ligand or receptor involved in CAF signaling, thereby preventing a signaling event.

[0152] In some embodiments, the CAF inhibitor blocks AC electric field-induced upregulation of FAP, aSMA, hedgehog, hyaluronic acid, and / or PDGFRb expression. In some embodiments, the CAF inhibitor blocks AC electric field-induced signaling via FAP, aSMA, hedgehog, hyaluronic acid, and / or PDGFRb.

[0153] In some embodiments, the CAF inhibitor prevents PDGFRb from interacting with or binding to PDGF, e.g., in some embodiments, the CAF inhibitor binds to or interacts with PDGFRb, thus preventing PDGFRb from interacting with or binding to its ligand, PDGF.

[0154] In some embodiments, the CAF inhibitor prevents Hsp90 ATPase activity. In some embodiments, Hsp90 ATPase activity upregulates fibroblast activation, and therefore, preventing or inhibiting Hsp90 ATPase activity can inhibit CAF activation.

[0155] In some embodiments, extracellular Hsp90α interacts with ER stress (which can be induced by an AC electric field) to promote fibroblast activation. Thus, in some embodiments, CAF inhibitors that are Hsp90 inhibitors can prevent or inhibit CAF activation.

[0156] In some embodiments, CAF inhibitors prevent Lox or Loxl from covalently cross-linking collagen and elastin in the extracellular matrix (ECM). The lysyl oxidase (LOX) family of enzymes plays an important role in the formation, maturation, and remodeling of the extracellular matrix (ECM), which supports tumor growth and metastasis. Thus, in some embodiments, inhibiting signaling via Lox or Loxl can treat cancer.

[0157] In some embodiments, the Hedgehog pathway intersects with CAF heterogeneity to promote fibroblast activation. Thus, in some embodiments, Hedgehog inhibitors can prevent or inhibit CAF activation.

[0158] In some embodiments, inhibiting hyaluronic acid formation inhibits the binding of hyaluronic acid to tumor cells, thereby inhibiting the promotion of CAFs that metastasize and invade tumors and promote tumorigenesis.

[0159] In some embodiments, inhibition of angiotensin inhibits hyaluronic acid formation, which inhibits the binding of hyaluronic acid to tumor cells, thereby encouraging CAFs to metastasize and invade tumors and promote tumorigenesis.

[0160] In some embodiments, inhibition of IL-1R inhibits binding to IL-1R and inhibits signaling that leads to activation of CAFs.

[0161] In some embodiments, the frequency of the AC electric field is 100 kHz to 500 kHz. In some embodiments, the frequency of the AC electric field is between 50 kHz and 1 MHz. In some embodiments, the frequency of the AC electric field is about 150 kHz, about 200 kHz, or about 250 kHz. The frequency of the AC electric field can be, but is not limited to, 50 to 500 kHz, 100 to 500 kHz, 25 kHz to 1 MHz, 50 to 190 kHz, 25 to 190 kHz, 150 to 300 kHz, 180 to 220 kHz, or 210 to 400 kHz. In some embodiments, the frequency of the AC electric field can be 50 kHz, 100 kHz, 150 kHz, 200 kHz, 250 kHz, 300 kHz, 350 kHz, 400 kHz, 450 kHz, 500 kHz, or any frequency therebetween. In some embodiments, the frequency of the AC electric field is about 200 kHz to about 400 kHz, about 250 kHz to about 350 kHz, or may be about 300 kHz. In some embodiments, the AC electric field can be in any of the ranges described herein.

[0162] In some embodiments, the AC electric field has a field strength of 0.1-10 V / cm RMS. In some embodiments, the AC electric field has a field strength of 0.5-4 V / cm RMS. In some embodiments, the field strength can be about or at least 1.75 V / cm RMS. In some embodiments, the field strength is about or at least 1 V / cm RMS. In some embodiments, the AC electric field has a field strength of about or at least 0.9 V / cm RMS. In some embodiments, the AC electric field has any of the field strengths described herein. In other embodiments, a combination of field strengths is applied, for example, combining two or more frequencies simultaneously or applying two or more frequencies at different times.

[0163] In some embodiments, the AC electric field can be applied at various intervals ranging from 0.5 hours to 72 hours. In some embodiments, different durations can be used (e.g., 0.5 hours to 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied for 2 hours per day. For example, the AC electric field can be applied for at least 4 hours per day, at least 8 hours per day, at least 12 hours per day, at least 16 hours per day, or at least 20 hours per day. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 2 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 3 days. In some embodiments, the AC electric field can be applied for at least 4, 8, 12, 16, or 20 hours per day for at least 7 days.

[0164] In some embodiments, the continuous exposure may continue for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or more of continuous exposure.

[0165] In some embodiments, the cumulative exposure may last for at least 42 hours, at least 84 hours, at least 168 hours, at least 250 hours, at least 400 hours, at least 500 hours, at least 750 hours, or more.

[0166] In some embodiments, the disclosed methods of reducing tumor growth, reducing tumor invasion, reducing tumor angiogenesis, and / or reducing tumor metastasis can further comprise administering a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a known cancer therapeutic agent other than a CAF inhibitor. For example, the cancer therapeutic agent can be, but is not limited to, chemotherapy, radiation therapy, immunotherapy, or hormone therapy. In some embodiments, the cancer therapeutic agent can be, but is not limited to, a tyrosine kinase inhibitor, a PI3K inhibitor, an Akt inhibitor, an anti-VEGF inhibitor, or an FGF inhibitor.

[0167] In some embodiments, AC electric field is applied before, after or simultaneously with administration of cancer therapeutic agent.In some embodiments, CAF inhibitor is applied before, after or simultaneously with administration of cancer therapeutic agent.In some embodiments, CAF inhibitor and cancer therapeutic agent are administered simultaneously, and AC electric field is applied before or after CAF activity inhibitor or CAF signaling inhibitor and cancer therapeutic agent.

[0168] In some embodiments, after applying the AC electric field and before administering the CAF inhibitor, the method further comprises detecting an increase in αSMA, FAP, PDGFRβ, and / or CD26 expression in the subject. In some embodiments, this detecting step allows confirmation that an increase in αSMA, FAP, PDGFRβ, hyaluronic acid, Hsp90, hedgehog, LLRC15, and / or CD26 expression is detected in response to the AC electric field, thus requiring administration of a CAF inhibitor.

[0169] In some embodiments, administration of the CAF inhibitor occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after application of the AC electric field. In some embodiments, the amount of time to wait after application of the AC electric field and before administering the CAF inhibitor can depend on the amount of time it takes for the AC electric field to cause fibroblast activation or increase expression of CAF markers.

[0170] In some embodiments, the subject in need thereof is a subject with cancer. G. Composition

[0171] Compositions and formulations comprising one or more CAF activity inhibitors or CAF signaling inhibitors, or combinations thereof, are disclosed. In some embodiments, the formulations further comprise a pharmaceutically acceptable carrier or diluent. For example, pharmaceutical compositions comprising a CAF activity inhibitor or CAF signaling inhibitor and a pharmaceutically acceptable carrier are disclosed. For example, pharmaceutical compositions comprising a FAP inhibitor, a selective PDGFRb inhibitor, a selective hedgehog inhibitor, a selective hyaluronic acid secretion inhibitor, an IL-1R inhibitor, a CXCR4 inhibitor, an HSP90 inhibitor, or an LLRC15 inhibitor and a pharmaceutically acceptable carrier are disclosed. Pharmaceutical compositions comprising a CAF activity inhibitor or CAF signaling inhibitor and a pharmaceutically acceptable diluent are also disclosed.

[0172] In some embodiments, the CAF activity inhibitor or CAF signaling inhibitor can be administered in any of the disclosed methods with a pharmaceutically acceptable carrier and / or diluent.

[0173] For example, the compositions described herein can include a pharmaceutically acceptable carrier. As is well known to those skilled in the art, "pharmaceutically acceptable" refers to a material or carrier selected to minimize degradation of the active ingredient and minimize adverse side effects in the subject. Examples of carriers include dimyristoylphosphatidylcholine (DMPC), phosphate-buffered saline, or multivesicular liposomes. For example, PG:PC:cholesterol:peptide or PC:peptide may be used as a carrier in the present invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.), ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, dextrose solution, etc. The pH of the solution can be about 5 to about 8, or about 7 to about 7.5. Further carriers include sustained-release formulations, such as semipermeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, such as films, stents (implanted in blood vessels during angioplasty), gels (including hydrogels), liposomes, or microparticles. Those skilled in the art will appreciate that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition being administered. These are typically standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0174] Pharmaceutical compositions may also contain carriers, thickeners, diluents, buffers, preservatives, etc., as long as the intended activity of the polypeptides, peptides, nucleic acids, and vectors of the present invention is not impaired. In addition to the compositions of the present invention, pharmaceutical compositions may also contain one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, and anesthetics. In the methods described herein, delivery of the disclosed compositions to cells can occur via a variety of mechanisms. Pharmaceutical compositions may be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. 1. Delivery of the Composition

[0175] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, fixed oils, and the like. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0176] Formulations for optical administration may include ointments, lotions, creams, gels, eye drops, suppositories, sprays, liquids, powders, etc. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0177] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some compositions may be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono-, di-, and tri-alkylamines, arylamines, and substituted ethanolamines. H. Kit

[0178] The above-mentioned materials and other materials can be packaged together in any suitable combination as a kit useful for carrying out or supporting the implementation of the disclosed method.A particular kit is useful when the kit components are designed and adapted to be used together in the disclosed method.For example, a kit is disclosed that includes one or more CAF inhibitors and one or more materials for delivering an alternating current electric field, such as the Optune system.For example, a kit is disclosed that includes one or more CAF activity inhibitors or CAF signaling inhibitors and one or more materials for delivering an alternating current electric field, such as the Optune system.In some embodiments, the kit also includes a cancer therapeutic agent. [Example]

[0179] The current data show that TT fields increase fibroblast activation in vivo and in vitro.

[0180] CAFs are fibroblasts present in tumor tissue that express and secrete proteins, thus influencing tumor remodeling. CAF activation can promote tumor growth. CAFs typically express FAP (fibroblast activation protein), alpha-smooth muscle actin (aSMA), platelet-derived growth factor receptor beta (PDGFRb), integrin beta 1 (ITDB1), CD26, and caveolin-1 (CAV1).

[0181] Activated CAFs can promote tumor growth, angiogenesis, invasion, and metastasis through multiple pathways. CAF activation can support tumor growth by secreting various cytokines and interacting with tumor and immune cells.

[0182] Application of a TT field for 48 hours in MRC5 lung fibroblasts resulted in the upregulation of PDGFRb (Figure 1A is a schematic diagram of the experiment, Figure 1B is FACS analysis, and Figure 1C is the percentage of PDGFRb cells as well as quantification of PDGFRb mean fluorescence intensity (MIF) and aSMA (Figure 2A is a schematic diagram of the experiment, and Figure 2B is a graph of MFI). Application of a TT field for 48 hours in MRC5 lung fibroblasts resulted in the upregulation of alpha SMA (aSMA) as seen by FACS analysis and MFI graph. Figure 2C shows the quantification of aSMA mean fluorescence intensity expression in lung fibroblasts (MRC-5) after 48 hours of treatment (results shown are from two separate experiments), and Figure 3 shows that application of a TT field for 48 hours in MRC-5 lung fibroblasts causes an increase in the percentage of fibroblasts expressing aSMA in Figure 3A, and Figure 3B shows the upregulation of alpha-SMA (aSMA), as seen from the FACS analysis and MFI graphs in Figure 3C.

[0183] Thus, as Figure 3 details the results shown in Figure 2C, not only did the amount of cells expressing aSMA increase, but the mean fluorescence intensity (MFI) per cell also increased, indicating that the cells expressed more aSMA. Confocal images of fibroblasts showing increased aSMA expression after 48 hours of TT field application, as shown in Figure 4A and quantified in Figure 4B, are shown.

[0184] aSMA expression increased in fibroblasts after incubation with conditioned medium (CM) derived from TT field-treated cancer cells (A549 lung adenocarcinoma cells). Figure 5A shows a schematic diagram of the experiment. Figure 5B shows the increase in aSMA mean fluorescence intensity in TT field-treated fibroblasts compared to controls.

[0185] Figure 6 shows the process of isolation and characterization of fibroblasts from the lungs of LLC2-bearing mice after 10 days of TT field application.

[0186] Figure 7 shows a scheme of the FACS analysis gating strategy to distinguish activated fibroblasts in a sample of cells derived from a tumor dissociation.

[0187] Figure 8B shows that aSMA expression is increased in CD45- / CD31- / PDGFR+CD29+ cells derived from tumors after TT field application, and Figure 8A shows that statistically significantly more CD45- / CD31- / PDGFR+CD29+ cells derived from tumors after TT field express aSMA compared to controls.

[0188] FIG. 9 shows that aSMA expression is increased in tumor-derived CD45− / CD31− / PDGFR+CD29+ cells after TT field application in two separate experiments.

[0189] The disclosed data show that inhibition of CAF signaling can be achieved by inhibiting PDGFRb. Targeting specific types of CAFs can be used for inhibition. Inhibiting FAP, Hsp90 hedgehog, hyaluronan secretion, IL-1R, CXCR4-CXCL12, and / or Lox and Loxl can all be used as strategies for CAF inhibition.

[0190] Furthermore, secreted cytokine array assay of conditioned medium from MRC5 after 72 h of TT field application at 150 kHz revealed secretion of MCP1 and CD26, a CAF marker indicative of differentiation of fibroblasts into CAFs (see Figure 10).

[0191] In vivo, fibroblasts isolated from the lungs of mice bearing LLC2 after TT field treatment exhibit a CAF phenotype, with highly expressed aSMA.

[0192] Therefore, treatment with TT fields while inhibiting the activation of cancer-associated fibroblasts may result in better patient outcomes.

[0193] ECM remodeling after TT field application was investigated in an orthotopic model of ovarian cancer (MOSE-L-FFL). TT fields were applied continuously for 10 days at a frequency of 200 kHz to ovarian tumor-bearing mice. Treatment with TT fields significantly increased LOX and collagen 4 protein expression, as visualized and quantified in tumor sections, without significant changes in collagen 1 and fibronectin protein expression within the tumor sections (Figures 13 and 14).

[0194] For TT field application to mice, the TT field was applied by two pairs of arrays placed on the torso of the mouse (ovarian mouse model).

[0195] For ex vivo immunohistochemistry, tumors were harvested from the ovarian bursa of female C57Bl / 6 mice inoculated with MOSE-L-FFL ovarian cancer cells and treated with either sham heating or a 200 kHz TT field for 10 consecutive days. Tumor sections were fixed in 4% PFA, embedded in ovarian OCT, and immunohistochemically examined using anti-LOX (1:100, NB100-2530), collagen 1 (1:100, Southern Biotech 1310-01), collagen 4 (1:100, Novus NBP1-26549), and fibronectin (1:100, Abcam ab2413). Slides were scanned using an automated slide scanner, 3DHistech Panoramic259 Flash III. Analysis was performed on an average of five images per animal using Image J software.

[0196] Some methods include tissue extracts and acid activation of serum and tissue extracts.

[0197] For tissue extract preparation, isolated solid tissue was rapidly rinsed in sterile saline to remove blood and briefly placed on a sterile cloth to dry. To obtain tissue lysates, approximately 30–50 mg of tissue was minced and sonicated on ice in 500 μl of lysis buffer (50 mM Tris-HCl pH 7.5) containing 100 mM sodium fluoride, 30 mM sodium pyrophosphate, 2 mM sodium molybdate, 1 mM sodium orthovanadate, 1 mM glycerophosphate, and 1x protease inhibitor cocktail. Samples were centrifuged at 13,000 rpm for 20 min at 4°C. The clear supernatant was collected and stored in aliquots at -70°C. Protein quantification in the lysates was performed by the bicinchoninic acid (BCA) method.

[0198] For acid activation of serum and tissue extracts, acid activation was used to isolate free TGF-β molecules from latent complexes to assess the amount of total TGF-β. Briefly, 30 μl of serum or protein lysate (equivalent to 200 μg of protein) was added to 100 μl of RIPA, followed by the addition of 10 μl of 1N HCl. Samples were rocked at 4°C for 1 hour. Acid activation was stopped by neutralization with 10 μl of 1N NaOH. Once acid activated, samples were stored on ice and used the same day.

[0199] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein which equivalents are intended to be encompassed by the following claims. Embodiment

[0200] Embodiment 1: A method of treating a subject in need thereof, comprising applying an alternating current electric field to a target site in the subject in need thereof and administering a cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof.

[0201] Embodiment 2: The method of embodiment 1, wherein the CAF inhibitor is a CAF activation inhibitor or a CAF signaling inhibitor.

[0202] Embodiment 3: The method of embodiment 2, wherein the CAF activation inhibitor is a fibroblast activation protein (FAP) inhibitor.

[0203] Embodiment 4: The method of embodiment 3, wherein the FAP inhibitor is talabostat, FAP-2286, or TAM558.

[0204] Embodiment 5: The method of embodiment 2, wherein the CAF activation inhibitor is a selective PDGFRb inhibitor.

[0205] Embodiment 6: The method of embodiment 5, wherein the selective PDFGRb inhibitor is sPDGFRbIg, crenolanib, orantinib, CP-673451, SU16f, Tyrphostin AG 1296, Tyrphostin AG1433, or seraltinib.

[0206] Embodiment 7: The method of embodiment 2, wherein the CAF activation inhibitor is an Hsp90 inhibitor.

[0207] Embodiment 8: The method of embodiment 7, wherein the Hsp90 inhibitor is XL888, geldanamycin, gedannin, tanspimycin, luminespib, BIIV021, CCT 018159, or 17-AAG.

[0208] Embodiment 9: The method of embodiment 2, wherein the CAF activation inhibitor is an LRRC15 inhibitor.

[0209] Embodiment 10: The method of embodiment 9, wherein the LRRC15 inhibitor is an ABBV-085 antibody.

[0210] Embodiment 11: The method of embodiment 2, wherein the CAF signaling inhibitor is a lysyl oxidase (LOX) inhibitor.

[0211] Embodiment 12: The method of embodiment 2, wherein the CAF signaling inhibitor is a lysyl oxidase-like (LOXL) inhibitor.

[0212] Embodiment 13: A method of reducing or preventing cancer-associated fibroblast (CAF) activation, the method comprising applying an alternating electric field to a cell population comprising one or more fibroblasts, and contacting the cell population with a CAF activation inhibitor.

[0213] Embodiment 14: A method of reducing cancer-associated fibroblast (CAF) signaling, comprising applying an alternating electric field to a cell population comprising one or more fibroblasts, and contacting the cell population with a CAF signaling inhibitor.

[0214] Embodiment 15: The method of embodiment 13, wherein the CAF activation inhibitor is a fibroblast activation protein (FAP) inhibitor.

[0215] Embodiment 16: The method of embodiment 15, wherein the FAP inhibitor is talabostat, FAP-2286, or TAM558.

[0216] Embodiment 17: The method of embodiment 13, wherein the CAF activation inhibitor is a selective PDGFRb inhibitor.

[0217] Embodiment 18: The method of embodiment 17, wherein the selective PDFGRb inhibitor is sPDGFRbIg, crenolanib, orantinib, CP-673451, SU16f, Tyrphostin AG 1296, Tyrphostin AG1433, or seraltinib.

[0218] Embodiment 19: The method of embodiment 13, wherein the CAF activation inhibitor is an Hsp90 inhibitor.

[0219] Embodiment 20: The method of embodiment 19, wherein the Hsp90 inhibitor is XL888, geldanamycin, gedannin, tanspimycin, luminespib, BIIV021, CCT 018159, or 17-AAG.

[0220] Embodiment 21: The method of embodiment 13, wherein the CAF activation inhibitor is an LRRC15 inhibitor.

[0221] Embodiment 22: The method of embodiment 21, wherein the LRRC15 inhibitor is an ABBV-085 antibody.

[0222] Embodiment 23: The method of embodiment 14, wherein the CAF signaling inhibitor is a lysyl oxidase (LOX) inhibitor.

[0223] Embodiment 24: The method of embodiment 14, wherein the CAF signaling inhibitor is a lysyl oxidase (LOX) inhibitor.

[0224] Embodiment 25: The method of any one of embodiments, wherein the target site comprises one or more cancer cells.

[0225] Embodiment 26: The method of any one of embodiments, wherein the alternating electric field is applied before, after, or simultaneously with administration of the CAF inhibitor.

[0226] Embodiment 27: The method of any one of the preceding claims, wherein the CAF inhibitor is administered intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intrathecally, intravascularly, intravenously, intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, topically, via intratumoral injection, or via inhalation.

[0227] Embodiment 28: The method of any one of embodiments 13 to 27, wherein the cell population is in vivo.

[0228] Embodiment 29: The method of any one of embodiments 13 to 27, wherein the cell population is in a subject.

[0229] Embodiment 30: The method of any one of embodiments, wherein the CAF activation inhibitor inhibits or reduces fibroblast activation protein (FAP), alpha smooth muscle actin (aSMA), or platelet-derived growth factor receptor beta (PDGFRb) expression.

[0230] Embodiment 31: The method of any one of embodiments, wherein the CAF activation inhibitor blocks upregulation of FAP, aSMA and / or PDGFRb expression in response to an alternating electric field.

[0231] Embodiment 32: The method of any one of embodiments, wherein the CAF activation inhibitor prevents PDGFRb from interacting with or binding to PDGF.

[0232] Embodiment 33: The method of any one of embodiments, wherein the CAF activation inhibitor prevents Hsp90 from ATPase activity, which upregulates fibroblast activation.

[0233] Embodiment 34: The method of any one of embodiments, wherein the CAF inhibitor prevents Lox or Loxl from covalently cross-linking collagen and elastin in the extracellular matrix (ECM).

[0234] Embodiment 35: The method of any one of embodiments, wherein the alternating electric field has a frequency of 50 kHz to 1 MHz.

[0235] Embodiment 36: The method of any one of embodiments, wherein the alternating electric field has a frequency of about 150 or 250 kHz.

[0236] Embodiment 37: The method of any one of embodiments, wherein the alternating electric field has an electric field strength of 0.5 to 10 V / cm RMS.

[0237] Embodiment 38: The method of any one of embodiments, wherein the alternating electric field has a field strength of about 0.9 V / cm RMS.

[0238] Embodiment 39: The method of any one of embodiments, further comprising administering a cancer therapeutic agent.

[0239] Embodiment 40: The method of any one of embodiments 1 to 39, wherein after step a) and before step b), increased expression of αSMA, FAP, PDGFRβ, hedgehog, hyaluronic acid, Hsp90, or CD26 is detected in the subject or cells.

[0240] Embodiment 41: The method of any one of embodiments 1 to 40, wherein step b) is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after step a) is performed.

[0241] Embodiment 42: A method of reducing tumor growth in a subject in need thereof, comprising: a) applying an alternating current electric field to a target site in the subject in need thereof; and b) administering a cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof.

[0242] Embodiment 43: A method of reducing tumor invasion in a subject in need thereof, comprising: a) applying an alternating current electric field to a target site in the subject in need thereof; and b) administering a cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof.

[0243] Embodiment 44: A method of reducing tumor angiogenesis in a subject in need thereof, comprising: a) applying an alternating current electric field to a target site in the subject in need thereof; and b) administering a cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof.

[0244] Embodiment 45: A method of reducing tumor metastasis in a subject in need thereof, comprising: a) applying an alternating current electric field to a target site in the subject in need thereof; and b) administering a cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof.

[0245] Embodiment 46: The method of any one of embodiments 42 to 45, wherein the target site comprises a cancer cell.

[0246] Embodiment 47: The method of any one of embodiments 1-2, 13-14, or 25-46, wherein the CAF activation inhibitor is a selective hedgehog inhibitor, a selective hyaluronic acid secretion inhibitor, or an IL-1R inhibitor.

[0247] Embodiment 48: The method of any one of embodiments 1-2, 13-14, or 25-46, wherein the CAF signaling inhibitor is a CXCR4 inhibitor or a CXCR12 inhibitor.

[0248] Embodiment 49: A cancer-associated fibroblast inhibitor in combination with an alternating electric field in a method of treating a subject in need thereof.

[0249] Embodiment 50: An alternating electric field in combination with a cancer-associated fibroblast inhibitor for use in a method of treating a subject in need thereof.

[0250] Embodiment 51: The method of embodiments 49-50, wherein the CAF inhibitor is a CAF activation inhibitor or a CAF signaling inhibitor.

[0251] Embodiment 52: The method of embodiment 51, wherein the CAF activation inhibitor is a fibroblast activation protein (FAP) inhibitor.

[0252] Embodiment 53: The method of embodiment 52, wherein the FAP inhibitor is talabostat, FAP-2286, or TAM558.

[0253] Embodiment 54: The method of embodiment 51, wherein the CAF activation inhibitor is a selective PDGFRb inhibitor.

[0254] Embodiment 55: The method of embodiment 54, wherein the selective PDFGRb inhibitor is sPDGFRbIg, crenolanib, orantinib, CP-673451, SU16f, Tyrphostin AG 1296, Tyrphostin AG1433, or seraltinib.

[0255] Embodiment 56: The method of embodiment 51, wherein the CAF activation inhibitor is an Hsp90 inhibitor.

[0256] Embodiment 57: The method of embodiment 56, wherein the Hsp90 inhibitor is XL888, geldanamycin, gedannin, tanspimycin, luminespib, BIIV021, CCT 018159, or 17-AAG.

[0257] Embodiment 58: The method of embodiment 51, wherein the CAF activation inhibitor is an LRRC15 inhibitor.

[0258] Embodiment 59: The method of embodiment 58, wherein the LRRC15 inhibitor is an ABBV-085 antibody.

[0259] Embodiment 60: The method of embodiment 51, wherein the CAF signaling inhibitor is a lysyl oxidase (LOX) inhibitor.

[0260] Embodiment 61: The method of embodiment 51, wherein the CAF signaling inhibitor is a lysyl oxidase-like (LOXL) inhibitor.

[0261] Embodiment 62: The method of any one of embodiments 49 to 61, wherein the target site comprises one or more cancer cells.

[0262] Embodiment 63: The method of any one of embodiments 49 to 62, wherein the alternating electric field is applied before, after, or simultaneously with administration of the CAF inhibitor.

[0263] Embodiment 64: The method of any one of claims 49 to 63, wherein the CAF inhibitor is administered intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intrathecally, intravascularly, intravenously, intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, topically, via intratumoral injection, or via inhalation.

[0264] Embodiment 65: The method of any one of embodiments 49 to 64, wherein the CAF activation inhibitor inhibits or reduces fibroblast activation protein (FAP), alpha smooth muscle actin (aSMA), or platelet-derived growth factor receptor beta (PDGFRb) expression.

[0265] Embodiment 66: The method of any one of embodiments 49 to 65, wherein the CAF activation inhibitor blocks the upregulation of FAP, aSMA and / or PDGFRb expression in response to an alternating electric field.

[0266] Embodiment 67: The method of any one of embodiments 49 to 66, wherein the CAF activation inhibitor prevents PDGFRb from interacting with or binding to PDGF.

[0267] Embodiment 68: The method of any one of embodiments 49 to 67, wherein the CAF activation inhibitor prevents Hsp90 from ATPase activity, which upregulates fibroblast activation.

[0268] Embodiment 69: The method of any one of embodiments 49 to 68, wherein the CAF inhibitor prevents Lox or Loxl from covalently cross-linking collagen and elastin in the extracellular matrix (ECM).

[0269] Embodiment 70: The method of any one of embodiments 49 to 69, wherein the alternating electric field has a frequency of 50 kHz to 1 MHz.

[0270] Embodiment 71: The method of any one of embodiments 49 to 70, wherein the alternating electric field has a frequency of about 150 or 250 kHz.

[0271] Embodiment 72: The method of any one of embodiments, wherein the alternating electric field has an electric field strength of 0.5 to 10 V / cm RMS.

[0272] Embodiment 73: The method of any one of embodiments 49 to 72, wherein the alternating electric field has a field strength of about 0.9 V / cm RMS.

[0273] Embodiment 74: The method of any one of embodiments 49 to 73, further comprising administering a cancer therapeutic agent.

[0274] Embodiment 75: The method described in embodiments 49 to 74, wherein after step a) and before step b), increased expression of αSMA, FAP, PDGFRβ, hedgehog, hyaluronic acid, Hsp90, or CD26 is detected in the subject or cells.

[0275] Embodiment 76: The method described in embodiments 49 to 75, wherein step b) is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after step a) is performed.

Claims

1. 1. A cancer-associated fibroblast (CAF) inhibitor for use with an alternating electric field in a method of treating a subject in need thereof, comprising: The method includes applying the alternating electric field to a target site in the subject in need thereof, and administering the cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof.

2. 1. An alternating current electric field in combination with a cancer-associated fibroblast inhibitor for use in a method of treating a subject in need thereof, comprising: The method includes applying the alternating current electric field to a target site in the subject in need thereof, and administering the cancer-associated fibroblast (CAF) inhibitor to the subject in need thereof.

3. The CAF inhibitor of claim 1 or the AC electric field in combination with the CAF inhibitor of claim 2, wherein the target site comprises one or more cancer cells.

4. 3. The AC electric field used in combination with the CAF inhibitor of claim 1 or the CAF inhibitor of claim 2, wherein the CAF inhibitor is a CAF activation inhibitor or a CAF signaling inhibitor.

5. The CAF inhibitor according to claim 4 or an AC electric field used in combination with the CAF inhibitor according to claim 4, wherein the CAF activation inhibitor is a fibroblast activation protein (FAP) inhibitor, a selective PDGFRb inhibitor, an Hsp90 inhibitor, an LRRC15 inhibitor, a selective hedgehog inhibitor, a selective hyaluronic acid secretion inhibitor, or an IL-1R inhibitor.

6. The CAF inhibitor according to claim 5 or the AC electric field used in combination with the CAF inhibitor according to claim 5, wherein the FAP inhibitor is talabostat, FAP-2286, or TAM558.

7. The CAF inhibitor according to claim 5, wherein the selective PDFGRb inhibitor is sPDGFRbIg, crenolanib, orantinib, CP-673451, SU16f, Tyrphostin AG 1296, Tyrphostin AG1433, or seraltinib; or an AC electric field used in combination with the CAF inhibitor according to claim 5.

8. The CAF inhibitor of claim 5 or the AC electric field used in combination with the CAF inhibitor of claim 5, wherein the Hsp90 inhibitor is XL888, geldanamycin, gedunin, tanspimycin, luminespib, BIIV 021, CCT 018159, or 17-AAG.

9. The CAF inhibitor according to claim 5 or the AC electric field used in combination with the CAF inhibitor according to claim 5, wherein the LRRC15 inhibitor is an ABBV-085 antibody.

10. 5. The CAF inhibitor of claim 4, or an AC electric field used in combination with the CAF inhibitor of claim 4, wherein the CAF signaling inhibitor is a lysyl oxidase (LOX) inhibitor, a lysyl oxidase-like (LOXL) inhibitor, a CXCR4 inhibitor, or a CXCR12 inhibitor.

11. The CAF inhibitor of any one of claims 1 to 10 or an AC electric field in combination with a CAF inhibitor of any one of claims 2 to 10, wherein the target site comprises one or more cancer cells.

12. The CAF activation inhibitor inhibits or reduces fibroblast activation protein (FAP), alpha smooth muscle actin (aSMA), or platelet-derived growth factor receptor beta (PDGFRb) expression, and / or Prevents PDGFRb from interacting with or binding to PDGF, and / or A CAF inhibitor according to claims 1 to 11 or an AC electric field in combination with a CAF inhibitor according to claims 2 to 11, which prevents Hsp90 from ATPase activity, which upregulates fibroblast activation.

13. The CAF inhibitor according to claims 1 to 12 or the AC electric field used in combination with the CAF inhibitor according to claims 2 to 12, wherein the AC electric field has a frequency of 50 kHz to 1 MHz.

14. The CAF inhibitor according to any one of claims 1 to 13, or the AC electric field used in combination with the CAF inhibitor according to any one of claims 2 to 13, wherein the AC electric field has a field strength of 0.5 to 10 V / cm RMS.

15. 15. The CAF inhibitor of claims 1 to 14 or an AC electric field in combination with the CAF inhibitor of claims 2 to 14, wherein the CAF inhibitor prevents Lox or Loxl from covalently cross-linking collagen and elastin in the extracellular matrix (ECM).

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