Systems and methods for treating cancer using ultrasound

Abstract

A novel localized mechanical HIFU (LM-HIFU) transcatheter device that can ablate cancer cells and disrupt the stromal barrier in the tumor, thereby enhancing the efficacy of therapeutic agents and increasing immune cell infiltration. A miniaturized dual lumen catheter device configured to deliver an M-HIFU to a tumor, the M-HIFU not having the anatomical limitations of conventional HIFUs, and being capable of entering a primary or metastatic tumor regardless of its location.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a non-provisional application of and claims the benefit of U.S. Provisional Patent Application No. 63 / 324,479 filed on March 28, 2022, the contents of which are incorporated herein by reference.

[0003] background

[0004] Solid tumors make up the vast majority of cancers, with more than 1.5 million new cases diagnosed each year in the U.S. Common treatments include surgery, ablation, radiation therapy, chemotherapy, and recent advances in immunotherapy and other targeted therapies, which can be delivered alone or as a combination therapy.

[0005] Immunotherapy has emerged as a treatment for cancer and mainly consists of immune checkpoint blockade or cell therapy. Although systemic immunotherapy has promise, it is effective in less than 15% of patients and has significant side effects that affect patients' quality of life. Therefore, most patients with metastatic disease still cannot benefit from immunotherapy and combination therapies.

[0006] Cancer immunotherapy using immune checkpoint blockade ("ICB") has revolutionized cancer therapy and is a major focus of cancer research. However, this treatment approach still has several disadvantages, including relatively low response rates. Therefore, there is a need in immunotherapy to convert immunologically "cold tumors" into inflamed "hot tumors." Because most patients do not respond to ICB alone, combinations of systemically delivered immunotherapies have been studied, and these not only increase tumor response rates, but also increase toxicity and treatment-related mortality.

[0007] While many attempts are being explored to separate systemic toxicity from the increase in anti-tumor immune response, this has not been achieved except using one approach - intralesional therapy. Intralesional therapy, or local delivery of agents into tumors, including oncolytic viruses and other immunotherapeutics, has been shown to stimulate anti-tumor responses with minimal, if any, systemic toxicity. Unfortunately, intratumoral delivery of these agents is typically performed via needle injection, and this has several limitations: 1) needle injection has several disadvantages, such as failed injections (e.g., missing the tumor, drug leakage into surrounding tissues, little drug delivered to the tumor, etc.) and 2) low response rates, typically less than 33%.

[0008] Furthermore, persistent barriers to treatment include the complex and dense nature of the tumor stroma and high interstitial tissue pressure within primary and metastatic tumors, which inhibit the delivery of therapeutics and limit the infiltration of activated immune cells.

[0009] Local ablation techniques, such as high-intensity focused ultrasound (HIFU), are being used to thermally destroy cancer cells and are increasingly being explored to enhance cancer immunotherapy. HIFU is currently delivered via sound waves originating outside the body, but sound waves cannot cross air-fluid interfaces. Therefore, current HIFU strategies have anatomical limitations that prevent them from treating common organs to which advanced cancer metastasizes, such as tumors within the lungs, airways, or gastrointestinal (GI) tract.

[0010] Therefore, there is a continuing need for improved methods of treating cancer.

[0011] Overview

[0012] As disclosed herein, microtransducer-based catheters use frequencies and energy levels configured to pierce tumor cell membranes, disrupt tumor stroma, improve uptake of therapeutic agents, and promote immune cell infiltration. To address the anatomical limitations of conventional HIFU, and the unmet need for more effective means of tumor ablation and delivery of anticancer therapeutics directly into tumors, microtransducer-based catheters are configured to deliver high-intensity focused ultrasound (HIFU) to disrupt the tumor microenvironment and simultaneously deliver therapeutic agents into the tumor, taking advantage of the synergy between tumor ablation immunotherapy. HIFU-induced mechanical destruction of target tissue by acoustic cavitation and disruption of cell membranes is known as histotripsy, and is achieved by high-pressure bursting of microbubbles induced by ultrasound therapy.

[0013] In one embodiment, the present disclosure provides a novel local mechanical HIFU (LM-HIFU) transcatheter device that can ablate cancer cells and disrupt the stromal barrier in tumors, thereby enhancing the efficacy of therapeutic agents and increasing immune cell infiltration. The delivery of M-HIFU using a miniaturized device as described herein does not have the anatomical limitations of traditional HIFU and enables access to primary or metastatic tumors regardless of their location.

[0014] One aspect of the present disclosure provides a system for treating a target (e.g., tumor tissue, peritumoral tissue, non-malignant tissue, blood cells, or immune cells), the system comprising (a) an ultrasound energy source; (b) a device coupled to the ultrasound energy source and configured and arranged to: (i) direct the energy to a desired location; (ii) release one or more microbubbles (an ultrasound contrast agent); and (iii) release one or more therapeutic agents, wherein the microbubbles burst upon receiving the energy, thereby disrupting the target and the surrounding extracellular matrix (ECM) and allowing the one or more therapeutic agents to be delivered into the target.

[0015] In some embodiments, one or more microbubbles are configured and arranged to contain one or more therapeutic agents, wherein the one or more microbubbles burst upon receiving energy, thereby releasing the one or more therapeutic agents into the target.

[0016] In another embodiment, the device further comprises configuration and arrangement for (iv) releasing one or more contrast agents.

[0017] In one embodiment, the ultrasound comprises high intensity focused ultrasound (HIFU).In another embodiment, the ultrasound comprises tissue fragmentation.

[0018] Another aspect of the present disclosure provides a method for delivering a drug, protein, nucleic acid or gene to a target, the method comprising using an ultrasound energy delivery system as provided herein, the ultrasound energy delivery system being configured and arranged to produce an energy output; supplying one or more microbubbles and one or more therapeutic agents, wherein the microbubbles burst upon receiving the energy, thereby destroying the target and the surrounding extracellular matrix (ECM) and allowing one or more therapeutic agents to be delivered into the target, thereby treating the target.

[0019] In some embodiments, one or more microbubbles are configured and arranged to contain one or more therapeutic agents, wherein the one or more microbubbles burst upon receiving energy, thereby releasing the one or more therapeutic agents into the target.

[0020] In another embodiment, the method further comprises releasing one or more contrast agents.

[0021] In another embodiment, one or more therapeutic agents are selected from the group consisting of cytokines, chemokines and other biological proteins (such as IL-12, etc.), oncolytic viruses, CAR-T cells, TILs, other cells, subcellular vesicles (including exosomes), cDNA, mRNA, self-replicating RNA, proteins, antibodies, single-chain antibodies, nanobodies, phages, immunosuppressants, anti-inflammatory agents, anti-proliferative agents, anti-migratory agents, anti-fibrotic agents, pro-apoptotic agents, vasodilators, calcium channel blockers, anti-tumor agents, anti-cancer agents, anti-thrombotic agents, anti-platelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, and combinations thereof.

[0022] In one embodiment, the one or more therapeutic agents include a cytokine. In one embodiment, the cytokine includes IL-12, IL-15, or a fusion protein of a cytokine.

[0023] In one embodiment, the present invention provides a catheter. The catheter includes an elongated hollow tube, a first lumen in the elongated hollow tube, a second lumen in the elongated hollow tube, a transducer located in the elongated hollow tube and adjacent to the first lumen or the second lumen, and a needle. The ultrasonic transducer is configured to transmit ultrasonic waves to a target through the lumen, and the needle is located in the elongated hollow tube and is configured to extend from the first lumen or the second lumen into the target to deliver treatment to the target.

[0024] In another embodiment, the present disclosure provides a method for treating a malignant tumor. The method comprises: inserting the catheter described above into a subject and toward the malignant tumor; activating the transducer to deliver energy to the malignant tumor through a first lumen, resulting in applying an acoustic peak negative pressure in the range of 10 MPa to 40 MPa to the malignant tumor; and activating the needle to extend from the second lumen into the malignant tumor, thereby delivering a therapeutically effective amount of a pharmaceutical composition.

[0025] In another embodiment, the present disclosure provides a system for treating a target. The system includes an ultrasonic energy source and a device coupled to the ultrasonic energy source. The device is configured and arranged to direct the ultrasonic energy to the target, release one or more microbubbles, and release one or more therapeutic agents, wherein the microbubbles burst after receiving the ultrasonic energy, thereby destroying the target and the extracellular matrix (ECM) surrounding the target, and allowing one or more therapeutic agents to be delivered into the target.

[0026] In another embodiment, the present disclosure provides a method for delivering a drug to a target. The method includes: inserting a catheter into a subject and toward the target, the catheter including the device described above; generating an ultrasonic energy output near the target; supplying one or more microbubbles near the target; and supplying one or more therapeutic agents through the catheter, wherein the microbubbles burst after receiving the ultrasonic energy, thereby destroying the target and the surrounding extracellular matrix (ECM) and allowing one or more therapeutic agents to be delivered into the target, thereby treating the target.

[0027] Other aspects of the invention will become apparent by consideration of the detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A is a schematic diagram illustrating a system including an ultrasound energy source for delivering a therapeutic agent to a target tissue according to some embodiments.

[0030] Figure 1B is a schematic diagram illustrating a system including an ultrasound energy source for delivering a therapeutic agent to a target tissue according to some embodiments.

[0031] Figure 2A According to some embodiments Figure 1A and 1BAn example of a transducer for a system.

[0032] Figure 2B is a diagram depicting a method according to some embodiments Figure 2A Diagram of the sensitivity of the transducer.

[0033] Figure 2C According to some embodiments, Figure 2A Image of the transducer's sonoporation drug delivery testing apparatus.

[0034] Figure 2D is a fluorescent image of GFP+ sonoporation treated cells according to some embodiments, as well as negative control cells and Figure 2A Luciferase activity of cells treated with sonoporation of the transducer.

[0035] Figure 3A is an example of a tubular HIFU transducer according to some embodiments.

[0036] Figure 3B According to some embodiments Figure 3A Schematic diagram of the transducer.

[0037] Figure 3C According to some embodiments, Figure 3A The simulated sound field profile generated by the transducer.

[0038] Figure 3D According to some embodiments, Figure 3A The measured acoustic profiles generated by the transducer in both the side and front viewing directions.

[0039] Figure 4A is a miniaturized HIFU transducer according to some embodiments.

[0040] Figure 4B is a diagram depicting a method according to some embodiments Figure 4A A graph of the sound pressure output of the transducer.

[0041] Figure 4C According to some embodiments, Figure 4A Resulting image of a bubble cloud generation test performed with a transducer.

[0042] Figure 5A Various views of a HIFU transducer are shown according to some embodiments.

[0043] Figure 5B According to some embodiments Figure 5A Simulated sound pressure field profile of the transducer operating at a frequency of 3.5 MHz.

[0044] Figure 5CAccording to some embodiments, Figure 5A Diagram of the transducer setup for generating cavitation.

[0045] Figure 5D According to some embodiments, Figure 5A Diagram of the transducer setup for generating cavitation.

[0046] Fig. 6A is a pair of graphs depicting tumor volume size and survival rate of mice treated with TAVO and control plasmid according to some embodiments.

[0047] Figure 6B is a pair of graphs depicting fold change in tumor volume for treated and untreated mice according to some embodiments.

[0048] Figure 6C Depicted is a method for classifying all samples into cell types or by Fig. 6A and 6B t-SNE plot of cells divided by treatment groups.

[0049] Fig.6D Depicted according to some embodiments, Fig. 6A and 6B Quantitative plot of the frequency of each clone for each treatment group.

[0050] Fig. 6E Depicted according to some embodiments, Fig. 6A and 6B Quantification of the activation signature scores for all T cells in each treatment group.

[0051] Fig. 7A is a circus plot that depicts, according to some embodiments, Fig. 6A and 6B Receptor-ligand interactions between receptors on CD8 T cells and ligands on macrophages in the treatment groups.

[0052] Figure 7B is a graph depicting a 50-gene CXCR3 gene signature score quantified in all cells according to some embodiments.

[0053] Fig. 8A According to some embodiments, Figure 4A and 5A Tumor curves for combination therapies performed with the transducer.

[0054] Figure 8B According to some embodiments, Figure 4A and 5A Tumor curves for combination therapies performed with the transducer.

[0055] Figure 8C According to some embodiments, Figure 4A and 5A Tumor curves for combination therapies performed with the transducer.

[0056] Fig.8D According to some embodiments, Figure 4A and 5A Tumor curves for combination therapies performed with the transducer.

[0057] Fig. 9 is a pair of images depicting a baseline image and a post-cycle image of lung metastases according to some embodiments.

[0058] Fig.10 Depicted are multiple images of fibrotic tumor stroma in breast cancer, according to some embodiments.

[0059] Fig.11 Images depicting drug distribution following intratumoral injection of PV-10 drug, according to some embodiments.

[0060] Fig.12 are images depicting how a tumor may be treated with different HIFU regimens according to some embodiments.

[0061] Fig.13 are histological photographs and graphs depicting HIFU treatment data and control (no treatment) data of T cell infiltration of tumors according to some embodiments.

[0062] Fig.14 Included are graphs depicting the combination of M-HIFU and PD-L1 treatment according to some embodiments.

[0063] Fig.15 Included are graphs depicting the results of combination treatment of IT-IL-12 gene therapy with M-HIFU according to some embodiments.

[0064] Fig.16A According to some embodiments, a composition for treating metastatic breast cancer comprises Figure 2A transducer system.

[0065] Fig. 16B According to some embodiments, a composition for treating metastatic breast cancer comprises Figure 2A transducer system.

[0066] Fig.17 is a graph depicting expression of ESR1 mutants that confer constitutive estrogen signaling and enhanced growth in precancerous murine mammary epithelial cells, according to some embodiments.

[0067] Fig.18 According to some embodiments Figure 1A and 1B Another example of a transducer for a system.

[0068] Fig.19 According to some embodiments and Fig.18 Table of acoustic properties of cell culture plates used together with the transducers.

[0069] Fig. 20 According to some embodiments, it is used for testing Fig.18 The transducer functions of the system.

[0070] Fig.21 is a diagram depicting a method according to some embodiments Fig.18 A graph of the sound pressure output of the transducer.

[0071] Fig. 22 According to some embodiments, it is used for testing Fig.18 The function of the transducer is to simulate the sound pressure field of an 800kHz ultrasonic beam.

[0072] Fig.23 is depicted according to some embodiments for testing Fig.18 Figure 2 Graph of the luciferase activity of the control and experimental groups of the transducer function.

[0073] Fig.24 is a diagram for testing at various sonication parameters according to some embodiments. Fig.18 A series of graphs showing luciferase activity of the sonoporation test of the transducer's functionality.

[0074] Fig.25 is a series of graphs depicting growth inhibition of local and distant tumors and enhanced tumor antigen-specific cellular immune responses according to some embodiments.

[0075] Fig.26 is a series of graphs showing enhanced intratumoral infiltration by M-HIFU treatment, according to some embodiments.

[0076] Fig.27A is a series of graphs of GO enrichment analysis and KEGG pathway analysis of DEGs in macrophages after M-HIFU treatment according to some embodiments.

[0077] Fig.27B is a series of graphs of GO enrichment analysis and KEGG pathway analysis of DEGs in macrophages after M-HIFU treatment according to some embodiments.

[0078] Fig.28 is a series of graphs depicting enhanced expression of immune checkpoint molecules by tumor-infiltrating immune cells after M-HIFU treatment according to some embodiments.

[0079] 29 is a series of graphs depicting that M-HIFU and PD-L1 blockade synergize to reject localized tumors, according to some embodiments.

[0080] Fig.30 is a series of graphs depicting that the combination of M-HIFU and anti-PD-L1 antibody induces upregulation of unique DEGs in tumor-infiltrating CD8 T cells and macrophage luciferase activity, according to some embodiments.

[0081] Fig.31 is a series of graphs depicting the results of the combination of M-HIFU and PD-1 / PD-L1 blockade according to some embodiments.

[0082] Fig.32 An exemplary acoustoporation transducer used in Example 7 is shown.

[0083] Fig.33 TABLE 7 Test conditions for the test group and control group used in Example 7.

[0084] Figure 34-37 Data from experimental test results of Example 7 are shown.

[0085] Details

[0086] Before any embodiments of the invention are explained in detail, it should be understood that the invention is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0087] The articles "a" and "an" are used herein to refer to one or more (ie, at least one) of the grammatical object of the article. For example, "an element" means at least one element and may include more than one element.

[0088] "About" is used to provide flexibility to a numerical range endpoint by providing that a given value can be "a little above" or "a little below" the endpoint without affecting the desired result.

[0089] The use of the terms "including," "comprising," or "having" and variations thereof herein is intended to encompass the elements listed thereafter and their equivalents as well as additional elements. As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in an alternative manner ("or").

[0090] As used herein, the transitional phrase "consisting essentially of (and grammatical variations) should be interpreted to cover the recited materials or steps "as well as those that do not materially affect the basic and novel characteristics of the claimed invention." Therefore, the term "consisting essentially of" as used herein should not be interpreted as being equivalent to "comprising."

[0091] In addition, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. For illustration, if the specification states that a composite comprises components A, B, and C, it is specifically intended that any one or combination of A, B, or C may be omitted and abandoned, either individually or in any combination.

[0092] Unless otherwise specified herein, the ranges of values ​​recited herein are intended only to be used as a shorthand method of individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is specified as 1% to 50%, it is intended that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% are explicitly recited in this specification. These are merely examples of specific intent, and all possible combinations of values ​​between and including the lowest and highest values ​​recited are considered to be explicitly stated in this disclosure.

[0093] As used herein, "treatment," "therapy," and / or "therapeutic regimen" refers to a clinical intervention performed in response to a disease, disorder, or physiological condition exhibited by a patient or to which the patient may be susceptible. The purposes of treatment include alleviation or prevention of symptoms, slowing or stopping the progression or deterioration of a disease, disorder, or condition, and / or alleviating a disease, disorder, or condition. As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the probability of a disease, disorder, or condition occurring in a subject who does not suffer from the disease, disorder, or condition but is at risk of or susceptible to the disease, disorder, or condition. The terms "effective amount" or "therapeutically effective amount" refer to an amount sufficient to achieve a beneficial or desired biological and / or clinical outcome.

[0094] As used herein, the term "disease" includes, but is not limited to, any abnormal condition and / or disorder that affects the structure or function of a part of an organism. It can be caused by external factors (such as infectious diseases) or by internal dysfunction (such as cancer, cancer metastasis, etc.).

[0095] As known in the art, cancer is generally considered to be uncontrolled cell growth. The method of the present invention can be used to treat any cancer and any metastasis thereof, including but not limited to carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, salivary gland cancer, mesothelioma, kidney cancer, vulvar cancer, pancreatic cancer, thyroid cancer, liver cancer, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma and peripheral neuroepithelioma.

[0096] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to both humans and non-human animals. The term "non-human animal" disclosed herein includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The methods and compositions disclosed herein can be used in vitro on samples (e.g., on isolated cells or tissues) or in vivo on subjects (i.e., living organisms, such as patients).

[0097] As used herein, "therapeutic agent" includes any molecular species and / or biological agent that is therapeutic when introduced into a subject being treated or becomes therapeutic after introduction into a subject being treated (e.g., by reaction with a natural or non-natural substance or condition or any other introduced substance). Examples of natural conditions include pH (e.g., acidity), chemicals, temperature, salinity, osmotic pressure, and conductivity; non-natural conditions include those such as magnetic fields, electromagnetic fields (such as radiofrequency and microwaves), and ultrasound. In the present disclosure, the chemical name of any therapeutic agent is used to refer to the compound itself and prodrugs (precursor substances that are converted into the active form of the compound in vivo) and / or their drug derivatives, analogs, or metabolites (biologically active compounds that the compound is converted into in vivo directly or upon introduction of other agents or conditions (e.g., enzymatic, chemical, energetic) or environments (e.g., pH)).

[0098] The scope of the present disclosure includes the use of any therapeutic agent whose medicinal effect can be enhanced by using ultrasonic energy as described herein. For the purpose of illustration, many therapeutic agent categories are identified to convey the understanding of the present disclosure. These categories of agents and specifically listed agents are not intended to limit the scope or practice of the present invention in any way; the scope of the present disclosure includes any therapeutic agent that can be considered to be beneficial to the treatment of patients. In addition, these agents can be delivered / administered in any suitable manner. As used herein, the term "administering" an agent (e.g., a therapeutic entity) to an animal or cell is intended to refer to the distribution, delivery or application of a substance to an intended target. With respect to therapeutic agents, the term "administering" is intended to refer to contacting a therapeutic agent with a subject or distributing, delivering or applying a therapeutic agent to a subject by any suitable route for delivering a therapeutic agent to a desired location in an animal, including delivery by parenteral or oral routes, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by intranasal or respiratory routes.

[0099] In some embodiments, examples of therapeutic agents may include cytokines, chemokines and other biological proteins (such as IL-12, etc.), oncolytic viruses, CAR-T cells, TIL, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory agents, anti-proliferative agents, anti-migratory agents, anti-fibrotic agents, pro-apoptotic agents, vasodilators, calcium channel blockers, anti-tumor agents, anti-cancer agents, antibodies, anti-thrombotic agents, anti-platelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, non-immunosuppressive agents, and combinations thereof.

[0100] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0101] Aspects of the present disclosure combine an energy source with microbubbles and one or more therapeutic agents to treat a selected area (e.g., a tumor) of a subject. As used herein, the terms "selected area," "treatment site," "desired target," and "target area" are used interchangeably and refer to an area in a subject where targeted therapy is desired. The treatment site may include tissue associated with a body cavity, an organ, or a local tumor. In one embodiment, the device and method of the present invention reduce the formation or progression of a tumor or hyperplastic growth. A "lumen" may be any blood vessel in a subject's vascular system, including veins, arteries, aortas, and particularly include coronary and peripheral arteries, as well as previously implanted grafts, shunts, fistulas, etc. In other embodiments, the systems and methods described herein may also be applied to other body cavities that experience excessive tumor cell growth, such as bile ducts. Examples of internal body tissue and organ applications include various organs, nerves, glands, ducts, etc.

[0102] According to one aspect of the present disclosure, a system for treating a tumor is provided. In such aspects, the system includes: (a) an ultrasonic energy source; (b) a device coupled to the energy source and configured and arranged to: (i) direct energy to a desired location; (ii) release one or more microbubbles; and (iii) release one or more therapeutic agents. As used herein, the term "microbubble" (a type of ultrasound contrast agent) is also referred to herein as "cavitating bubbles" or "cavitation bubbles", which are tiny inflated bubbles that can be injected into a subject, and they remain inactive in the subject unless stimulated by the energy generated by the energy source. In practice, the energy source directs energy to the microbubble, causing it to vibrate and rupture, thereby destroying surrounding tissue (e.g., ECM, tumor, etc.) and allowing one or more therapeutic agents to be delivered to the tumor.

[0103] An example of a system for delivering energy from an ultrasound energy source to microbubbles (and in some embodiments, for delivering a therapeutic agent to a desired target (e.g., a tumor)) according to some embodiments of the present disclosure is described in Figure 1A and 1B Shown in. Figure 1A and 1B Each shows an example of a system 100A, 100B for delivering energy from an ultrasound energy source to microbubbles and then to a desired target, according to some embodiments. Figure 1A In the example shown, the system 100 includes a device 102 that includes a simple or single-source ultrasonic energy source 101 . Figure 1B Another example is shown in which the device 102 includes an ultrasonic energy source 101 that includes a composite ultrasonic device (eg, a probe or transducer).

[0104] It should be understood that although Figure 1A and 1B Different types of ultrasound energy sources 101 are shown, but any ultrasound source suitable for therapeutic applications can be used in the systems and methods provided herein. Suitable examples include, but are not limited to, HIFU, tissue fragmentation, and the like.

[0105] In any case, the device 102 includes one or more adjacent tubes 104 for releasing microbubbles 106 and / or therapeutic agents 108. Once released from the device 102, energy 110 is released from an energy source 101 within the device 102 and delivered to the microbubbles 106. The microbubbles 106 are then activated, which allows for the destruction of target tissue 112 and / or the subsequent delivery of one or more therapeutic agents 108 to the target tissue 112. It should be understood that although Figure 1A and 1BThe examples shown in depict microbubbles and one or more therapeutic agents being delivered in a longitudinal direction, but in some embodiments, the microbubbles and / or one or more therapeutic agents may alternatively or additionally be delivered in one or more other directions (e.g., in a transverse direction).

[0106] In some embodiments, microvesicles and therapeutic agents are delivered to the subject simultaneously. In other embodiments, microvesicles are delivered to the subject before delivering one or more therapeutic agents. In other embodiments, microvesicles are delivered to the subject after one or more therapeutic agents.

[0107] In other embodiments again, one or more therapeutic agents are loaded in microbubbles, wherein they can be broken or "activated" to release its content by slight interaction with applied energy. By "loading" these microbubbles with one or more specific therapeutic agents, and then activating microbubbles to release its content at target tissue, it is possible to limit its side effect by only one or more therapeutic agents being delivered to desired position (or substantially so). In such embodiments, microbubbles can include tiny gas-filled lipids or fat. As a result, in target tissue (for example, tumor), there is one or more therapeutic agents of high concentration to destroy target tissue. However, despite the presence of medicine in blood, other tissues in vivo are spared, because medicine is attached to microbubbles and is inactive.

[0108] In another embodiment, system also provides and makes target tissue visualization.Inflated microbubble (for example, size / diameter is in micron level) can be applied by ultrasonic energy and visualize or image.For example, in one embodiment, system can utilize energy source, and described energy source directs energy on microbubble, makes microbubble return unique echo in blood flow, and it produces significant difference or high " contrast " between blood vessel and surrounding tissue, thereby enables clinician to visualize target area.In other embodiments, visualization can be realized by using contrast agent.In either case, the contrast characteristics of medicament and / or microbubble allow the visualization of target tissue.

[0109] The system provided herein can be used in a variety of methods for treating diseases such as cancer. On the other hand, the present disclosure provides a method for delivering a drug to a tumor, the method comprising using an energy delivery system as provided herein, the energy delivery system being configured and arranged to produce an energy output; supplying one or more microbubbles and one or more therapeutic agents, wherein the microbubbles burst after receiving energy, thereby destroying the tumor and the surrounding extracellular matrix ("ECM") and allowing one or more therapeutic agents to be delivered into the tumor, thereby treating the tumor.

[0110] In some embodiments, one or more microbubbles are configured and arranged to contain one or more therapeutic agents, wherein the one or more microbubbles burst upon receiving energy, thereby releasing the one or more therapeutic agents into the tumor.

[0111] In some embodiments, the methods include introducing anti-cancer therapeutic agents by exposing blood vessel wall cells to energy to facilitate intracellular activation to allow entry of these agents into target tissues.

[0112] For example, suitable drugs within the scope of the present disclosure include, but are not limited to, the following drugs: Adriamycin PFS Injection (Pharmacia & Upjohn); Adriamycin RDF for Injection (Pharmacia & Upjohn); Alkeran for Injection (GlaxoWellcome Oncology / HIV); Aredia for Injection (Novartis); BiCNU (Bristol-Myers Squibb Oncology / Immunology); Blenoxane (Bristol-Myers Squibb Oncology / -Immunology); Camptosar Injection (Pharmacia & Upjohn); Celestone Soluspan Suspension (Schering); Cerubidine for Injection (Bedford); Cosmegen for Injection (Merck); Cytoxan for Injection (Bristol-Myers Squibb Oncology / Immunology); DaunoXome (NeXstar); Depo-Provera Sterile Aqueous Suspension (Pharmacia & Upjohn); Didronel Intravenous Infusion (MGI); Doxil Injection (Sequus); Doxorubicin Hydrochloride for Injection, USP (Astra); Doxorubicin Hydrochloride Injection, USP (ASTRA); DTIC-Dome (Bayer); Elspar (Merck); Epogen for Injection (Amgen); Ethyol for Injection (Alza); Etopophos for Injection (Bristol-Myers Squibb Oncology / Immunology); Etoposide Injection (Astra); Fludara for Injection (Berlex); Fluorouracil Injection (Roche Laboratories);Gemzar for Injection (Lilly); Hycamtin for Injection (SmithKline Beecham); Idamycin for Injection (Pharmacia & Upjohn); Ifex for Injection (Bristol-Myers Squibb Oncology / Immunology); Intron A for Injection (Schering); Kytril Injection (SmithKline Beecham); Leucovorin Calcium for Injection (Immunex); Leucovorin Calcium for Injection, Wellcovorin Brand (Glaxo Welcome Oncology / HIV); Leukine (Immunex); Leustatin Injection (Ortho Biotech); Lupron Injection (Tap); Mesnex Injection (Bristol-Myers Squibb Oncology / Immunology; Methotrexate Sodium Tablets, Injection, for Injection and LPF Injection (Immunex); Mithracin for intravenous use (Bayer); Mustargen for Injection (Bristol-Myers Squibb Oncology / Immunology); Mutamycin for Injection (Bristol-Myers Squibb Oncology / -Immunology); Navelbine Injection (Glaxo Wellcome Oncology / HIV); Neupogen for Injection (Amgen); Nipent for Injection (SuperGen); Novantrone for Injection (Immunex); Oncaspar (Rhone-Poulenc Rorer); Oncovin Solution Vials and Hyporets (Oncovin Solution Vials & Hyporets)(Lilly);Paraplatin for injection (Bristol-Myers Squibb Oncology / Immunology); Photofrin for injection (Sanofi); Platinol for injection (Bristol-Myers Squibb Oncology / Immunology); Platinol-AQ injection (Bristol-Myers Squibb Oncology / Immunology); Procrit for injection (Ortho Biotech); Proleukin for injection (Chiron Therapeutics); Roferon-A Injection (Roche Laboratories); Rubex for injection (Bristol-Myers Squibb Oncology / Immunology); Sandostatin Injection (Novartis); Sterile FUDR (Roche Laboratories); Taxol Injection (Bristol-Myers Squibb Oncology / Immunology); Taxol Abraxane-ABI-007 (Abraxis Bioscience); Taxotere for Injection Concentrate (Rhone-Poulenc Rorer); TheraCys BCG Live (Intravesical) (Pasteur Merieux Connaught); Thioplex for injection (Immunex); Tice BCG vaccine, USP (Organon); Velban vial (Lilly); Vumon for injection (Bristol-Myers Squibb Oncology / Immunology); Zinecard for injection (Pharmacia & Upjohn); Zofran injection (Glaxo Wellcome Oncology / HIV); Zofran premixed injection (Zofran Injection Premixed) (Glaxo Wellcome Oncology / HIV); Zoladex (Zeneca).

[0113] Other classes of drugs within the scope of the present disclosure include alkylating agents, which target DNA and are cytotoxic, mutagenic, and carcinogenic. All alkylating agents produce alkylation through the formation of intermediates. Alkylating agents impair cellular function by transferring alkyl groups to amino, carboxyl, sulfhydryl, or phosphate groups of biologically important molecules. Such drugs include, but are not limited to, Busulfan (Myleran), Chlorambucil (Leukeran), Cyclophosphamide (Cytoxan, Neosor, Endoxus), Ifosfamide (Isophosphamide, Ifex), Melphalan (Alkeran, Phenylalanine Mustargen, L-Pam, L-Sarcolysin), Nitrogen Mustargen (Mechlorethamine, Mustarden, HIV2), Nitrosonceas (Carmustine CBCNV, Bischlorethyl, Nitrosourea), Lomustine (CCNV, Cyclohexylchloroethyl nitrosourea), ChlorethylNitrosouren), CeeNV), semustine (methyl-CCNV) and streptozocin (Streptozotocin), streptozocin (Streptozoticin, Zanosan), Thiotepa (Theo-TEPA and Triethylenethrophosphoranide).

[0114] Agents with alkylating activity include a group of compounds that include heavy metal alkylating agents (platinum complexes) that act primarily through covalent bonding, and "non-classical alkylating agents" are also within the scope of the present disclosure. Such agents typically contain a chloromethyl group and a significant N-methyl group. Such other agents include, but are not limited to, Amsacrine (m-AMSA, msa, Acridinylanisidiale, 4'-)(9-acridinylamine)methanesulfin-m-anesidide (4'-)(9-acridinylamins)methanesulfin-m-anesidide), Carboplatin (Paraplatin, Carboplatinum, CBDCA), Cisplatin (Cesplatinum), Dacarbazine (DTIC, DIC dimethyltricizenormidazoleconboxamide), Hexamethylmelanine (HMM, Altretanine, Hexylin) and Procarbazine (Matulane, Natulanan).

[0115] Antimetabolites are also included within the scope of the present disclosure and include, but are not limited to, examples such as Azacitidine (5-azacylidine, ladakamycin), Cladribine (2-CdA, CdA, 2-chloro-2-deoxyadenosine), Cytarabine (Cytosine Arabinoside, Cytosar, Tarabine), Fludarabine (2-fluoroadenine arabinoside-5-phosphate, fludara). Fluorouracil (5-FV, Adrucil, Efuctex), Hydroxyurea (hydroxycarbamide, Hydrea), Leucovorin (Leucovorin Calcium), Mercaptopurine (G-MP, Purinethol), Methotrexate (Amethopterin), Mitoguazone (Methyl-GAG), Pentostatin (2'-deoxycoformycin), and Thioguanine (6-TG, aminopurine-6-thiol-hemihydrate).

[0116] Antitumor antibiotics usually interfere with DNA by intercalation, whereby the drug inserts itself between DNA base pairs. The introduction of ultrasound enhances this interference. Such drugs include but are not limited to actinomycin DC Cosmegen, Dactinomycin, Bleomycin (Bleomycin) (Blenoxane), Daunoxubin (Daunoxubibin) (rubidomycin), Doxorubicin (Doxorubicin) (Adriamycin, Hydroxydaunorubicin, Hydroxydaunorubicin, Rubex), Idarubicin (Idarubicin) (44-demethylorydan norubicin, Idamycin), Mithramycin (Mithracin, Plicamycin), Milomycin C (Milomycin C) and Mitoxantrone (Mitorantione) (Novantrone).

[0117] Plant alkaloids bind to tubulin, thereby inhibiting microtubule assembly; and energy such as laser or ultrasound can enhance this binding. Such alkaloids include, but are not limited to, Etoposide, Paclitaxel (Taxol), Teniposide (Treniposide), Vinblastine (Velban, Velsar, Alkaban), Vincristine (Oncovin, Vincasar, Leurocristine) and Vindesine (Eldisine).

[0118] Hormonal agents include steroids and related agonists and antagonists such as, but not limited to, adrenocortical steroids, adrenocortical steroid inhibitors, mitolane, androgens, antiandrogens, antiestrogens, estrogens, LHRH agonists, progestins.

[0119] Anti-angiogenic agents include, but are not limited to, the fumagillin derivative TNP-470, platelet factor 4, interleukin-12, the metalloproteinase inhibitor Batimastat, Carboryaminatriarzole, Thalidomide, Interferon α-2a, Linomide, and Sulfated Polysaccharide Tecogalan (DS-4152).

[0120] Specific examples of therapeutic agents that can be used in various embodiments include, but are not limited to, mycophenolic acid, mycophenolic acid derivatives (e.g., 2-methoxymethyl derivatives and 2-methyl derivatives), VX-148, VX-944, mycophenolate mofetil, mizoribine, methylprednisolone, dexamethasone, CERTICAN™ (e.g., everolimus, RAD), rapamycin, ABT-773 (Abbot Labs), ABT-797 (Abbot Labs), TRIPTOLIDE™, METHOTREXATE™, phenylalkylamines (e.g., verapamil), benzothiazepines (e.g., diltiazem), 1,4-dihydropyridines (e.g., benidipine, nifedipine, nicarrdipine, isradipine, felodipine, amlodipine, nilvadipine, nisoldipine, manidipine, nitrendipine, barnidipine) (H YPOCATM), ascomycin (ASCOMYCINTM), wortmannin (WORTMANNINTM), LY294002, camptothecin (CAMPTOTHECINTM), flavopiridol, isoquinoline, HA-1077 (1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride), TAS-301 (3-bis(4-methoxyphenyl)methylene-2-indolone), topotecan (TOPOTECANTM), hydroxyurea, tacrolimus (TACROLIMUSTM) (FK 506), cyclophosphamide, cyclosporine, daclizumab, azathioprine, prednisone, diferuloymethane, diferuloylmethane, diferulylmethane, gemcitabine (GEMCITABINE™), cilostazol (PLETAL™), tranilast, enalapril, quercetin, suramin, estradiol, cycloheximide, tiazofurin, zafurin, AP23573, rapamycin derivatives, non-immunosuppressive analogs of rapamycin (e.g., rapalog, AP21967, rapalog derivatives), CCI-779 (a rapamycin analog available from Wyeth), mycophenolate sodium, benidipine hydrochloride hydrochloride), sirolimus, rapamine, their metabolites, derivatives and / or combinations.

[0121] In other embodiments, the therapeutic agent may include cytokines, including but not limited to IL-2, TNF-, IL-12, and the like.

[0122] The systems and devices of the present disclosure can be configured to release or make available therapeutic agents in one or more treatment phases, wherein the one or more phases have similar or different performance (e.g., delivery) profiles. The therapeutic agent can be used by the tissue in a sustainable, intermittent or continuous amount, in one or more phases and / or at a delivery rate. Any of the at least one therapeutic agent can perform one or more functions, including preventing or reducing proliferative activity, reducing or inhibiting tumor formation and / or growth, etc.

[0123] The total amount of therapeutic agent available for use by the tissue depends in part on the level and amount of the desired therapeutic outcome. The therapeutic agent may be available in one or more stages, each stage having a release rate and duration similar or different from other stages. The release rate may be predefined. In one embodiment, the release rate may provide a sustainable level of therapeutic agent to the treatment site. In another embodiment, the release rate is substantially constant. The rate may be reduced and / or increased as desired.

[0124] These therapeutic agents can be provided and / or delivered to a subject or target in any conventional therapeutic form or formulation, such as, by way of example only: liquids, powders, particles, microbubbles, microspheres, nanospheres, liposomes, and / or combinations thereof.

[0125] Some embodiments of the present disclosure may also include delivering at least one therapeutic agent and / or optional compound in a subject or target simultaneously with or after the interventional treatment. More specifically, the therapeutic agent may be delivered to a targeted site (including a treatment site) simultaneously with or after the interventional treatment. For example: (a) in tumor treatment, the therapeutic agent may be delivered to the treatment site as an independent therapy without any other concurrent treatment (e.g., concurrent treatment provided by physical or mechanical intervention); (b) the therapeutic agent may be delivered to the treatment site as the only therapy for treating a disease (e.g., a tumor); (c) the therapeutic agent may be delivered to the treatment site after any suitable interventional procedure; (d) the therapeutic agent may be delivered to the treatment site before, during, after, or a combination thereof.

[0126] The therapeutic agent may be provided to the treatment site in a sustained, intermittent or continuous amount, in one or more stages and / or at a delivery rate.

[0127] Disclosed herein is a microtransducer-based catheter that uses different acoustic wave energy levels to a) overcome the anatomical barriers of current HIFU therapy (as described above) by directly impacting the tumor and ablating primary and metastatic lesions, b) disrupt the tumor extracellular matrix with or without contrast agents to facilitate intratumoral delivery of therapeutic agents, and c) produce a sonoporation effect (pulsed disruption of tumor cell membranes to enhance permeability) to allow nucleotide drugs such as mRNA to enter living tumor cells. The microtransducer-based catheter is configured to deliver therapeutic agents directly into tumors using an image-guided microtransducer HIFU catheter, using currently available imaging equipment and techniques. The device is configured to deliver a series of high-pressure acoustic bursts of focused ultrasound to produce intratumoral acoustic cavitation, wherein the expansion and collapse of microbubbles releases high-pressure cavitation energy that disrupts physical barriers in tumors and cancer cells through membrane rupture, thereby allowing a series of ablative and / or effective intratumoral retention and uptake of therapeutic agents.

[0128] Figure 2A A forward-looking ultrasound (US) transducer 200 is shown according to some embodiments. The transducer 200 includes multiple layers. For example, as shown, the transducer 200 includes a double layer of PZT-5A layer 202A, a matching (Al2O3 / epoxy) layer 202B, and a backing (bubble / epoxy) layer 202C. The hole area is about 2×2 mm 2 , and the operating frequency is configured to be approximately 0.8 MHz. Additionally, in other embodiments, the piezoelectric material may vary, the size of the aperture area may vary, and the operating frequency may also vary.

[0129] In some embodiments, the US transducer 200 can be configured as a tubular HIFU transducer. For example, Figure 3A A tubular HIFU transducer 300 is depicted for sonoporation. Figure 3B A schematic diagram 302 of a transducer 300 is depicted, the transducer 300 including a tubular piezoelectric device 304A and a matching layer 304B.

[0130] Figure 4A A miniaturized HIFU transducer 400 is depicted in accordance with some embodiments. The miniaturized HIFU transducer 400 is configured to have a center frequency of 5 MHz and includes five active elements 404 (e.g., PZT-4 plates) in the illustrated embodiment. In other embodiments, the transducer 400 may include more than five or less than five active elements 404. Additionally, in other embodiments, the size of each element 404 may vary.

[0131] The US transducer 200 may be configured as a transcatheter HIFU device, such as those described herein (e.g., Figure 4Atransducer 400) and provides, for example, local delivery of M-HIFU and IL-12 gene delivery to the tumor (as explained in more detail below). For example, Figure 5A Another exemplary embodiment of a HIFU transducer 400 is shown, which may be used with Figure 1A , 1B The hollow tube 104 of the device 102 is integrated. For example, the transducer 400 can be integrated into a 6-10Fr catheter with a drug delivery lumen. The transducer 400 includes a plurality of active elements 404, each of which includes a plurality of layers, the plurality of layers including, for example, a PZT-5H material layer 406A and a matching layer 406B. In a non-limiting example, PZT-5H can be used instead of PZT-4 material because the tissue fragmentation device 400 can be operated with relatively short pulse signals (i.e., about 30 cycles and <1% duty cycle). The thickness of each active element 404 can range from less than about 50 μm to about 500 μm. The hole of each element 404 is 1.4×1.8 mm 2 , and can range from less than about 1 mm x 1 mm to about 3 mm x 3 mm, depending on the energy requirements and application. In other embodiments, the transducer 400 can include more than five or less than five active elements 404. Additionally, in other embodiments, the size of each element 404 can vary.

[0132] The US transducer 200, 400 is introduced through a hollow tube or catheter 104, such as Fig. 16B As shown. The catheter 104 includes a first lumen 210 and a second lumen 212. The lumens 210, 212 may be positioned laterally along the length of the catheter or at the distal end of the catheter. Fig. 16B As shown, one of the lumens 210, 212 is located laterally on the catheter, and the other of the lumens 210, 212 is located at the distal end of the catheter. The US transducer 200, 400 is aligned with one of the lumens 210, 212 to transmit US waves through the lumens 210, 212. The catheter 102 passes through the subject to reach a target site (e.g., tissue, tumor, etc.), where the US transducer 200, 400 transmits US waves to the target. The parameters of the US transducer 200, 400 are appropriately selected for the purpose of applying ablation, sonoporation or tissue fragmentation or HIFU procedures to the target.

[0133] Example 1 - Miniaturized HIFU transducer [HJA(1]

[0134] like Figure 2AThe forward-looking ultrasound (US) transducer 200 shown is designed for acoustic porosity studies. The transducer 200 includes a double layer of PZT-5A layer 202A, a matching (Al2O3 / epoxy) layer 202B, and a backing (bubble / epoxy) layer 202C. The hole area is about 2×2 mm 2 , and the operating frequency is 0.8MHz.

[0135] Figure 2B is a graph 250 showing the transmit sensitivity of the transducer 200 as a function of input voltage (Vpp), wherein the sound pressure is measured at a distance of 2 mm from the aperture surface of the transducer 200 . Figure 2C Depicted is the use of the US transducer 200 to treat HEK293 cells in the presence of plasmid DNA (pCDH-GFP-LUC, 5 μg / mL) in a 96-well plate. Figure 2D is a fluorescent image of GFP+ sonoporation treated cells according to some embodiments, as well as negative control cells and Figure 2A Luciferase activity of cells treated with sonoporation by the transducer. Figure 2D As shown in images 275A-275D, focal GFP expression was observed only in sonoporation treated cells, but not in negative control cells (no US exposure). In sonoporation treated cells, LUC activity levels were as high as 3,600 units (846.0 + / - 786.5 units), while in negative controls were less than 170 (76.4 + / - 78.6 units), indicating successful transfection of HEK cells with pCDH-GFP-LUC by sonoporation treatment. Sonoporation can be further improved in terms of relevant ultrasound parameter selection, appropriate contrast agent dosage, and appropriate treatment time.

[0136] Tubular HIFU transducers have also been designed for sonoporation testing. Figure 3A A tubular HIFU transducer 300 is depicted. Figure 3B A schematic diagram 302 of a transducer 300 is depicted, the transducer 300 including a tubular piezoelectric device 304A and a matching layer 304B. Figure 3C The simulated sound field profile 325 generated by the transducer 300 is shown. pp The simulations were performed with an input voltage of 1000 MHz and an operating frequency of 0.85 MHz. It was found that at sub-MHz (e.g., 0.85 MHz), the peak negative pressure (PNP) in both the transverse and longitudinal directions can lead to cavitation, which is necessary for the sonoporation process. Figure 3DThe acoustic profiles measured in the side and front viewing directions (profiles 350A and 350B, respectively) are shown in . The PNP values ​​obtained by measurement (side viewing PNP is about 0.95 MPa and front viewing PNP is about 0.55 MPa) are similar to the values ​​obtained from simulation (0.81 MPa and 0.47 MPa for the side and front viewing directions, respectively).

[0137] Acoustoporation techniques result in the gradual suppression of a tumor or cancer. In contrast, tissue fragmentation and tissue ablation techniques aim to immediately remove or ablate malignant tumor tissue. Interstitial tissue ablation devices are relatively mature technologies; this approach essentially utilizes long US wave pulses to induce thermal necrosis in the target lesion. However, miniaturization of HIFU transducers remains a challenge for catheter-guided tissue ablation. Miniaturized tissue fragmentation transducers are quite unusual. Compared to typical thermally induced ablation techniques, tissue fragmentation utilizes very short (<1-2% duty cycle) US wave pulses with high rarefaction pressure output (>10MPa). However, most tissue fragmentation transducers operate on the outer surface of the human body. Recently, some researchers have proposed a relatively small-sized (approximately 5mm) prospective tissue fragmentation transducer for the treatment of brain tumors. However, interstitial or transcatheter HIFU capable of tissue fragmentation is challenging because high rarefaction pressure needs to be output from a small (<2mm) HIFU hole.

[0138] Figure 4A A miniaturized HIFU transducer 400 is depicted in accordance with some embodiments. The catheter HIFU transducer 400 is configured to have a center frequency of 5 MHz and includes five active elements 404 (eg, PZT-4 plates) in the illustrated embodiment. The aperture of each element 404 is 1.4 x 1.8 mm 2 In other embodiments, transducer 400 can include more than five or fewer than five active elements 404. Additionally, in other embodiments, the size of each element can vary. Figure 4B The acoustic pressure output 425 produced by the HIFU transducer 400 is shown (line 426A indicates the peak-to-peak pressure, and line 426B indicates the peak negative pressure). The quadratic regression curve shows that 300V is required pp The input was used to generate negative pressure in excess of 13 MPa. Next, a bubble generation test was performed to confirm the efficacy of the HIFU transducer 400 for possible tissue fragmentation treatment. The transducer 400 was sonicated in distilled degassed water, and vaporization during sonication was detected using a portable ultrasound imaging probe (iQ+, Butterfly Network, Guilford, CT) (see below for details). Figure 5C described in more detail). Figure 4CWhite spots 452 in captured image 450 of FIG. 4 show water vaporization induced by the HIFU transducer 400 , demonstrating the ability of tissue fragmentation therapy.

[0139] Typically, the center frequency of a transcatheter HIFU transducer used for tissue ablation and tissue fragmentation may range from about 100 kHz to 7.5 MHz.

[0140] The US transducer 200 may be configured as a transcatheter HIFU device, such as those described herein (e.g., Figure 4A transducer 400) and provides, for example, local delivery of M-HIFU and IL-12 gene delivery to the tumor (as explained in more detail below). For example, Figure 5A Another exemplary embodiment of a HIFU transducer 400 is shown, which may be used with Figure 1A , 1B The tube 104 of the device 102 is integrated. For example, the transducer 400 can be integrated into a 6-10Fr catheter with a drug delivery lumen. The transducer 400 includes a plurality of active elements 404, each of which includes a plurality of layers, for example, including a PZT-5H material layer 406A and a matching layer 406B. In a non-limiting example, PZT-5H can be used instead of PZT-4 material because the tissue fragmentation device 400 can be operated with a relatively short pulse signal (i.e., about 30 cycles and <1% duty cycle), and mechanical losses associated with transducer self-heating may not be a problem. The thickness of each active element 404 can be about 400 μm. The 3D printed mount can be used as a confocal geometry as well as an optical backing. Electrodes between individual piezoelectric plates (not shown) of the active element 404 can be connected using, for example, coaxial cables and conductive silver epoxy.

[0141] Figure 5B The acoustic pressure field distribution 525 of the transducer 400 operating at a frequency of 3.5 MHz is shown. In the linear regime, the maximum rarefaction pressure level is expected to exceed 24 MPa. The -6 dB focal region is estimated to be approximately For the acoustic characterization of the prototype LM-HIFU transducer 400, a hydrophone (HNA-0400, Onda Corp., Sunnyvale, CA) was used to record the acoustic pressure field by using a 3D motion console.

[0142] After acoustic characterization, according to Figure 5C and 5D The illustrated setups 550A and 550B use transducer 400 to generate cavitation, which results in Figure 4CIn addition to ultrasonic imaging of vaporized bubbles 552, a high-speed camera and ultrasonic imaging probe 554 (eg, iQ+, Butterfly Network, Guilford, CT) is used to visualize / capture images of bubble generation within the distilled degassed water medium.

[0143] In some embodiments, the transducer 400 provides rarefaction pressure levels exceeding 15 MPa for vaporization of general organ tissue. Tissue fragmentation treatments are performed in relatively short durations (<2 minutes) and exhibit a high degree of accuracy in precise treatment volumes. In situ tumor removal. Based on the accumulated data from gene transfer tests and tissue fragmentation tests performed with prototypes, the tissue fragmentation transducer 400 is configured to have strong ablation for tissue fragmentation, which is expected to induce apoptotic death of all tumor cells in the treatment area. Optionally, PMN-PT single crystals with much higher piezoelectric constants (d33>2,500pC / N) than PZT-based composites (d33<700pC / N) are used to provide high negative pressures, and larger pore sizes (e.g., 8-10Fr) are considered.

[0144] In another exemplary treatment method, the addition of systemic administration of ICB (anti-PD-1) to LM-HIFU+IT-IL-12 therapy enhanced the anti-tumor efficacy of ICB. In a previous study, anti-PD-1 antibodies were combined with IT-IL-12 treatment, which not only significantly inhibited tumor growth in multiple TNBC tumor models, but also led to complete tumor regression and long-term tumor-free survival in some treated mice. Based on the interaction group analysis of TILs in IT-IL-12 therapy, interactions between antigen presentation, myeloid populations, and CD8 T cells were enriched, and the CXCR3 gene signature was enhanced after IT-IL-12. The expression of the CXCR3 gene signature was found in tumors rich in CD8 T cells, and the expression was also associated with improved PFS and OS in TNBC patients.

[0145] For ease of description, the transducer described in the exemplary systems and methods below is referred to as transducer 200 and devices 102A, 102B. However, it should be understood that any suitable embodiment of transducer 200 (e.g., transducers 300 and 400 described above) can be used in such systems and methods.

[0146] Example 2 - Methods of use for cancer treatment [HJA(2]

[0147] Exemplary embodiments of HIFU devices including transducers 200, 400 described above can be used in conjunction with immunotherapy for a variety of treatments, including, for example, treatment of some types of cancer. In one exemplary embodiment, the treatment method includes using a miniaturized transcatheter LM-HIFU device (e.g., Figure 4A and 5A transducer 400), which can destroy tumor tissue and induce efficient gene expression in tumor cells or tumor-infiltrating immune cells through efficient gene transduction. As described herein, it has been found that intratumoral administration of plasmid IL-12 (IT-pIL12-EP, in Figures 6A-6E , 7A-7B and 8A-8D, which are described in more detail below) significantly inhibited the growth of triple-negative BC (TNBC) in mice and prolonged survival (e.g. Fig. 6A 600A and 602A in FIG. ). Specifically, Fig. 6A Figures 600A and 602A in the Figures depict data from BALB / c mice bearing murine 4T1 tumors (the mice received intratumoral injection of mIL12-P2A plasmid or control plasmid followed by in vivo electroporation on day 0), and the growth inhibition of 4T1 tumors and survival of mice by treatment (IL-12 group: 13 mice, control group: 14 mice). In addition, as Figure 6B As shown in Figure 600B (treated tumors) and Figure 602B (untreated tumors), treatment provided protection in untreated lesions as well as treated lesions by inducing systemic anti-tumor immunity. In particular, Figures 600B and 602B depict the abscopal effects of IT-pIL-12 administration. Tumor volumes were normalized to the size at the start of treatment (day 0) (n=4 per group). The left figure depicts data for treated tumors, while the right figure depicts data for tumors on the untreated side. Single-cell RNA sequencing (scRNA-seq) on TILs revealed that IT-pIL12-EP treatment induced a significant increase in T cells and dendritic cells, as well as a decrease in neutrophil infiltration into tumors, as shown Figure 6C As shown in Figure 600C in Figure 600C (t-SNE plots of cells classified into cell types or by treatment group for all samples, including analysis of the proportion of each cell type in each treatment group). The degree of T cell activation and clonal expansion was greater in IT-pIL12-EP-treated tumors compared with control-treated tumors, supporting the hypothesis that local delivery of IL-12 induces an adaptive immune response (respectively). Fig.6D and 6EFigure 600D and Figure 600E in ). Figure 600D depicts (Lt) quantification of the frequency of each clone, with the top 50 clones for each treatment shown as colored bars. (Rt) UMAP shows re-clustering of all TCR+ cells colored by clonal expansion (<10=blue; >10=red). Figure 600E depicts (Lt) quantification of activation signature scores for all T cells in each treatment. (Rt) UMAP is colored by 50-gene activation signature scores. All error bars represent mean ± SEM*p<0.05***p<0.001. More than 1,600 genes were significantly upregulated after IT-pIL12-EP treatment, including genes associated with increased CD8 T cell infiltration and activation, trafficking, antigen presentation, and exhaustion. KEGG pathway analysis further highlighted the enrichment of antigen presentation, cytokine, chemokine, and PDL1 pathways in IT-pIL12-EP treated tumors. Interactome analysis based on scRNA-seq data highlighted the CXCL9 / 10 / 11 / CXCR3 axis as the top interactions between receptors on macrophages and ligands on CD8 T cells ( Fig. 7A 700A in Figure 700A). Fig. 7A is a Circos diagram depicting receptor-ligand interactions between receptors on CD8 T cells and ligands on macrophages. Connections shown in red represent the top 25% of interactions between these cell types. A CXCR3 gene signature and the top 50 genes with the highest correlation with CXCR3 were identified, and it was found that IT-pIL12-EP treated tumors had significantly higher CXCR3 signature scores than cells from control treated tumors (Figure 2A). Figure 7B Figure 700B shows the 50-gene CXCR3 gene signature score quantified in all cells. All error bars represent mean ± SEM ***p<0.001.

[0148] Altogether, these analyses demonstrated that IT-pIL12-EP treatment increased the infiltration and activation of CD8 T cells, however the mixed expression of activation and exhaustion markers and the enrichment of pathways such as PD-1 / PD-L1 suggested that this therapy could be rationally combined with checkpoint inhibitors to maximize therapeutic efficacy. To test this, tumor-bearing mice were treated with IT-pIL12-EP in combination with an anti-PD-1 antibody. Figures 8A-8DFigures 800A-800D in show the tumor curves from the combination therapy, respectively. Figure 800A depicts the growth inhibition of 4T1 tumors by combination therapy. Tumor volume was measured every other day (control: 7 mice, anti-PD-1: 9 mice, IL-12: 7 mice, combination: 10 mice). Figure 800B depicts the growth inhibition of JC-HER3 tumors by combination therapy. Tumor size was measured every other day (control: 7 mice, anti-PD-1: 6 mice, IL-12: 8 mice, combination: 7 mice). Figures 800C and 800D depict the mouse survival rate for each treatment arm (arm) shown for the 4T1 tumor model (C) and the JC-HER3 tumor model (D). All error bars represent mean ± SEM*P<0.05, **P<0.01, ***P<0.001. Not only did the combination significantly slow tumor growth in multiple TNBC models, but it also resulted in complete tumor regression and long-term tumor-free survival in two different TNBC models (28.6% in the 4T1 model and 75.0% in the JC-HER3 model).

[0149] The clinical safety of intratumoral pIL-12 (TAVO) monotherapy (OMS-140) was tested in a single-arm prospective clinical trial of pretreated locally advanced or recurrent TNBC. In addition to demonstrating the safety and feasibility of this pIL-12 monotherapy, increased CD8+ T cell infiltration and decreased suppressive myeloid populations were observed in tumor biopsies obtained from patients after pIL-12 monotherapy in preclinical studies.

[0150] Systemic anti-tumor immune responses against distant untreated tumors were also observed without any evidence of cytokine storm or other toxicity. Importantly, one patient who was previously unresponsive to anti-PD1 and demonstrated an increased CXCR3 gene signature score with pIL-12 treatment underwent anti-PD-L1 therapy as his immediate next treatment and demonstrated a significant clinical response ( Fig. 9 900A and 900B in , where 900A depicts an exemplary baseline image of lung metastases and 900B depicts lung metastases after 1 cycle of IL-12 treatment and 3 months of nivolumab therapy). These data identified gene signatures that represent functional biomarkers that convert non-responders to responders.

[0151] Example 3 - Combining Tumor Microenvironment Disruption with Local Immunotherapy [HJA(3]

[0152] One obstacle to the use of therapeutic agents is the tumor microenvironment (TME), which contains cancer cells as well as a stromal compartment that includes fibroblasts, myofibroblasts, leukocytes, endothelial cells, macrophages, adipocytes, and extracellular matrix (ECM). Breast cancer has different levels of stroma, such as Fig.10 (which includes images 1000A-1000C, each of which depicts a fibrotic tumor stroma in breast cancer at different stages), and the ECM formed by matrix proteins (collagen, elastin, fibronectin, and laminin), which creates a physical barrier that prevents intratumoral drug distribution and direct contact between tumor cells and drugs or tumor-infiltrating immune effector cells. Fig.11 As shown in images 1100A and 1100B in , intratumoral injection of drugs often fails, the drug leaks into the surrounding tissue, and little drug is delivered into the tumor. Image 1100A depicts nearly complete filling, while image 1100B depicts minimal drug retention with leakage.

[0153] It has been demonstrated that non-invasive HIFU and IT-IL-12 gene delivery for solid tumors can be used to enhance immune infiltration and elicit systemic anti-tumor immune responses. HIFU can also help disrupt the stromal barrier in tumors and can enhance IT-IL-12 gene therapy by improving the distribution of IL-12 expression vectors in tumors.

[0154] To disrupt the stromal barrier as well as tumor cells and enhance immune infiltration, a novel HIFU strategy was identified and described herein that mechanically destroys cells and matrix, unlike conventional thermal HIFU (T-HIFU) therapy that results in coagulative necrosis.

[0155] An ex vivo HIFU system (VIFU2000 wet system for small animals, Alpinion) was used, and mechanical disruption was achieved by acoustic cavitation of the TME, which was achieved using high-pressure burst exposure (termed mechanical HIFU, M-HIFU, or tissue fragmentation). M-HIFU induces mechanical damage to tumor tissue and tumor stroma and blood vessels, resulting in cavitation and internal bleeding in an immunocompetent model of BC ( Fig.12 , which includes images 1200A and 1200B showing how a murine MC38 tumor growing on the thigh was treated by M-HIFU and T-HIFU). Histological analysis of tumor tissue harvested 1, 7, or 11 days after M-HIFU treatment showed enhanced immune infiltration, including T cell infiltration into the tumor (e.g. Fig.131300A and 1300B in Figure 1300A). H&E staining of tumor tissue one day after M-HIFU treatment is shown in Figure 1300A. Arrows indicate mechanically damaged areas with immune cell infiltration. CD8+ T cell infiltration increased significantly on day 7 after M-HIFU. As depicted in Figure 1300B, the mean of CD4, CD8 or FoxP3 stained+ cells for each treatment (mean of 5HPF for each tumor) is shown. Bars represent mean ± SE, n = 3. *: p value < 0.05.

[0156] MM3MG-HER2 cells were implanted into the right thigh and left flank of female BALB / c mice. After 7 days, M-HIFU was performed on the right thigh tumor in the M-HIFU monotherapy or combination treatment groups. Anti-PD-L1 antibody (100 μg / injection) or isotype control IgG (100 μg / injection) was injected intraperitoneally (ip) on days 10, 13, and 16. Tumor growth curves of HIFU-treated thigh tumors and untreated distant tumors are shown. The data in Figures 1400A and 1400B represent the results from two combined experiments; n=13 to 19 per group. Error bars: mean ± SD. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

[0157] The superior antitumor efficacy of M-HIFU with tumor growth inhibition and survival improvement compared with conventional T-HIFU was demonstrated in an immunocompetent MM3MG-HER2 murine breast cancer model. Modest abscopal effects on untreated distant tumors by M-HIFU monotherapy were observed, which could be enhanced by combination therapy of M-HIFU and PD-1 / PD-L1 blockade, as Fig.14 As shown in Figures 1400A and 1400B.

[0158] Since both IT-IL-12 gene therapy and M-HIFU treatment can induce antitumor efficacy through non-overlapping mechanisms and both modalities showed synergy with anti-PD-L1 antibody therapy, it was hypothesized that combining M-HIFU with IT-IL-12 gene therapy would enable a wider distribution of the IL-12 expression vector into the tumor and induce more intratumoral T cell infiltration by disrupting the stromal barrier. To test this hypothesis, E0771 tumor-bearing mice were treated with IL-12 gene therapy alone, M-HIFU alone, or a combination of the two. Fig.15 Figure 1500A in Figure 1500A shows the strongest tumor growth inhibition in the combination treatment group, although no statistical difference was confirmed between IL-12 alone and the combination treatment due to the relatively early initiation of intervention. 3When E0771 tumor-bearing mice were intervened relatively late (28 days) (Figures 1500B and 1500C), it was found that the combination of IL-12 gene therapy and M-HIFU induced stronger tumor growth inhibition and higher mouse survival compared with IL-12 gene therapy alone.

[0159] Referring to Figure 1500A, 28 days after tumor cell implantation, E0771 tumor-bearing C57BL / 6 mice were treated with IT-IL-12 gene therapy alone, M-HIFU alone, or a combination thereof. Tumor volume was monitored every two days. n = 4-5 mice. The combined treatment was significantly superior to HIFU alone in inhibiting tumor growth (p < 0.01). Referring to Figure 1500B, intervention was initiated 28 days after E0771 cell implantation. Mice received IL-12 gene therapy alone or a combination of IL-12 and HIFU. N = 8 mice. The combined treatment induced significantly stronger tumor growth inhibition (AUC analysis, t-test: p < 0.0001). Finally, referring to Figure 1500C, mouse survival is shown. Log-rank test: p < 0.005.

[0160] If respectively Fig.16A and 16B As shown in Figures 1600A and 1600B in Figure 1600A and 1600B, an exemplary treatment strategy for, for example, metastatic breast cancer (BC) is shown. The disclosed method includes: with a catheter (e.g., Figure 1A and 1B The device 102) is integrated with a transcatheter HIFU transducer (e.g., transducer 200, 400), which is inserted into a metastatic tumor (e.g., liver, lung, brain, or bone metastasis) under ultrasound imaging or MR imaging 1602, performs tissue fragmentation (M-HIFU) to destroy tumor cells and tumor matrix barriers, and locally delivers an IL-12 expression vector to induce enhanced expression of IL-12 in the TME. The miniaturized HIFU transducer 200 can be directly inserted into primary or metastatic BC, even if they are located deep or in a site that is difficult to target by in vitro HIFU, such as the liver dome, behind the ribs, near large blood vessels, or deep in the lungs.

[0161] In particular, as shown in Figures 1600A and 1600B, a transcatheter HIFU transducer is inserted into the tumor site with a guide needle under ultrasound imaging or MR imaging. Tissue fragmentation (M-HIFU) is performed to destroy tumor cells and tumor matrix barriers, and the IL-12 expression vector is infused into the tumor through another lumen of the catheter. Tumor destruction and induced inflammatory TME and raised cytotoxic immune cells eradicate the treated tumor. The induced systemic anti-tumor immunity (which is enhanced by IT-IL-12) is expected to eradicate distant metastatic tumors.

[0162] In order to enhance IT-IL-12-induced systemic anti-tumor immunity, in vitro M-HIFU was combined with IT-IL-12, and the combined treatment was found to be more effective than IT-IL-12 alone. Therefore, this combination strategy is proposed to treat metastatic BC. This local therapy can be applied to metastatic breast tumors located in or on visceral organs such as the liver, lungs, and brain. Transcatheter HIFU devices, such as those described herein (e.g., device 102 including transducers 200, 400), provide local delivery of M-HIFU and delivery of IL-12 genes to tumors.

[0163] Example 4 - In vivo functionality of a miniaturized HIFU transducer [HJA(4]

[0164] A combination of pathological analysis and scRNA sequencing was used to analyze the TME altered by LM-HIFU+IT-IL-12 therapy alone and in combination with ICB to confirm the in vivo functionality of the catheter LM-HIFU device 102. Next, the anti-tumor efficacy and induced tumor-specific immune responses achieved by LM-HIFU+IT-IL-12 combination therapy with / without anti-PD-1 mAb were evaluated, and abscopal effects and inhibition of lung metastasis were demonstrated in the TNBC model. Finally, pathological analysis and scRNA-seq analysis were used to evaluate whether there were significant changes in the TME in LM-HIFU+IT-IL-12 treated tumors and whether there was an increase in the CXCR3 gene signature in TILs.

[0165] E0771 BC cells were implanted subcutaneously into the flank of female C57BL / 6 mice. Once the tumor size reached 7-8 mm, the tumor was treated with LM-HIFU using the transcatheter device 102, while Ad-IL-12 (10 μL in 50 μL saline) was delivered via a double lumen catheter. 10 Virus particles / tumor) were infused into the tumor. The following groups were formed: a) no treatment, b) LM-HIFU alone, c) IT-IL-12 alone, and d) LM-HIFU + IT-IL-12. Group b mice had only LM-HIFU without Ad-IL-12 infusion, and group c mice had only Ad-IL-12 infusion without HIFU energy. (n=8 mice per group, 4 mice for 3-day termination and 4 mice for 7-day termination). On days 3 and 7 after the procedure, 4 mice per group were euthanized daily, and tumors and blood were collected for assays.

[0166] Half of the tumor tissue was used for pathological analysis to evaluate tumor tissue destruction and T cell infiltration into the tumor by IHC, and IL-12 expression by in situ hybridization (RNAscope). One quarter of the tumor tissue was enzymatically digested, and the isolated immune cells were analyzed by flow cytometry. The level of IL-12 protein was analyzed by IL-12 ELISA using tumor lysate and serum. It was confirmed whether LM-HIFU treatment destroyed tumor tissue and enhanced T cell infiltration into the tumor at an earlier time point, and increased IL-12 levels in IT-IL-12 treated tumors, but did not increase IL-12 levels in the blood. Single-cell secretomes were used to determine how LM-HIFU+IT-IL-12 modifies tumor-infiltrating immune cells.

[0167] The systemic tumor-specific immune response and antitumor efficacy induced by LM-HIFU+IT-IL-12 combination therapy against murine TNBC were demonstrated by the following method. E0771-OVA murine BC cell line was implanted into the flank of female mice (C57BL / 6). When the tumor size in the right flank reached approximately 7-8 mm in diameter, LM-HIFU or IT-IL-12 treatment was performed as a monotherapy or combination therapy for the tumor. A catheter with a miniaturized HIFU transducer and drug infusion lumen was inserted into the tumor, and LM-HIFU and / or IT-IL-12 was performed.

[0168] The following groups were tested: a) no treatment, b) LM-HIFU alone, c) IT-IL-12 alone, and d) LM-HIFU + IT-IL-12 (n = 14 mice per group, 4 mice for 7-day termination). Four mice in each arm were euthanized 7 days after treatment. Tumors, spleens, and blood were collected and subjected to IFN-γ ELISPOT assay (OVA peptide (SIINFEKL) as stimulating antigen), ELISA (antibody for OVA protein), and flow cytometry using H-2Kb / OVA (SIINFEKL) MHC tetramers. Tumor samples were analyzed for IL-12 production by in situ hybridization (RNAscope) and IL-12 ELISA using tumor lysates as samples. The number of tumor-infiltrating leukocytes was analyzed by flow cytometry and immunohistochemistry (IHC). After digestion of tumor tissue with triple enzyme buffer (collagenase III, hyaluronidase, DNase), cells isolated from tissue will be subjected to multiparameter flow cytometry to characterize immune cell populations for the following cells: CD4 T helper cells (Th1: CD4 / CCR5, Th2: CD4 / CCR4), CD8 CTLs (central memory: CD8 / CD62L, effector memory: CD8 / KLRG1), dendritic cells (CD11c / CD80 / CD86), B cells (CD19) and NK cells (CD49b / NKG2D), regulatory T (Treg) cells (CD4 / CD25 / FoxP3), tumor-associated macrophages (TAMs) (CD11b / F4-80 / CSF-1R), myeloid-derived suppressor cells (MDSCs) (CD11b / Gr-1). The activation status of T cells will be analyzed with anti-CD25, CD69, ICOS, PD-1, CD107, and granzyme-B. The expression of immune checkpoint molecules CTLA-4, PD-L1, TIM3, LAG3, and TIGIT was also analyzed as a marker of cell exhaustion. The expression of immune checkpoint molecules CTLA-4, PD-L1, TIM3, LAG3, and TIGIT will also be analyzed as a marker of cell exhaustion. Cells from the spleen were also analyzed by flow cytometry and compared with TILs. In addition, fluorescence microscopy analysis was performed by staining tumor tissues with fluorescently labeled anti-CD3, CD4, CD8, CD11b, CD11c, CD19, CD49b, F4 / 80, Ly6G, FoxP3 antibodies and antibodies against immune checkpoint molecules. The interaction of different immune cell types and the total number of immune cells in the TME were evaluated. Photos were taken and the number of stained cells was analyzed using Image J software. Multiple cell lineages (CD4+T, CD8+T, Treg, NK, DC, macrophages, MDSC) were identified and quantified.

[0169] For scRNA-seq, tumor tissue was digested as described above and live tumor-infiltrating immune cells (viability dye-, CD45+ cells) were FACS sorted. 10X libraries were created using the Chromium Single Cell 5'Library Construction Kit (v1.1) following the manufacturer's protocol. Gene expression and V(D)J enrichment libraries were created for each sample. The generated cDNA and final GEX / TCR library were quality checked using an Agilent Bioanalyzer 2100 and sequenced on a NovaSeq S4 instrument. Genomics Software (Version 9.0.20, Copyright Fastq files from 10X library sequencing were processed using UMAP plots using the top 15 principal components, a minimum distance of 0.4, and 30 neighbors 42. Differentially expressed genes were identified using the GSA algorithm in Partek Flow. KEGG pathway enrichment analysis was performed on genes found to be differentially expressed at FDR 5% and with a fold change >|2|. Clustering, cell type identification, and differential gene expression analysis were performed on single-cell data, and treatment groups were de-identified for blinding. Samples from the four groups were compared. Because an increased CXCR3 gene signature with enhanced antigen presentation was identified in a previous study to systemically expand and license T cells in IT-IL-12-treated TNBC, it was evaluated whether combined LM-HIFU+IT-IL-12 treatment using a catheter-based LM-HIFU device would induce similar changes in the TME.

[0170] For the other 10 mice, tumor size was measured three times a week until they reached the humane endpoint (tumor volume >2,000 mm 3 ) and assessed the survival of the mice. The percent tumor growth inhibition (%TGI), defined as the percentage difference between the median tumor volume (MTV) of treated and control mice, was also compared. To confirm the induction of potent systemic anti-tumor immunity, long-term survivors after tumor eradication will receive tumor re-challenge 12 weeks after LM-HIFU + IT-IL-12 treatment. Tumor growth / tumor-free survival will be monitored for up to two months. This task aims to confirm that LM-HIFU + IT-IL-12 combination therapy is superior to IT-IL-12 monotherapy or LM-HIFU monotherapy in inducing anti-tumor immunity and inhibiting tumor growth.

[0171] In the bilateral TNBC implantation model, the abscopal effect induced by LM-HIFU+IT-IL-12 therapy and the anti-tumor efficacy enhanced by the combination with anti-PD-1 antibody were demonstrated as follows. E0771-OVA cells were implanted bilaterally into the flanks of female C57BL / 6 mice. When the tumor size in the right flank reached approximately 7-8 mm in diameter, the tumor in the right flank was treated with LM-HIFU+IT-IL-12 with or without anti-PD-1 antibody, but the contralateral tumor was not treated.

[0172] The following groups were tested: a) no treatment, b) anti-PD-1 mAb alone, c) LM-HIFU + IT-IL-12 + control IgG, and d) LM-HIFU + IT-IL-12 + anti-PD-1 mAb (n = 14 mice per group, 4 mice for 7-day termination). Anti-PD-1 Ab or control IgG (200 μg / injection) was administered intraperitoneally on day 3 after LM-HIFU + IT-IL-12 treatment and repeated twice a week for two weeks.

[0173] As described above, 4 mice from each group were euthanized 7 days after treatment, and tumor samples collected from treated local sites and untreated distant sites were analyzed. Collected splenocytes and serum were used for IFN-γELISPOT determination, tetramer determination and ELISA. scRNA-seq analysis and multi-parameter flow cytometry were performed on TIL. The TILs isolated from treated tumors and untreated distant tumors in the four treatment groups were analyzed and compared (only Group A had 1 TIL sample). It was assessed whether the LM-HIFU+IT-IL-12 therapy combined with anti-PD-1 showed more significant TME changes compared with the LM-HIFU+IT-IL-12 therapy alone. The differences in TME changes between treated tumors and untreated distant tumors were also analyzed.

[0174] For the other 10 mice, tumor size (both HIFU-treated local tumors and untreated distant tumors) was monitored three times a week until they reached the humane endpoint (tumor volume >2,000 mm 3 ), and the survival of mice between the arms was compared. %TGI was also compared. To confirm the induction of potent systemic anti-tumor immunity, long-term survivors after tumor eradication received tumor re-challenge 12 weeks after LM-HIFU+IT-IL-12 treatment. Tumor growth / tumor-free survival was monitored for up to two months.

[0175] The task was to confirm that the combination of anti-PD-1 antibody and LM-HIFU+IT-IL-12 therapy was superior to LM-HIFU+IT-IL-12 therapy or anti-PD-1 antibody alone in inducing systemic anti-tumor immunity and inhibiting distant tumor growth (abscopal effect).

[0176] In the highly metastatic E0771-LX3-OVA model, inhibition of spontaneous lung metastasis by LM-HIFU+IT-IL-12 therapy with anti-PD-1 antibodies was demonstrated as follows. A highly metastatic variant, named E0771-CH3-MX3, was established in the laboratory after three rounds of orthotopic implantation of cells isolated from lung metastases of E0771 cells in C57BL / 6 mice. For all mice tested, this metastatic variant was confirmed to produce lung metastases 5-6 weeks after orthotopic implantation (5×10E5 cells / injection). E0771-CH3-MX3 cells were implanted into the 4th mammary fat pad of female C57BL / 6 mice. When the tumor grew to approximately 7-8 mm in diameter, LM-HIFU+IT-IL-12 treatment with or without anti-PD-1 antibodies was performed. The following groups were tested: a) no treatment, b) anti-PD-1 mAb alone, c) LM-HIFU + IT-IL-12 + control IgG, and d) LM-HIFU + IT-IL-12 + anti-PD-1 mAb (n = 10 mice per group). Anti-PD-1 Ab or control IgG (200 μg / injection) was administered intraperitoneally on day 3 after LM-HIFU + IT-IL-12 treatment and repeated twice a week for two weeks. Tumor growth as well as mouse activity, respiratory rate, appetite, and mouse physical condition were monitored. Mice were euthanized 6 weeks after tumor cell implantation, and lung metastasis was assessed macroscopically and microscopically on the collected lungs. Tissue sections were analyzed by H&E staining and IHC using anti-p53 antibody (E0771 cell line: p53+), and metastatic foci were quantified using Image J software. Fluorescence microscopy analysis was performed to identify effector immune cells in metastatic nodules by staining frozen tumor tissues with fluorescently labeled antibodies against CD3, CD4, CD8, CD49b, and granzyme B. Pictures were taken and the number of stained cells was analyzed using Image J software.

[0177] The optimized intralesional immunotherapy was used to investigate the antitumor efficacy of LM-HIFU+IT-IL-12 treatment against ER+BC and HER2+BC with low ICB responsiveness, and the induction of systemic antitumor immunity and abscopal effects of this combination therapy were also confirmed.

[0178] The ovalbumin-expressing murine triple-negative BC cell line E0771 (E0771-OVA) was established using a lentiviral vector as described previously. MM3MG-HER2 was established by retroviral transduction of the receptor tyrosine protein kinase erbB-2 (HER2) using an untransformed BALB / c mammary epithelial cell line (MM3MG), a HER2-driven BC cell line. OVA and HER2 antigens were used in the assay to evaluate the induction of systemic antitumor immunity.

[0179] Using DNA deep sequencing technology, recent studies have identified several conservative mutations in the DNA binding domain of ESR1 in a significant proportion (approximately 30-40%) of patients with endocrine-resistant metastatic ER+ breast cancer. Since there is no established model of endocrine-resistant ER+ breast cancer in immunocompetent mice, an ER+ endocrine-resistant breast cancer cell line model that will grow in immunocompetent syngeneic animals was generated. MM3MG cells were artificially transformed with wild-type or mutated human ESR1 (Y537N, Y537S, D538G).

[0180] like Fig.17 As depicted in Figure 1700A in Figure 1700A, lentiviral stably transduced precancerous MM3MG cells (BALB / c background) were transfected with ERE luciferase reporter together with transfection controls under estrogen-free conditions or positive (20 nM E2) conditions and cells were harvested at 24 hours (N=6). Fig.17 In Figure 1700B, these cells were implanted into female mice (no estrogen pellets) and tumor growth was assessed over time by caliper measurement. In all figures, * indicates p<0.05 from ESR1-WT control, and ** indicates p<0.01.

[0181] In this model, these ESR1 mutants were found to confer enhanced estrogen signaling in these cell lines ( Fig.17 1700A in Figure 1700A) and in fact "transformed" them, allowing them to form more aggressive tumors that were successfully transplanted into mice compared to their control and wild-type ESR1 counterparts ( Fig.17 1700B in Figure 1700B). Crucially, these mice do not require exogenous estrogen supplementation to allow estrogen-mediated signaling or tumor growth. These cells were also found to be completely resistant to standard endocrine therapies (e.g., tamoxifen and aromatase inhibitors (data not shown)). Thus, the established murine breast cancer cell line MM3MG-ESR1mut (Y537N, Y537S, D538G) mimics metastatic ER+ breast cancer in patients, which is endocrine resistant, has ESR1 gene mutations, and behaves more aggressively.

[0182] The systemic tumor-specific immune response and anti-tumor efficacy of LM-HIFU+IT-IL-12 combination therapy for ER+ and HER2+ murine breast cancer are demonstrated as follows. The murine BC cell lines MM3MG-ESR1mut (experiment 3.1) and MM3MG-HER2 (experiment 3.2) were implanted into the right flank of female BALB / c mice. As described above for TNBC, LM-HIFU or IT-IL-12 treatment was performed as a monotherapy or combination therapy for tumors. A catheter LM-HIFU device with a drug infusion lumen was inserted into the tumor, and LM-HIFU+IT-IL-12 combination therapy was performed with optimized settings.

[0183] The following groups were tested: a) no treatment, b) LM-HIFU alone, c) IT-IL-12 alone, and d) LM-HIFU + IT-IL-12 (n = 14 mice per group, 4 mice for 7-day termination). 7 days after treatment, 4 mice in each arm were euthanized. Tumors, spleens, and blood were collected and IFN-γ ELISPOT assays (ESR1 peptide mixture or HER2 peptide mixture as stimulation antigens for experiments 3.1 and 3.2, respectively) and cell-based ELISAs were performed (parental E0771 / E0771-ESR1 cells or parental 4T1 / 4T1-HER2 cells were used for experiments 3.1 and 3.2, respectively, and both cell lines were available). Tumor lysates were used as samples to analyze the IL-12 production of tumor samples by in situ hybridization (RNAscope) and IL-12 ELISA. In order to analyze the cell type and activation / maturation status of TIL, flow cytometry analysis was performed as described above. Tumor infiltrating leukocytes were further analyzed by fluorescence microscopy. scRNA-seq analysis was also performed. Pathological analysis, flow cytometry, and scRNA-seq analysis were performed to determine whether ER+ and HER2+ tumors responded to LM-HIFU+IT-IL-12 treatment in a similar manner compared with TNBC, such as increased intratumoral T cell infiltration, changes in the TME, and induction of antigen-specific immune responses.

[0184] For the other 10 mice, tumor size was measured three times a week until they reached the humane endpoint (tumor volume >2,000 mm 3 ) and assessed the survival of mice. Comparison of tumor growth inhibition percentage (%TGI). To confirm the induction of potent systemic anti-tumor immunity, long-term survivors after tumor eradication received tumor re-challenge 12 weeks after LM-HIFU + IT-IL-12 treatment. Tumor growth / tumor-free survival was monitored for up to 2 months.

[0185] In the bilateral ER+ and HER2+ model, the abscopal effect induced by LM-HIFU+IT-IL-12 therapy and the anti-tumor efficacy enhanced by the combination with anti-PD-1 antibody were demonstrated as follows. MM3MG-ESR1mut or MM3MG-HER2 cells were bilaterally implanted into the flanks of female BALB / c mice (Experiments 3.3, 3.4). As described above, tumors in the right flank were treated with LM-HIFU+IT-IL-12 with or without anti-PD-1 antibody, but the contralateral tumors were not treated.

[0186] The following groups were tested: a) no treatment, b) anti-PD-1 mAb alone, c) LM-HIFU + IT-IL-12 + control IgG, and d) LM-HIFU + IT-IL-12 + anti-PD-1 mAb (n = 14 mice per group). Anti-PD-1 Ab or control IgG (200 μg / injection) was administered intraperitoneally on day 3 after LM-HIFU + IT-IL-12 treatment and repeated twice a week for two weeks.

[0187] Four mice from each group were euthanized 7 days after treatment, and tumor samples collected from treated local sites and untreated distant sites were analyzed. Collected splenocytes and serum were used for IFN-γ ELISPOT assays and ELISA. scRNA-seq analysis was performed on TILs isolated from treated tumors and untreated distant tumors in each group. It was assessed whether LM-HIFU+IT-IL-12 therapy combined with anti-PD-1 showed more significant TME changes compared to LM-HIFU+IT-IL-12 therapy alone. CXCR3 gene signature levels were also assessed. The differences in TME changes between treated tumors and untreated distant tumors in each group were also assessed.

[0188] For the other 10 mice, tumor size (both HIFU-treated local tumors and untreated distant tumors) was measured three times a week until they reached the humane endpoint (tumor volume >2,000 mm 3 ), and compare the survival of mice between the arms. %TGI was also compared. In order to confirm the induction of potent systemic anti-tumor immunity, long-term survivors after tumor eradication received tumor re-challenge. Tumor growth / tumor-free survival was monitored for up to two months. In this task, it was intended to confirm that the combination of anti-PD-1 antibodies and LM-HIFU+IT-IL-12 therapy would induce the strongest anti-tumor effects, systemic anti-tumor immunity, and abscopal effects in ER+ and HER2+BC models. It was also studied whether ER+ and HER2+BC would show different changes in the TME compared to TNBC after combined treatment.

[0189] Example 5 - In vivo sonoporation-induced drug / gene delivery using a miniaturized HIFU transducer[HJA(5]

[0190] As mentioned above, typical US transducers for sonoporation (e.g., single-element focusing US transducers) are extracorporeal, which is not suitable for intratumoral immunotherapy. Several limitations of extracorporeal US transducers include that it cannot effectively sonicate tumors behind bone and fat due to ultrasound attenuation and absorption, and it cannot precisely inject MBs and nucleic acids simultaneously into the US treatment zone, which may miss the target. These problems reduce the effectiveness of drug / gene delivery and increase the undesirable systemic toxicity of cancer immunotherapy.

[0191] Thus, the systems and methods described herein relate to a miniaturized US transducer (eg, transducer 400 described above) integrated into a catheter (ie, a catheter US transducer) for in vivo sonoporation-induced drug / gene delivery in intratumoral immunotherapy.

[0192] Design and manufacture hole size 2×2mm 2 An 800 kHz US transducer (similar to transducer 400 of FIG. 5E ) was used for in vivo sonoporation studies. Fig.18 The transducer 400 in the illustrated embodiment includes the Figure 4A and 5A The described double layer of PZT-5A, matching layer (Al2O3 / epoxy resin) and backing layer (bubble / epoxy resin) (not shown). In order to inject microbubbles into the sonication area, the lumen was embedded in the catheter, located next to the US transducer. The lumen size was about 0.9 mm (outer diameter), while the catheter size was about 3.0 mm (outer diameter). The output sound pressure of the developed transducer was characterized by a calibrated hydrophone (HNA-0400, ONDA Corporation, CA, USA) in degassed water.

[0193] Since US standing waves may affect the efficiency of the in vitro acoustic porosity effect, acoustic simulations were first performed using the k-Wave toolbox to explore the propagation of 800kHz US waves in a 384-well cell culture plate. Considering the axisymmetric boundary conditions of each well in the cell culture plate, the computational domain was set to a 3.6mm×7.6mm 2D plane filled with water. The material of the culture plate is polystyrene, and its acoustic properties (including density, sound velocity, and absorption coefficient) are summarized in Fig.19 Table 1900. The US transducer is placed at the top of the hole, the US frequency is 800kHz, and the input pressure is 0.4MPa.

[0194] Human embryonic kidney (HEK) 293T cells and plasmid DNA encoding green fluorescent protein-luciferase (GFPLUC) were prepared. HEK 293T cells (12,000 cells / well, 3×105 cells / mL, 40 μL / well) and plasmid DNA (0.4 μg / well, 20 μg / mL, 20 μL / well) were plated in 384-well plates for sonoporation testing. MB (SonoVol, Inc., NC, USA) was used as cavitation nuclei. MB was mixed with phosphate buffered saline (PBS) solution in a 10 mL syringe for tube injection and the MB concentration was diluted to 8.73 × 10 8 During the sonoporation test, 30 μL of MB solution was infused into each well through the syringe, and the total solution volume in each well was about 90 μL.

[0195] like Fig. 20 As shown in the test system 2000, a pulsed electrical waveform first generated by a function generator 2002 (e.g., 33250A, Agilent Technologies, Inc., CA, USA) and then amplified by a radio frequency power amplifier 2004 (e.g., 75A250A, Amplifier Research Corporation, PA, USA) is used to drive the catheter US transducer 200. The catheter US transducer 200 is inserted into a cell culture plate 2006 to inject the MB solution. The MB injection is controlled by a syringe pump 2008 (e.g., NE-1010, New Era Pump Systems, Inc. NY, USA) with an infusion rate of 0.1 mL / min. The cells are then sonicated for 30 seconds at a duty cycle of 5%. Various sonication parameters were tested, including peak negative pressure (PNP) (0.1-0.7 MPa) and number of cycles (CN) (20-2000 cycles).

[0196] After sonication, the tested cells were incubated in a CO2 incubator at 37°C for 24 hours. To evaluate the efficiency of the sonoporation effect, cells were harvested from each well and lysed in lysis buffer. Luciferase activity was measured by adding luciferin using a luminometer (e.g., GloMax, Promega Corporation, WI, USA) and reported in relative light units (RLU).

[0197] In this work, six independent experimental runs were performed for each group (i.e., n = 6). One-way analysis of variance (ANOVA) was used to compare luciferase activities between the control and experimental groups. P values ​​< 0.05 were considered statistically significant.

[0198] Fig.21Measurements of the PNP and mechanical index (MI) of a prototype 800kHz catheter US transducer are shown in Graph 2100 in , indicating that the fabricated transducer can produce the required PNP (about 0.5 MPa) for sonoporation applications. In addition, the focal length of the catheter transducer is about 1 mm close to the transducer surface.

[0199] Simulate ultrasonic beam ( Fig. 22 The beam 2200) shows that the standing wave induced by the bottom wall of the cell culture plate can increase the acoustic pressure and deform the acoustic pressure field. The maximum acoustic pressure increased by 20% and was 0.48MPa in the cell culture plate. Although the catheter US transducer has a focal length of about 1mm, it can be observed that a high acoustic pressure area appears near the bottom wall of the cell culture plate due to US wave reflection. This phenomenon shows that cells can be placed on the bottom surface of each well for effective cell sonication.

[0200] Comparison of luciferase activity between the negative control group and the experimental group (see Fig.23 2300). Negative controls consisted of a "no plasmid" group, a "plasmid" group, a "MB only" group, and a "US only" group, and the experimental group was referred to as the "MB+US" group. In addition, a positive control for gene transfer by lipofectamine was run in parallel to ensure that the assay performed well (data not shown). For sonication parameters, the "US only" group had 0.2 MPa of PNP, and 20 cycles of CN, while the "MB+US" group had 0.4 MPa of PNP, and 200 cycles of CN. Luciferase assay ( Fig.23 The graph 2300 in Figure 2300 shows that the "plasmid" group has only 10 2 level of RLU, while the “MB+US” group reached 10 5 The corresponding maximum fold change in luciferase activity was approximately 1500-fold, indicating a significant enhancement of transfection. Subsequently, preliminary parameter sensitivity studies were performed to investigate how PNP and CN affect the efficiency of sonoporation. Fig.24 (which shows luciferase activity of sonoporation test under various sonication parameters including PNP: 0.1-0.7 MPa and CN: 20-2000 cycles (n=6)) shows that 20 (graph 2400A) or 200 cycles (graph 2400B) are better than 2000 cycles (graph 2400C), and the optimal PNP range is 0.3 MPa to 0.5 MPa. Fig.24 , it is speculated that excessive sound pressure and long pulse duration (PD, equal to CN / US frequency) can induce cell death and reduce the efficiency of the sonoporation effect. Fig.24, considering that the PD of 20 cycles of 800kHz US is only 25μs, the bubble-cell contact probability is relatively low in such a short period of time. Therefore, the trend of PNP-RLU is different from that of Fig.24 2400B and 2400C. In addition, it is interesting to observe that for the 20 cycle condition, 0.3-0.4 MPa has a relatively low RLU (Chart 2400A), and for the 200 cycle condition, 0.3 MPa has a relatively low RLU (Chart 2400B). It is speculated that this may be related to the conversion of stable cavitation and inertial cavitation. In fact, inertial cavitation can form larger holes in the cell membrane to achieve higher sonoporation efficiency, but it also causes the MB to collapse suddenly. This may explain why the sonoporation efficiency decreases when PNP is higher than 0.3 MPa in Chart 2400A and 0.2 MPa in Chart 2400B. Afterwards, inertial cavitation takes effect, and the sonoporation efficiency rises with the increase of PNP until cell death occurs.

[0201] Therefore, the results of the developed 800 kHz front-view catheter US transducer suggest that it is promising for in vivo sonoporation-induced drug / gene delivery in intratumor immunotherapy.

[0202] Example 6 - Modification of the tumor microenvironment using M-HIFU [HJA(6]

[0203] Despite the initial local manifestations and multimodal treatment, triple-negative and human ErbB-2 (HER2) positive breast cancer (BC) still have a high metastatic spread rate. Cancer immunotherapy in the form of immune checkpoint blockade (ICB) has a moderate activity limited to a small percentage of triple-negative BC. The reasons for the limited efficacy of ICB therapy in BC include relatively low somatic mutation rates, tumors cannot attract immune infiltrates (especially tumor-infiltrating lymphocytes), the expression of other immune checkpoint molecules that inhibit adaptive immune responses in the tumor microenvironment (TME), and inhibitory cell types such as regulatory T cells (Treg), tumor-associated macrophages (TAM) and myeloid-derived suppressor cells to the suppression of innate immunity in tumors. In fact, in preclinical and clinical studies, invasive BC growth and invasion are related to TAM. Therefore, changing the polarization of TAM in TME has become the focus of attempts to improve the efficacy of BC immunotherapy. Unfortunately, pharmacological strategies that specifically regulate TAM without systemic changes to non-TAM have not been successful, and alternatives such as local delivery or ablation are being considered.

[0204] Local ablation of tumor cells in the TME by image-guided delivery of various energies (including high-intensity focused ultrasound (HIFU), radiofrequency, microwaves, and cryoprobes) has been used clinically as a minimally invasive therapy for localized prostate, breast, liver, kidney, bone, and brain tumors. These ablative therapies use different energy sources to produce tumor cell destruction, but also trigger antitumor immune responses against antigens within in situ tumor fragments. Conventional HIFU (thermal high-intensity focused ultrasound (T-HIFU)) induces rapid coagulative necrosis of tissue at the target lesion to which the energy is applied. Although the tissue close to the target lesion is not coagulated, it is subjected to thermal stress sufficient to cause cell apoptosis. As T-HIFU-treated cells release endogenous danger signals, causing immune cells to secrete interferon gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α), it has been observed that the accumulation of CD4+ and CD8+ cells in T-HIFU-treated tumors increases. Nonetheless, the limitations of T-HIFU's efficacy in larger tumors have led to the investigation of alternative forms of HIFU to destroy tumors, such as high-pressure bursts that induce acoustic cavitation, which has been termed mechanical high-intensity focused ultrasound (M-HIFU).

[0205] Compared with T-HIFU, M-HIFU increased the accumulation of dendritic cells in treated tumors, exhibited stronger antitumor efficacy, produced enhanced antitumor immunity, and reduced the risk of metastasis. Notably, M-HIFU also increased the infiltration of T cells in treated tumors. In order to better describe the mechanism of stronger antitumor immunity induced by M-HIFU, single-cell RNA sequencing of tumors was used, and the TME after no treatment or traditional T-HIFU and M-HIFU was analyzed. Changes in the local and distant TME of mouse BC were observed, and the conversion of macrophage subtypes in M-HIFU-treated tumors was also observed, while the conversion was not present after T-HIFU treatment. In addition, upregulation of immune checkpoint molecules such as programmed cell death-1 (PD-1) / programmed cell death ligand 1 (PD-L1), lymphocyte activation gene 3, or TIM-3 was observed, which can deplete activated T cells in the TME, leading to the suppression of antitumor immunity. Therefore, the combination of M-HIFU and anti-PD-L1 therapy was investigated, which showed upregulation of gene expression by CD8+ T cells in type I interferon-mediated signaling pathways, T cell proliferation, and chemokine / cytokine secretion, and was associated with a significant increase in local and distant anti-tumor effects, including complete responses in the majority of treated animals.

[0206] Our laboratory used the human HER2 oncogene to transduce MM3MG (a murine precancerous mammary epithelial cell line) to express HER2 (referred to as MM3MG-HER2 cells) via retroviral vectors and polybrene. JC cells (a murine BC cell line) were transduced with the human HER3 gene (referred to as JC-HER3). 28 4T1-HER2 cells were also obtained.

[0207] The VIFU 2000 system (Alpinion Medical Systems, Bothell, Washington, USA) was used for HIFU treatment. A 1.5MHz HIFU transducer was used to treat cells or tumors in two different schemes (50% duty cycle, 1Hz pulse repetition frequency, 20W, 10 seconds, or 2% duty cycle, 5Hz pulse repetition frequency, 200W, 20 seconds) to produce thermal necrosis or mechanical lysis of tumor cells. The former is defined as T-HIFU, while the latter is defined as M-HIFU. T-HIFU raises the temperature inside the tumor tissue to >60°C within a few seconds, while the temperature inside the tumor tissue during M-HIFU is <42°C. M-HIFU is similar to boiling tissue fragmentation, which produces cavitation activity in vivo, which can damage tumor tissue and cells through shear stress generated by complex bubble oscillations and bubble-bubble-tissue-cell interactions. The -6dB focal size of the HIFU transducer measured at a low power level of 10W is 0.72mm×7.22mm in the lateral and axial directions, respectively. At high power levels for T-HIFU (20 W) and M-HIFU (200 W), the corresponding focal spot sizes based on numerical simulations were estimated to be 0.74 mm × 6.50 mm and 0.60 mm × 5.69 mm, respectively. Regarding the focal spots of treatment, both the X-axis and Y-axis intervals were 2 mm, and a total of 9 points were selected in in vitro and in vivo studies. A shorter interval of 1 mm was used only when the size of the tumor was not large enough to use 2 mm for all intervals. The same interval strategy was used for M-HIFU and T-HIFU. To avoid damage to the skin or bones by HIFU treatment, tumor tissue just below the skin or close to the thigh bone was spared from exposure to focused ultrasound; therefore, based on macroscopic evaluation, approximately 20%-40% of the tumor tissue was ablated by HIFU treatment.

[0208] Female BALB / c mice or SCID-beige mice (Jackson Labs, Bar Harbor, Maine, USA) aged 5-8 weeks were raised and maintained. Human HER2-transgenic mice were also used. F1 hybrid HER2 transgenic mice were established by crossing with BALB / c mice. Human HER3-transgenic mice (MMTV-neu / MMTV hHER3) with FVB background were also used. FVB mice homozygous for the hHER3 gene were established at Duke University and then crossed with BALB / c mice to establish BALB / c mice homozygous for the hHER3 gene.

[0209] When the local tumor volume reaches 2000mm 3 To test the immunogenicity of HIFU-treated tumor cells, BALB / c mice were treated with in vitro T-HIFU-treated or M-HIFU-treated MM3MG-HER2 cells (1×10 6 On day 0, some mice were euthanized and spleens, draining lymph nodes, and blood were collected for in vitro assays: IFN-γ ELISpot, flow cytometry, and cell-based ELISA. Other mice (10 mice / group) were inoculated with 1×10 6 MM3MG-HER2 cells. Tumor size was measured continuously, and tumor volume was calculated using the following formula: long axis × (short axis) 2 ×0.5.

[0210] For the treatment model using MM3MG-HER2 tumors, MM3MG-HER2 cells were subcutaneously inoculated into the left leg of mice on day 0 (1 × 10 6 In the bilateral tumor model, 1×10 5 or 5×10 5 The cells were inoculated into the right flank. The established leg tumors were treated with T-HIFU or M-HIFU on day 7. For the rechallenge experiment, mice cured by M-HIFU treatment received MM3MG-HER2 cells (1×10 6Cells were injected subcutaneously into the flank, and tumor size and mouse survival were monitored. For combination treatment with anti-PD-L1 antibodies, mice received an intraperitoneal injection of 100 μg of anti-PD-L1 antibody (clone 10F.9G2, Bio X Cell, West Lebanon, NH) or isotype control IgG (clone LTF-22, Bio XCell) on days 12, 15, and 18 in the unilateral tumor model or on days 10, 13, and 16 in the bilateral tumor model. For the JC-HER3 tumor model, cells were inoculated into the left leg (1×10 6 cells)±inoculated into the right flank (5×10 5 Cells). Leg tumors were treated with M-HIFU on day 8 (for combined treatment experiments) or day 11 (for single M-HIFU treatment experiments). For combined treatment, mice received intraperitoneal injections (200 μg / injection) of anti-PD-L1 antibodies or isotype control IgG on days 8, 11, and 15 in unilateral tumor models or on days 8, 11, 15, and 18 in bilateral tumor models. For immune cell depletion, mice received 250 μg of antibodies against CD4 (clone GK1.5, Bio X Cell), antibodies against CD8a (clone 53-6.72, Bio XCell) or 10 μL of antibodies against natural killer (NK) cells (anti-Asialo GM1 antibodies; Wako Pure Chemical Corporation, Osaka, Japan) 1 day before the first HIFU treatment and 2 days after the first HIFU treatment, and then the same amount was injected every 5 days throughout the experiment.

[0211] Mouse IFNγ-ELISpot assay (e.g., Mabtech, Cincinnati, Ohio, USA) was performed according to the manufacturer's instructions. Cells were stimulated with HER2 intracellular domain (ICD) peptide, HER2 extracellular domain (ECD) peptide (25 μg / mL; JPT Peptide Technologies, Berlin, Germany) or unrelated HIV-gag peptide mixture (2.6 μg / mL, JPT Peptide Technologies). KS ELISpot V.4.2 software was used to count the number of IFN-γ spots in a high-resolution automatic ELISpot reader system (e.g., Carl Zeiss, White Plains, New York, USA).

[0212] The plate was filled with 3 × 10 4 4T1 parental cells or 6×10 44T1-HER2 cells were coated overnight. Serial dilutions of serum were added (final titration 1:50-1:6400) and incubated on ice for 1 hour. The plates were washed and fixed with 1% formalin and then incubated with IRDye 800CW donkey anti-mouse (1:2000; LI-COR Biosciences, Lincoln, Nebraska, USA) for 60 minutes. Fluorescence intensity was measured using the 800nm ​​channel using an Odyssey CLx LI-COR reader (LI-COR).

[0213] The tumors were weighed and homogenized on ice for up to 20 seconds in a cell lysis buffer (Cat. No. 9803, Cell Signaling, 9 volumes of tumor weight) supplemented with PMSF (1 mM) using a Qiagen TissueRupter (e.g., Qiagen, Germantown, Maryland, USA). The tumor homogenate was then sonicated for 10 seconds on ice using a Branson Ultrasonic SLPe Digital Sonifier Cell Disruptor (e.g., Branson Ultrasonics, Danbury, Connecticut, USA). The sonicated samples were centrifuged at 13,000 rpm for 20 minutes at 4°C, and the collected supernatant was used as a tumor lysate for ELISA. Tumor lysates were assessed for levels of IFN-γ, TNF-α, and TGF-β1 using commercially available ELISA kits for IFN-γ (Catalog No. ab46081; Abcam, Cambridge, Massachusetts, USA), TNF-α (BMS607-3; Invitrogen, Waltham, Massachusetts, USA), and transforming growth factor β1 (TGF-β1) (BMS608-4, Invitrogen) and assayed according to the manufacturer's instructions.

[0214] Tumors were manually destroyed with a razor and then enzymatically digested to obtain a single cell suspension of tumor tissue. Single cell suspensions of spleen and lymph nodes were obtained by manual crushing and filtering through a 70 μm cell strainer (BD Biosciences, San Jose, California, USA). Cells were stained using a live / dead fixable Aqua Dead Cell Stain Kit (LIVE / DEADFixable Aqua Dead Cell Stain Kit) (e.g., Thermo Fisher Scientific, Rockford, Illinois, USA) and then stained with surface marker antibodies (Supplementary Table 1 online) at room temperature for 30 minutes. Isotype IgG or fluorescence minus one (FMO) controls were used as negative staining controls. Anti-CD16-32 antibodies (Thermo Fisher) were used to block FcγIII / II receptors. Intracellular staining was performed using fixation / permeabilization and permeabilization buffer (Fixation / Permeabilization and Permeabilization Buffer) (ThermoFisher) according to the manufacturer's instructions. Stained cells were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software IX (BD Biosciences).

[0215] Single cell suspensions dissociated from untreated or HIFU treated tumors (8 days after HIFU treatment) were obtained as described above. For combination therapy experiments, mice received intraperitoneal injections of 100 μg anti-PD-L1 antibodies or isotype control IgG antibodies 3 days and 6 days after HIFU treatment. CD45+ leukocytes were sorted from tumor digests by flow cytometry, and cDNA libraries were prepared using Bio-Rad single cell separators (ddSEQ) and SureCell WTA 3'Library Prep kits from Illumina. Raw sequencing data was generated in the form of FastQ files and uploaded to BaseSpace sequencing centers (Illumina, San Diego, California, USA) to be expressed quantitatively using automated pipelines. PartekFlow software (Partek, St. Louis, Missouri, USA) was used to analyze the resulting gene expression matrix files for each treatment condition. Unsupervised clustering was performed to separate cell types, and markers of cell types were identified using differential gene expression. These markers were then used to identify cell subsets within CD45+ sorted immune cells. Preprocessed gene counts were used to generate a Uniform Manifold Approximation and Projection (UMAP) for visualization of cell types under different treatment conditions. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and differential gene expression analysis were performed using Partek Flow software.

[0216] Formalin-fixed paraffin-embedded tumor tissue sections were stained by horseradish peroxidase (HRP) method using rabbit anti-mouse CD3ε antibody (1:150, D4V8L; Cell Signaling Technology, Danvers, Massachusetts, USA), CD4 antibody (1:100, D7D2Z; Cell Signaling) or CD8a antibody (1:400, D4W2Z; Cell Signaling). Antigen retrieval was performed with citrate unmasking solution (Cell Signaling). DAB Substrate Kit (VectorLabs, Burlingame, California, USA) was used to visualize the signal before incubation with HRP (Histofine Simple Stain Mouse MAX PO (R), NichireiBiosciences, Tokyo, Japan). Isotype-matched rabbit IgG was used as a negative staining control. The stained slides were scanned on a DP80 microscope (OLYMPUS, Tokyo, Japan), and digital images were observed using cell-Sens (OLYMPUS).

[0217] Data are expressed as mean ± SEM in tumor growth graphs or as mean ± SD in in vitro assays and flow cytometry data. Tumor volume, flow cytometry, ELISA and ELISPOT data from experiments in three or more treatment groups were analyzed by one-way ANOVA and Tukey's multiple comparison test. For experiments with only two groups, a two-tailed unpaired Student's t test was used. Unless otherwise stated, tumor volume was analyzed only at the endpoint. Statistical analysis was performed using Prism (e.g., GraphPad, San Diego, California, USA). Kaplan-Meier survival curves for tumor-bearing mice were generated and log-rank tests were performed using JMP Pro V.11.0 software (e.g., SAS Institute, Cary, North Carolina, USA). P values ​​of 0.05 or less were considered statistically significant. Not all significant differences are shown in each figure (*p<0.05, **p<0.01, ***p<0.001).

[0218] Implanted MM3MG-HER2 BC in BALB / c mice were treated with M-HIFU or T-HIFU, and the antitumor effects on local (treated) and distant tumor masses were evaluated. M-HIFU had better control of treated tumors as well as untreated distant tumors compared with T-HIFU ( Fig.25 Figures 2500A-2500C in Figure 2500A-2500C). In two other murine BC models, E0771-OVA and JC-HER3, M-HIFU demonstrated tumor growth inhibition of treated and untreated disease sites. When compared to untreated controls, M-HIFU induced significantly stronger cellular immune responses to HER2 ECD, HER2 ICD, and mixed peptide antigens, while T-HIFU was able to induce only slightly stronger responses to HER2 ECD antigen ( Fig.25 2500D). Compared with T-HIFU, M-HIFU induced a significant increase in the cellular immune response. However, M-HIFU and T-HIFU induced similar levels of humoral immunity against HER2-expressing tumor cells ( Fig.25 2500E in Figure 2500). Also in the JC-HER3 tumor model, the induction of HER3 antigen-specific cellular immune responses in HER3 transgenic mice was confirmed after M-HIFU treatment, indicating the powerful ability of M-HIFU to induce anti-tumor immunity by breaking tolerance. Importantly, after M-HIFU treatment, long-lived immune memory responses were induced in mice that had previously rejected their tumors ( Fig.25 This memory response could not be measured in T-HIFU treated mice, as none of the treated tumors completely regressed ( Fig.25 Figure 2500B in Figure 2500B). One potential explanation for this difference in anti-tumor response is a baseline difference in tumor cell immunogenicity after treatment with M-HIFU compared to T-HIFU. To test this hypothesis, mice were inoculated with ex vivo HIFU-treated MM3MG-HER2 cells and assessed for immunogenicity. Despite the difference in the type of cell death induced by M-HIFU versus T-HIFU (apoptosis versus necrosis), there were no significant differences in systemic T cell activation, tumor antigen-specific antibody production, or tumor control elicited by tumor cells killed by inoculation with M-HIFU or T-HIFU.

[0219] about Fig.25 , Graphs 2500A-2500F depict data regarding superior growth inhibition of local and distant tumors and enhanced tumor antigen-specific cellular immune responses by M-HIFU compared to T-HIFU. For Graph 2500A, 1×10 6MM3MG-HER2 cells were injected into the legs of BALB / c mice. Established leg tumors were treated with M-HIFU or T-HIFU on day 7 after tumor inoculation. A comparison of their tumor growth curves is shown. Graph 2500B depicts the survival curve. When the tumor volume reached 2,000 mm 3 Mice were euthanized at 50 days or on day 50. n = 9 mice (no treatment), 10 (M-HIFU) or 13 (T-HIFU) (A, B), 32 mice in total. Log-rank test was performed. For the data in graph 2500C, MM3MG-HER2 cells were injected into the left thigh of HER2 transgenic mice (1×10 6 cells) and right flank (1×10 5 cells). Leg tumors were treated with M-HIFU or T-HIFU on day 7. A comparison of flank tumor growth curves is shown. n=13 mice (no treatment) or 14 (M-HIFU and T-HIFU), a total of 41 mice. For the data in Figure 2500D, lymphocytes were isolated from the spleen on day 18 after tumor inoculation (day 11 after HIFU treatment), and IFN-γ secretion was detected by ELISpot assay. The average number of spots of HER2 peptides for ECD, ICD and mixture (ECD+ICD) is shown. n=4 per group. For the data in Figure 2500E, serum was collected from mice on day 11 after HIFU treatment. The level of anti-HER2 antibodies in mouse serum was assessed using a cell-based ELISA. n=3 mice (no treatment) or 8 (M-HIFU and T-HIFU), a total of 19 mice. For the data of Figure 4500F, MM3MG-HER2 cells (1×10 6 cells / mouse) and then challenged mice cured of MM3MG-HER2 tumors by M-HIFU treatment. Age-matched naive female BALB / c mice were used as a control group. The survival rate of mice is shown, and the log-rank test was performed. n = 5 mice / group, 10 mice in total. (A–C) Error bars represent SE. (D,E) Error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ECD, extracellular domain; HER2, human ErbB-2; ICD, intracellular domain, IFN-γ, interferon gamma; M-HIFU, mechanical high-intensity focused ultrasound; T-HIFU, thermal high-intensity focused ultrasound.

[0220] Since tumor cells treated with M-HIFU or T-HIFU in vitro had similar immunogenicity, we hypothesized that the differential alterations of the TME by the two HIFU treatments accounted for the differences in the induction (and therefore efficacy) of systemic antitumor immunity in the BC model. By immunohistochemistry, we observed greater CD4 and CD8 T cell infiltration in M-HIFU-treated tumors compared with no-treatment controls, and slightly increased T cell infiltration in T-HIFU-treated tumors ( Fig.26 This was confirmed by flow cytometry of digested tumors, which showed an increase in both CD4 and CD8 T cells and NK cells after M-HIFU treatment ( Fig.26 2600B in Figure 2600B). To confirm the changes in immune cell profiles, the absolute number of tumor-infiltrating immune cells was determined for each treatment group. Increased numbers of CD4 T cells, CD8 T cells, and NK cells were confirmed in M-HIFU-treated tumors, while no differences in granulocyte and macrophage populations were observed. By Ki67 staining, enhanced T cell proliferation was observed in M-HIFU-treated tumors compared to control tumors ( Fig.26 In addition to increased infiltration and active proliferation, CD8 T cells present in M-HIFU-treated tumors had increased expression of T cell activation markers and granzymes ( Fig.26 In M-HIFU treated tumors, dendritic cell (DC) maturation was increased, as evidenced by upregulation of major histocompatibility complex (MHC) class II and CD80 ( Fig.26 Importantly, M-HIFU affects the polarization state of tumor-infiltrating macrophages, such as Fig.26 As shown in Figure 2600F in Figure 2600F. Compared with control tumors, the positive expression of CD206 by macrophages in M-HIFU-treated tumors was significantly reduced, and the expression level of MHC class II was significantly increased, indicating that M-HIFU can induce macrophage repolarization and produce more anti-tumor-TME. In support of this finding, significantly lower levels of TGF-β1 and higher levels of IFN-γ were observed in M-HIFU-treated tumors compared with control tumors ( Fig.26 Figure 2600G).

[0221] about Fig.26 M-HIFU enhanced the infiltration of activated CD4+ and CD8+ cells and M1-polarized macrophages into tumors. 6MM3MG-HER2 cells were injected into the legs of BALB / c mice. Established leg tumors were treated with M-HIFU or T-HIFU on day 7 after tumor inoculation. Regarding Figure 2600A, immunohistochemical staining of T cells in tumor sections. Tumors on day 13 after HIFU treatment were collected, fixed with formalin and stained with anti-mouse CD4 and CD8 monoclonal antibodies. Representative images are shown (left: no treatment, middle: T-HIFU, right: M-HIFU). The scale bar is 50 μm. Quantification of positive cells in high-power fields (HPF) is shown in the right figure. Error bars represent SD, n=3 per group. Regarding Figure 2600B, 7 days after HIFU treatment of MM3MG-HER2 tumors in mice, tumors were collected and digested for flow cytometry analysis. The percentage of CD4+, CD8+, CD3+, CD49b+, Ly6G+, CD11c+ and F4 / 80+ cells in live CD45+ cells of each HIFU treatment group was analyzed. n=4 per group. For Figure 2600C, the expression of the proliferation marker Ki67 of each cell type in tumor-infiltrating immune cells was analyzed by flow cytometry, and the Ki67 positive percentage of each cell type was shown. n=4 per group. For Figure 2600D, the expression of CD69+, inducible T cell co-stimulator (ICOS)+, and granzyme B+ by CD8+ cells in each HIFU treatment group was analyzed by flow cytometry and displayed. n=4 per group. For Figure 2600E, the MFI of MHC class II (left) and CD80 (right) expression on CD11c+ dendritic cells in each HIFU treatment group is shown. For Figure 2600F, the expression of CD206 and MHC class II by the CD11b+F4 / 80+ macrophage population in each treatment group was analyzed. Representative dot plots of CD206 and MHC class II staining are shown in the left figure. The percentage of CD206-positive macrophages and the mean fluorescence intensity of MHC class II expression are shown. n=4 per group. For the 2600G panel, ELISAs for IFN-γ, TNF-α, and TGF-β1 were performed with tumor lysates prepared from untreated, T-HIFU, or M-HIFU-treated MM3MG-HER2 tumors. n = 5 per group. Error bars represent SD. *P < 0.05, **P < 0.01, ****P < 0.0001. IFN-γ, interferon gamma; MFI, mean fluorescence intensity; M-HIFU, mechanical high-intensity focused ultrasound; TGF-β1, transforming growth factor β1; T-HIFU, thermal high-intensity focused ultrasound; TNF-α, tumor necrosis factor alpha.

[0222] To investigate the effects of HIFU on the TME in a more detailed manner, single-cell RNA sequencing (scRNA-seq) analysis was performed on tumor-infiltrating leukocytes from untreated tumors to compare gene expression with those from tumors treated with M-HIFU and T-HIFU. GO enrichment analysis and KEGG pathway analysis were performed on the differentially expressed genes (DEGs) within the identified macrophage populations from both HIFU strategies and compared with macrophages from untreated tumors. Ten significantly upregulated GO terms in the biological process category were shown in M-HIFU-treated macrophages. Fig.27A Figure 2700A in Figure 2700A. The upregulated genes are mainly involved in the regulation of immune response, inflammatory response, leukocyte activation, immune system processes, cell chemotaxis, endocytosis, and apoptosis signaling pathways. A similar analysis of the top 10 differentially expressed GO terms in T-HIFU treated tumors did not contain any immune related terms, but instead centered around biosynthetic processes.

[0223] The top 10 GO terms for cellular components and molecular function categories of M-HIFU-treated and T-HIFU-treated tumors compared with untreated tumors are shown in Fig.27A Figures 2700B and 2700C in Figure 2700B and 2700C. There was no overlap in the top terms upregulated after these different forms of HIFU, again demonstrating fundamental differences in the molecular consequences of each therapy. KEGG pathway analysis of M-HIFU treated tumors showed that the top 10 upregulated pathways were enriched in antigen processing / presentation, cytokine-cytokine receptor interactions, chemokine signaling pathways, and phagosome / lysosome pathways, consistent with the inflammatory anti-tumor phenotype of macrophages after M-HIFU ( Fig.27B Figure 2700D in Figure 2700D). Tumors treated with T-HIFU had only 7 KEGG pathways significantly upregulated compared to untreated tumors. These pathways are all involved in metabolic processes. The difference in significantly upregulated pathways between M-HIFU treated and T-HIFU treated tumors highlights the immunogenicity of our M-HIFU treatment at this time point. Differential gene expression analysis of tumor infiltrating macrophages identified 298 genes that were expressed at levels statistically higher than untreated tumors (FDR corrected p value < 0.05) ( Fig.27B2700E in Figure 2700E). Many genes (Irg1, Ccl2, Maff, Ier3, Lcp2, Ptpn2, Cd14, Cxcl16, Ier3, Nfkbia, Ccrl2, and Tlr2) are characteristic genes of M1 / classically activated macrophages,34 while other genes (Tnfsf10Ctsc, Gzme, Cd8a, and Pdcd1) are typically associated with M1 macrophages. These findings confirm a highly activated M1-biased macrophage population within the TME of M-HIFU-treated tumors. Therefore, scRNA-seq analysis suggests that M-HIFU induces an inflammatory, M1-biased macrophage population, which may contribute to enhanced antitumor immunity in M-HIFU-treated tumors.

[0224] about Fig.27A and 27B , GO enrichment analysis and KEGG pathway analysis of DEGs in macrophages after M-HIFU treatment. Regarding Figures 2700A-2700C, GO enrichment analysis of upregulated DEGs in macrophages from M-HIFU treated tumors compared to macrophages from untreated tumors. Enrichment scores of GO terms for the categories of (A) biological process (Figure 2700A), (B) cellular component (Figure 2700B), and (C) molecular function (Figure 2700C) are shown. Regarding Figure D, KEGG pathway analysis of upregulated DEGs in macrophages from M-HIFU treated tumors compared to macrophages from untreated tumors. The top 10 KEGG pathways in terms of enrichment scores are shown. Regarding Figure 2700E, differential gene expression analysis in tumor infiltrating macrophages from M-HIFU treated tumors compared to tumor infiltrating macrophages from untreated tumors. Representative genes significantly upregulated in macrophages from the M-HIFU treatment group (log2(fold change)>1, FDR<0.05) are shown in red. DEG, differentially expressed gene; FDR, false discovery rate; GO, gene ontology; M-HIFU, mechanical high-intensity focused ultrasound.

[0225] Although a productive antitumor immune response was generated in nearly half of the M-HIFU-treated mice, many eventually developed progressive disease ( Fig.25 2500A and 2500B in Figures 2500A and 2500B). Therefore, the etiology of this tumor escape was investigated, beginning with the evaluation of the expression of immune checkpoint molecules in M-HIFU treated tumors versus T-HIFU treated tumors. At 7 days post-treatment, PD-L1 expression on neutrophils (Ly6G+), DCs (CD11c+), and macrophages (F4 / 80+) was increased only in M-HIFU treated tumors ( Fig.282800A and 2800B in Figures 2800A and 2800B). However, analysis only 3 days after HIFU treatment showed that neither HIFU treatment mode increased PD-L1 expression in these tumor-associated cell populations. This suggests that HIFU itself does not cause an immediate upregulation of PD-L1, but is instead a result of a sustained immune response caused by M-HIFU treatment. Increased IFN-γ ( Fig.26 2600G in Figure 2600G) may play an important role in this upregulation of PD-L1. Other immune checkpoint molecules including PD-1 and TIM3 did not change significantly after M-HIFU, but the expression of lymphocyte activation gene 3 (LAG-3) on CD8+ cells was significantly higher after M-HIFU than that after T-HIFU ( Fig.28 2800C and 2800D in Figure ). Taken together, these data demonstrate enhanced anti-tumor immunity within the TME caused by M-HIFU, which was not observed by T-HIFU but was likely suppressed by the concomitant increase in immune checkpoint molecules.

[0226] about Fig.28 , Figures 2800A-2800D show the expression of immune checkpoint molecules enhanced by tumor-infiltrating immune cells after M-HIFU treatment. With respect to Figure 2800A, representative flow cytometry histograms showing PD-L1 expression on Ly6G+ cells, CD11c+ cells, and F4 / 80+ cells are shown. Blue: M-HIFU, red: T-HIFU, black: no treatment, gray fill: isotype control. With respect to Figure 2800B, the percentage of PD-L1+ cells in Ly6G+ cells, CD11c+ cells, and F4 / 80+ cells in live CD45+ tumor-infiltrating immune cells of each HIFU treatment group is shown. n=4 per group. With respect to Figures 2800C and 2800D, the expression of immune checkpoint molecules PD-1, TIM-3, and LAG-3 on tumor-infiltrating CD4+ (Figure 2800C) and CD8+ (Figure 2800D) cells is shown. n=4 per group. Error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001. LAG-3, lymphocyte activation gene 3; M-HIFU, mechanical high-intensity focused ultrasound; PD-1, programmed death-1; PD-L1, programmed cell death ligand 1; T-HIFU, thermal high-intensity focused ultrasound.

[0227] Based on the upregulation of immune checkpoint molecules after M-HIFU, it was hypothesized that PD-1 / PD-L1 blockade could enhance the anti-tumor efficacy of M-HIFU treatment. To test this, M-HIFU treatment was combined with anti-PD-L1 antibody administration. The combination treatment cured 72.2% of mice implanted with MM3MG-HER2 tumors, compared to 26.3% and 52.6% when anti-PD-L1 or M-HIFU was given alone ( Fig.29A 2900A and 2900B in Figures 2900A and 2900B). Using another BC model, JC-HER3, this combination also resulted in tumor eradication and long-term survival in 37.5% of mice. Analysis of IFN-γ ELISpot assays of splenocytes showed that the number of HER2-specific IFN-γ secreting cells was significantly increased in mice treated with M-HIFU + anti-PD-L1 compared to mice treated with M-HIFU or anti-PD-L1 alone ( Fig.29A 2900C in Figure 2900C). Flow cytometric analysis of digested tumors showed an increase in the proportion of CD4+ T cells, CD8+ T cells, and NK cells in tumors treated with the combination therapy ( Fig.29A 2900D in Figure 2900D), and the activation of both CD4+ and CD8+ T cells was enhanced in the combination therapy. Based on the enhanced expression of granzyme B, M-HIFU monotherapy and combination therapy showed that tumor-infiltrating CD8+ T cells had stronger cytolytic activity. In addition, CD4+Foxp3+Treg cells were increased in tumors treated with combination therapy compared to control tumors or tumors treated with M-HIFU monotherapy, although the difference was not statistically significant.

[0228] To demonstrate which infiltrating cells were responsible for the observed antitumor efficacy, MM3MG-HER2 tumors were treated with the combination therapy in the presence of depleting antibodies against CD4+, CD8+, or NK cells. Depletion of CD8+ or NK cells abolished the antitumor efficacy of the combination therapy, but depletion of CD4+ cells did not result in a significant change in the antitumor effect ( Fig.29B Figure 2900E and Figure 2900F).

[0229] With respect to Figure 29, M-HIFU and PD-L1 blockade synergistically reject local tumors. With respect to Figure 2900A, established MM3MG-HER2 tumors in the legs of BALB / c mice were treated with M-HIFU on day 7. Anti-PD-L1 antibody (100 μg / 100 μL) or isotype control IgG (100 μg / 100 μL) was injected intraperitoneally 5, 8, and 11 days after M-HIFU treatment. Survival curves are shown and log-rank tests were performed. n=13 mice (isotype control), 19 (monotherapy group) or 18 (combination group), a total of 50 mice. With respect to Figure 2900B, individual tumor growth curves for each treatment group are shown. The numbers in each figure show mice in mice / groups where tumors were eradicated. With respect to Figure 2900C, spleens were collected for immunoassays 9 days after starting M-HIFU treatment with / without anti-PD-L1 antibodies. The induction of HER2 antigen-specific cellular responses was analyzed by IFN-γ ELISpot assay using harvested splenocytes and HER2 peptide mixture as stimulating antigens. n = 4 per group. For Figure 2900D, the percentage of CD4+, CD8+, and CD49b+ cells in tumor-infiltrating CD45+ cells was analyzed by flow cytometry analysis. n = 4 (treatment group) or 3 (control group). For Figures 2900E and 2900F, as Fig.28 Mice treated with a combination of M-HIFU and anti-PD-L1 antibodies in Figure 2800A were administered or not with depleting antibodies against CD4+, CD8a+, and NK cells on days 6, 9, and 14, and every 5 days until the end of the experiment. n = 5 mice (isotype control) or 6 (other groups), a total of 29 mice. Regarding Figure 2900E, survival curves are shown and log-rank tests are performed. Regarding Figure 2900F, individual tumor growth curves for each cell depletion group are shown. Error bars represent SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. HER2, human ErbB-2; IFN-γ, interferon gamma; M-HIFU, mechanical high-intensity focused ultrasound; PD-L1, programmed cell death ligand 1.

[0230] To explain the enhanced anti-tumor efficacy of adding anti-PD-L1 treatment, leukocyte populations were further characterized by scRNAseq analysis of tumor-infiltrating CD45+ immune cells derived from mice that were untreated, treated with M-HIFU alone, anti-PD-L1, or the combination therapy. DEGs that were significantly upregulated in the treatment groups compared to the no-treatment control were identified in CD8 T cells or macrophages, and the number of DEGs is shown in Fig.30In Figures 3000A-3000F in Figure 3000A-3000F. In CD8 T cells, 1690 DEGs were identified in the three treatment groups, and 333 genes were unique to the combination therapy, while 231 genes and 365 genes were unique to M-HIFU and anti-PD-L1 monotherapy, respectively, indicating that the combination therapy not only exerted the additive effect of single therapy ( Fig.30 The most significantly enriched GO terms of the upregulated DEGs in the combination treatment are shown as enrichment scores in the heatmap with the other two treatments ( Fig.30 3000B in Figure 3000B). In CD8 T cells, DEGs were mainly enriched in type I interferon-mediated signaling pathways, activated T cell proliferation, chemokine secretion, cellular response to interferon γ, interleukin (IL)-12 production, and cytokine secretion. KEGG pathway analysis showed that the upregulated DEGs were significantly enriched in pathways such as complement and coagulation cascades, cytokine-cytokine receptor interactions, and NF-κB signaling pathways (Online Supplementary Figure 3000B). Fig.10 A). Thus, the combination therapy appears to activate CD8 T cells and enhance anti-tumor immunity by modulating multiple pathways.

[0231] To elucidate the genes involved in the enhanced antitumor efficacy of the combination therapy compared with M-HIFU monotherapy, the gene expression levels of CD8 T cells in the combination therapy and M-HIFU monotherapy were compared ( Fig.30 3000C in Figure 3000C). Genes associated with CD8 T cell activation, such as Cd33, Cx3cr1, Cxcl3, Cxcl11, and Cxcl16, were more than twice as strongly expressed after combination treatment compared with M-HIFU monotherapy, confirming the enhanced activation of CD8 T cells by combining anti-PD-L1 with M-HIFU treatment, which corresponds to the excellent antitumor efficacy against distant tumors observed in the bilateral tumor model. In macrophages, among the 822 DEGs significantly upregulated in treated samples, 335 genes were unique to the combination treatment ( Fig.30 3000D in Figure 3000D), mainly enriched in lymphocyte migration and defense response to tumor cells, as well as in multiple pathways related to extracellular matrix / fibroblasts ( Fig.30 3000E in Figure 3000E). KEGG pathway analysis revealed gene enrichment in protein digestion and absorption, extracellular matrix-receptor interactions, cell adhesion molecules, PI3K-Akt signaling pathway, and T cell receptor signaling pathway in the combination therapy. These results may indicate that macrophages treated with the combination therapy are more actively involved in the enhanced antitumor activity and tissue remodeling after ultrasound destruction of the tumor. Interestingly, as Fig.30As shown in Figure 3000F, when compared with tumor-infiltrating macrophages in M-HIFU-treated tumors, tumor-infiltrating macrophages in the combination treatment group showed relative upregulation of gene signatures of M1 / classically activated macrophages (Dcn, Acpp, Csf1, Adgrl2, and Col4a2) and M2 / alternatively activated macrophages (Il1rl1, Mmp9, Mfsd6, Angptl2, Spint2, and Sft2d2).

[0232] With respect to Figure 29, the combination of M-HIFU and anti-PD-L1 antibody induces upregulation of unique DEGs in tumor-infiltrating CD8 T cells and macrophages. Established MM3MG-HER2 tumors in the legs of BALB / c mice were treated with M-HIFU, followed by intraperitoneal injection of 100 μg of anti-PD-L1 antibody or isotype control IgG antibody 3 and 6 days after HIFU treatment. Live CD45+ leukocytes were isolated from enzymatically digested tumors by flow-based sorting and used for scRNA-seq. Data analysis was performed using Partek Flow software. With respect to Figure 3000A, the number of DEGs of CD8 T cells upregulated in the treatment group compared to the untreated control is shown in a Venn diagram. With respect to Figure 3000B, GO enrichment analysis was performed on the upregulated DEGs in CD8 T cells in each treatment group and summarized in a heat map. The top 17 GO terms in the combination treatment are shown together with the enrichment data for the M-HIFU and anti-PD-L1 monotherapy groups. Regarding 3000C, differential gene expression analysis was performed on the DEGs upregulated in CD8 T cells between the combination therapy and M-HIFU monotherapy. Representative DEGs significantly upregulated (log2 (fold change)>1, FDR<0.05) in the combination group are shown in red letters. Regarding 3000D, the number of DEGs of macrophages upregulated in the treatment group compared to the untreated control is shown in the Venn diagram. Regarding 3000E, GO enrichment analysis was performed on the DEGs upregulated in macrophages in each treatment group and summarized in a heat map. The top 11 GO terms in the combination therapy are shown together with the enrichment data in the M-HIFU and anti-PD-L1 monotherapy groups. Regarding 3000F, differential gene expression analysis was performed on the DEGs upregulated in macrophages between the combination therapy and M-HIFU monotherapy. Representative DEGs that were significantly upregulated in the combined group (log2(fold change)>1, FDR<0.05) and contributed to the activated KEGG pathways or were referred to as M1 or M2 signature genes are shown in red letters. DEG, differentially expressed gene; GO, gene ontology; M-HIFU, mechanical high-intensity focused ultrasound; PD-L1, programmed cell death ligand 1.

[0233] Based on enhancing systemic tumor antigen-specific immune responses by adding anti-PD-L1 Fig.29A2900C in Figure 2), it was hypothesized that PD-1 / PD-L1 blockade would also improve the abscopal effects of M-HIFU. When MM3MG-HER2 tumors were treated with M-HIFU and intraperitoneal anti-PD-L1 antibody ( Fig.31 In the more stringent bilateral tumor model, greater growth inhibition of distant untreated tumors was observed when treated with either treatment alone, in which a larger number of tumor cells ( Fig.29A Five times the 2900C in the figure) were implanted into the flank of mice ( Fig.31 3100B in Figure 3100B). To elucidate the changes in the TME in distant tumors not treated with HIFU induced by the combination therapy, distant tumors were analyzed by flow cytometry. Fig.31 As shown in Figure 3100C in Figure 3100C, M-HIFU monotherapy failed to increase CD4 T / CD8 T / NK cell infiltration in rapidly growing distant tumors, which may be the main reason for the weak antitumor effect on distant tumors. However, compared with untreated controls and M-HIFU monotherapy, combination therapy as well as anti-PD-L1 monotherapy increased the proportion of CD4+, CD8+, and NK cell populations in distant tumors ( Fig.31 3100C in Figure 3100C). Again, in these groups, CD4 T cells again expressed higher levels of activation markers, and in the combination treatment group, granzyme B expression in CD8 T cells was significantly higher in distant tumors ( Fig.31 3100D in Figure 3100D), indicating that the CD8 T cells in the combination group had the most enhanced cytotoxic function, which led to the strongest abscopal effect ( Fig.31 Figure 3100B in the figure).

[0234] Finally, to confirm which immune cells are responsible for efficacy against distant tumors in the bilateral tumor model, mice were treated with M-HIFU + anti-PD-L1 therapy in the presence of CD4+, CD8+, or NK cell depleting antibodies ( Fig.31 In both leg and flank tumors, depletion of CD8+ or NK cells significantly abolished the anti-tumor efficacy ( Fig.313100F in Figure 3100F). The greater efficacy of the combination therapy against distant tumors in a CD8+ cell-dependent manner was also confirmed in the JC-HER3 tumor model. In order to clarify how the depletion of immune cells affects the TME of distant tumors in mice treated with combination therapy, the profile of tumor-infiltrating immune cells in distant tumors was analyzed by flow cytometry. Interestingly, systemic depletion of CD8+ or NK cells resulted in an intratumoral immune cell profile similar to that of untreated control tumors after M-HIFU+ anti-PD-L1, and reduced ICOS expression and granzyme B production of CD8 T cells, indicating that these two cell types are not only key effectors, but may also affect the immune composition of TME. On the other hand, the depletion of CD4+ cells induced a significant increase and activation of CD8 T cells, as well as a decrease in macrophages and Tregs in distant tumors, which may explain the stronger distant effect observed in the CD4+ depletion group. These results show that both CD8+ cells and NK cells play an important role in the immune-based attack of distant tumors and HIFU-treated tumors in the M-HIFU+ anti-PD-L1 combination therapy.

[0235] about Fig.31 , the combination of M-HIFU and PD-1 / PD-L1 blockade synergistically inhibited distant tumor growth, which was dependent on CD8+ and NK cells. Regarding Figure 3100A, MM3MG-HER2 cells were injected into the left leg of BALB / c mice (1×10 6 cells) and right ventral side (5×10 5 cells). Fig.25 Compared to Figure 2500C in Figure 3100, a larger number of cells were implanted in the flank to produce a more stringent model. Established leg tumors were treated with M-HIFU on day 7. Anti-PD-L1 antibody (100 μg / 100 μL) or isotype control IgG (100 μg / 100 μL) was injected intraperitoneally on days 10, 13, and 16. Regarding Figure 3100B, tumor growth curves of distant flank tumors are shown. n=10 mice per group, for a total of 40 mice. Regarding Figures 3100C and 3100D, on day 18, distant flank tumors (HIFU untreated side) were collected and infiltrating immune cells were analyzed by flow cytometry. n=3 (combination group) or 4 (other groups). Regarding Figure 3100C, the percentages of CD4+, CD8+, and CD49b+ cells in CD45+ cells are shown. Regarding Figure 3100D, the expression of ICOS and Foxp3 on CD4+ cells and the expression of ICOS and granzyme B on CD8+ cells. Fig.28The same treatment regimen as in Figure 2800A was used to treat mice with a combination of M-HIFU and anti-PD-L1 antibodies, with or without CD4+, CD8a+, and NK cell depletion antibodies administered on days 6, 9, and 14, and every 5 days until the end of the experiment. As for Figure 3100F, tumor growth curves for leg tumors (left) and flank tumors (right) are shown. n = 5 mice per group, 25 mice in total. The error bars in Figures 3100B and 3100F represent SE. The error bars in Figures 3100C and 3100D represent SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. HER2, human ErbB-2; M-HIFU, mechanical high-intensity focused ultrasound; NK, natural killer; PD-1, programmed death-1; PD-L1, programmed cell death ligand 1.

[0236] Many approaches seeking to alter the TME to induce systemic anti-tumor immunity are under development, including intratumoral delivery of cytokines and oncolytic viruses, and physical tumor destruction using focused ultrasound, etc. The traditional focused ultrasound method produces coagulative necrosis (T-HIFU) by heating the tissue; however, due to the denaturation of tumor proteins, it may not optimally induce systemic anti-tumor immunity. Traditional T-HIFU changes the TME but does not induce a memory systemic immune response. Therefore, M-HIFU, which causes cell death and extracellular matrix destruction through acoustic microcavitation rather than heating the tissue, has been studied, and it has previously been observed to improve antigen presentation, leading to a more favorable immune response.

[0237] In this study, it was demonstrated that M-HIFU induced local and systemic immunity against BC, which was more effective than T-HIFU in the model. This was demonstrated by stronger antigen presentation, induction of antigen-specific T cells in spleen cells, and increased T cell infiltration at the M-HIFU ablation site and contralateral tumors. A potential criticism is that there are multiple T-HIFU regimens, which may induce very different anti-tumor effects or anti-tumor immune responses compared to the T-HIFU technology disclosed herein. Some existing systems report data on the use of a form of T-HIFU (thermal ablation focused ultrasound) to treat 4T1 tumors with poor immunogenicity. Although thermal ablation alone has limited effects on the intratumoral accumulation of activated T cells (attributed to immunosuppressive TME), the combination with gemcitabine leads to enhanced tumor control and improved survival of mice in a CD8 and CD4 T cell-dependent manner. It is reported that T-HIFU for BC in patients enhances the infiltration of immune cells.

[0238] Recent studies have demonstrated enhanced immunogenicity in BC treated with T-HIFU combined with systemic ICB or topically applied toll-like receptor 9 agonist CpG. The timing of immunoassays may also affect the results of comparative analyses between different treatments. For example, in our study, T-HIFU-treated tumors may have been in the adaptive resistance / wound healing phase at the time of tissue collection. Even taking these into account, it is believed that the immunoassay data (which corresponded well with in vivo antitumor efficacy) still indicate the overall ability of these two HIFU modes in inducing antitumor immunity. More preclinical and clinical studies may be needed to evaluate and optimize the antitumor efficacy of HIFU treatment for BC.

[0239] Although M-HIFU can significantly inhibit the growth of treated tumors and prolong the survival of mice bearing a single tumor, the antitumor activity produced against untreated distant tumors is modest. One explanation for the observation is that in M-HIFU-treated tumors, myeloid cell populations, including macrophages, upregulate PD-L1, which may lead to the induction of insufficient systemic antitumor immunity. PD-L1 has been shown to produce constitutive negative signals in macrophages and induce an immunosuppressive phenotype. Anti-PD-L1 treatment itself reshapes the macrophage compartment in the tumor to a more pro-inflammatory phenotype, which mainly leads to enhanced T cell activity by increasing IFN-γ levels in the TME. In the above-mentioned published studies, the administration of anti-PD-L1 antibodies together with M-HIFU enhanced the antitumor efficacy against distant tumors. Importantly, the data showed that the net effect of the combined treatment was a more favorable T and NK cell attraction and activation pattern not only at the HIFU-treated tumor site, but also at distant untreated tumors. As mentioned earlier, others have combined HIFU with pathogen-associated molecular patterns such as CpG to produce a distant effect. These data, combined with the demonstration that tumor control is CD8+ T cell-dependent, support the contention that HIFU induces antigen-specific CD8+ T cell responses that can be enhanced by nonspecific immune stimulants.

[0240] Despite the significant antitumor effect of the combination therapy, some distant tumors grew after the initial control period. There are two potential explanations for the hypothesized lack of complete control. First, after tumor apoptosis in response to M-HIFU plus anti-PD-L1, tissue repair within the altered TME may involve repolarization of macrophages to M2 as previously observed. In the combined M-HIFU and anti-PD-L1 model, in addition to genes typically associated with the M1 phenotype, macrophage genes involved in cell-matrix adhesion and extracellular matrix organization (important for wound healing) were also upregulated. However, in anti-CD47 antibody-treated murine BC that experienced sustained tumor regression, an increase in intratumoral phagocytic macrophages has been observed, accompanied by upregulation of gene signatures of wound healing, ECM remodeling, and anti-inflammatory.

[0241] Therefore, once the initial antitumor immune response is activated by M1 macrophages, the trend of upregulated gene expression consistent with a reparative phenotype may not be detrimental. Second, intratumoral Tregs derived from infiltrating CD4+ T cells may counteract effector T cell function. Flow cytometric analysis of M-HIFU-treated tumors showed that the combination strategy increased the infiltration of CD4+ T cells, including a population with enhanced Foxp3 expression, which is consistent with Treg cells. Depletion of CD4+ Tregs enhanced the antitumor efficacy of the combination therapy in local and HIFU-untreated distant tumors. An additional observation is that human BC is not traditionally considered an inflamed immunogenic tumor and is less responsive to immunotherapy such as ICB compared to other immunogenic solid tumors such as melanoma and lung cancer. In this study, tumor models engineered to express OVA, HER2, or HER3 antigens were used to monitor the induction of tumor antigen-specific immune responses by HIFU treatment. Unlike human BC cases, the forced expression of these foreign antigens makes tumor cells more immunogenic, and therefore requires careful interpretation of the antigen-specific immune responses induced in these models. Importantly, however, in a BC model, JC-HER3 tumors were implanted into HER3 transgenic mice that were immune-tolerant to HER3 antigens, and it was confirmed that anti-HER3 cellular immune responses were induced by M-HIFU treatment, with significant antitumor efficacy and abscopal effects. These data suggest that M-HIFU is able to break immune tolerance to self-antigens.

[0242] In summary, the systems and methods described herein demonstrate that the TME is favorably altered by M-HIFU and that systemic anti-tumor efficacy is enhanced by the combination of M-HIFU and anti-PD-L1 antibodies, which results in significantly stronger growth inhibition of distant tumors compared to either M-HIFU or anti-PD-L1 monotherapy. To further enhance anti-tumor efficacy and eradicate distant tumors by induced anti-tumor immunity, clinically applicable strategies can be tested in combination with M-HIFU plus anti-PD-L1, such as depletion of Tregs by targeted antibodies and enhancement of M1-biased TME by intratumoral IL-12 gene therapy.

[0243] Example 7 - Gene delivery using sonoporation energy [HJA(7]

[0244] This example demonstrates the delivery of genes / mRNA into tumor and non-tumor tissues (including normal tissues) by sonoporation. In vivo testing of sonoporation was performed using murine CT26 cells, bilateral flank injection, BALB / c mice. Fig.32 An exemplary sonoporation transducer is shown in . The ultrasonic treatment parameters were frequency 1.1 MHz, peak negative pressure (PNP): 0.1 MPa, 0.4 MPa, 0.7 MPa, and 200 cycles.

[0245] In the first sequence, the transducer was inserted into the target, the solution was injected into the target for 60 seconds, and then sonication was applied for 30 seconds. In the second sequence, the transducer was inserted into the target, the solution was injected for 60 seconds, and then sonication was applied for 30 seconds, the solution was injected again for 30 seconds, and then sonication was applied for 30 seconds.

[0246] The injected solution contained 7.68×10 8 The microbubble concentration was 10 bubbles / mL and the plasmid DNA concentration was 20 μg / mL. The solution was injected at a rate of 0.1 mL / min.

[0247] There were 12 test groups, consisting of three (multiple PNPs) x two (different order) x two (replicate). There were three control groups (i.e., no treatment, plasmid only, plasmid and MB). The test conditions are shown in Fig.33 middle. Fig.33 Number 1 in the figure is the negative-negative control group, i.e., no treatment; Fig.33 No. 2 in the figure is a negative control group, i.e., having only plasmid; and Fig.33 Number 3 in the figure is the negative control group, i.e., having plasmid and MB.

[0248] Positive controls included intratumoral injection of Ad-luciferase and GFP-luciferase plasmid injection and electroporation (eg, same concentration of plasmid, same plasmid, same time point). To eliminate the risk of missing a portion of the tumor expressing the plasmid, the entire tumor was lysed.

[0249] The data were measured using a photometer and are shown in Figure 34-37 middle. Fig.35 Data are shown for patients injected intratumorally with Ad-luciferase or saline. Fig.36 Data using electroporation are shown. Fig.37 Data for application of the sonoporation effect are shown.

[0250] It should be understood that the foregoing detailed description and the accompanying examples are illustrative only and should not be taken as limiting the scope of the present invention.

[0251] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the present invention, including but not limited to changes and modifications associated with the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the present invention.

[0252] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0253] Item 1. A catheter comprising: an elongated hollow tube; a first lumen in the elongated hollow tube; a second lumen in the elongated hollow tube; a transducer located within the elongated hollow tube and adjacent to the first lumen or the second lumen, the ultrasonic transducer being configured to transmit ultrasonic waves through the lumen toward a target; and a needle located within the elongated hollow tube and configured to extend from the first lumen or the second lumen into the target to deliver treatment to the target.

[0254] Clause 2. The catheter of Clause 1, wherein the therapy is delivered to the target simultaneously with the ultrasound waves being delivered to the target.

[0255] Clause 3. The catheter of Clause 1 or 2, wherein the transducer is configured to deliver an in vivo sonoporation effect to the target.

[0256] Clause 4. The catheter of any one of Clauses 1 to 3, wherein the in vivo sonoporation generates acoustic cavitation at the target to induce the formation of pores in a cell membrane of the target, thereby increasing the permeability of the target.

[0257] Clause 5. The catheter of any one of Clauses 1 to 4, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cells, or immune cells.

[0258] Clause 6. The catheter of any one of Clauses 1 to 5, wherein the needle is configured to deliver immunotherapy directly into the target.

[0259] Clause 7. The catheter of any one of Clauses 1 to 5, wherein the needle is configured to deliver gene therapy directly into the target.

[0260] Clause 8. The catheter of any one of Clauses 1 to 5, wherein the needle is configured to deliver one or more therapeutic agents directly into the target.

[0261] Clause 9. The catheter of Clause 1, wherein the transducer is configured to deliver a high pressure acoustic burst of focused ultrasound toward the target to generate acoustic cavitation at the target.

[0262] Clause 10. The catheter of Clause 9, wherein the acoustic cavitation at the target provides expansion and collapse of microbubbles to release high pressure cavitation energy, thereby disrupting the extracellular matrix of the target.

[0263] Clause 11. The catheter of Clause 9 or 10, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cells, or immune cells.

[0264] Clause 12. The catheter of any one of Clauses 9 to 11, wherein the needle is configured to deliver immunotherapy directly into the target.

[0265] Clause 13. The catheter of any one of Clauses 9 to 11, wherein the needle is configured to deliver gene therapy directly into the target.

[0266] Clause 14. The catheter of any one of Clauses 9 to 11, wherein the needle is configured to deliver one or more therapeutic agents directly into the target.

[0267] Clause 15. The catheter of any one of Clauses 1 to 14, wherein the transducer is configured to deliver energy to the target resulting in application of an acoustic pressure (negative peak) in the range of 5 MPa to greater than 50 MPa to the target.

[0268] Clause 16. The catheter of Clause 15, wherein the acoustic pressure applied to the target is in the range of 15 MPa to 30 MPa.

[0269] Clause 17. The catheter of Clause 15 or 16, wherein the acoustic pressure applied to the target is in the range of 20 MPa to 45 MPa.

[0270] Clause 18. The catheter of any one of Clauses 1 to 17, wherein the transducer is configured to ablate the target.

[0271] Clause 19. The catheter of any one of Clauses 1 to 18, wherein the target is adjacent to or within a gas-filled anatomical organ.

[0272] Clause 20. The catheter of Clause 19, wherein the gas-filled anatomical organ is a lung, intestine, airway, or bladder.

[0273] Clause 21. The catheter of any of Clauses 1 to 20, wherein the transducer comprises a plurality of electrodes positioned adjacent to one another in a non-linear orientation.

[0274] Clause 22. The catheter of Clause 21, wherein the plurality of electrodes are positioned to form a radius of curvature in the range of 5 mm to 10 mm.

[0275] Clause 23. The catheter of Clause 21 or 22, wherein each of the plurality of electrodes has a hole size of approximately 1.4 mm x 1.8 mm.

[0276] Clause 24. The catheter of any one of Clauses 21 to 23, wherein at least one of the plurality of electrodes comprises a piezoelectric plate having a thickness of less than about 50 μm to about 500 μm.

[0277] Clause 25. The catheter of any one of Clauses 1 to 24, wherein the catheter is a 6-7 Fr catheter.

[0278] Clause 26. The catheter of any one of Clauses 1 to 25, wherein the catheter is a 8-10 Fr catheter.

[0279] Clause 27. A method for treating a malignant tumor, the method comprising: inserting a catheter according to claim 1 into a subject and toward the malignant tumor; activating the transducer to deliver energy to the malignant tumor through the first lumen, resulting in applying an acoustic peak negative pressure in the range of 10 MPa to 40 MPa to the malignant tumor; and activating the needle to extend from the second lumen into the malignant tumor, thereby delivering a therapeutically effective amount of a drug composition.

[0280] Clause 28. A method according to Clause 27, wherein the pharmaceutical composition comprises at least one selected from the group consisting of: cytokines, chemokines and other biological proteins (IL-12, etc.), oncolytic viruses, CAR-T cells, TIL, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory agents, antiproliferative agents, anti-migratory agents, anti-fibrotic agents, pro-apoptotic agents, vasodilators, calcium channel blockers, anti-tumor agents, anti-cancer agents, antibodies, anti-thrombotic agents, anti-platelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors and non-immunosuppressive agents.

[0281] Clause 29. The method according to clause 27 or 28, wherein the pharmaceutical composition comprises an alkylating agent for targeting DNA.

[0282] Item 30. A system for treating a target, the system comprising: an ultrasonic energy source; a device coupled to the ultrasonic energy source and configured and arranged to: direct the ultrasonic energy to the target, release one or more microbubbles, and release one or more therapeutic agents, wherein the microbubbles burst after receiving the ultrasonic energy, thereby destroying the target and the extracellular matrix (ECM) surrounding the target and allowing the one or more therapeutic agents to be delivered into the target.

[0283] Clause 31. The system of clause 30, wherein the one or more microbubbles are configured and arranged to contain the one or more therapeutic agents, wherein the one or more microbubbles burst upon receiving the ultrasound energy, thereby releasing the one or more therapeutic agents into the target.

[0284] Clause 32. The system of clause 30 or 31, wherein the device further comprises a configuration and arrangement for releasing one or more contrast agents.

[0285] Clause 33. The system of any one of clauses 30 to 32, wherein the ultrasound energy comprises high intensity focused ultrasound (HIFU).

[0286] Clause 34. The system of any one of clauses 30 to 32, wherein the ultrasound energy comprises tissue fragmentation.

[0287] Clause 35. The system according to any one of clauses 30 to 34, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cell or immune cell.

[0288] Clause 36. A method for delivering a drug to a target, the method comprising: inserting a catheter into a subject and directed toward the target, the catheter comprising a device according to clause 30; generating an ultrasonic energy output near the target; supplying one or more microbubbles near the target; and supplying one or more therapeutic agents through the catheter, wherein the microbubbles burst upon receiving the ultrasonic energy, thereby destroying the target and the surrounding extracellular matrix (ECM) and allowing the one or more therapeutic agents to be delivered into the target, thereby treating the target.

[0289] Clause 37. The method of Clause 36, wherein the one or more microbubbles are configured and arranged to contain the one or more therapeutic agents, wherein the one or more microbubbles burst upon receiving the ultrasound energy, thereby releasing the one or more therapeutic agents into the target.

[0290] Clause 38. The method according to clause 36 or 37, wherein the method further comprises releasing one or more contrast agents.

[0291] Clause 39. A method according to any one of clauses 36 to 38, wherein the one or more therapeutic agents are selected from the group consisting of: cytokines, chemokines and other biological proteins (IL-12, etc.), oncolytic viruses, CAR-T cells, TIL, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory agents, antiproliferative agents, anti-migratory agents, anti-fibrotic agents, pro-apoptotic agents, vasodilators, calcium channel blockers, anti-tumor agents, anti-cancer agents, antibodies, anti-thrombotic agents, anti-platelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, non-immunosuppressive agents and combinations thereof.

[0292] Clause 40. The method according to any one of clauses 36 to 39, wherein the one or more therapeutic agents comprises a cytokine, chemokine or other biological protein.

[0293] Clause 41. The method according to Clause 40, wherein the cytokine comprises IL-12.

Claims

1. A catheter, comprising: A long, thin hollow tube; a first lumen in the elongated hollow tube; a second lumen in the elongated hollow tube; a transducer located within the elongated hollow tube and adjacent to the first lumen or the second lumen, the ultrasonic transducer being configured to transmit ultrasonic waves through the lumen toward a target; as well as A needle is positioned within the elongated hollow tube and is configured to extend from the first lumen or the second lumen into the target to deliver therapy to the target.

2. The catheter of claim 1, wherein the therapy is delivered to the target simultaneously with the ultrasound waves being delivered to the target.

3. The catheter of claim 1 or 2, wherein the transducer is configured to deliver an in vivo sonoporation effect to the target. 4 . The catheter according to claim 1 , wherein the in vivo sonoporation generates acoustic cavitation at the target to induce pores in a cell membrane of the target, thereby increasing the permeability of the target.

5. The catheter of any one of claims 1 to 4, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cells or immune cells.

6. The catheter of any one of claims 1 to 5, wherein the needle is configured to deliver immunotherapy directly into the target.

7. The catheter of any one of claims 1 to 5, wherein the needle is configured to deliver gene therapy directly into the target.

8. The catheter of any one of claims 1 to 5, wherein the needle is configured to deliver one or more therapeutic agents directly into the target.

9. The catheter of claim 1, wherein the transducer is configured to deliver a high pressure acoustic burst of focused ultrasound toward the target to generate acoustic cavitation at the target.

10. The catheter of claim 9, wherein the acoustic cavitation at the target provides expansion and collapse of microbubbles to release high pressure cavitation energy, thereby disrupting the extracellular matrix of the target.

11. The catheter of claim 9 or 10, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cells or immune cells.

12. The catheter of any one of claims 9 to 11, wherein the needle is configured to deliver immunotherapy directly into the target.

13. The catheter of any one of claims 9 to 11, wherein the needle is configured to deliver gene therapy directly into the target.

14. The catheter of any one of claims 9 to 11, wherein the needle is configured to deliver one or more therapeutic agents directly into the target.

15. The catheter of any one of claims 1 to 14, wherein the transducer is configured to deliver energy to the target resulting in application of an acoustic pressure (negative peak) in the range of 5 MPa to greater than 50 MPa to the target.

16. The catheter of claim 15, wherein the acoustic pressure applied to the target is in the range of 15 MPa to 30 MPa.

17. The catheter of claim 15 or 16, wherein the acoustic pressure applied to the target is in the range of 20 MPa to 45 MPa.

18. The catheter of any one of claims 1 to 17, wherein the transducer is configured to ablate the target.

19. The catheter of any one of claims 1 to 18, wherein the target is adjacent to or within a gas-filled anatomical organ.

20. The catheter of claim 19, wherein the gas-filled anatomical organ is a lung, intestine, airway, or bladder.

21. The catheter of any one of claims 1 to 20, wherein the transducer comprises a plurality of electrodes positioned adjacent to each other in a non-linear orientation.

22. The catheter of claim 21, wherein the plurality of electrodes are positioned to form a radius of curvature in the range of 5 mm to 10 mm.

23. The catheter of claim 21 or 22, wherein each of the plurality of electrodes has a hole size of approximately 1.4 mm x 1.8 mm.

24. The catheter of any one of claims 21 to 23, wherein at least one of the plurality of electrodes comprises a piezoelectric plate having a thickness of less than about 50 μm to about 500 μm.

25. The catheter of any one of claims 1 to 24, wherein the catheter is a 6-7 Fr catheter.

26. The catheter of any one of claims 1 to 25, wherein the catheter is a 8-10 Fr catheter.

27. A method for treating a malignant tumor, the method comprising: inserting the catheter according to claim 1 into a subject toward the malignant tumor; activating the transducer to deliver energy to the malignant tumor through the first lumen, resulting in application of an acoustic peak negative pressure in the range of 10 MPa to 40 MPa to the malignant tumor; as well as The needle is activated to extend from the second lumen into the malignant tumor, thereby delivering a therapeutically effective amount of the pharmaceutical composition.

28. The method of claim 27, wherein the pharmaceutical composition comprises at least one selected from the group consisting of cytokines, chemokines and other biological proteins (IL-12, etc.), oncolytic viruses, CAR-T cells, TILs, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory agents, antiproliferative agents, anti-migratory agents, anti-fibrotic agents, pro-apoptotic agents, vasodilators, calcium channel blockers, anti-tumor agents, anti-cancer agents, antibodies, anti-thrombotic agents, anti-platelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, and non-immunosuppressive agents.

29. The method of claim 27 or 28, wherein the pharmaceutical composition comprises an alkylating agent for targeting DNA.

30. A system for treating a target, the system comprising: Ultrasonic energy source; device, the device being coupled to the ultrasonic energy source and being configured and arranged to: Directs ultrasound energy to the target, releasing one or more microbubbles, and One or more therapeutic agents are released, wherein the microbubbles burst upon receiving the ultrasound energy, thereby disrupting the target and the extracellular matrix (ECM) surrounding the target and allowing the one or more therapeutic agents to be delivered into the target.

31. The system of claim 30, wherein the one or more microbubbles are configured and arranged to contain the one or more therapeutic agents, wherein the one or more microbubbles burst upon receiving the ultrasound energy, thereby releasing the one or more therapeutic agents into the target.

32. The system of claim 30 or 31, wherein the device further comprises a configuration and arrangement for releasing one or more contrast agents.

33. The system of any one of claims 30 to 32, wherein the ultrasound energy comprises high intensity focused ultrasound (HIFU).

34. The system of any one of claims 30 to 32, wherein the ultrasonic energy comprises tissue fragmentation.

35. The system of any one of claims 30 to 34, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cell, or immune cell.

36. A method for delivering a drug to a target, the method comprising: inserting a catheter into a subject and toward the target, the catheter comprising the device according to claim 30; generating an ultrasonic energy output in the vicinity of the target; supplying one or more microbubbles in proximity to the target; and One or more therapeutic agents are supplied through the catheter, wherein the microbubbles burst upon receiving the ultrasound energy, thereby disrupting the target and surrounding extracellular matrix (ECM) and allowing the one or more therapeutic agents to be delivered into the target, thereby treating the target.

37. The method of claim 36, wherein the one or more microbubbles are configured and arranged to contain the one or more therapeutic agents, wherein the one or more microbubbles burst upon receiving the ultrasound energy, thereby releasing the one or more therapeutic agents into the target.

38. The method of claim 36 or 37, wherein the method further comprises releasing one or more contrast agents.

39. The method according to any one of claims 36 to 38, wherein the one or more therapeutic agents are selected from the group consisting of: cytokines, chemokines and other biological proteins (IL-12, etc.), oncolytic viruses, CAR-T cells, TIL, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory agents, antiproliferative agents, anti-migratory agents, anti-fibrotic agents, pro-apoptotic agents, vasodilators, calcium channel blockers, anti-tumor agents, anti-cancer agents, antibodies, antithrombotic agents, antiplatelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, non-immunosuppressive agents, and combinations thereof.

40. The method of any one of claims 36 to 39, wherein the one or more therapeutic agents comprise a cytokine, chemokine, or other biological protein.

41. The method of claim 40, wherein the cytokine comprises IL-12.

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