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 tumors, enhancing efficacy of therapeutic drugs and increasing immune cell infiltration. A miniaturized dual lumen catheter device is configured to deliver M-HIFU to tumors, eliminating the anatomical limitations of conventional HIFU and allowing access to primary or metastatic tumors regardless of their location.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a nonprovisional application and claims the benefit of U.S. Provisional Patent Application No. 63 / 324,479, filed March 28, 2022, the contents of which are incorporated herein by reference. [Background technology]

[0002] Solid tumors constitute the majority of cancers, with over 1.5 million new cases diagnosed annually in the U.S. Common treatments include surgery, ablation, radiation therapy, chemotherapy, and more recently advanced immunotherapy and other targeted therapies, which are offered alone or in combination. Immunotherapy has emerged as a treatment for cancer and mainly consists of immune checkpoint blockade or cell therapy. Although systemic immunotherapy shows promise, it is effective in less than 15% of patients and has significant side effects that affect the quality of life of patients. Therefore, the majority of patients with metastatic disease still cannot benefit from immunotherapy and combination therapies. Cancer immunotherapy using immune checkpoint blockade ("ICB") is a game-changing cancer therapy and has become a major focus of cancer research. Nevertheless, this method of treatment still suffers from several drawbacks, such as a relatively low response rate. Thus, there is a need for immunotherapy to transform immunologically "cold tumors" into inflammatory "hot tumors". As the majority of patients do not respond to ICB alone, combinations of systemically delivered immunotherapies have been investigated, but these not only increase tumor response rates but also toxicity and treatment-related mortality.

[0003] Numerous attempts have been explored to uncouple systemic toxicity from the increase in antitumor immune responses, but have not been achieved with the exception of one modality: the use of intralesional therapy. Intralesional therapy, or local delivery of drugs to tumors, includes oncolytic viruses and other immune agents that have shown the ability to stimulate antitumor responses with minimal, if any, systemic toxicity. Unfortunately, intratumoral delivery of these drugs is typically by needle injection, which has several limitations: 1) needle injection has several drawbacks, such as injection failure (e.g., tumor removal, drug leakage into surrounding tissue, less drug delivered to tumor, etc.), and 2) response rates are low, often less than 33%. Additionally, persistent barriers to treatment include the complex and dense nature of the tumor stroma and high stromal tissue pressure within primary and metastatic tumors, which impede the delivery of therapeutic agents and limit the entry of activated immune cells. Localized ablation techniques, such as high intensity focused ultrasound (HIFU), have been used to thermally destroy cancer cells and are increasingly being investigated to enhance cancer immunotherapy. HIFU is currently delivered by acoustic waves generated outside the body, but acoustic waves cannot cross the air-liquid interface. Thus, current HIFU strategies have anatomical limitations that prevent them from treating tumors in common organs of advanced cancer metastasis, such as the lungs, airways, or gastrointestinal (GI) tract. Thus, there is a continuing need for improved approaches to treat cancer. Summary of the Invention

[0004] As disclosed herein, microtransducer-based catheters use frequencies and energy levels set to puncture tumor cell membranes, disrupt tumor stroma, improve therapeutic drug uptake, 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 anti-cancer therapeutic drugs directly to tumors, microtransducer-based catheters are configured to deliver high intensity focused ultrasound (HIFU) to disrupt the tumor microenvironment and simultaneously deliver therapeutic drugs to tumors, utilizing synergy with tumor ablation immunotherapy. The mechanical destruction of target tissue and cell membrane disruption by HIFU-induced acoustic cavitation is known as histotripsy and is achieved by high pressure bursts of microbubbles induced by ultrasound treatment. In one embodiment, the present disclosure provides a novel localized mechanical HIFU (LM-HIFU) transcatheter device that can ablate cancer cells and disrupt stromal barriers in tumors, enhancing efficacy of therapeutic agents and increasing immune cell infiltration. The use of a miniaturized device for delivering M-HIFU described herein is free of the anatomical limitations of conventional HIFU, allowing access to primary or metastatic tumors regardless of their location.

[0005] One aspect of the present disclosure provides a system for treatment of a target (e.g., tumor tissue, tissue surrounding a tumor, non-malignant tissue, blood cells, or immune cells) comprising: (a) an ultrasound energy source; (b) a device coupled to the ultrasound energy source and configured and arranged to (i) direct energy to a desired location; (ii) release one or more microbubbles (an ultrasound contrast agent); and (iii) release one or more therapeutic agents, wherein upon receipt of energy, the microbubbles burst, thereby disrupting the target and surrounding extracellular matrix (ECM) and allowing the one or more therapeutic agents to be delivered into the target. In some embodiments, the one or more microbubbles are constructed and arranged to contain one or more therapeutic agents, where the one or more microbubbles burst upon receiving energy, thereby releasing the one or more therapeutic agents into the target. In another embodiment, the device further comprises (iv) constructed and arranged to release one or more imaging agents. In one embodiment, the ultrasound comprises high intensity focused ultrasound (HIFU). In another embodiment, the ultrasound comprises histotripsy.

[0006] Another aspect of the present disclosure provides a method for delivering a drug, protein, nucleic acid, or gene to a target, comprising using an ultrasonic energy delivery system provided herein, configured and arranged to produce an energy output; providing one or more microbubbles and one or more therapeutic agents, wherein the microbubbles burst upon receiving 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. In some embodiments, the one or more microbubbles are constructed and arranged to contain one or more therapeutic agents, where the one or more microbubbles burst upon receiving energy, thereby releasing the one or more therapeutic agents into the target. In another embodiment, the method further comprises for the release of one or more imaging agents.

[0007] In another embodiment, the one or more therapeutic agents are selected from the group consisting of cytokines, chemokines, and other biological proteins (such as, for example, IL-12), oncolytic viruses, CAR-T cells, TILs, other cells, sub-intracellular vesicles such as exosomes, cDNA, mRNA, self-replicating RNA, proteins, antibodies, single chain antibodies, nanobodies, phages, immunosuppressants, anti-inflammatory drugs, anti-proliferative drugs, anti-migratory agents, anti-fibrotic agents, pro-apoptotic drugs, vasodilators, calcium channel blockers, antineoplastic agents, anti-cancer agents, anti-thrombotic agents, anti-platelet agents, IIb / IIIa agents, anti-viral agents, mTOR (mammalian target of rapamycin) inhibitors, and combinations thereof. In one embodiment, the one or more therapeutic agents comprises a cytokine, hi one embodiment, the cytokine comprises IL-12, IL-15, or a fusion protein of a cytokine.

[0008] In one embodiment, the present invention provides a catheter comprising an elongated hollow tube, a first lumen within the elongated hollow tube, a second lumen within the elongated hollow tube, a transducer located within the elongated hollow tube adjacent the first lumen or the second lumen, and a needle, the ultrasound transducer configured to emit ultrasound waves through the lumen to a target, and the needle located within the elongated hollow tube and configured to extend from the first lumen or the second lumen to enter the target and deliver a therapy to the target. In another embodiment, the present disclosure provides a method of treating a malignant tumor, the method comprising inserting the above-mentioned catheter into a subject toward the malignant tumor, actuating the transducer to deliver energy to the malignant tumor through the first lumen resulting in an acoustic peak negative pressure in the range of 10 MPa to 40 MPa being applied to the malignant tumor, and actuating the needle to extend from the second lumen into the malignant tumor to deliver a therapeutically effective amount of a pharmaceutical composition.

[0009] In another embodiment, the present disclosure provides a system for treatment of a target, the system including an ultrasonic energy source and a device coupled to the ultrasonic energy source, the device configured and arranged to direct ultrasonic energy to the target, release one or more microbubbles, and release one or more therapeutic agents, where upon receipt of the ultrasonic energy, the microbubbles burst, 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. In another embodiment, the present disclosure provides a method for delivering a drug to a target, the method includes inserting a catheter including the above-mentioned device into a subject toward the target, generating ultrasonic energy output near the target, providing one or more microbubbles near the target, and providing one or more therapeutic agents through the catheter, where the microbubbles burst upon receiving the ultrasonic energy, thereby disrupting the target and surrounding extracellular matrix (ECM), allowing the one or more therapeutic agents to be delivered into the target, thereby treating the target. Other aspects of the invention will become apparent by consideration of the detailed description and accompanying drawings. [Brief description of the drawings]

[0010] [Figure 1A] FIG. 1 is a schematic diagram illustrating a system including an ultrasonic energy source for delivery of a therapeutic agent to a target tissue according to some embodiments. [Figure 1B] FIG. 1 is a schematic diagram illustrating a system including an ultrasonic energy source for delivery of a therapeutic agent to a target tissue according to some embodiments. [Figure 2A] FIG. 2 illustrates an example of a transducer of the system of FIGS. 1A and 1B according to some embodiments. [Figure 2B] 2B is a graph illustrating the sensitivity of the transducer of FIG. 2A according to some embodiments. [Figure 2C] 2B is an image of a sonoporation drug delivery test setup of the transducer of FIG. 2A according to some embodiments. [Figure 2D] 2B shows a fluorescence image of GFP+ sonoporation-treated cells treated with the transducer of FIG. 2A according to some embodiments, as well as fluorescence images of luciferase activity in negative control cells and sonoporation-treated cells. [Figure 3A] FIG. 2 illustrates an example of a tubular HIFU transducer according to some embodiments. [Figure 3B] 3B is a schematic diagram of the transducer of FIG. 3A according to some embodiments. [Figure 3C] FIG. 3B illustrates a simulated sound field profile produced by the transducer of FIG. 3A according to some embodiments. [Figure 3D] 3B shows measured acoustic profiles in both the side and forward looking directions produced by the transducer of FIG. 3A in accordance with some embodiments. [Figure 4A] FIG. 1 illustrates a miniaturized HIFU transducer according to some embodiments. [Figure 4B] 4B is a graph illustrating the sound pressure output of the transducer of FIG. 4A according to some embodiments. [Figure 4C] 4B is an image of the results of a bubble cloud generation test performed by the transducer of FIG. 4A according to some embodiments. [Figure 5A] 1A-1D illustrate multiple views of a HIFU transducer according to some embodiments. [Figure 5B] FIG. 5B illustrates a simulated sound pressure field profile of the transducer of FIG. 5A under 3.5 MHz frequency operation according to some embodiments. [Figure 5C] 5B is a diagram of a setup for cavitation generation using the transducer of FIG. 5A according to some embodiments. [Figure 5D] 5B is a diagram of a setup for cavitation generation using the transducer of FIG. 5A according to some embodiments. [Figure 6A]1 is a pair of plots depicting tumor volume size and survival rates for mice treated with TAVO and a control plasmid according to some embodiments. [Figure 6B] 1 is a pair of plots depicting fold tumor volume change in treated and untreated mice according to some embodiments. [Figure 6C] FIG. 6B depicts a t-SNE plot of cells classified into cell types for all samples of FIGS. 6A and 6B or separated by treatment group according to some embodiments. [Figure 6D] FIG. 6C depicts a quantification plot of the frequency of each clone in each treatment group of FIGS. 6A and 6B according to some embodiments. [Figure 6E] FIG. 6C depicts a quantification plot of activation signature scores across all t-cells in each treatment group of FIGS. 6A and 6B according to some embodiments. [Figure 7A] FIG. 6C is a circos plot depicting receptor-ligand interactions between receptors on CD8 T cells and ligands on macrophages for the treatment groups of FIGS. 6A and 6B according to some embodiments. [Figure 7B] 1 is a plot depicting a 50-gene CXCR3 gene signature score quantified across all cells according to some embodiments. [Figure 8A] 4A and 5B are tumor curves from a combination therapy performed with the transducer of FIGS. 4A and 5A according to some embodiments. [Figure 8B] 4A and 5B are tumor curves from a combination therapy performed with the transducer of FIGS. 4A and 5A according to some embodiments. [Figure 8C] 4A and 5B are tumor curves from a combination therapy performed with the transducer of FIGS. 4A and 5A according to some embodiments. [Figure 8D] 4A and 5B are tumor curves from a combination therapy performed with the transducer of FIGS. 4A and 5A according to some embodiments. [Figure 9] 1 is a pair of images showing baseline and post-cycle images of lung metastases according to some embodiments. [Figure 10]FIG. 1 depicts multiple images of fibrous tumor stroma of breast cancer, according to some embodiments. [Figure 11] FIG. 1 depicts images of drug distribution after intratumoral injection of the PV-10 drug according to some embodiments. [Figure 12] 1A-1D are images illustrating how a tumor is treated with different HIFU protocols according to some embodiments. [Figure 13] 13A-13C are histology graphs and plots depicting HIFU-treated and control (no treatment) data of T cell infiltration of tumors according to some embodiments. [Figure 14] FIG. 1 includes graphs depicting the combination of M-HIFU and PD-L1 treatment according to some embodiments. [Figure 15] FIG. 1 includes plots depicting the results of combined IT-IL-12 gene therapy and M-HIFU treatment according to some embodiments. [Figure 16A] FIG. 2B illustrates a system including the transducer of FIG. 2A for the treatment of metastatic breast cancer according to some embodiments. [Figure 16B] FIG. 2B illustrates a system including the transducer of FIG. 2A for the treatment of metastatic breast cancer according to some embodiments. [Figure 17] 1 is a graph depicting ESR1 mutant expression resulting in constitutive estrogen signaling and enhanced proliferation in precancerous mouse mammary epithelial cells according to some embodiments. [Figure 18] FIG. 2 illustrates another example of a transducer of the system of FIGS. 1A and 1B according to some embodiments. [Figure 19] 19 is a table of acoustic properties of cell culture plates for use with the transducer of FIG. 18 according to some embodiments. [Figure 20] FIG. 19 illustrates a system for testing the functionality of the transducer of FIG. 18 according to some embodiments. [Figure 21] 19 is a graph illustrating the sound pressure output of the transducer of FIG. 18 in accordance with some embodiments. [Figure 22]FIG. 19 illustrates the acoustic pressure field of a simulated 800 kHz ultrasound beam for testing the functionality of the transducer of FIG. 18 according to some embodiments. [Diagram 23] 19 is a table showing the luciferase activity of control and experimental groups for testing the functionality of the transducer of FIG. 18 according to some embodiments. [Figure 24] 19 is a series of graphs depicting luciferase activity of sonoporation experiments under various sonication parameters to test the functionality of the transducer of FIG. 18 according to some embodiments. [Diagram 25] 1 is a series of tables depicting inhibition of local and distant tumor growth and enhancement of tumor antigen-specific cellular immune responses according to several embodiments. [Figure 26] 1 is a series of data plots regarding enhanced intratumoral infiltration by M-HIFU treatment according to some embodiments. [Figure 27A] 1 is a series of plots of GO enrichment analysis and KEGG pathway analysis of DEGs in macrophages after M-HIFU treatment according to some embodiments. [Figure 27B] 1 is a series of plots of GO enrichment analysis and KEGG pathway analysis of DEGs in macrophages after M-HIFU treatment according to some embodiments. [Figure 28] 1 is a series of plots depicting increased expression of immune checkpoint molecules by tumor-infiltrating immune cells following M-HIFU treatment according to some embodiments. [Figure 29A] 1 is a series of plots illustrating that M-HIFU and PD-L1 inhibition synergize to reject local tumors, according to some embodiments. [Figure 29B] 1 is a series of plots illustrating that M-HIFU and PD-L1 inhibition synergize to reject local tumors, according to some embodiments. [Diagram 30] 1 is a series of plots showing that combination of M-HIFU and anti-PD-L1 antibody induces up-regulation of unique DEGs in tumor-infiltrating CD8 T cells and macrophages, according to some embodiments. [Diagram 31] 1 is a series of plots depicting the results of combined M-HIFU and PD-1 / PD-L1 inhibition according to some embodiments. [Diagram 32] FIG. 1 illustrates an example sonoporation transducer used in Example 7. [Diagram 33] 1 is a table showing the test conditions for the test group and the control group used in Example 7. [Diagram 34] FIG. 1 illustrates data from the experimental test results of Example 7. [Diagram 35] FIG. 1 illustrates data from the experimental test results of Example 7. [Diagram 36] FIG. 1 illustrates data from the experimental test results of Example 7. [Figure 37] FIG. 1 illustrates data from the experimental test results of Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Before describing any embodiments of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or carried out in various ways. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can include more than one element. "About" is used to provide flexibility to the endpoints of numerical ranges by specifying that a given value may be "slightly above" or "slightly below" the endpoints without affecting the desired result. The use of the terms "including," "comprising," or "having," and variations thereof herein are meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations interpreted in the alternative ("or").

[0012] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be interpreted to include the recited materials or steps and those "that do not materially affect the basic and novel characteristics" of the claimed invention. Thus, the term "consisting essentially of" as used herein should not be interpreted as the equivalent of "comprising." Further, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or excluded. For illustrative purposes, if the specification describes a composite as comprising components A, B, and C, it is expressly intended that any or combination of A, B, or C, singly or in any combination, may be excluded and negated. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1%-50%, values ​​such as 2%-40%, 10%-30%, or 1%-3%, are intended to be expressly recited in this specification. These are merely examples of what is expressly intended, and all possible combinations of numerical ranges between and including the minimum and maximum values ​​should be considered to be expressly recited in this disclosure.

[0013] As used herein, "treatment", "therapy" and / or "therapeutic regimen" refer to a clinical intervention administered in response to a disease, disorder, or physiological condition exhibited by or to which a patient is predisposed. The objective of treatment includes reducing or preventing symptoms, slowing or halting the progression or worsening of a disease, disorder, or condition, and / or ameliorating a disease, disorder, or condition. As used herein, terms such as "prevent", "preventing", "prevention", "prophylactic treatment", and the like refer to reducing the likelihood of onset of a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition but is at risk of developing or predisposed to the disease, disorder, or condition. The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce beneficial or desired biological and / or clinical results. The term "disease" as used herein includes, but is not limited to, any abnormal state and / or disorder of structure or function affecting a part of an organism, which may be caused by an external factor, such as an infection, or an internal malfunction, such as cancer, cancer metastasis, etc.

[0014] As known in the art, cancer is generally considered to be uncontrolled cell proliferation. The method of the present invention can be used to treat any cancer, including but not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia, and any metastasis thereof. 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, hepatoma, colorectal cancer, uterine cervical cancer, endometrial cancer, salivary gland cancer, mesothelioma, kidney cancer, vulvar cancer, pancreatic cancer, thyroid cancer, hepatocellular carcinoma, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphocytic leukemia, acute myeloid leukemia, Ewing's sarcoma, and peripheral neuroepithelioma. As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to both human and non-human animals. The term "non-human animal" in the present disclosure includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The methods and compositions disclosed herein can be used on samples either in vitro (e.g., isolated cells or tissues) or in vivo in a subject (i.e., a living organism, such as a patient).

[0015] As used herein, a "therapeutic agent" includes any molecular species and / or biological agent that is therapeutic when introduced into a subject being treated, or that becomes therapeutic after being introduced into a subject being treated, for example, by reaction with a native or non-native substance or condition, or other introduced substance. Examples of native conditions include pH (e.g., acidity), chemicals, temperature, salinity, osmolality, and conductivity, and examples of non-native conditions include magnetic fields, electromagnetic fields (e.g., radio frequency and microwave), and ultrasound, etc. In this disclosure, the chemical name of any therapeutic agent is used to refer to the compound itself and to prodrugs (precursors that are converted into the active form of the compound in the body) and / or its pharmaceutical derivatives, analogs, or metabolites (biologically active compounds that the compound converts into in the body directly or upon the introduction of other agents or conditions (e.g., enzymatic, chemical, energy) or environments (e.g., pH)).

[0016] The scope of the present disclosure includes the use of any therapeutic agent whose efficacy may be enhanced by the use of ultrasound energy as described herein. For illustrative purposes, several therapeutic agent classes are identified for the understanding of the present disclosure. These classes of agents and the 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 may be considered beneficial in treating a patient. Furthermore, these agents may be delivered / administered by any suitable modality. As used herein, the term "administering" an agent, such as a therapeutic entity, to an animal or cell is intended to refer to dosing, delivering, or applying a substance to a desired target. In the context of a therapeutic agent, the term "administering" is intended to refer to contacting or dispensing, delivering or applying a therapeutic agent to a subject by any route suitable for delivering the therapeutic agent to a desired location in an animal, including delivery by either parenteral or oral routes, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by nasal or airway routes. In some embodiments, exemplary therapeutic agents include cytokines, chemokines, and other biological proteins (e.g., IL-12, etc.), oncolytic viruses, CAR-T cells, TILs, 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, antineoplastic agents, anti-cancer agents, antibodies, anti-thrombotic agents, anti-platelet agents, IIb / IIIa agents, anti-viral agents, mTOR (mammalian target of rapamycin) inhibitors, non-immunosuppressive agents, and combinations thereof.

[0017] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Aspects of the present disclosure combine an energy source with microbubbles and one or more therapeutic agents to treat a selected region of a subject (e.g., a tumor). As used herein, "selected region," "treatment site," "desired target," and "target region" are used interchangeably and refer to a region within a subject where targeted treatment is desired. Treatment sites may include tissues, organs, or localized tumors associated with a body lumen. In one embodiment, the present devices and methods inhibit the formation or progression of tumors or hyperplastic growth. A "lumen" may be any vessel in the vasculature of a subject, including veins, arteries, aortas, particularly coronary and peripheral arteries, as well as previously implanted grafts, shunts, fistulas, and the like. In other embodiments, the systems and methods described herein may also be applied to other body lumens, such as the bile duct, that undergo excessive neoplastic cell growth; examples of application to internal body tissues and organs include various organs, nerves, glands, ducts, and the like.

[0018] According to one aspect of the present disclosure, a system for the treatment of tumors is provided. In such an aspect, the system includes: (a) an ultrasonic energy source; (b) a device coupled to the energy source, 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 "microbubbles", which are a type of ultrasound contrast agent and are also referred to herein as "cavitating bubbles" or "cavitation bubbles", are small, gas-filled bubbles that can be injected into a subject, where they remain inactive unless stimulated by energy generated by an energy source. In effect, the energy source directs energy to the microbubbles, causing them to vibrate and burst, thereby disrupting the surrounding tissue (e.g., ECM, tumor, etc.) and allowing one or more therapeutic agents to be delivered into the tumor. An example of a system for delivery of energy from an ultrasonic energy source to microbubbles (and in some embodiments, delivery of a therapeutic agent to a desired target, such as a tumor) according to some embodiments of the present disclosure is shown in FIGS. 1A and 1B. FIGS. 1A and 1B illustrate example systems 100A, 100B, respectively, for delivery of energy from an ultrasonic energy source to microbubbles to a desired target, according to some embodiments. According to the example illustrated in FIG. 1A, the system 100 includes a device 102 that includes a simple or single-source ultrasonic energy source 101. FIG. 1B illustrates another example, where the device 102 includes an ultrasonic energy source 101 that includes a multiple ultrasound device (e.g., a probe or transducer).

[0019] 1A and 1B illustrate different types of ultrasound energy sources 101, it should be understood that any ultrasound suitable for therapeutic applications can be used in the systems and methods provided herein. Suitable examples include, but are not limited to, HIFU, histotripsy, etc. In either case, the device 102 includes one or more adjacent tubes 104 for the emission of the microbubbles 106 and / or therapeutic agent 108. Once emitted from the device 102, energy 110 is emitted from an energy source 101 within the device 102 and delivered to the microbubbles 106. The microbubbles 106 are then activated to enable destruction of the target tissue 112 and / or subsequent delivery of one or more therapeutic agents 108 to the target tissue 112. Although the examples illustrated in Figures 1A and 1B depict the microbubbles and therapeutic agent being delivered longitudinally, it should be understood that in some embodiments the microbubbles and / or therapeutic agent may alternatively or additionally be delivered in one or more other directions (e.g., transversely). In some embodiments, the microbubbles and the therapeutic agent are delivered to the subject simultaneously. In other embodiments, the microbubbles are delivered to the subject prior to delivery of the therapeutic agent. In yet other embodiments, the microbubbles are delivered to the subject after the therapeutic agent.

[0020] In yet other embodiments, the therapeutic agent is loaded into microbubbles, where it can burst or be "activated" to release its contents upon slight interaction with the applied energy. By "loading" these microbubbles with a particular therapeutic agent and then activating the microbubbles to release its contents at the target tissue, it is possible to limit the side effects of the therapeutic agent by delivering it only to the site of need (or substantially to the site of need). In such an embodiment, the microbubbles may be composed of small, gas-filled lipids or fats. As a result, there is a high concentration of the therapeutic agent in the target tissue (e.g., tumors) and such destruction takes place. Despite the presence of the drug in the blood, other tissues of the body are not affected because the drug is attached to the microbubbles and is inert. In another embodiment, the system further provides for visualization of the target tissue. Gas-filled microbubbles (e.g., micron-sized / diameter) can be visualized or imaged by applying ultrasound energy. For example, in one embodiment, the system can utilize an energy source that directs energy to the microbubbles, causing them to return characteristic echoes within the bloodstream, creating a dramatic difference, or high "contrast," between the blood vessels and the surrounding tissue, thereby allowing the clinician to visualize the target area. In other embodiments, visualization can be achieved by administration of a contrast agent. In either case, the contrast properties of the agent and / or the microbubbles allow visualization of the target tissue.

[0021] The systems provided herein can be used in numerous ways to treat diseases such as cancer. In another aspect, the disclosure provides a method for delivering drugs to a tumor, comprising using an energy delivery system provided herein, configured and arranged to produce an energy output; providing one or more microbubbles and one or more therapeutic agents, wherein the microbubbles burst upon receiving energy, thereby disrupting the tumor and surrounding extracellular matrix (ECM), allowing the one or more therapeutic agents to be delivered into the tumor, thereby treating the tumor. In some embodiments, the one or more microbubbles are constructed and arranged to contain one or more therapeutic agents, where the one or more microbubbles burst upon receiving energy, thereby releasing the one or more therapeutic agents into the tumor. In some embodiments, the methods include promoting intracellular activation by exposing vascular wall cells to energy and introducing anti-cancer therapeutic agents to cause translocation of these agents to target tissues.

[0022] For example, suitable drugs within the scope of the present disclosure include, but are not limited to: Adriamycin PFS Injection (Pharmacia & Upjohn); Adriamycin RDF for Injection (Pharmacia & Upjohn); Alkeran for Injection (Glaxo Wellcome 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 IV 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, Injectable, 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&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); Aseptic 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 Vials (Lilly); Vumon for Injection (Bristol-Myers Squibb Oncology / Immunology); Zinecard for Injection (Pharmacia&Upjohn); Zofran Injection (Glaxo Wellcome Oncology / HIV); Zofran Injection Premixed (Glaxo Wellcome Oncology / HIV); Zoladex (Zeneca).;

[0023] Other classes of drugs within the scope of this disclosure include DNA-targeting, cytotoxic, mutagenic, and carcinogenic alkylating agents. All alkylating agents result in alkylation through the formation of intermediates. Alkylating agents impair cellular function by transferring alkyl groups to amino, cartoryl, sulfhydryl, or phosphate groups of biologically important molecules. Such drugs include busulfan (Myleran), chlorambucil (Leukeran), cyclophosphamide (Cytoxan, Neosor, Endoxus), ifosfamide (isophosphamide, Ifex), melphalan (Alkeran, Phenylalanine Mustargen, L-Pam, L-Sarcolysin), Nitrogen Mustargen (mechlorethamine, Mustargen, HIV2), Nitrosonceas (carmustine, CBCNV, bischlorethyl, nitrosourea), lomustine (CCNV, cyclohexyl chlorethyl, These include, but are not limited to, Nitrosouren, CeeNV), Semustine (Methyl-CCNV), and Streptozotocin (Streptozotocin, Streptozoticin, Zanosar (Zanosan), Thiotepa (Thio-TEPA, and Triethylenethrophosphoranide).

[0024] Agents with alkylating agent activity include a class of compounds that include heavy metal alkylating agents (platinum complexes) that act primarily by covalent bonding, and "non-classical alkylating agents" are also within the scope of this disclosure. Such agents typically contain a chloromethyl group and a critical N-methyl group. Such other agents include, but are not limited to, Amsacrine (m-AMSA, msa, Acridinylanisidiale, 4'-)(9-Acridinylamines)methanesulfine-m-anesizide, Carboplatin (Paraplatin, Carboplatinum, CBDCA), Cisplatin (Cesplatinum), Dacarbazine (DTIC, DIC, dimethyltricizenormidazoleconboxamide), Hexamethylmelanine (HMM, Altretanine, Hexylin), and Procarbazine (Matulane, Natulanan).

[0025] Also included within the scope of this disclosure are antimetabolites, such as azacitidine (5-azacylidine, radakamycin), 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), and the like. Examples include, but are not limited to, cyclosporine (cyclosporine), hydroxyurea (hydroxycarbamide, Hydrea), leucovorin (leucovorin calcium), mercaptopurine (G-MP, Purinethol), methotrexate (amethopterin), mitoguazone (methyl-GAG), pentostatin (2'-deorycoformycin), and thioguanine (6-TG, aminopurine-6-thiol-hemihydrate).

[0026] Antitumor antibiotics usually interfere with DNA by intercalation, which allows the drug to fit between DNA base pairs. The introduction of ultrasound enhances this interference. Such drugs include, but are not limited to, Actinomycin DC Cosmegen, Dactinomycin), Bleomycin (Blenoxane), Daunoxubibin (Rubidomycin), Doxorubicin (Adriamycin, Hydroxydaunorubicin, Hydroxydaunomycin, Rubex), Idarubicin (44-demethylorydan norubicin, Idamycin), Mithracin (Mithracin, Plicamycin), Mitomycin C, and Mitorantione (Novantrone). Plant alkaloids bind to microtubule proteins, thereby inhibiting microtubule assembly, and energy such as laser or ultrasound may enhance such binding. Such alkaloids include, but are not limited to, Etoposide, Paclitaxel (Taxol), Treniposide, Vinblastine (Velban, Velsar, Alkaban), Vincristine (Oncovin, Vincasar, Leulocristine), and Vindesine (Eldisine).

[0027] Hormonal agents include steroids and related agonists and antagonists, such as, but not limited to, corticosteroids, corticosteroid inhibitors, mitotane, androgens, antiandrogens, antiestrogens, estrogens, LHRH agonists, and progesterone. Angiogenesis inhibitors include, but are not limited to, the fumagillin derivative TNP-470, platelet factor 4, interleukin-12, the metalloproteinase inhibitor batimastat, carboryaminatriarzole, thalidomide, interferon alpha-2a, linomide, and the sulfated polysaccharide Tecogalan (DS-4152).

[0028] Specific examples of therapeutic agents that can be used in various embodiments include mycophenolic acid, mycophenolic acid derivatives (e.g., 2-methoxymethyl 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 (HYPOCA™), ASCOMYCIN™, WORTMANNIN™, LY294002, CAMPTOTHECIN™, flavopiridol, isoquinolines, HA-1077 (1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride), TAS-301 (3-bis(4-methoxyphenyl)methylene-2-indolinone), TOPOTECAN™, hydroxyurea, TACROLIMUS™ (FK 506), cyclophosphamide, cyclosporine, daclizumab, azathioprine, prednisone, diferuloymethane, diferulylmethane, GEMCITABINE™, cilostazol (PLETAL™), tranilast, enalapril, quercetin, suramin, estradiol, cycloheximide, tiazofurin, zafurin, AP23573, rapamycin derivatives, non-immunosuppressant analogs of rapamycin (e.g., rapalogs, AP21967, derivatives of rapalogs), CCI-779 (analogs of rapamycin available from Wyeth), sodium mycophenolate acid), benidipine hydrochloride, sirolimus, rapamine, metabolites, derivatives, and / or combinations thereof.

[0029] In other embodiments, therapeutic agents may include cytokines, including, but not limited to, IL-2, TNF-, IL-12, and the like. The systems and devices of the present disclosure can be configured to release or make available therapeutic agents in one or more treatment phases, the one or more treatment phases having the same or different performance (e.g., delivery) profiles. The therapeutic agents can be made available to the tissue in sustained, intermittent, or continuous amounts; in one or more phases and / or rates of delivery. Any one of the at least one therapeutic agent can perform one or more functions, including preventing or inhibiting proliferative activity, inhibiting or inhibiting tumor formation and / or growth, etc.

[0030] The total amount of therapeutic agent made available to the tissue depends in part on the level and amount of therapeutic result desired. The therapeutic agent can be made available in one or more phases, with each phase having a similar or different release rate and duration than the other phases. The release rate can be predefined. In embodiments, the rate of release can provide a sustainable level of therapeutic agent to the treatment site. In another embodiment, the rate of release is substantially constant. The rate can be decreased and / or increased as desired. These therapeutic agents may be provided and / or delivered to a subject or target in any conventional therapeutic form or formulation, such as, by way of example only: liquid, powder, particles, microbubbles, microspheres, nanospheres, liposomes, and / or combinations thereof. Some embodiments of the present disclosure may also include delivering at least one therapeutic agent and / or optional compound into a subject or target simultaneously or following an interventional procedure. More specifically, the therapeutic agent can be delivered to a target site, including a treatment site, simultaneously or following an interventional procedure. For example: (a) the therapeutic agent can be delivered to a treatment site as the sole therapy for the treatment of a tumor, without other concurrent treatments such as those provided by physical or mechanical intervention; (b) the therapeutic agent can be delivered to a treatment site as the sole therapy for the treatment of a disease (e.g., a tumor); (c) the therapeutic agent can be delivered to a treatment site following any suitable interventional procedure; (d) the therapeutic agent can be delivered to a treatment site before, during, after an interventional procedure, or a combination thereof.

[0031] Therapeutic agents can be made available to the treatment site in sustained, intermittent, or continuous amounts; in one or more phases; and / or rates of delivery. Disclosed herein is a microtransducer-based catheter that overcomes current anatomical barriers (as described above) for HIFU therapy by a) directly impacting the tumor and ablating primary and metastatic lesions, b) disrupting tumor extracellular matrix with or without contrast agents to facilitate intratumoral delivery of therapeutic agents, and c) using different acoustic energy levels to sonoporate (pulsed blocking of tumor cell membranes to increase permeability) and allow nucleotide drugs such as mRNA to enter living tumor cells. The microtransducer-based catheter is configured to deliver therapeutic agents directly to tumors using image-guided microtransducer HIFU catheters using currently available imaging equipment and technology. The device is configured to deliver a range of high-pressure acoustic bursts of focused ultrasound to form intratumoral acoustic cavitation, where the expansion and collapse of microbubbles releases high-pressure cavitation energy that disrupts physical barriers in tumors and cancer cells by membrane disruption, allowing for a variety of ablations and / or effective intratumoral retention and uptake of therapeutic agents.

[0032] 2A illustrates a forward-facing ultrasound (US) transducer 200 according to some embodiments. The transducer 200 includes multiple layers. For example, as illustrated, the transducer 200 includes a dual-layer PZT-5A layer 202A, a matching (Al2O3 / epoxy) layer 202B, and a backing (foam / epoxy) layer 202C. The aperture area is approximately 2×2 mm 2 and the operating frequency is set to approximately 0.8 MHz. Furthermore, in other embodiments, the piezoelectric material may vary, the size of the aperture area may vary, and the operating frequency may vary. In some embodiments, the US transducer 200 can be configured as a tubular HIFU transducer. For example, Fig. 3A shows a tubular HIFU transducer 300 for sonoporation. Fig. 3B shows a schematic diagram 302 of the transducer 300 including a tube-type piezo 304A and a matching layer 304B. 4A depicts a miniaturized HIFU transducer 400 according to 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 six or less than four active elements 404. Additionally, in other embodiments, each element 404 may vary in size.

[0033] The US transducer 200 may be configured as a transcatheter HIFU device such as those described herein (e.g., transducer 400 of FIG. 4A), providing, for example, localized delivery of M-HIFU and IL-12 gene delivery to tumors (described in more detail below). For example, FIG. 5A shows another example embodiment of a HIFU transducer 400 that may be integrated with the hollow tube 104 of the device 102 of FIGS. 1A, 1B. For example, the transducer 400 may be integrated into a 6-10 Fr catheter with a drug delivery lumen. The transducer 400 includes multiple active elements 404, each of which includes multiple layers, including, for example, a PZT-5H material layer 406A and a matching layer 406B. In a non-limiting example, PZT-5H may be used in place of PZT-4 material, since the histotripsy device 400 may operate with a relatively short pulse signal (i.e., about 30 cycles and a duty cycle of less than 1%). The thickness of each active element 404 may range from less than about 50 μm to about 500 μm. The opening of each element 404 is 1.4×1.8 mm 2and may range from about 1 mm by less than 1 mm to about 3 mm by 3 mm depending on the energy needs and application. In other embodiments, the transducer 400 may include more than six or less than four active elements 404. Additionally, in other embodiments, each element 404 may vary in size.

[0034] The US transducer 200, 400 is introduced through a hollow tube or catheter 104 as illustrated in FIG. 16B. The catheter 104 includes a first lumen 210 and a second lumen 212. The lumens 210, 212 can be located laterally along the length of the catheter or at the distal end of the catheter. For example, as illustrated in FIG. 16B, one of the lumens 210, 212 is located laterally in 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 and emits US waves through the lumen 210, 212. The catheter 102 traverses the subject to reach the target site (e.g., tissue, tumor, etc.), where the US transducer 200, 400 emits US waves into the subject. The parameters of the US transducers 200, 400 are preferably selected for the purpose of ablation, sonoporation or histotripsy, or application of a HIFU procedure to a target.

[0035] Example 1 Miniaturized HIFU transducer A forward-facing ultrasound (US) transducer 200 illustrated in Figure 2A was designed for sonoporation studies. The transducer 200 included a dual-layer PZT-5A layer 202A, a matching (Al2O3 / epoxy) layer 202B, and a backing (foam / epoxy) layer 202C. The aperture area was approximately 2 x 2 mm. 2 and the operating frequency was 0.8MHz. FIG. 2B is a graph 250 illustrating the transmitting sensitivity of the transducer 200 as a function of input voltage (Vpp), where the acoustic pressure was measured at a distance of 2 mm from the aperture surface of the transducer 200. FIG. 2C depicts the application of the US transducer 200 to treat HEK293 cells in a 96-well plate in the presence of plasmid DNA (pCDH-GFP-LUC, 5 μg / mL). FIG. 2D is a fluorescent image of GFP+ sonoporation-treated cells treated with the transducer of FIG. 2A according to some embodiments, as well as fluorescent images of luciferase activity in negative control cells and sonoporation-treated cells. As shown in images 275A-275D of FIG. 2D, focal GFP expression was observed only in sonoporation-treated cells and not in negative control cells (no US exposure). The LUC activity level was up to 3,600 units (846.0 + / - 786.5 units) in sonoporation-treated cells, while it was less than 170 (76.4 + / - 78.6 units) in the negative control, indicating successful transfection of HEK cells with pCDH-GFP-LUC by sonoporation treatment. Sonoporation can be further improved in terms of the selection of relevant ultrasound parameters, appropriate dose of contrast agent, and appropriate treatment time.

[0036] A tubular HIFU transducer was also designed for sonoporation testing. Figure 3A shows a tubular HIFU transducer 300. Figure 3B shows a schematic diagram 302 of the transducer 300, including a tube-type piezo 304A and a matching layer 304B. Figure 3C illustrates the simulated acoustic field profile 325 generated by the transducer 300. The simulated experiment was performed with an input voltage of 80V. ppand an operating frequency of 0.85 MHz. It was found that peak negative pressure (PNP) in both lateral and longitudinal directions at sub-MHz (e.g., 0.85 MHz) can induce cavitation, which is essential for the sonoporation process. Measured acoustic profiles in both side-looking and forward-looking directions (profiles 350A and 350B, respectively) are illustrated in FIG. 3D. The measurements yielded PNP values ​​(approximately 0.95 MPa for side-looking PNP and 0.55 MPa for forward-looking PNP) similar to those obtained from the simulated experiments (0.81 MPa for side-looking direction and 0.47 MPa for forward-looking direction).

[0037] With sonoporation techniques, tumors or cancers are gradually suppressed. In contrast, histotripsy and tissue ablation techniques aim to immediately remove or ablate malignant tumor tissue. Interstitial tissue ablation devices are a relatively mature technology, and this modality basically utilizes long US wave pulses to induce thermal necrosis in the target lesion. However, miniaturization of HIFU transducers remains a challenge for catheter-directed tissue ablation. Miniaturized histotripsy transducers are very rare. In contrast to typical thermal ablation techniques, histotripsy utilizes very short (less than 1-2% duty cycle) US wave pulses and high rarefactional pressure output (more than 10 MPa). However, most histotripsy transducers operate on the external surface of the human body. In recent years, several researchers have presented a relatively small size (approximately 5 mm) forward-facing histotripsy transducer for treating brain tumors. However, interstitial or transcatheter HIFU capable of histotripsy is challenging due to the requirement of high expanded acoustic pressure output from a small (<2 mm) HIFU orifice.

[0038] 4A depicts a miniaturized HIFU transducer 400 according to some embodiments. The catheter HIFU transducer 400 is configured to have a center frequency of 5 MHz and, in the illustrated embodiment, includes five active elements 404 (e.g., PZT-4 plates). The aperture of each element 404 is 1.4×1.8 mm 2 In other embodiments, the transducer 400 may include more than six or less than four active elements 404. Additionally, in other embodiments, each element may vary in size. FIG. 4B shows the acoustic pressure output 425 generated by the HIFU transducer 400 (line 426A shows the peak-to-peak pressure, and line 426B shows the peak negative pressure). The quadratic regression curve shows that at 300V pp It shows that input power is necessary to generate a negative pressure of more than 13 MPa. Next, a bubble generation test was performed to confirm the effectiveness of the HIFU transducer 400 for potential histotripsy treatment. The transducer 400 was sonicated in distilled degassed water, and a portable ultrasound imaging probe (iQ+, Butterfly Network, Guilford, CT) was used to detect evaporation during sonication (described in more detail below with respect to FIG. 5C). The whitened spot 452 in the captured image 450 of FIG. 4C indicates water evaporation induced by the HIFU transducer 400, demonstrating the capability of histotripsy treatment. In general, the center frequency of transcatheter HIFU transducers for tissue ablation and histotripsy can range from about 100 kHz to 7.5 MHz.

[0039] The US transducer 200 may be configured as a transcatheter HIFU device such as those described herein (e.g., transducer 400 of FIG. 4A), providing, for example, localized delivery of M-HIFU and IL-12 gene delivery to tumors (described in more detail below). For example, FIG. 5A shows another example embodiment of a HIFU transducer 400 that may be integrated with the tube 104 of the device 102 of FIGS. 1A, 1B. For example, the transducer 400 may be integrated into a 6-10 Fr catheter with a drug delivery lumen. The transducer 400 includes multiple active elements 404, each of which includes multiple 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 in place of PZT-4 material because the histotripsy device 400 operates with a relatively short pulse signal (i.e., about 30 cycles and less than 1% duty cycle) and mechanical losses associated with transducer self-heating may not be an issue. The thickness of each active element 404 can be about 400 μm. A 3D printed mount can act as the confocal geometry and light backing. Electrodes between the individual piezoelectric plates (not shown) of the active element 404 can be connected using, for example, coaxial cables and conductive silver epoxy.

[0040] 5B shows the sound pressure field profile 525 of the transducer 400 under 3.5 MHz frequency operation. In the linear region, the maximum extended sound pressure level is expected to exceed 24 MPa. The -6 dB focal zone is approximately φ0.6×3.8 mm. 3 For 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 three-dimensional motion-controlled stage. Following acoustic characterization of cavitation generation using transducer 400, setups 550A and 550B shown in Figures 5C and 5D, the results of which are illustrated in Figure 4C. In addition to ultrasound imaging of evaporated bubbles 552, a high-speed camera and ultrasound imaging probe 554 (e.g., iQ+, Butterfly Network, Guilford, CT) was used to visualize / capture images of bubble generation in distilled, degassed water media.

[0041] In some embodiments, the transducer 400 provides a maximum expansion sound pressure level of more than 15 MPa for vaporization of general organ tissue. The histotripsy procedure was performed within a relatively short time (less than 2 minutes) and removed the tumor in situ within a precise treatment volume (less than φ1×5 mm3). The histotripsy transducer 400 was configured to have strong ablation for histotripsy, and was expected to induce apoptotic death of all tumor cells in the treatment area based on accumulated data from gene transfer and histotripsy tests using the prototype. Alternatively, a PMN-PT single crystal with a piezoelectric constant (d33, greater than 2,500 pC / N) much higher than that of PZT-based composites (d33, less than 700 pC / N) was used to provide high negative pressure and allow for a larger aperture (e.g., 8-10 Fr).

[0042] In another exemplary method of treatment, the addition of systemic administration of ICB (anti-PD-1) to LM-HIFU+IT-IL-12 therapy enhances the antitumor effect of ICB. In a previous study, anti-PD-1 antibody was combined with IT-IL-12 treatment, which not only significantly suppressed tumor growth in multiple TNBC tumor models, but also led to complete tumor regression and long-term tumor-free survival in some of the treated mice. Interactome analysis of TILs in IT-IL-12 therapy showed that interactions between antigen presentation, myeloid populations, and CD8 T cells were enriched, and the CXCR3 gene signature was enhanced after IT-IL-12. Expression of the CXCR3 gene signature was found in tumors enriched in CD8 T cells and was also associated with improved PFS and OS in patients with TNBC. For ease of explanation, the transducers described in the example systems and methods below will be 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) may be utilized in such systems and methods.

[0043] Example 2 Methods of Use for the Treatment of Cancer Exemplary embodiments of HIFU devices including transducers 200, 400 described herein above may be used in combination with immunotherapy for multiple types of treatment, including, for example, several types of cancer. In an exemplary embodiment, the method of treatment includes using a miniaturized transcatheter LM-HIFU device (e.g., transducer 400 of Figs. 4A and 5A) that can destroy tumor tissue and induce efficient gene expression in tumor cells or tumor-infiltrating immune cells by efficient gene transfer. As described herein, we discovered that intratumoral administration of plasmid IL-12 with electroporation (IT-pIL12-EP, shown as TAVO in Figs. 6A-6E, 7A-7B, 8A-8D, described in more detail below) significantly suppressed the growth of triple-negative BC (TNBC) in mice and extended survival time (as illustrated in plots 600A and 602A of Fig. 6A). In particular, plots 600A and 602A in FIG. 6A represent data for BALB / c mice bearing murine 4T1 tumors that received intratumoral administration of mIL12-P2A or control plasmids followed by in vivo electroporation on day 0, and the inhibition of 4T1 tumor growth and survival of the mice by treatment (IL-12 group: 13 mice, control group: 14 mice). Furthermore, as shown in plots 600B (treated tumors) and 602B (untreated tumors) in FIG. 6B, the treatment provided protection in untreated and treated lesions by induction of systemic antitumor immunity. In particular, plots 600B and 602B represent the abscopal effect 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 graph represents data for treated tumors, whereas the right graph represents data for tumors on the untreated side. Single-cell RNA sequencing (scRNA-seq) of TILs revealed that IT-pIL12-EP treatment resulted in a significant increase in T cells and dendritic cells, as well as a decrease in neutrophil infiltration into the tumor, as shown in plot 600C of Figure 6C (t-SNE plot of cells categorized by cell type for all samples or separated by treatment group, including a proportion analysis of each cell type in each treatment group).T cells were more activated and clonally expanded in IT-pIL12-EP treated tumors compared to control treated tumors, supporting the hypothesis of an adaptive immune response induced by local delivery of IL-12 (plots 600D and 600E in Figures 6D and 6E, respectively). Plot 600D represents the (Lt) quantification of the frequency of each clone, with the top 50 clones for each treatment shown in colored bars. (Rt) UMAP showing reclustering of all TCR+ cells from those colored by clonal expansion (<10=blue; >10=red). Plot 600E represents the (Lt) quantification of the activation signature score across all T cells in each treatment. (Rt) UMAP colored by 50 gene activation signature score. All error bars represent the mean ± SEM. * p<0.05 *** p<0.001. Over 1600 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 enrichment of antigen presentation, cytokine, chemokine, and PDL1 pathways in IT-pIL12-EP treated tumors. Interactome analysis of scRNA-seq data showed that the top interactions between receptors on macrophages and ligands on CD8 T cells highlight the CXCL9 / 10 / 11 / CXCR3 axis (plot 700A in Figure 7A). Plot 700A is a Circos plot depicting receptor-ligand interactions between receptors on CD8 T cells and ligands on macrophages. Bonds shown in red represent the top 25% of interactions between these cell types. The CXCR3 gene signature and the top 50 genes most highly associated with CXCR3 were identified, and IT-pIL12-EP treated tumors were found to have a significantly higher CXCR3 signature score than cells from control treated tumors (as shown in plot 700B of FIG. 7B). Plot 700B illustrates the 50-gene CXCR3 gene signature quantified across all cells. All error bars represent the mean ± SEM. *** p<0.001.

[0044] Overall, these analyses demonstrate increased infiltration and activation of CD8 T cells with IT-pIL12-EP treatment, while the mixed expression of markers of activation and exhaustion and enrichment of pathways such as PD-1 / PD-L1 indicate 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. Plots 800A-800D in Figures 8A-8D illustrate tumor curves from the combination therapy, respectively. Plot 800A represents the growth inhibition of 4T1 tumors with the combination therapy. Tumor volumes were measured every other day (control: 7 mice, anti-PD-1: 9 mice, IL-12: 7 mice, combination: 10 mice). Plot 800B represents the growth inhibition of JC-HER3 tumors with the combination therapy. Tumor sizes were measured every other day (control: 7 mice, anti-PD-1: 6 mice, IL-12: 8 mice, combination: 7 mice). Plots 800C and 800D represent mouse survival in each treatment group for the 4T1 tumor model (C) and the JC-HER3 tumor model (D). All error bars represent the mean ± SEM. * P < 0.05, ** P < 0.01, *** P<0.001. The combination not only significantly delayed tumor growth in multiple TNBC models, but also led to complete tumor regression and long-term tumor-free survival in two TNBC models (28.6% in the 4T1 model and 75.0% in the JC-HER3 model).

[0045] The clinical safety of the therapy was examined in a single-arm, prospective clinical trial of intratumoral pIL-12 (TAVO) monotherapy (OMS-140) in previously treated 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 following pIL-12 monotherapy in preclinical studies. A systemic antitumor immune response to distant, untreated tumors was also observed, without evidence of cytokine storm or other toxicity. Importantly, one patient who had previously failed to respond to anti-PD-1 and showed an increase in CXCR3 gene signature score with pIL-12 treatment immediately received anti-PD-L1 therapy as a next-line treatment and showed a large clinical response (as shown in images 900A and 900B in Figure 9, where image 900A represents an exemplary baseline image of lung metastases and image 900B represents lung metastases after one cycle of IL-12 treatment and three months of nivolumab therapy). These data identify gene signatures that represent functional biomarkers that convert non-responders into responders.

[0046] Example 3 Combining disruption of the tumor microenvironment with localized immunotherapy One barrier to the use of therapeutics is the tumor microenvironment (TME), which is composed of cancer cells and stromal compartments including fibroblasts, myofibroblasts, leukocytes, endothelial cells, macrophages, adipocytes, and extracellular matrix (ECM). Breast cancer has various levels of stroma, as shown in FIG. 10, which includes images 1000A-1000C, which respectively represent fibrous tumor stroma in breast cancer at different stages and ECM formed by stromal proteins (collagen, elastin, fibronectin, and laminin), which form a physical barrier that prevents intratumoral drug distribution and direct contact of tumor cells with drugs or tumor-infiltrating immune effector cells. As shown in images 1100A and 1100B of FIG. 11, intratumoral injections of drugs often failed, with the drug leaking into the surrounding tissue and very little drug delivered into the tumor. Image 1100A represents a nearly complete injection, while image 1100B represents minimal drug retention with leakage. It has been demonstrated that non-invasive HIFU and IT-IL-12 gene delivery to solid tumors can be used to enhance immune infiltration and induce systemic antitumor immune responses. HIFU may also be useful in disrupting the stromal barrier in tumors, enhancing IT-IL-12 gene therapy by improving the distribution of IL-12 expression vectors in tumors.

[0047] In order to disrupt the interstitial barrier and tumor cells and promote immune infiltration, a novel HIFU strategy that mechanically destroys cells and stroma, in contrast to conventional thermal HIFU (T-HIFU) therapy that results in coagulation necrosis, has been determined and described herein. An external HIFU system was used (VIFU2000 Wet System for Small Animals, Alpinion) to achieve mechanical disruption by acoustic cavitation of the TME enabled by the use of high pressure burst exposure, termed mechanical HIFU, M-HIFU, or histotripsy. M-HIFU induced mechanical damage to tumor tissue and tumor stroma and blood vessels, resulting in cavitation with internal bleeding in an immune-competent model of BC (FIG. 12, including images 1200A and 1200B illustrating how murine MC38 tumors grown in the thigh were treated by M-HIFU and T-HIFU). Histological analysis of tumor tissues taken 1, 7, or 11 days after M-HIFU treatment revealed enhanced immune infiltration, including T cell infiltration, into the tumor (as shown in graph 1300A and table 1300B of FIG. 13). H&E staining of tumor tissues 1 day after M-HIFU treatment, as illustrated in graph 1300A. Arrows indicate mechanically disrupted areas with immune cell infiltration. Increased CD8+ T cell infiltration was evident 7 days after M-HIFU. As presented in Table 1300B, the mean of CD4, CD8, or FoxP3 staining+ cells (average of 5 HPFs for each tumor) is shown for each treatment. Bars represent mean ± SE, n=3. * :p-value<0.05.

[0048] MM3MG-HER2 cells were implanted into the right thigh and left flank of female BALB / c mice. Seven days later, thigh tumors were subjected to M-HIFU in the right M-HIFU monotherapy group or combination group. Anti-PD-L1 antibody (100 μg / injection) or isotype control IgG (100 μg / injection) was injected intraperitoneally on days 10, 13, and 16. Tumor growth curves of HIFU-treated thigh tumors and untreated distant tumors are shown. Data in graphs 1400A and 1400B represent the results from two pooled experiments, n=13-19 per group. Error bars: mean ± SD. * :p<0.05, ** :p<0.01, *** :p<0.001, **** :p<0.0001. The superior antitumor efficacy of M-HIFU in tumor growth inhibition and improved survival compared to conventional T-HIFU was demonstrated in immune-competent MM3MG-HER2 mouse breast cancer model. As shown in plots 1400A and 1400B of Figure 14, a moderate abscopal effect on untreated distant tumors was observed with M-HIFU monotherapy, which could be enhanced by combined treatment of M-HIFU and PD-1 / PD-L1 blockade.

[0049] Because both IT-IL-12 gene therapy and M-HIFU treatment can induce antitumor effects by non-overlapping mechanisms and both modalities appear to synergize with anti-PD-L1 antibody therapy, we hypothesized that the combination of M-HIFU and IT-IL-12 gene therapy would allow a broader distribution of IL-12 expression vectors into tumors and induce better T cell infiltration into tumors by disrupting the interstitial barrier. To test this hypothesis, mice bearing E0771 tumors were treated with IL-12 gene therapy alone, M-HIFU alone, or a combination of both. As shown in plot 1500A of Figure 15, tumor growth inhibition was strongest in the combination treatment group, but no statistical difference was observed between IL-12 alone and the combination treatment at this relatively early onset of intervention. Next, when tumors grew to approximately 500 mm 3When relatively late intervention (28 days) was performed in E0771 tumor-bearing mice (plots 1500B and 1500C), it was found that the combination of IL-12 gene therapy and M-HIFU induced stronger tumor growth inhibition and prolonged mouse survival compared to IL-12 gene therapy alone. With reference to plot 1500A, C57BL / 6 mice bearing E0771 tumors were treated with IT-IL-12 gene therapy alone, M-HIFU alone, or their combination 28 days after tumor cell implantation. Tumor volumes were monitored every 2 days. n=4-5 mice. The combination treatment inhibited tumor growth significantly better than HIFU alone (p<0.01). With reference to plot 1500B, interventions were started 28 days after E0771 cell implantation. Mice received either IL-12 gene therapy alone or IL-12 in combination with HIFU. N=8 mice. The combination treatment induced significantly stronger tumor growth inhibition (AUC analysis, t-test: p<0.0001). Finally, with reference to plot 1500C, mouse survival is shown. Log-rank test: p<0.005.

[0050] As shown in images 1600A and 1600B of Figures 16A and 16B, respectively, an exemplary treatment strategy is illustrated for metastatic breast cancer (BC). The disclosed method includes a transcatheter HIFU transducer (e.g., transducer 200, 400) integrated with a catheter (e.g., device 102 of Figures 1A and 1B) that is inserted into a metastatic tumor (such as liver, lung, brain, or bone metastasis) under ultrasound imaging or MR imaging 1602, and performs histotripsy (M-HIFU) to destroy tumor cells and tumor-stroma barrier, and an IL-12 expression vector is delivered locally to induce increased expression of IL-12 in the TME. The miniaturized HIFU transducer 200 can be inserted directly into primary or metastatic BC, even if the primary or metastatic BC is deep-seated or located in areas that are difficult to target by HIFU outside the body, such as the hepatic dome, behind the ribs, near large blood vessels, or deep in the lungs.

[0051] In particular, as illustrated in images 1600A and 1600B, a transcatheter HIFU transducer was inserted into the tumor site using a guiding needle under ultrasound imaging or MR imaging. Histotripsy (M-HIFU) was performed to destroy tumor cells and tumor stromal barrier, and an IL-12 expression vector was injected into the tumor through another lumen of the catheter. The treated tumor was eradicated by tumor destruction and induction of inflammatory TME and recruitment of cytotoxic immune cells. The induced systemic antitumor immunity, enhanced by IT-IL-12, was expected to eradicate distant metastatic tumors. To enhance the systemic antitumor immunity induced by IT-IL-12, ex vivo M-HIFU was combined with IT-IL-12 and the combined treatment was found to be more potent than IT-IL-12 alone. Therefore, this combination strategy was proposed to treat metastatic BC. This localized therapy can be applied to metastatic breast tumors located in or on visceral organs such as liver, lung, and brain. Transcatheter HIFU devices, such as those described herein (e.g., device 102 including transducers 200, 400), provide localized delivery of M-HIFU and IL-12 gene delivery to tumors.

[0052] Example 4 IN VIVO FUNCTIONALITY OF MINIATURED HIFU TRANSDUCER The TME altered by LM-HIFU+IT-IL-12 therapy alone and in combination with ICB was analyzed using a combination of pathological analysis and scRNA sequencing to confirm the in vivo functionality of the catheter LM-HIFU device 102. Next, the antitumor effect and induced tumor-specific immune response by LM-HIFU+IT-IL-12 combination therapy with and without anti-PD-1 mAb were evaluated, and the abscopal effect and inhibition of lung metastasis were demonstrated in a TNBC model. Finally, pathological and scRNA-seq analyses were used to evaluate whether there were significant changes in the TME in LM-HIFU+IT-IL-12 treated tumors and an increase in the CXCR3 gene signature in TILs. E0771 BC cells were subcutaneously implanted into the flank of female C57BL / 6 mice. When the tumors reached a size of 7-8 mm, the tumors were treated with LM-HIFU using a transcatheter device 102, and Ad-IL-12 (1010 viral particles / tumor in 50 μL saline) was simultaneously injected into the tumor through a dual lumen catheter. The following groups were created; a) no treatment, b) LM-HIFU alone, c) IT-IL-12 alone, and d) LM-HIFU+IT-IL-12. Mice in group b received only LM-HIFU without Ad-IL-12 injection, and mice in group c received only Ad-IL-12 injection without HIFU energy. (n=8 mice per group, 4 mice were terminated on day 3, and 4 mice were terminated on day 7). On days 3 and 7 after the procedure, 4 mice in each group were euthanized on the respective days, and tumors and blood were collected for assay.

[0053] Half of the tumor tissue was used for pathology analysis to assess 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 isolated immune cells were analyzed by flow cytometry. Tumor lysates and serum were used to analyze IL-12 protein levels by IL-12 ELISA. We determined whether LM-HIFU treatment enhanced tumor tissue destruction and T cell infiltration into the tumor at early time points and whether IL-12 levels increased in IT-IL-12 treated tumors but not in blood. We determined how LM-HIFU+IT-IL-12 altered tumor-infiltrating immune cells using single cell secretome.

[0054] The induction of systemic tumor-specific immune responses and antitumor effects of combined LM-HIFU+IT-IL-12 therapy on mouse TNBC were demonstrated by the following method. E0771-OVA mouse BC cell line was implanted into the flank of female mice (C57BL / 6). When the tumor size on the right flank reached approximately 7-8 mm in diameter, LM-HIFU or IT-IL-12 treatment was performed on the tumor as a monotherapy or combination therapy. A catheter equipped with a miniaturized HIFU transducer and a drug injection lumen was inserted into the tumor, and LM-HIFU and / or IT-IL-12 were performed.

[0055] 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, of which 4 mice were terminated on day 7). Four mice from each group were euthanized 7 days after treatment. Tumors, spleens, and blood were harvested and subjected to IFN-γ ELISPOT assay (OVA peptide (SIINFEKL) as stimulatory antigen), ELISA (antibody against OVA protein), and flow cytometry using H-2Kb / OVA (SIINFEKL) MHC tetramer. 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). Tumor tissues were digested with a triple enzyme buffer (collagenase III, hyaluronidase, and DNase) and the immune cell populations were characterized by multiparameter flow cytometry on cells isolated from the tissues, including CD4 helper T 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 (TAM) (CD11b / F4-80 / CSF-1R), and myeloid-derived suppressor cells (MDSC) (CD11b / Gr-1). The activation status of T cells is analyzed using anti-CD25, CD69, ICOS, PD-1, CD107, and granzyme-B. Expression of immune checkpoint molecules CTLA-4, PD-L1, TIM3, LAG3, and TIGIT is also analyzed as a marker of cell exhaustion. Expression of immune checkpoint molecules CTLA-4, PD-L1, TIM3, LAG3, and TIGIT is also analyzed as a marker of cell exhaustion. Cells from the spleen were also analyzed by flow cytometry and compared to TILs.Furthermore, fluorescence microscopy analysis was performed by staining tumor tissues with fluorescently labeled anti-CD3, anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-CD19, anti-CD49b, anti-F4 / 80, anti-Ly6G, anti-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. Pictures 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.

[0056] For scRNA-seq, tumor tissues were digested as described above and live tumor-infiltrating immune cells (viability dye, CD45+ cells) were FACS sorted. 10x libraries were generated using the Chromium Single Cell 5' Library Construction Kit (v1.1) following the manufacturer's protocol. Both gene expression and V(D)J enriched libraries were generated for each sample. Generated cDNA and final GEX / TCR libraries were quality checked using an Agilent Bioanalyzer 2100 and sequenced on a NovaSeq S4 instrument. Fastq files from 10x library sequencing were processed using Partek® Genomics Suite® software (Version 9.0.20, Copyright; 2018 Partek Inc). Identified clusters were visualized using UMAP plots using the first 15 principal components, a minimum distance of 0.4, and 30 neighbors42. 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 5% FDR with a fold change > |2|. Clustering of single-cell data, cell type identification, and differential gene expression analysis were performed blinded to treatment group for blinding. Samples from four groups were compared. As previous studies have identified an increased CXCR3 gene signature with enhanced antigen presentation, systemic T cell expansion and licensing in IT-IL-12-treated TNBC, we assessed whether combined LM-HIFU+IT-IL-12 treatment using a catheter LM-HIFU device would induce similar changes in the TME.

[0057] The other 10 mice were treated with CT scans at a humane endpoint (tumor volume of 2,000 mm 3Tumor size was measured three times a week until it reached a median tumor volume (>100 μg / kg) and mouse survival was assessed. Percentage of tumor growth inhibition (TGI%), defined as the percentage difference between median tumor volume (MTV) of treated and control mice, was also compared. To confirm the induction of potent systemic antitumor immunity, long-term surviving mice after tumor eradication were subjected to tumor rechallenge 12 weeks after LM-HIFU+IT-IL-12 treatment. Tumor growth / tumor-free survival was monitored up to 2 months. This task was intended to confirm that combined LM-HIFU+IT-IL-12 treatment was superior to IT-IL-12 or LM-HIFU monotherapy in terms of induction of antitumor immunity and tumor growth inhibition. The abscopal effect induced by LM-HIFU+IT-IL-12 therapy and the enhanced antitumor effect by combination with anti-PD-1 antibody in a bilateral TNBC transplantation model were demonstrated as follows: E0771-OVA cells were bilaterally transplanted into the flanks of female C57BL / 6 mice. When the tumor size in the right flank reached approximately 7-8 mm in diameter, LM-HIFU+IT-IL-12 treatment with or without anti-PD-1 antibody was performed on the tumor in the right flank, while the tumor in the contralateral side was left untreated.

[0058] 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, of which 4 mice were terminated on day 7). Anti-PD-1 Ab or control IgG (200 μg / injection) were administered intraperitoneally on day 3 after LM-HIFU+IT-IL-12 treatment and repeated twice weekly for 2 weeks. As described herein above, four mice from each group were euthanized 7 days after treatment and tumor samples taken from both treated local and untreated distant sites were analyzed. Harvested splenocytes and serum were used for IFN-γ ELISPOT assay, tetramer assay, and ELISA. scRNA-seq analysis and multiparameter flow cytometry were performed on TILs. TILs isolated from treated and untreated distant tumors were analyzed and compared for the four treatment groups (only group A had one TIL sample). It was evaluated whether LM-HIFU+IT-IL-12 therapy in combination with anti-PD-1 showed more pronounced TME changes compared to LM-HIFU+IT-IL-12 therapy alone. The difference in TME changes between treated and untreated distant tumors was also analyzed.

[0059] The other 10 mice were treated with CT scans at a humane endpoint (tumor volume of 2,000 mm 3 Tumor size (both HIFU-treated local tumors and untreated distant tumors) was measured three times a week until tumor size reached >100 nm (>100 nm) and mouse survival was compared between groups. TGI% was also compared. To confirm the induction of potent systemic antitumor immunity, long-term surviving mice after tumor eradication were subjected to tumor re-challenge 12 weeks after LM-HIFU+IT-IL-12 treatment. Tumor growth / tumor-free survival was monitored up to 2 months. This objective was to determine whether the combination of anti-PD-1 antibody and LM-HIFU+IT-IL-12 therapy is superior to LM-HIFU+IT-IL-12 therapy or anti-PD-1 antibody alone in terms of inducing systemic anti-tumor immunity and inhibiting distant tumor growth (abscopal effect).

[0060] Inhibition of spontaneous lung metastasis by LM-HIFU+IT-IL-12 therapy and anti-PD-1 antibody was demonstrated in the highly metastatic E0771-LX3-OVA model as follows: After three repeated orthotopic implantations of cells isolated from lung metastases of E0771 cells into C57BL / 6 mice, a highly metastatic variant named E0771-CH3-MX3 was established in the laboratory. This metastatic variant was confirmed to develop lung metastasis in all mice tested by 5-6 weeks after orthotopic implantation (5×10E5 cells / injection). E0771-CH3-MX3 cells were implanted into the fourth mammary fat pad of female C57BL / 6 mice. When the tumors grew to approximately 7-8 mm in diameter, LM-HIFU+IT-IL-12 treatment with or without anti-PD-1 antibody 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 2 weeks. Tumor growth and mouse activity, respiratory rate, appetite, and body condition of the mice were monitored. Mice were euthanized 6 weeks after tumor cell implantation and harvested lungs were evaluated macroscopically and microscopically for lung metastasis. Tissue sections were analyzed by H&E staining and IHC with anti-p53 antibody (E0771 cell line: p53+), and metastatic foci were quantified using Image J software. Fluorescence microscopy analysis was performed by staining frozen tumor tissues with fluorescently labeled anti-CD3, anti-CD4, anti-CD8, anti-CD49b, and anti-granzyme B antibodies to determine effector immune cells in metastatic nodules. Photographs were taken and the number of stained cells was analyzed using Image J software.

[0061] Using optimized intralesional immunotherapy, we investigated the antitumor effect of LM-HIFU+IT-IL-12 treatment on ER+BC and HER2+BC that were poorly responsive to ICB, and confirmed the induction of systemic antitumor immunity and abscopal effects by this combination treatment. The ovalbumin-expressing murine triple-negative BC cell line E0771 (E0771-OVA) was established using lentiviral vectors as previously described. Utilizing a non-transformed BALB / c mammary epithelial line (MM3MG), a HER2-driven BC cell line, MM3MG-HER2, was established by retroviral transduction of the receptor tyrosine-protein kinase erbB-2 (HER2). OVA and HER2 antigens were used in the assay to evaluate the induction of systemic antitumor immunity. Using DNA deep sequencing techniques, recent studies have identified several conserved mutations to the DNA-binding domain of ESR1 in a significant proportion (approximately 30-40%) of endocrine-resistant metastatic ER+ breast cancer patients. Because no established models of endocrine-resistant ER+ breast cancer exist in immunocompetent mice, an ER+ endocrine-resistant breast cancer cell line model was generated that grows in immunocompetent syngeneic animals. MM3MG cells were artificially transformed with wild-type or mutant human ESR1 (Y537N, Y537S, D538G).

[0062] As depicted in plot 1700A of FIG. 17, pre-cancerous MM3MG cells (BALB / c background) stably transduced with lentivirus were transfected with ERE luciferase reporter in estrogen-free or positive (20 nM E2) conditions along with transfection controls and harvested at 24 hours (N=6). In plot 1700B of FIG. 17, these cells were implanted into female mice (no estrogen pellets) and tumor growth was assessed over time by caliper measurements. In all panels, * represents p<0.05 from ESR1-WT control; ** indicates p<0.01. In this model, these ESR1 mutants were found to result in enhanced estrogen signaling in these cell lines (plot 1700A in FIG. 17), and indeed to "transform" them, enabling them to form more aggressive tumors that successfully engraft in mice when compared to their control and wild-type ESR1 counterparts (plot 1700B in FIG. 17). Crucially, these mice did not require exogenous estrogen supplementation to enable estrogen-mediated signaling or tumor growth. These cells were also found to be completely resistant to standard endocrine therapy (e.g., tamoxifen and aromatase inhibitors (data not shown)). Thus, the established mouse breast cancer cell line, MM3MG-ESR1mut (Y537N, Y537S, D538G), mimics the patient's metastatic ER+ breast cancer, which is endocrine resistant, harbors an ESR1 gene mutation, and behaves more aggressively.

[0063] The induction of systemic tumor-specific immune responses and antitumor effects of combined LM-HIFU+IT-IL-12 therapy on ER+ and HER2+ mouse breast cancer were demonstrated as follows. Mouse 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. LM-HIFU or IT-IL-12 treatment was performed on the tumor as a monotherapy or combination therapy, as described herein above in TNBC. A catheter LM-HIFU device with a drug injection lumen was inserted into the tumor, and combined LM-HIFU+IT-IL-12 treatment was performed with optimized settings.

[0064] 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, of which 4 mice were terminated on day 7). Four mice from each group were euthanized 7 days after treatment. Tumors, spleens, and blood were harvested and IFN-γ ELISPOT assays (ESR1 peptide mix or HER2 peptide mix as stimulating antigens in experiments 3.1, 3.2, respectively) and cell-based ELISA (parental E0771 / E0771-ESR1 cells or parental 4T1 / 4T1-HER2 cells in experiments 3.1 and 3.2, respectively, both cell lines available) were performed. Tumor samples were analyzed for IL-12 production by in situ hybridization (RNAscope) and IL-12 ELISA using tumor lysates as samples. To analyze the cell type and activation / maturation status of TILs, flow cytometry analysis was performed as described herein above. Tumor-infiltrating leukocytes were further analyzed by fluorescence microscopy. scRNA-seq analysis was also performed. Pathological, flow cytometry, and scRNA-seq analyses were used to determine whether ER+ and HER2+ tumors respond similarly to LM-HIFU+IT-IL-12 treatment, including increased intratumoral T cell infiltration, changes in the TME, and induction of antigen-specific immune responses, compared to TNBC.

[0065] The other 10 mice were treated with CT scans at a humane endpoint (tumor volume of 2,000 mm 3 Tumor size was measured three times a week until tumor size reached >100% (>100%), and mouse survival was assessed. Tumor growth inhibition percentage (TGI%) was compared. To confirm the induction of potent systemic antitumor immunity, long-term surviving mice after tumor eradication were subjected to tumor rechallenge 12 weeks after LM-HIFU+IT-IL-12 treatment. Tumor growth / tumor-free survival was monitored for up to 2 months. The abscopal effect induced by LM-HIFU+IT-IL-12 therapy and the enhanced anti-tumor effect by combination with anti-PD-1 antibody in bilateral ER+ and HER2+ models can be demonstrated as follows: MM3MG-ESR1mut or MM3MG-HER2 cells were implanted bilaterally into the flanks of female BALB / c mice (Experiment 3.3, 3.4). As described herein above, LM-HIFU+IT-IL-12 treatment with or without anti-PD-1 antibody was performed on the tumors on the right flank, while the tumors on the contralateral side were left untreated. 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) were administered intraperitoneally on day 3 after LM-HIFU+IT-IL-12 treatment and repeated twice a week for 2 weeks.

[0066] Four mice from each group were euthanized 7 days after treatment, and tumor samples harvested from both treated local and untreated distant sites were analyzed. Harvested splenocytes and serum were used for IFN-γ ELISPOT assay and ELISA. scRNA-seq analysis was performed on TILs isolated from both treated and untreated distant tumors in each group. We evaluated whether LM-HIFU+IT-IL-12 therapy in combination with anti-PD-1 showed more pronounced TME changes compared with LM-HIFU+IT-IL-12 therapy alone. CXCR3 gene signature levels were also evaluated. Differences in TME changes between treated and untreated distant tumors in each group were also analyzed. The other 10 mice were treated with CT scans at a humane endpoint (tumor volume of 2,000 mm 3Tumor size (both HIFU-treated local tumors and untreated distant tumors) was measured three times a week until it reached a tumor size of >1000 (>10 ...

[0067] Example 5 Drug / gene delivery by in vivo sonoporation using a miniaturized HIFU transducer As mentioned above, typical US transducers for sonoporation (e.g., single-element focused US transducers) are extracorporeal and not suitable for intratumoral immunotherapy. Some limitations of extracorporeal US transducers include the inability to efficiently sonicate tumors behind bone and fat due to ultrasound attenuation and absorption, and the inability to precisely inject MB and nucleic acid simultaneously into the US treatment zone, which may result in off-targeting. These issues reduce the efficacy of drug / gene delivery and increase unwanted systemic toxicity for cancer immunotherapy. Thus, the systems and methods described herein are directed to a miniaturized US transducer (e.g., transducer 400 described above) integrated into a catheter (i.e., a catheter US transducer) for drug / gene delivery by internal sonoporation in intratumoral immunotherapy.

[0068] Aperture size is 2 x 2 mm 2A 800 kHz US transducer (similar to transducer 400 in FIG. 5E) was designed and fabricated for in vivo sonoporation studies. Transducer 400 in the embodiment illustrated in FIG. 18 includes a dual-layer PZT-5A, a matching (Al2O3 / epoxy) layer, and a backing (bubbles / epoxy) layer (not shown) as described above with respect to FIGS. 4A and 5A. A lumen was embedded in a catheter next to the US transducer to inject microbubbles into the sonication area. The lumen size was about 0.9 mm (outer diameter), while the catheter size was about 3.0 mm (outer diameter). The sound pressure output of the developed transducer was characterized by a calibrated hydrophone (HNA-0400, ONDA Corporation, CA, USA) in degassed water. Because US standing waves may affect sonoporation efficiency in vitro, acoustic simulation experiments were first performed using the k-Wave toolbox to investigate the propagation of 800 kHz 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 3.6 mm × 7.6 mm two-dimensional plane filled with water. The material of the culture plate was polystyrene, and its acoustic properties, including density, sound speed, and absorption coefficient, were summarized in Table 1900 in Figure 19. The US transducer was placed above the well, and the US frequency was 800 kHz and the input pressure was 0.4 MPa.

[0069] Human embryonic kidney (HEK) 293T cells and plasmid DNA encoding green fluorescent protein-luciferase (GFPLUC) were prepared. HEK293T 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 a 384-well plate for sonoporation testing. VesselVue® MBs (SonoVol, Inc., NC, USA) with an average diameter of 1.01±0.59 μm were used as cavitation nuclei in this study. MBs were mixed with phosphate-buffered saline (PBS) solution in a 10 mL syringe for ductal injection, and the MB concentration was diluted to 8.73×108 bubbles / mL. During sonoporation testing, 30 μL of MB solution was injected into each well through the injection duct, and the total volume of the solution in each well was approximately 90 μL.

[0070] As shown in the test system 2000 of FIG. 20, a pulsed electrical waveform was first generated by a function generator 2002 (e.g., 33250A, Agilent Technologies, Inc., CA, USA) and then amplified by an RF power amplifier 2004 (e.g., 75A250A, Amplifier Research Corporation, PA, USA) to drive a catheter US transducer 200. The catheter US transducer 200 was inserted into a cell culture plate 2006 and MB solution was injected. MB injection was controlled by a syringe pump 2008 (e.g., NE-1010, New Era Pump Systems, Inc. NY, USA) with an injection rate of 0.1 mL / min. The cells were then sonicated for 30 seconds with a duty cycle of 5%. Various sonication parameters including peak negative pressure (PNP) (0.1-0.7 MPa) and number of cycles (CN) (20-2000 cycles) were tested. After sonication, the test cells were incubated in a CO2 incubator at 37°C for 24 hours. To evaluate sonoporation efficiency, cells were harvested from each well and lysed in lysis buffer. By adding luciferin, luciferase activity was measured using a luminometer (e.g., GloMax, Promega Corporation, WI, USA) and reported in relative light units (RLU).

[0071] In this study, six independent experiments (i.e., n = 6) were performed for each group. Luciferase activity was compared between the control and experimental groups using one-way analysis of variance (ANOVA). A p value < 0.05 was considered statistically significant. Measurements of the PNP and mechanical index (MI) of a prototype 800 kHz catheter US transducer are illustrated in Table 2100 of Figure 21 and show that the fabricated transducer is capable of generating the PNP (~0.5 MPa) required for sonoporation applications. Furthermore, the focal length of the catheter transducer was ~1 mm close to the transducer surface. The simulated ultrasound beam (beam 2200 in FIG. 22) 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% to 0.48 MPa at the cell culture plate. Although the catheter US transducer has a focal length of about 1 mm, it can be observed that the reflection of the US wave caused a high acoustic pressure area near the bottom wall of the cell culture plate. This phenomenon indicates that cells can be placed on the bottom surface of each well for effective cell sonication.

[0072] The negative control and experimental groups were compared in terms of luciferase activity (see series 2300 in Figure 23). The negative control groups consisted of "no plasmid", "with plasmid", "MB alone" and "US alone" groups, while the experimental group refers to the "MB+US" group. A positive control of gene transfer using Lipofectamine was also performed in parallel to confirm that the assay worked well (data not shown). The sonication parameters were 0.2 MPa PNP and 20 cycles CN for the "US alone" group, whereas 0.4 MPa PNP and 200 cycles CN for the "MB+US" group. The luciferase assay (table 2300 in Figure 23) demonstrated that the "MB+US" group achieved 105 levels of RLU, whereas the "with plasmid" group achieved only 102 levels of RLU. The corresponding maximum fold change in luciferase activity was approximately 1500-fold, indicating a significant enhancement of transfection. Following this, a preliminary parameter sensitivity study was performed to investigate how PNP and CN affect sonoporation efficiency. Figure 24 (illustrating luciferase activity of sonoporation tests under various sonication parameters including PNP: 0.1-0.7 MPa, and CN: 20-2000 cycles (n=6)) shows that 20 cycles (Table 2400A) or 200 cycles (Table 2400B) are superior to 2000 cycles (Table 2400C), and the optimal PNP is in the range of 0.3 MPa-0.5 MPa. According to Figure 24, it was speculated that too high acoustic pressure and too long pulse duration (PD equal to CN / US frequency) may induce cell death and reduce sonoporation efficiency. Meanwhile, Figure 24 shows that given that the PD of 20 cycles of 800 kHz US is only 25 μs, the possibility of bubble-cell contact was relatively low in such a short time. Therefore, the trends of PNP-RLU were different from Tables 2400B and 2400C in Figure 24. Furthermore, it was interesting to find that the RLU was relatively low at 0.3-0.4 MPa for 20 cycles (Table 2400A) and relatively low at 0.3 MPa for 200 cycles (Table 2400B). It was speculated that this may be related to the transformation from stable cavitation and inertial cavitation.In fact, inertial cavitation can create larger holes in the cell membrane and 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 Table 2400A and higher than 0.2 MPa in Table 2400B. Then inertial cavitation plays its role and the sonoporation efficiency increases with increasing PNP until cell death occurs.

[0073] Thus, the results for the developed 800 kHz forward-facing catheter US transducer showed that it is promising for drug / gene delivery by in vivo sonoporation in intratumoral immunotherapy.

[0074] Example 6 Changes in the tumor microenvironment by M-HIFU Triple-negative and human ErbB-2 (HER2)-positive breast cancer (BC) have a high probability of metastatic spread despite early localized symptoms and multimodality treatment. Cancer immunotherapy in the form of immune checkpoint blockade (ICB) has modest activity limited to a small proportion of triple-negative BC. Reasons for the limited efficacy of ICB therapy in BC include a relatively low somatic mutation rate, the inability of tumors to attract immune infiltration, especially tumor-infiltrating lymphocytes, the expression of additional immune checkpoint molecules in the tumor microenvironment (TME) that suppress adaptive immune responses, and the suppression of intratumoral innate immunity by inhibitory cell types, such as regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells. Indeed, aggressive BC proliferation and invasion have been associated with TAMs in both preclinical and clinical trials. Consequently, altering the polarization of TAMs in the TME has become the focus of attempts to increase the efficacy of BC immunotherapy. Unfortunately, pharmacological strategies to specifically modulate TAMs without systemic changes in non-TAMs have been unsuccessful, and options such as local delivery or ablation are being considered.

[0075] Localized ablation of tumor cells in the TME by image-guided delivery of various energies, including high-intensity focused ultrasound (HIFU), radiofrequency, microwave, and cryoprobe, has been used clinically as a minimally invasive treatment for localized tumors in the prostate, breast, liver, kidney, bone, and brain. Such ablation therapies use different energy sources to result in tumor cell destruction, but also induce an antitumor immune response against antigens in tumor debris in situ. Conventional HIFU (thermal high-intensity focused ultrasound (T-HIFU)) rapidly induces coagulation necrosis of tissue at the target lesion where the energy is applied. Tissue proximal to the target lesion does not coagulate, but is subjected to sufficient thermal stress to induce apoptosis. Increased accumulation of CD4+ and CD8+ cells in T-HIFU-treated tumors has been observed, as T-HIFU-treated cells release endogenous danger signals, leading to the secretion of interferon gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α) from immune cells. Nevertheless, due to the limited effectiveness of T-HIFU in larger tumors, alternative forms of HIFU for tumor destruction have been investigated, such as high pressure bursts that cause acoustic cavitation, called mechanical high-intensity focused ultrasound (M-HIFU).

[0076] Compared with T-HIFU, M-HIFU increased the accumulation of dendritic cells in treated tumors, exhibiting stronger antitumor effects, enhancing antitumor immunity, and reducing the risk of metastasis. It should be noted that M-HIFU also increased the infiltration of T cells in treated tumors. To better delineate the mechanism of greater antitumor immunity induced by M-HIFU, single-cell RNA sequencing of tumors and TME without treatment or after conventional T-HIFU and M-HIFU was used. Changes in the local and distant TME of mouse BC were observed, as well as a conversion of macrophage subtypes in M-HIFU-treated tumors that was not seen after T-HIFU treatment. In addition, increased expression 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 could exhaust activated T cells in the TME, resulting in suppression of antitumor immunity. Thus, the combination of M-HIFU and anti-PD-L1 therapy was studied and demonstrated increased gene expression by CD8+ T cells in type I interferon-mediated signaling pathways, T cell proliferation, and chemokine / cytokine secretion, which was accompanied by a dramatic increase in local and distant antitumor effects, including complete responses in the majority of treated animals.

[0077] In our laboratory, MM3MG, a mouse premalignant mammary epithelial cell line, was transduced with the human HER2 oncogene by retroviral vector and polybrene to express HER2 (referred to as MM3MG-HER2 cells). JC cells, a mouse BC cell line, were transduced with the human HER3 gene (referred to as JC-HER3). 28 4T1-HER2 cells were also obtained in the same way. A VIFU2000 system (Alpinion Medical Systems, Bothell, Washington, USA) was used for HIFU treatment. Cells or tumors were treated using a 1.5 MHz HIFU transducer under two different protocols (50% duty cycle, 1 Hz pulse repetition frequency, 20 W, 10 s or 2% duty cycle, 5 Hz pulse repetition frequency, 200 W, 20 s), resulting in either thermal necrosis or mechanical lysis of tumor cells. The former was defined as T-HIFU and the latter as M-HIFU. In T-HIFU, the temperature in tumor tissue rose to over 60°C within a few seconds, while during M-HIFU the temperature in tumor tissue was below 42°C. M-HIFU is similar to boiling histotripsy, which can produce cavitation activity in vivo and damage tumor tissue and cells through shear stress generated by complex bubble oscillation and bubble-bubble-tissue-cell interactions. The -6 dB focal dimensions of the HIFU transducer were measured at a low power level of 10 W and were 0.72 mm × 7.22 mm in the lateral and axial directions, respectively. At the higher power levels used in T-HIFU (20 W) and M-HIFU (200 W), the corresponding focal dimensions based on numerical simulation were estimated to be 0.74 mm × 6.50 mm and 0.60 mm × 5.69 mm, respectively. For the treatment focal points, the spacing in both the X-axis and Y-axis was 2 mm, and a total of 9 points were selected for both the in vitro and in vivo studies. The short spacing of 1 mm was used only when the size of the tumor was not sufficient to place 2 mm for all spacings. The same spacing strategy was used for both M-HIFU and T-HIFU. To avoid skin or bone damage due to the HIFU treatment, the tumor tissue just under the skin or close to the femur was not exposed to focused ultrasound; therefore, based on macroscopic assessment, approximately 20%-40% of the tumor tissue was ablated by the HIFU treatment.

[0078] Five- to eight-week-old female BALB / c or SCID-beige mice (Jackson Labs, Bar Harbor, Maine, USA) were bred and maintained. Human HER2 transgenic mice were also utilized. F1 hybrid HER2 transgenic mice were established by mating with BALB / c mice. Human HER3 transgenic mice (MMTV-neu / MMTV hHER3) on an FVB background were also used. FVB mice homozygous for the hHER3 gene were established at Duke University and then mated with BALB / c mice to establish BALB / c homozygous for the hHER3 gene.

[0079] Local tumor volume 2000mm 3 Mice were euthanized when tumor size reached 100 μg / kg / day. To test the immunogenicity of HIFU-treated tumor cells, BALB / c mice were injected intradermally on the back with in vitro T-HIFU-treated or M-HIFU-treated MM3MG-HER2 cells (1×106 cells) on days −14 and −7. On day 0, some mice were euthanized and spleens, draining lymph nodes, and blood were harvested for in vitro assays: IFN-γ ELISpot, flow cytometry, and cell-based ELISA. Other mice (10 mice / group) were inoculated with 1×106 MM3MG-HER2 cells in the left lower leg. Tumor size was measured serially, and tumor volume was calculated using the formula: major axis × (minor axis) 2 × 0.5.

[0080] In the therapy model using MM3MG-HER2 tumors, MM3MG-HER2 cells were inoculated subcutaneously (1×106 cells) into the left lower leg of mice on day 0. In the bilateral tumor model, 1×105 or 5×105 cells were also inoculated into the right flank on day 0. Established lower limb tumors were treated with T-HIFU or M-HIFU on day 7. In the re-challenge experiment, mice cured by M-HIFU treatment were injected subcutaneously with MM3MG-HER2 cells (1×106 cells) into the flank on day 35 (28 days after M-HIFU), and tumor size and mouse survival were monitored. For combination treatment with anti-PD-L1 antibody, mice were peritoneally injected with 100 μg of anti-PD-L1 antibody (clone 10F.9G2, Bio X Cell, West Lebanon, NH) or isotype control IgG (clone LTF-22, Bio X Cell) on days 12, 15, and 18 for unilateral tumor models and on days 10, 13, and 16 for bilateral tumor models. For JC-HER3 tumor models, cells were inoculated into the left lower leg (1×106 cells) with or without inoculation into the right flank (5×105 cells) on day 0. Lower leg tumors were treated with M-HIFU on day 8 (for combination treatment experiments) or day 11 (for single M-HIFU treatment experiments). For combination treatment, mice were intraperitoneally injected with anti-PD-L1 antibody or isotype control IgG (200 μg / injection) on days 8, 11, and 15 in the unilateral tumor model, and on days 8, 11, 15, and 18 in the bilateral tumor model. For immune cell depletion, mice were peritoneally injected with 250 μg of an antibody against CD4 (clone GK1.5, Bio X Cell), an antibody against CD8a (clone 53-6.72, Bio X Cell), or 10 μL of an antibody against natural killer (NK) cells (anti-asialo GM1 antibody; Wako Pure Chemical Industries, Osaka, Japan) 1 day before and 2 days after the first HIFU treatment, and then the same amount was injected every 5 days for the duration of the experiment.

[0081] Mouse IFNγ-ELISpot assays (e.g., Mabtech, Cincinnati, Ohio, USA) were 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 an irrelevant HIV-gag peptide mix (2.6 μg / mL, JPT Peptide Technologies). The number of IFN-γ spots was counted using a high-resolution automated ELISpot reader system (e.g., Carl Zeiss, White Plains, New York, USA) using KS ELISpot V.4.2 software. Plates were coated overnight with 3 × 104 parental 4T1 cells or 6 × 104 4T1-HER2 cells per well. Serially diluted sera (final titration 1:50 to 1:6400) were added and incubated on ice for 1 h. Plates were washed, fixed with 1% formalin, and then incubated with IRDye 800CW donkey anti-mouse (1:2000; LI-COR Biosciences, Lincoln, Nebraska, USA) for 60 min. Fluorescence intensity was determined using the 800 nm channel with an Odyssey CLx LI-COR reader (LI-COR).

[0082] Tumors were weighed and homogenized in cell lysis buffer (cat. no. 9803, Cell Signaling, 9x tumor mass) supplemented with PMSF (1mM) using a Qiagen TissueRupter (e.g., Qiagen, Germantown, Maryland, USA) for up to 20 seconds on ice. Tumor homogenates were then sonicated for 10 seconds on ice using a Branson Ultrasonic SLPe Digital Sonifier Cell Disruptor (e.g., Branson Ultrasonics, Danbury, Connecticut, USA). Sonicated samples were centrifuged at 13,000 rpm for 20 minutes at 4°C, and the collected supernatant was used as tumor lysate for ELISA. Tumor lysates were assessed for levels of IFN-γ, TNF-α, and transforming growth factor beta 1 (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 TGF-β1 (BMS608-4, Invitrogen), and the assays were performed according to the manufacturer's instructions.

[0083] Single cell suspensions of tumor tissue were obtained by manual disruption of tumors using a razor blade followed by enzymatic digestion. Single cell suspensions of spleen and lymph nodes were obtained by manual grinding and filtration 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 (e.g., Thermo Fisher Scientific, Rockford, Illinois, USA) and then stained with surface marker antibodies (online supplemental table 1) for 30 min at room temperature. Isotype IgG or fluorescence minus one (FMO) controls were used as negative staining controls. Anti-CD16-32 antibody (Thermo Fisher) was used to block FcγIII / II receptors. Intracellular staining was performed using fixation / permeabilization and permeabilization buffer (Thermo Fisher) 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).

[0084] Dissociated single cell suspensions from untreated or HIFU-treated tumors (8 days after HIFU treatment) were obtained as described above. For combination therapy experiments, mice were injected intraperitoneally with 100 μg of anti-PD-L1 antibody or isotype control IgG antibody both 3 and 6 days after HIFU treatment. CD45+ leukocytes were sorted by flow cytometry from tumor digests, and cDNA libraries were prepared using a Bio-Rad single cell isolator (ddSEQ) and Illumina's SureCell WTA 3'Library Prep kit. Raw sequencing data were generated in the form of FastQ files and uploaded to BaseSpace sequencing Hub (Illumina, San Diego, California, USA) for expression quantification using an automated pipeline. The resulting gene expression matrix files for each treatment condition were analyzed using Partek Flow software (Partek, St. Louis, Missouri, USA). 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 subpopulations within CD45+ sorted immune cells. Preprocessed gene counts were used to generate Uniform Manifold Approximation and Projection (UMAP) for visualization of cell types in 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.

[0085] Staining of formalin-fixed paraffin-embedded tumor tissue sections was performed 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). A DAB substrate kit (Vector Labs, Burlingame, California, USA) was used for visualization of signals before incubation with HRP (Histofine Simple Stain Mouse MAX PO®, Nichirei Biosciences, Tokyo, Japan). Isotype-matched rabbit IgG was used as a negative staining control. Stained slides were scanned under a DP80 microscope (Olympus, Tokyo, Japan), and digital images were viewed using cell-Sens (Olympus).

[0086] Data are presented as mean ± SEM for tumor growth graphs or mean ± SD for in vitro assays and flow cytometry data. Tumor volume, flow cytometry, ELISA, and ELISPOT data from experiments with three or more treatment groups were analyzed by one-way ANOVA with Tukey's multiple comparison test. In experiments with only two groups, a two-tailed unpaired Student's t test was used. Tumor volumes were analyzed only at terminal endpoints unless otherwise indicated. Statistical analysis was performed using Prism (e.g., GraphPad, San Diego, California, USA). Kaplan-Meier survival curves of 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). A p value of 0.05 or less was considered statistically significant. Not all significant differences are shown in all graphs (* p<0.05、 ** p<0.01、 *** p<0.001)。

[0087] Implanted MM3MG-HER2 BC in BALB / c mice were treated with either M-HIFU or T-HIFU, and antitumor effects were evaluated in both local (treated) and distant tumor burdens. Compared with T-HIFU, M-HIFU showed greater control of treated and untreated distant tumors (Tables 2500A-2500C in FIG. 25). Tumor growth inhibition in both treated and untreated diseased sites by M-HIFU was confirmed in two other mouse BC models, E0771-OVA and JC-HER3. M-HIFU was able to induce significantly stronger cellular immune responses with HER2 ECD, HER2 ICD, and mixed peptide antigens when compared with untreated controls, whereas T-HIFU was only able to induce a moderately stronger response with HER2 ECD antigen (Table 2500D in FIG. 25). There was a clear trend toward stronger cellular immune responses induced by M-HIFU compared with T-HIFU. However, M-HIFU and T-HIFU induced similar levels of humoral immunity against HER2-expressing tumor cells (Table 2500E in Figure 25). In the JC-HER3 tumor model, induction of HER3 antigen-specific cellular immune responses in HER3 transgenic mice was also confirmed after M-HIFU treatment, suggesting the strong ability of M-HIFU to induce antitumor immunity by breaking tolerance. Importantly, a long-lasting immune memory response was induced in mice that had previously rejected tumors after M-HIFU treatment (Table 2500F in Figure 25). This memory response could not be measured in T-HIFU-treated mice, as none of the treated tumors regressed completely (Table 2500B in Figure 25). A possible explanation for this difference in antitumor response is the baseline difference in immunogenicity of tumor cells after treatment with M-HIFU compared to T-HIFU. To test this hypothesis, mice were vaccinated with MM3MG-HER2 cells treated in vitro and their immunogenicity was evaluated. Despite the different types of cell death induced by M-HIFU versus T-HIFU (apoptosis versus necrosis), there were no significant differences in systemic T cell activation, production of tumor antigen-specific antibodies, or tumor control elicited by inoculation of M-HIFU or T-HIFU-killed tumor cells.

[0088] With reference to FIG. 25, Tables 2500A-2500F present data on superior growth inhibition of local and distant tumors and enhancement of tumor antigen-specific cellular immune responses by M-HIFU compared to T-HIFU. In Table 2500A, 1×10 6 MM3MG-HER2 cells were injected into the lower limbs of BALB / c mice. Established lower limb tumors were treated with M-HIFU or T-HIFU on day 7 after tumor inoculation. A comparison of the tumor growth curves is shown. Table 2500B presents the survival curves. For tumors with a tumor volume of 2,000 mm 3Mice were euthanized at day 50 or when the tumor reached a tumor size of 100 μg / ml. n=9 mice (no treatment), 10 mice (M-HIFU), or 13 mice (T-HIFU) (A, B), totaling 32 mice. Log-rank test was performed. For data in Table 2500C, MM3MG-HER2 cells were injected into the left hind leg (1×106 cells) and right flank (1×105 cells) of HER2 transgenic mice on day 0. Limb tumors were treated with M-HIFU or T-HIFU on day 7. Comparison of flank tumor growth curves is shown. n=13 mice (no treatment) or 14 mice (M-HIFU and T-HIFU), totaling 41 mice. For data in Table 2500D, lymphocytes were isolated from spleens on day 18 after tumor inoculation (11 days after HIFU treatment) and IFN-γ secretion was detected by ELISpot assay. The mean number of spots for ECD, ICD, and mixed (ECD+ICD) HER2 peptides is shown. n=4 per group. For data in Table 2500E, serum was collected from mice on day 11 after HIFU treatment. The levels of anti-HER2 antibodies in mouse serum were evaluated by cell-based ELISA. n=3 mice (no treatment) or 8 mice (M-HIFU and T-HIFU), total 19 mice. For data in Figure 4500F, mice cured of MM3MG-HER2 tumors by M-HIFU treatment were rechallenged with subcutaneous injection of MM3MG-HER2 cells (1x106 cells / mouse) 28 days after M-HIFU treatment. Age-matched naive female BALB / c mice were used as control group. Mouse survival rate is shown and log-rank test was performed. n=5 mice per group, total 10 mice. (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.

[0089] Since tumor cells treated with M-HIFU or T-HIFU in vitro were similarly immunogenic, we hypothesized that differential changes in the TME by the two HIFU treatments were responsible for the difference in the induction of systemic antitumor immunity, and therefore efficacy, in the BC model. Compared to untreated controls, greater CD4 and CD8 T cell infiltration was observed by immunohistochemistry in M-HIFU-treated tumors, with a slight increase in T cell infiltration in T-HIFU-treated tumors (plot 2600A in FIG. 26). This was confirmed by flow cytometry of digested tumors, which demonstrated an increase in both CD4 and CD8 T cells and NK cells after M-HIFU treatment (plot 2600B in FIG. 26). To confirm changes in immune cell profile, absolute numbers of tumor-infiltrating immune cells were determined in each treatment group. Increased numbers of CD4 T cells, CD8 T cells, and NK cells were confirmed in the M-HIFU-treated group, but no differences in granulocyte and macrophage populations were observed. Enhanced T cell proliferation was seen in M-HIFU treated tumors compared to control tumors by Ki67 staining (plot 2600C in FIG. 26). In addition to increased infiltration and active proliferation, CD8 T cells present in M-HIFU treated tumors showed increased expression of T cell activation markers and granzymes (plot 2600D in FIG. 26). Dendritic cell (DC) maturation was increased in M-HIFU treated tumors as evidenced by elevated expression of major histocompatibility complex (MHC) class II and CD80 (plot 2600E in FIG. 26). Importantly, M-HIFU affected the polarization state of tumor infiltrating macrophages as shown in plot 2600F in FIG. 26. The positivity of CD206 expression was significantly lower and the expression level of MHC class II was significantly higher in M-HIFU treated tumors compared to those in control tumors, suggesting that M-HIFU can induce repolarization of macrophages to a more antitumor TME. In support of this finding, significantly lower TGF-β1 and higher IFN-γ levels were observed in M-HIFU treated tumors compared to control tumors (Table 2600G of FIG. 26).

[0090] For Fig. 26, M-HIFU enhances intratumoral infiltration of activated CD4+ and CD8+ cells and increases M1 polarized macrophages. 1x106 MM3MG-HER2 cells were injected into the lower limbs of BALB / c mice. Established lower limb tumors were treated with M-HIFU or T-HIFU on day 7 after tumor inoculation. For Plot 2600A, immunohistochemical staining of T cells in tumor sections. Tumors were harvested on day 13 after HIFU treatment, fixed in formalin, and stained with anti-mouse CD4 and CD8 monoclonal antibodies. Representative images are shown (left: no treatment, middle: T-HIFU, right: M-HIFU). Scale bar is 50 μm. Quantification of positive cells in high power fields (HPF) is shown in the right panel. Error bars represent SD, n=3 per group. For plot 2600B, 7 days after HIFU treatment of MM3MG-HER2 tumors in mice, tumors were harvested and digested for flow cytometry analysis. The percentages of CD4+, CD8+, CD3+, CD49b+, Ly6G+, CD11c+, and F4 / 80+ cells among viable CD45+ cells were analyzed for each HIFU treatment group. n=4 per group. For plot 2600C, expression of proliferation marker Ki67 was analyzed by flow cytometry for each cell type in tumor-infiltrating immune cells, and the percentage of Ki67 positive for each cell type is shown. n=4 per group. For plot 2600D, expression of CD69+, inducible T cell costimulatory molecule (ICOS)+, and granzyme B+ by CD8+ cells was analyzed by flow cytometry and shown for each HIFU treatment group. n=4 per group. For plot 2600E, MFI of MHC class II (left) and CD80 (right) expression on CD11c+ dendritic cells is shown for each HIFU treatment group. For plot 2600F, expression of CD206 and MHC class II by the CD11b+F4 / 80+ macrophage population was analyzed for each treatment group. Representative dot plots of CD206 and MHC class II staining are shown in the left panel. The percentage of CD206 positive macrophages and the mean fluorescence intensity of MHC class II expression are shown. n=4 per group.For plot 2600G, ELISAs for IFN-γ, TNF-α, and TGF-β1 were performed using tumor lysates made from MM3MG-HER2 tumors treated with no treatment, T-HIFU, or M-HIFU. 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 beta 1; T-HIFU, thermal high-intensity focused ultrasound; TNF-α, tumor necrosis factor alpha.

[0091] To study the effect 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 and compared with gene expression from tumors treated with M-HIFU and T-HIFU. GO enrichment analysis and KEGG pathway analysis of differentially expressed genes (DEGs) in macrophage populations identified from the two HIFU strategies were performed and compared with macrophages from untreated tumors. Ten significantly upregulated GO terms in biological process categories in M-HIFU-treated macrophages are shown in Table 2700A of Figure 27A. The upregulated genes were mainly involved in immune response, inflammatory response, leukocyte activation, immune system process, 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 include immune-related terms, but rather focused on biosynthetic processes.

[0092] The top 10 GO terms in the categories of cellular components and molecular functions for both M-HIFU and T-HIFU treated tumors compared to untreated tumors are shown in plots 2700B and 2700C of FIG. 27A. There was no overlap in the top terms upregulated after these different forms of HIFU, again demonstrating the fundamental differences in the molecular outcomes 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 / lysosomal pathways, consistent with the inflammatory, antitumor phenotype of macrophages after M-HIFU (plot 2700D of FIG. 27B). Only seven KEGG pathways were significantly upregulated in T-HIFU treated tumors compared to untreated tumors. All of these pathways were involved in metabolic processes. The difference in significantly upregulated pathways between M-HIFU and T-HIFU treated tumors highlights the immunogenic nature of our M-HIFU therapy at this time point. Differential gene expression analysis of tumor-infiltrating macrophages identified 298 genes that were expressed at statistically higher levels (FDR adjusted p-value <0.05) than untreated tumors (plot 2700E in Figure 27B). Several genes (Irg1, Ccl2, Maff, Ier3, Lcp2, Ptpn2, Cd14, Cxcl16, Ier3, Nfkbia, Ccrl2, and Tlr2) are signature genes of M1 / classically activated macrophages, 34 and other genes (Tnfsf10, Ctsc, Gzme, Cd8a, and Pdcd1) are commonly associated with M1 macrophages. These findings confirm the highly activated M1-biased macrophage population in the TME of M-HIFU-treated tumors. Thus, scRNA-seq analysis demonstrated that M-HIFU induces a proinflammatory M1-biased macrophage population, which may contribute to enhanced antitumor immunity in M-HIFU-treated tumors.

[0093] For Figures 27A and 27B, GO enrichment analysis and KEGG pathway analysis of DEGs in macrophages after M-HIFU treatment. For plots 2700A-2700C, GO enrichment analysis of DEGs upregulated in macrophages from M-HIFU treated tumors compared to macrophages from untreated tumors. Enrichment scores of GO terms are shown for categories of (A) biological process (plot 2700A), (B) cellular component (plot 2700B), and (C) molecular function (plot 2700C). For plot D, KEGG pathway analysis of DEGs upregulated in macrophages from M-HIFU treated tumors compared to macrophages from untreated tumors. The top 10 KEGG pathways in terms of enrichment score are demonstrated. For plot 2700E, differential gene expression analysis in tumor infiltrating macrophages from M-HIFU treated tumors compared to those from untreated tumors. Representative genes that were significantly upregulated (log2(fold change)>1, FDR<0.05) in macrophages from M-HIFU treatment group are shown in red. DEG, differentially expressed genes; FDR, false discovery rate; GO, gene ontology; M-HIFU, mechanical high intensity focused ultrasound.

[0094] Although nearly half of the M-HIFU-treated mice mounted a productive antitumor immune response, many eventually developed progressive disease (Tables 2500A and 2500B in FIG. 25). Therefore, we began by assessing the expression of immune checkpoint molecules in M-HIFU-treated versus T-HIFU-treated tumors to investigate the pathogenesis of this tumor evasion. At 7 days after treatment, PD-L1 expression was increased in neutrophils (Ly6G+), DCs (CD11c+), and macrophages (F4 / 80+) only in M-HIFU-treated tumors (plots 2800A and 2800B in FIG. 28). However, analysis only 3 days after HIFU treatment revealed that neither method of HIFU treatment 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 the result of an ongoing immune response caused by M-HIFU treatment. IFN-γ, which was increased in tumor tissues at day 5 after M-HIFU treatment (plot 2600G in FIG. 26), may play an important role in this upregulation of PD-L1. Other immune checkpoint molecules, including PD-1 and TIM3, were not significantly altered after M-HIFU, whereas lymphocyte activation gene 3 (LAG-3) expression on CD8+ cells was significantly higher after M-HIFU than after T-HIFU (plots 2800C and 2800D in FIG. 28). Together, these data demonstrate that the enhancement of antitumor immunity in the TME caused by M-HIFU is not seen in T-HIFU and may be suppressed by the concomitant increase in immune checkpoint molecules.

[0095] With respect to FIG. 28, plots 2800A-2800D illustrate the increased expression of immune checkpoint molecules by tumor-infiltrating immune cells following M-HIFU treatment. With respect to plot 2800A, representative flow cytometry histograms showing PD-L1 expression on Ly6G+, CD11c+, and F4 / 80+ cells are shown. Blue: M-HIFU, red: T-HIFU, black: no treatment, grey fill: isotype control. With respect to plot 2800B, the percentage of PD-L1+ cells among Ly6G+, CD11c+, and F4 / 80+ cells among viable CD45+ tumor-infiltrating immune cells is shown for each HIFU treatment group. n=4 per group. For plots 2800C and 2800D, the expression of immune checkpoint molecules, PD-1, TIM-3, and LAG-3 on tumor-infiltrating CD4+ cells (plot 2800C) and CD8+ cells (plot 2800D) 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.

[0096] Based on the increased expression of immune checkpoint molecules after M-HIFU, we hypothesized that PD-1 / PD-L1 blockade could enhance the antitumor effect of M-HIFU treatment. To test this, M-HIFU treatment was combined with anti-PD-L1 antibody administration. This combined treatment cured 72.2% of mice implanted with MM3MG-HER2 tumors, compared with 26.3% and 52.6% when anti-PD-L1 or M-HIFU was given alone, respectively (plots 2900A and 2900B in Figure 29A). This combination also led to tumor eradication and long-term survival in 37.5% of mice using another BC model, JC-HER3. IFN-γ ELISpot assay of splenocytes demonstrated a significant increase in the number of HER2-specific IFN-γ secreting cells in mice treated with M-HIFU+anti-PD-L1 compared to mice treated with M-HIFU or anti-PD-L1 alone (plot 2900C in Figure 29A), and flow cytometry analysis of digested tumors revealed increased percentages of CD4+ T cells, CD8+ T cells, and NK cells in tumors treated with combination therapy, with enhanced activation of both CD4+ and CD8+ T cells in combination therapy. Stronger cytolytic activity of tumor-infiltrating CD8+ T cells was suggested with M-HIFU monotherapy and combination therapy based on increased granzyme B expression. Furthermore, 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.

[0097] To demonstrate which infiltrating cells were responsible for the observed antitumor effect, MM3MG-HER2 tumors were treated with combination therapy in the presence of depleting antibodies against CD4+ cells, CD8+ cells, or NK cells. Depletion of CD8+ cells or NK cells abolished the antitumor effect of the combination therapy, whereas depletion of CD4+ cells did not appreciably change the antitumor effect (Figure 29B, plots 2900E and 2900F).

[0098] For Figure 29, M-HIFU and PD-L1 inhibition synergize to reject local tumors. For plot 2900A, MM3MG-HER2 tumors established in the lower limbs of BALB / c mice were treated with M-HIFU on day 7. Anti-PD-L1 antibody (100μg / 100μL) or isotype control IgG (100μg / 100μL) was injected intraperitoneally 5, 8, and 11 days after M-HIFU treatment. Survival curves are shown and log-rank test was performed. n=13 mice (isotype control), 19 mice (monotherapy group) or 18 mice (combination group), totaling 50 mice. For plot 2900B, individual tumor growth curves are shown for each treatment group. The numbers in each plot indicate the mice with tumor eradication / mice in that group. For plot 2900C, 9 days after the initiation of M-HIFU treatment with / without PD-L1 antibody, spleens were harvested for immunoassay. Induction of HER2 antigen-specific cellular responses was analyzed by IFN-γ ELISpot assay using harvested splenocytes and HER2 peptide mix as stimulatory antigen. n=4 per group. For plot 2900D, the percentage of CD4+ cells, CD8+ cells, and CD49b+ cells among tumor-infiltrating CD45+ cells was analyzed by flow cytometry analysis. n=4 (treated group) or 3 (control group). For plots 2900E and 2900F, mice were treated with a combination of M-HIFU and anti-PD-L1 antibody as in plot 2800A of Figure 28, with or without administration of depleting antibodies against CD4+ cells, CD8a+ cells, and NK cells on days 6, 9, 14, and every 5 days until the end of the experiment. n=5 mice (isotype control) or 6 mice (other groups), total 29 mice. For plot 2900E, survival curves are shown and log-rank test was performed. For plot 2900F, individual tumor growth curves are shown for each cell-depleted group. 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.

[0099] To explain the enhanced antitumor effect with the addition of anti-PD-L1 treatment, leukocyte populations were further characterized by performing scRNAseq analysis on tumor-infiltrating CD45+ immune cells from mice treated with no treatment, M-HIFU, a-PD-L1 alone, or the combination treatment. 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 numbers of DEGs are shown in plots 3000A-3000F of Figure 30. In CD8 T cells, 1690 DEGs were identified in the three treatment groups, of which 333 genes were unique to the combination treatment, whereas 231 and 365 genes were unique to M-HIFU and anti-PD-L1 monotherapy, respectively, suggesting that the combination treatment is not simply exerting an additive effect of the monotherapies (plot 3000A of Figure 30). The most significantly enriched GO terms of the DEGs upregulated in the combination treatment, together with the other two treatments, were shown as enrichment scores in a heatmap (plot 3000B in Fig. 30). In CD8 T cells, the DEGs were mainly enriched in type I interferon-mediated signaling pathway, activated T cell proliferation, chemokine secretion, cellular response to interferon gamma, interleukin (IL)-12 production, and cytokine secretion. KEGG pathway analysis shows that the upregulated DEGs were significantly enriched in pathways such as complement and coagulation cascades, cytokine-cytokine receptor interactions, and NF-kappa B signaling pathway (online supplemental Fig. 10A). Thus, the combination treatment appears to activate CD8 T cells and enhance antitumor immunity through the regulation of multiple pathways.

[0100] To clarify the genes involved in the enhanced antitumor effect of the combination treatment over M-HIFU monotherapy, gene expression levels by CD8 T cells in the combination treatment and M-HIFU monotherapy were compared (plot 3000C in FIG. 30). Genes related to CD8 T cell activation, such as Cd33, Cx3cr1, Cxcl3, Cxcl11, and Cxcl16, were more than two-fold more strongly expressed after combination treatment compared to M-HIFU monotherapy, confirming the enhanced activation of CD8 T cells by combining anti-PD-L1 with M-HIFU treatment, which corresponded to the superior antitumor effect against distant tumors observed in the bilateral tumor model. In macrophages, among the 822 DEGs that were significantly upregulated in the treated samples, 335 genes were unique to the combination treatment (plot 3000D in FIG. 30), and were enriched in multiple pathways mainly related to lymphocyte migration and defense responses to tumor cells, as well as extracellular matrix / fibroblasts (plot 3000E in FIG. 30). KEGG pathway analysis revealed gene enrichment in protein digestion and absorption, extracellular matrix-receptor interaction, cell adhesion molecules, PI3K-Akt signaling pathway, and T cell receptor signaling pathway in the combination treatment. These results may suggest that combination therapy-treated macrophages are more actively involved in the enhanced antitumor activity and tissue remodeling after tumor destruction by ultrasound. Interestingly, as shown in plot 3000F of FIG. 30, tumor-infiltrating macrophages in the combination treatment group showed a relative elevated expression of gene signatures in both M1 / classically activated macrophages (Dcn, Acpp, Csf1, Adgrl2, and Col4a2) and M2 / alternatively activated macrophages (Il1rl1, Mmp9, Mfsd6, Angptl2, Spint2, and Sft2d2) when compared with those in M-HIFU-treated tumors.

[0101] With respect to FIG. 29, the combination of M-HIFU and anti-PD-L1 antibody induces upregulation of unique DEGs in tumor-infiltrating CD8 T cells and macrophages. MM3MG-HER2 tumors established in the lower limbs 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 both 3 and 6 days after HIFU treatment. Viable 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 plot 3000A, the number of DEGs in CD8 T cells upregulated in the treatment group compared to the no-treatment control is shown in a Venn diagram. With respect to plot 3000B, GO enrichment analysis was performed on the DEGs upregulated in CD8 T cells in each treatment group and summarized in a heatmap. The top 17 GO terms in the combination treatment are shown along with enrichment data in the M-HIFU and anti-PD-L1 monotherapy groups. For plot 3000C, differential gene expression analysis was performed on the DEGs upregulated in CD8 T cells between the combination treatment and M-HIFU monotherapy. Representative DEGs significantly upregulated (log2(fold change)>1, FDR<0.05) in the combination group are shown in red text. For plot 3000D, the number of DEGs in macrophages upregulated in the treatment group compared to the no treatment control is shown in a Venn diagram. For plot 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 treatment are shown along with enrichment data in the M-HIFU and anti-PD-L1 monotherapy groups. For plot 3000F, differential gene expression analysis was performed on DEGs upregulated in macrophages between combination treatment and M-HIFU monotherapy. Representative DEGs that were significantly upregulated in the combination group (log2(fold change)>1, FDR<0.05) and contribute to activated KEGG pathways or are known as M1 or M2 signature genes are indicated in red letters.DEGs, differentially expressed genes; GO, gene ontology; M-HIFU, mechanical high-intensity focused ultrasound; PD-L1, programmed cell death ligand 1.

[0102] Based on the enhancement of systemic tumor antigen-specific immune responses by adding anti-PD-L1 (plot 2900C in FIG. 29A), we hypothesized that PD-1 / PD-L1 blockade would also improve the abscopal effect of M-HIFU. When MM3MG-HER2 tumors were treated with M-HIFU and anti-PD-L1 antibodies were administered intraperitoneally (plot 3100A in FIG. 31), greater growth suppression was observed against distant untreated tumors than either treatment alone in a more rigorous bilateral tumor model, where mice were implanted with a higher number of tumor cells (5-fold higher compared to plot 2900C in FIG. 29A) in the flank (plot 3100B in FIG. 31). To elucidate the changes in the TME in distant HIFU untreated tumors induced by the combined treatment, distant tumors were analyzed by flow cytometry. As shown in plot 3100C of FIG. 31, M-HIFU monotherapy failed to increase the infiltration of CD4 T / CD8 T / NK cells in the rapidly growing distant tumors, which may be the main reason for the weaker antitumor effect against distant tumors. However, combination treatment and anti-PD-L1 monotherapy increased the percentage of CD4+, CD8+, and NK cell populations in distant tumors compared with untreated control and M-HIFU monotherapy (plot 3100C of FIG. 31). Similarly, CD4 T cells again expressed higher levels of activation markers in these groups, and granzyme B expression in CD8 T cells was significantly higher in distant tumors in the combination treatment group (plot 3100D of FIG. 31), suggesting that the cytotoxic function of CD8 T cells was most enhanced in the combination treatment group, which resulted in the strongest abscopal effect (plot 3100B of FIG. 31).

[0103] Finally, to determine which immune cells were responsible for the effect on distant tumors in the bilateral tumor model, mice were treated with M-HIFU + anti-PD-L1 therapy in the presence of depleting antibodies against CD4+ cells, CD8+ cells, or NK cells (plot 3100E in FIG. 31). In both leg and flank tumors, depletion of CD8+ cells or NK cells significantly abolished the anti-tumor effect (plot 3100F in FIG. 31). The greater effect of combination therapy on distant tumors in a CD8+ cell-dependent manner was also confirmed in the JC-HER3 tumor model. To determine how immune cell depletion affects the TME of distant tumors in mice treated with combination therapy, we analyzed the profile of tumor-infiltrating immune cells in distant tumors by flow cytometry. Interestingly, systemic depletion of CD8+ cells or NK cells resulted in an intratumoral immune cell profile after M-HIFU + anti-PD-L1 that was similar to that of untreated control tumors, with reduced ICOS expression and granzyme B production by CD8 T cells, suggesting that these two cell types are not only the main effectors but may also affect the immune composition of the TME. Meanwhile, depletion of CD4+ cells induced a marked increase and activation of CD8 T cells and a decrease in macrophages and Tregs in distant tumors, which may explain the stronger abscopal effect observed in the CD4+ depleted group. These results indicate that both CD8+ cells and NK cells play important roles in immune-based attack of not only HIFU-treated tumors but also distant tumors in this M-HIFU + anti-PD-L1 combination therapy.

[0104] For FIG. 31, the combination of M-HIFU and PD-1 / PD-L1 blockade synergistically inhibited the growth of distant tumors dependent on CD8+ and NK cells. For plot 3100A, MM3MG-HER2 cells were injected into both the left lower leg (1×106 cells) and right flank (5×105 cells) of BALB / c mice on day 0. To generate a more rigorous model, a larger number of cells were implanted into the flank compared to plot 2500C in FIG. 25. Established lower 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. For plot 3100B, tumor growth curves of distant flank tumors are shown. n=10 mice per group, total of 40 mice. For plots 3100C and 3100D, on day 18, distant flank tumors (HIFU untreated side) were harvested and infiltrating immune cells were analyzed by flow cytometry. n=3 (combination group) or 4 (other groups). For plot 3100C, the percentages of CD4+ cells, CD8+ cells, and CD49b+ cells among CD45+ cells are shown. For plot 3100D, the expression of ICOS and Foxp3 on CD4+ cells and ICOS and Granzyme B by CD8+ cells. For plot 3100E, mice were treated with a combination of M-HIFU and anti-PD-L1 antibody on the same treatment schedule as plot 2800A in Figure 28, with or without depleting antibodies against CD4+ cells, CD8a+ cells, and NK cells on days 6, 9, 14, and every 5 days until the end of the experiment. For plot 3100F, tumor growth curves are shown for leg tumors (left) and flank tumors (right). n=5 mice per group, 25 mice total. In plots 3100B and 3100F, error bars represent SE. In plots 3100C and 3100D, error bars 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; programmed death-1, PD-L1, programmed cell death ligand 1.

[0105] Many approaches are under development to alter the TME to induce systemic antitumor immunity, including intratumoral delivery of cytokines and oncolytic viruses, as well as physical tumor destruction, particularly using focused ultrasound. Traditional focused ultrasound approaches induce coagulation necrosis by heating the tissue (T-HIFU); but may not optimally induce systemic antitumor immunity due to denaturation of tumor proteins. Although traditional T-HIFU alters the TME, it still does not induce a memory systemic immune response. Therefore, M-HIFU, which induces cell death and extracellular matrix destruction by acoustic microcavitation rather than heating the tissue, has been investigated, and it has previously been observed to improve antigen presentation and result in a more favorable immune response.

[0106] This study demonstrated that M-HIFU induces local and systemic immunity against BC and is more potent than T-HIFU in the model. This was evidenced by greater antigen presentation, induction of antigen-specific T cells in splenocytes, and increased T cell infiltration in the M-HIFU ablated site and in the contralateral tumor. A potential criticism is that there is a wide range of T-HIFU protocols that may induce entirely different antitumor effects or antitumor immune responses compared to the T-HIFU technique disclosed herein. Some of the existing systems have reported data using a form of T-HIFU (thermal ablative focused ultrasound) to treat poorly immunogenic 4T1 tumors. Thermal ablation alone had limited impact on intratumoral accumulation of activated T cells (due to the immunosuppressive TME), but combination with gemcitabine enhanced tumor control in a CD8 and CD4 T cell-dependent manner and improved mouse survival. T-HIFU of BC in patients has been reported to enhance immune cell infiltration. Recent studies have demonstrated enhanced immunogenicity of BC treated by T-HIFU in combination with systemic ICB or local application of the toll-like receptor 9 agonist CpG. It is also conceivable that the timing of immune assays may have influenced the results of comparative analysis between different treatment methods. For example, T-HIFU-treated tumors may be in the adaptive resistance / wound healing phase at the time of tissue collection in our study. Even with these considerations, the immune assay data, which corresponded well to the in vivo antitumor effects, may indicate the overall ability of these two HIFU methods in inducing antitumor immunity. More preclinical and clinical studies may be needed to evaluate and optimize the antitumor effects of HIFU for BC treatment.

[0107] M-HIFU was able to significantly inhibit the growth of treated tumors and extend the survival of mice bearing a single tumor, but the antitumor activity against untreated distant tumors was modest. One explanation for the observed results is the upregulation of PD-L1 by myeloid cell populations, including macrophages, in M-HIFU-treated tumors, which may have led to the induction of insufficient systemic antitumor immunity. PD-L1 has been shown to exert a constitutive negative signal in macrophages, inducing an immunosuppressive phenotype. Anti-PD-L1 treatment, by itself, remodeled the macrophage compartment in tumors to a more proinflammatory phenotype, mainly through increased IFN-γ levels in the TME, leading to enhanced T cell activity. In the study disclosed above, administration of anti-PD-L1 antibody and M-HIFU enhanced the antitumor effect against distant tumors. Importantly, the data suggest that the net effect of the combined treatment is a more favorable pattern of T cell and NK cell attraction and activation, not only at the site of the HIFU-treated tumor, but also in distant untreated tumors. As mentioned previously, there are other examples of combining HIFU with pathogen-associated molecular patterns such as CpG to produce an abscopal effect. These data, together with this demonstration that tumor control is CD8+ T cell dependent, support the contention that HIFU induces antigen-specific CD8+ T cell responses that can be augmented by nonspecific immune stimulators.

[0108] Despite the substantial antitumor effects of the combination therapy, some distant tumors grew after the initial period of control. We hypothesized that there are two potential explanations for the lack of complete control. First, the previously observed repolarization of macrophages to M2 may be involved in tissue repair within the altered TME after tumor apoptosis in response to M-HIFU and anti-PD-L1. In the M-HIFU and anti-PD-L1 model, macrophage genes involved in cell-matrix adhesion and extracellular matrix organization, which are important for wound healing, were upregulated in addition to genes typically associated with the M1 phenotype. However, we have observed this phenomenon of increased intratumoral phagocytic macrophages accompanied by upregulation of gene signatures of wound healing, ECM remodeling, and anti-inflammation in anti-CD47 antibody-treated BC mice with ongoing tumor regression.

[0109] Thus, the tendency to elevate gene expression consistent with a repair phenotype may not be detrimental once the initial antitumor immune response is activated by M1 macrophages. Second, intratumoral Tregs derived from infiltrating CD4+ T cells may counteract effector T cell function. Flow cytometry analysis of M-HIFU-treated tumors demonstrates that the combination strategy increased the infiltration of CD4+ T cells, including a population that exhibited increased Foxp3 expression, consistent with Treg cells. Depletion of CD4+ Tregs enhanced the antitumor effect of the combination treatment both locally and in distant tumors that were not HIFU-treated. One further observation is that human BC is not traditionally considered an inflammatory immunogenic tumor and is less responsive to immunotherapies such as ICB compared to other immunogenic solid tumors such as melanoma and lung cancer. In this study, we used tumor models engineered to express OVA, HER2, or HER3 antigens to monitor the induction of tumor antigen-specific immunity by HIFU treatment. The forced expression of these foreign antigens rendered the tumor cells more immunogenic, unlike in the case of human BC, so the induced antigen-specific immune responses in these models required careful interpretation. Importantly, however, in one of the BC models, JC-HER3 tumors were transplanted into HER3 transgenic mice that were immune-tolerant to the HER3 antigen, and M-HIFU treatment was found to induce anti-HER3 cellular immune responses with significant antitumor and abscopal effects. These data suggest that M-HIFU could break immune tolerance to self-antigens. In summary, the systems and methods described herein demonstrated beneficial alteration of the TME by M-HIFU and enhanced systemic anti-tumor effects by the combination of M-HIFU and anti-PD-L1 antibody, which resulted in significantly stronger growth inhibition of distant tumors compared with M-HIFU or anti-PD-L1 monotherapy. To further enhance the anti-tumor effects and eradicate distant tumors by induced anti-tumor immunity, clinically applicable strategies of combining M-HIFU with anti-PD-L1, such as depletion of Tregs by targeted antibodies and promotion of M1-biased TME by intratumoral IL-12 gene therapy, may be tested.

[0110] Example 7 Gene delivery using sonoporation energy This example demonstrates gene / mRNA delivery to non-tumor tissues, including tumor and normal tissues, by sonoporation. In vivo studies using sonoporation were performed using mouse CT26 cells, bilateral flank injections, and BALB / c mice. An exemplary sonoporation transducer is illustrated in FIG. 32. The sonication parameters were: frequency 1.1 MHz, peak negative pressure (PNP): 0.1 MPa, 0.4 MPa, 0.7 MPa, and 200 cycles. In the first procedure, the transducer was inserted into the target, the solution was injected into the target for 60 seconds, then sonicated for 30 seconds, and in the second procedure, the transducer was inserted into the target, the solution was injected into the target for 60 seconds, then sonicated for 30 seconds, the solution was injected again for 30 seconds, then sonicated for 30 seconds. The injected solution was 7.68 × 10 8 The solution consisted of microbubbles at a concentration of 10 ...

[0111] There were 12 test groups consisting of 3 (various PNPs) x 2 (different procedures) x 2 (replicates). There were 3 control groups (i.e., no treatment, with plasmid alone, with plasmid and with MB). The test conditions are shown in Figure 33. Number 1 in Figure 33 was the negative-negative control group, i.e., no treatment; number 2 in Figure 33 was the negative control group, i.e., with plasmid alone; number 3 in Figure 33 was the negative control group, i.e., with plasmid and with MB. Positive controls included intratumoral injection of Ad-luciferase and GFP-luciferase plasmid injection and electroporation (e.g., same plasmid concentration, same plasmid, same time point).To eliminate the risk of missing parts of the tumor expressing the plasmid, the entire tumor was lysed.

[0112] The data was measured with a luminometer and is shown in Figures 34-37. Figure 35 illustrates data using Ad-luciferase or saline as intratumoral injections. Figure 36 illustrates data using electroporation. Figure 37 illustrates data using sonoporation. It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be construed as limiting the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the invention, including without limitation those related to its chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use. For reasons of completeness, the various aspects of the invention are set out in the following numbered items.

[0113] Item 1. A catheter including an elongated hollow tube, a first lumen within the elongated hollow tube, a second lumen within the elongated hollow tube, a transducer located within the elongated hollow tube and adjacent the first lumen or the second lumen, the ultrasound transducer configured to emit ultrasound waves through the lumen to a target, and a needle located within the elongated hollow tube and configured to extend from the first lumen or the second lumen to enter the target and deliver a therapy to the target. Item 2. The catheter of item 1, wherein therapy is delivered to the target while ultrasound is delivered to the target. Item 3. The catheter of item 1 or 2, wherein the transducer is configured to deliver intracorporeal sonoporation to a target. Item 4. The catheter according to any one of items 1 to 3, wherein the in vivo sonoporation generates acoustic cavitation in the target, inducing the formation of pores in the target's cell membrane, and increasing the permeability of the target. Item 5. The catheter according to any one of Items 1 to 4, wherein the target is malignant tissue, tumor tissue, peritumor tissue, non-malignant tissue, blood cells, or immune cells. Item 6. The catheter of any one of items 1 to 5, wherein the needle is configured to deliver the immunotherapy directly into the target. Item 7. The catheter of any one of items 1 to 5, wherein the needle is configured to deliver the gene therapy directly into the target.

[0114] Item 8. The catheter of any one of items 1 to 5, wherein the needle is configured to deliver one or more therapeutic agents directly into the target. Item 9. The catheter of item 1, wherein the transducer is configured to deliver high pressure acoustic bursts of focused ultrasound toward the target to generate acoustic cavitation at the target. Item 10. The catheter described in Item 9, wherein acoustic cavitation at the target results in the expansion and collapse of microbubbles, releasing high pressure cavitation energy and disrupting the extracellular matrix of the target. Item 11. The catheter according to item 9 or 10, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cells, or immune cells. Item 12. The catheter of any one of items 9 to 11, wherein the needle is configured to deliver the immunotherapy directly into the target. Item 13. The catheter of any one of items 9 to 11, wherein the needle is configured to deliver the gene therapy directly into the target.

[0115] Item 14. The catheter of any one of items 9 to 11, wherein the needle is configured to deliver one or more therapeutic agents directly into the target. Item 15. The catheter of any one of items 1 to 14, wherein the transducer is configured to deliver energy to the target that results in an acoustic pressure (peak negative) in the range of 5 MPa to greater than 50 MPa applied to the target. Item 16. The catheter according to item 15, wherein the acoustic pressure applied to the target is within the range of 15 MPa to 30 MPa. Item 17. The catheter according to Item 15 or 16, wherein the acoustic pressure applied to the target is within the range of 20 MPa to 45 MPa. Item 18. The catheter of any one of items 1 to 17, wherein the transducer is configured to ablate a target. Item 19. The catheter of any one of items 1 to 18, wherein the target is adjacent to or within a gas-filled anatomical organ. Item 20. The catheter according to item 19, wherein the gas-filled anatomical organ is the lung, intestine, airway, or bladder. Item 21. The catheter of any one of items 1 to 20, wherein the transducer includes multiple electrodes positioned adjacent to one another in a non-linear direction. Item 22. The catheter according to item 21, wherein the multiple electrodes are positioned to form a radius of curvature in the range of 5 mm to 10 mm.

[0116] Item 23. The catheter of item 21 or 22, wherein each of the plurality of electrodes has an aperture size of approximately 1.4 mm x 1.8 mm. Item 24. The catheter of any one of items 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. Item 25. The catheter according to any one of items 1 to 24, wherein the catheter is a 6 to 7 Fr catheter. Item 26. The catheter according to any one of Items 1 to 25, wherein the catheter is an 8 to 10 Fr catheter. Item 27. A method of treating a malignant tumor, comprising: inserting the catheter of 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 an acoustic peak negative pressure in the range of 10 MPa to 40 MPa being applied to the malignant tumor; and activating the needle to extend from the second lumen into the malignant tumor to deliver a therapeutically effective amount of the pharmaceutical composition. Item 28. The method according to item 27, wherein the pharmaceutical composition comprises at least one selected from the group consisting of cytokines, chemokines, and other biological proteins (such as IL-12), oncolytic viruses, CAR-T cells, TILs, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory drugs, antiproliferative drugs, anti-migratory agents, antifibrotic agents, proapoptotic drugs, vasodilators, calcium channel blockers, antineoplastic agents, anticancer agents, antibodies, antithrombotic agents, antiplatelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, and non-immunosuppressants.

[0117] Item 29. The method according to item 27 or 28, wherein the pharmaceutical composition comprises an alkylating agent for targeting DNA. Item 30. A system for treatment of a target, comprising: an ultrasonic energy source; a device coupled to the ultrasonic energy source and configured and arranged to direct ultrasonic energy to the target, release one or more microbubbles, and release one or more therapeutic agents, wherein upon receipt of the ultrasonic energy, the microbubbles burst, thereby disrupting the target and the extracellular matrix (ECM) surrounding the target, and enabling the one or more therapeutic agents to be delivered into the target. Item 31. The system of item 30, wherein the one or more microbubbles are constructed and arranged to contain one or more therapeutic agents, and wherein the one or more microbubbles burst upon receiving ultrasound energy, thereby releasing the one or more therapeutic agents into the target.

[0118] Item 32. The system of items 30 or 31, wherein the device further comprises a configuration and arrangement for releasing one or more contrast agents. Item 33. The system of any one of items 30 to 32, wherein the ultrasound energy comprises high intensity focused ultrasound (HIFU). Item 34. The system of any one of items 30 to 32, wherein the ultrasound energy includes histotripsy. Item 35. The system according to any one of items 30 to 34, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cells, or immune cells. Item 36. A method for delivery of a drug to a target, comprising inserting a catheter including the device of item 30 into a subject toward the target, generating an ultrasonic energy output near the target, providing one or more microbubbles near the target, and providing one or more therapeutic agents through the catheter, wherein the microbubbles burst upon receiving the ultrasonic 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. Item 37. The method of item 36, wherein the one or more microbubbles are constructed and arranged to contain one or more therapeutic agents, and wherein the one or more microbubbles burst upon receiving ultrasound energy, thereby releasing the one or more therapeutic agents into the target. Item 38. The method according to item 36 or 37, wherein the method further comprises releasing one or more imaging agents.

[0119] Item 39. The method of any one of items 36 to 38, wherein the one or more therapeutic agents are selected from the group consisting of cytokines, chemokines, and other biological proteins (such as IL-12), oncolytic viruses, CAR-T cells, TILs, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory drugs, anti-proliferative drugs, anti-migratory agents, anti-fibrotic agents, pro-apoptotic drugs, vasodilators, calcium channel blockers, antineoplastic agents, anticancer agents, antibodies, antithrombotic agents, antiplatelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, non-immunosuppressants, and combinations thereof. Item 40. The method of any one of items 36 to 39, wherein the one or more therapeutic agents include a cytokine, a chemokine, or other biological protein. Item 41. The method of item 40, wherein the cytokine comprises IL-12.

Claims

1. A long, slender hollow tube; The first lumen in a long, slender hollow tube; The second lumen inside a long, slender hollow tube; A transducer located within an elongated hollow tube and adjacent to a first lumen or a second lumen, wherein the ultrasonic transducer is configured to emit ultrasonic waves to a target through the lumen; and A needle located within a slender, hollow tube, extending from a first or second lumen and configured to enter the target and deliver therapy to the target. A catheter containing a catheter.

2. The catheter according to claim 1, wherein a therapy is delivered to a target while ultrasound is delivered to the target.

3. The catheter according to claim 1 or 2, wherein the transducer is configured to deliver intracellular sonoporation to a target.

4. The catheter according to claim 1, wherein in vivo sonoporation generates acoustic cavitation at the target, induces the formation of pores in the target cell membrane, and increases the permeability of the target.

5. The catheter according to claim 1, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cells, or immune cells.

6. The catheter according to claim 1, wherein the needle is configured to deliver immunotherapy directly into a target.

7. The catheter according to claim 1, wherein the needle is configured to deliver gene therapy directly into a target.

8. The catheter according to claim 1, wherein the needle is configured to directly deliver one or more therapeutic agents into a target.

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

10. The catheter according to claim 9, wherein acoustic cavitation at the target causes expansion and collapse of microbubbles, releasing high-pressure cavitation energy and disrupting the extracellular matrix of the target.

11. The catheter according to 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 according to claim 9, wherein the needle is configured to deliver immunotherapy directly into a target.

13. The catheter according to claim 9, wherein the needle is configured to deliver gene therapy directly into a target.

14. The catheter according to claim 9, wherein the needle is configured to deliver one or more therapeutic agents directly into a target.

15. The catheter according to claim 1, wherein the transducer is configured to deliver energy to a target that produces a sound pressure (negative peak) in the range of 5 MPa to over 50 MPa applied to the target.

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

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

18. The catheter according to claim 1, wherein the transducer is configured to ablate a target.

19. The catheter according to claim 1, wherein the target is adjacent to or located within a gas-filled anatomical organ.

20. The catheter according to claim 19, wherein the anatomical organ filled with gas is the lung, intestine, airway, or bladder.

21. The catheter according to claim 1, wherein the transducer includes a plurality of electrodes positioned adjacent to each other in a non-linear direction.

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

23. The catheter according to claim 21 or 22, wherein each of the multiple electrodes has an opening size of approximately 1.4 mm × 1.8 mm.

24. The catheter according to claim 21, wherein at least one of the multiple electrodes includes a piezoelectric plate having a thickness of less than about 50 μm to about 500 μm.

25. The catheter according to claim 1, wherein the catheter is a 6-7 Fr catheter.

26. The catheter according to claim 1, wherein the catheter is an 8-10 Fr catheter.

27. A catheter used in a method for treating malignant tumors, wherein the method is Inserting a catheter into the target area towards the malignant tumor; To activate the transducer and deliver energy through the first lumen to the malignant tumor that produces an acoustic peak negative pressure in the range of 10 MPa to 40 MPa applied to the malignant tumor; and Activating the needle to extend it into the malignant tumor through the second lumen and deliver a therapeutically effective amount of the pharmaceutical composition. The catheter according to claim 1, including the following:

28. The catheter according to claim 27, wherein the pharmaceutical composition comprises at least one selected from the group consisting of cytokines, chemokines, and other biological proteins (such as IL-12), oncolytic viruses, CAR-T cells, TILs, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory drugs, antiproliferative drugs, anti-migration agents, anti-fibrotic agents, apoptosis promoters, vasodilators, calcium channel blockers, antineoplastic agents, anticancer agents, antibodies, antithrombotic agents, antiplatelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, and non-immunosuppressants.

29. The catheter according to claim 27 or 28, wherein the pharmaceutical composition comprises an alkylating agent for targeting DNA.

30. A system for treating targets, Ultrasonic energy source; Connected to an ultrasonic energy source, Directing ultrasonic energy towards the target, It releases one or more microbubbles, To release one or more therapeutic agents A device configured and arranged such that, upon receiving ultrasonic energy, microbubbles burst, thereby destroying the target and the extracellular matrix (ECM) surrounding the target, and enabling the delivery of one or more therapeutic agents into the target. A system that includes this.

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

32. The system according to claim 30 or 31, wherein the device further includes configurations and arrangements for releasing one or more contrast agents.

33. The system according to claim 30, wherein the ultrasonic energy includes high-density focused ultrasound (HIFU).

34. The system according to claim 30, wherein the ultrasonic energy includes histotripsy.

35. The system according to claim 30, wherein the target is malignant tissue, tumor tissue, peritumoral tissue, non-malignant tissue, blood cells, or immune cells.

36. A system used in a method for delivering a drug to a target, wherein the method is Inserting a catheter containing the aforementioned device into the target area, To generate ultrasonic energy output near the target, To supply one or more microbubbles near the target, and To deliver one or more therapeutic agents through a catheter. The system according to claim 30, comprising, wherein microbubbles burst upon receiving ultrasonic energy, thereby disrupting the target and surrounding extracellular matrix (ECM), allowing one or more therapeutic agents to be delivered into the target and thereby treat the target.

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

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

39. The system according to claim 36, wherein one or more therapeutic agents are selected from the group consisting of cytokines, chemokines, and other biological proteins (such as IL-12), oncolytic viruses, CAR-T cells, TILs, cDNA, mRNA, self-replicating RNA, proteins, immunosuppressants, anti-inflammatory drugs, antiproliferative drugs, anti-migration agents, anti-fibrotic agents, apoptosis promoters, vasodilators, calcium channel blockers, antineoplastic agents, anticancer agents, antibodies, antithrombotic agents, antiplatelet agents, IIb / IIIa agents, antiviral agents, mTOR (mammalian target of rapamycin) inhibitors, non-immunosuppressants, and combinations thereof.

40. The system according to claim 36, wherein one or more therapeutic agents include cytokines, chemokines, or other biological proteins.

41. The system according to claim 40, wherein the cytokine includes IL-12.

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