Controlled release of cell nuclei by histotripsy

Abstract

A histotripsy therapy system configured for the treatment of tissue is provided, which may include any number of features. Provided herein are systems and methods that provide efficacious non-invasive and minimally invasive therapeutic, diagnostic and research procedures. Methods of biological tissue modification are also provided. The systems and methods disclosed here are uniquely different in that they aim to target, plan and treat target tissue, and selectively destroy and modify cellular architecture, but with specific intent and treatment parameters specialized for retaining specific target physical biological structures within the cell itself.

Description

CONTROLLED RELEASE OF CELL NUCLEI BY HISTOTRIPSYPRIORITY CLAIM

[0001] This patent application claims priority to U.S. provisional patent application no. 63 / 718,447, titled “CONTROLLED RELEASE OF CELL NUCLEI BY HISTOTRIPSY,” and filed on November 8, 2024, which is herein incorporated by reference in its entirety.INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.GOVERNMENT RIGHTS

[0003] This invention was made with government support under CA269394 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0004] The present disclosure details novel histotripsy systems configured to produce acoustic cavitation, methods, devices and procedures for the minimally and non-invasive treatment of healthy, diseased and / or injured tissue. The histotripsy systems and methods described herein, also referred to Histotripsy, may include transducers, drive electronics, positioning robotics, imaging systems, and integrated treatment planning and control software to provide comprehensive treatment and therapy for soft tissues in a patient.BACKGROUND

[0005] Many medical conditions require invasive surgical interventions. Invasive procedures often involve incisions, trauma to muscles, nerves and tissues, bleeding, scarring, trauma to organs, pain, need for narcotics during and following procedures, hospital stays, and risks of infection. Non-invasive and minimally invasive procedures are often favored, if available, to avoid or reduce such issues. Unfortunately, non-invasive and minimally invasive procedures may lack the precision, efficacy or safety required for treatment of many types of diseases and conditions. Enhanced non-invasive and minimally invasive procedures are needed, preferably not requiring ionizing or thermal energy for therapeutic effect.

[0006] Histotripsy, or pulsed ultrasound cavitation therapy, is a technology where extremely short, intense bursts of acoustic energy induce controlled cavitation (microbubble- 1 -SG Docket No. 10860-536.600formation) within the focal volume. The vigorous expansion and collapse of these microbubbles mechanically homogenizes cells and tissue structures within the focal volume. This is a very different end result than the coagulative necrosis characteristic of thermal ablation. To operate within a non-thermal, Histotripsy realm; it is necessary to deliver acoustic energy in the form of high amplitude acoustic pulses with low duty cycle.

[0007] Compared with conventional focused ultrasound technologies, Histotripsy has important advantages: 1) the destructive process at the focus is mechanical, not thermal; 2) cavitation appears bright on ultrasound imaging thereby confirming correct targeting and localization of treatment; 3) treated tissue generally, but not always, appears darker (more hypoechoic) on ultrasound imaging, so that the operator knows what has been treated; and 4) Histotripsy produces lesions in a controlled and precise manner. It is important to emphasize that unlike thermal ablative technologies such as microwave, radiofrequency, and high- intensity focused ultrasound (HIFU), Histotripsy relies on the mechanical action of cavitation for tissue destruction.

[0008] Ultrasound imaging is used for targeting and monitoring histotripsy ablation. During therapy, cavitation appears as a bright, transient, localized echo indicating where ablation is occurring. In a homogeneous medium with known sound speed and attenuation, the exact location of the histotripsy focus can be predicted based on the system design. Real treatments require the sound field to pass through a water coupling bath and multiple tissue layers which aberrate and refract the acoustic beam shifting the true focal location. The large aperture of the therapeutic transducer compared to an imager greatly exacerbates these effects. The current clinical histotripsy system uses an estimated focal location for ultrasound guided targeting based on calibration treatments in a tissue phantom with the imager physically registered to the therapy transducer. There can still be a residual focal shift of several millimeters, hence the focal location is confirmed / corrected at the start of treatment by ramping the ultrasound pressure until cavitation is generated and visualized on ultrasound imaging. This procedure must be repeated at the center and margins of the target volume. Cavitation-based targeting has two major drawbacks. 1) Cavitation generation, even briefly, will cause damage to tissue at the focus. This is a safety concern if a refracted / aberrated focus targets a location outside the planned treatment volume prior to correction. 2) Some portions of the therapy transducer may be blocked by impenetrable structures (ribs, lung / bowel gas) that could be harmed by excess acoustic exposure.- 2 -SG Docket No. 10860-536.600BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0010] FIGS. 1 A-1B illustrate an ultrasound imaging and therapy system.

[0011] FIGS. 2A-2F show H&E histology slide of the in vivo mouse B16F10 tumors: A) control untreated and treated by histotripsy at B) 2, C) 8, D) 20, E) 40, and F) 100 ppi. At 2 and 8 ppi, partial cellular disruption is observed. At 20ppl and 40ppl, there are no intact cells with substantial extracellular free-flowing cell nuclei (dark dots; examples pointed by yellow arrows). At 100 ppi, there are reduced extracellular free-flowing cell nuclei, and nuclei look more shrunken (examples pointed by yellow arrows).

[0012] FIGS. 3 A-3C: A) Tumor growth curve (Tumor volume vs. days post inoculation) for untreated control, 8ppl, 20ppl, 40ppl, and lOOppl histotripsy treatment groups (n=4). The treatment groups had slower tumor growth compared to the control group. The 20ppl and 40ppl histotripsy treatment groups had the slower tumor growth compared to 8ppl and lOOppl . B)-C) IHC measurements show that the 40ppl groups produced significantly higher CD45 and CD8 T-cell infiltration in the local treated tumor compared to other groups.

[0013] FIGS. 4A-4E: H&E histology slide of the in vivo mouse MC 38 tumors A) control untreated and treated by histotripsy at B) 15, C) 50, and D) 100 ppi. There are extracellular free-floating cell nuclei (dark dots; examples pointed by yellow arrows) in 15ppl and 50ppl slides, but no nuclei are available at lOOppl.

[0014] FIGS. 5A-5B: 5 A) The increase of the cavitation lifespan plateaued at around 15PPL of the histotripsy dose. 5B) Kaplan-Meier survival curves for different histotripsy doses in the murine MC38 CRC model (n = 6-12 per group) showed the best survival results (complete tumor regression in all mice) for the 15PPL group.

[0015] FIG. 6: H&E histology slide of the in vivo mouse MC 38 tumors control untreated and treated by histotripsy at 10, 25, 50, 100, and 300 ppi. At lOppl and 25ppl, there is partial tumor cellular disruption. At 50ppl, most tumor cells are disrupted in the target region, with free floating tumor cell nuclei released. The free-floating cell nuclei reduced at lOOppl. At 300ppl, there is no observable cellular and subcellular components inside the treated region.

[0016] FIG. 7: Kaplan-Meier survival curves for two cohorts of experiments. Left: The first cohort includes four groups of mice: untreated control and 10, 25, and 50 ppi treatment- 3 -SG Docket No. 10860-536.600groups (n=5-6). Right: The second cohort includes four groups of mice: untreated control and 50, 100, and 300 ppi treatment groups (n=5-6).

[0017] FIGS. 8A-8C: 8A) Released HER2 levels of histotripsy -treated cell suspension in cell free fragments showing higher HER2 release with increasing histotripsy dose. 8B) Released HER2 levels of histotripsy -treated tumors in cell free fragments detected by UPLC at 285 nm showing increased HER2 release with increasing histotripsy dose. 8C) Western blot analysis of histotripsy cell free fragment (C.F.) probed with monoclonal HER2 antibody. Lanes 1 and 2 were ladder (13 pL) and rHER2 (0.012 pg), respectively as references. Lanes 3-6 were the analysis for cell lysis with a 15 pg of protein per lane. Lane 3 was untreated, lanes 4-6 were the low, moderate, and high dose histotripsy treatment, respectively. LH: lOppl; MH: 50ppl; HH: lOOppl.

[0018] FIGS. 9A-9D. HER2 detected after in vivo histotripsy treatment of the mouse HER2 breast cancer model. 9A) Representative ultrasound image of the histotripsy cavitation cloud during treatment. Analysis of in vivo histotripsy E0771E2 tumor cell free fragments / supernatants with 9B) UPLC assay, detected at 285 nm; 9C) BCA protein assay. 9D) Western blot assay probed with monoclonal HER2 antibody. Ladder was loaded with 15 pL / lane; Positive control rHER2 was loaded with 0.01 pg / lane; Samples were loaded with 50 pg / lane protein. n = 4. *** p < 0.001.SUMMARY OF THE DISCLOSURE

[0019] Histotripsy produces tissue fractionation through dense energetic bubble clouds generated by short, high-pressure, ultrasound pulses. When using pulses shorter than 2 cycles, the generation of these energetic bubble clouds only depends on where the peak negative pressure (P-) exceeds an intrinsic threshold for inducing cavitation in a medium (typically 26 - 30 MPa in soft tissue with high water content).

[0020] A method of providing histotripsy therapy is provided, comprising: delivering histotripsy pulses with a histotripsy therapy transducer to a target tissue volume to generate cavitation within the target tissue volume; obtaining B-mode ultrasound images of the target tissue volume while the histotripsy pulses are being delivered; evaluating one or more ultrasound backscatter parameters to predict a degree of cellular destruction within the target tissue volume; and stopping delivery of the histotripsy pulses based on the evaluation of the ultrasound backscatter parameters.

[0021] In some aspects, the one or more ultrasound backscatter parameters comprises backscatter intensity. In other aspects, the one or more ultrasound backscatter parameters- 4 -SG Docket No. 10860-536.600comprises backscatter texture. In some aspects, the one or more ultrasound backscatter parameters comprises backscatter isotropy.

[0022] Another method of providing histotripsy therapy is provided, comprising: delivering histotripsy pulses with a histotripsy therapy transducer to a target tissue volume to generate cavitation within the target tissue volume; receiving acoustic cavitation emission (ACE) signals from the cavitation with the histotripsy therapy transducer; calculating a time duration between initiation and collapse of the cavitation from the ACE signals to predict a degree of cellular destruction within the target tissue volume; and stopping delivery of the histotripsy pulses based on the calculated time duration of the cavitation.

[0023] In some aspects, the method includes evaluating changes in the time duration as additional histotripsy pulses are delivered.

[0024] In some aspects, the method includes identifying a plateau in the time duration of the cavitation.

[0025] In another aspect, the method includes associating complete cellular membrane disruption of the target tissue volume with the plateau in the time duration of the cavitation.

[0026] In some aspects, the methods above can include adjusting delivery of histotripsy pulses based on the degree of cellular destruction.

[0027] In some aspects, the target tissue volume comprises a tumor. In other aspects, the target tissue volume comprises liver tissue.

[0028] A histotripsy therapy system is provided, comprising: a B-mode ultrasound imaging system configured to obtain B-mode ultrasound images of the target tissue volume; a histotripsy therapy transducer array; a robotic positioning system coupled to the histotripsy therapy transducer array, the robotic positioning system being configured to place a focus of the histotripsy transducer array within the target tissue volume; and an electronic controller configured to: control the histotripsy therapy transducer to deliver histotripsy pulses to the target tissue volume to generate cavitation within the target tissue volume; control the B- mode ultrasound imaging system to obtain B-mode ultrasound images of the target tissue volume while the histotripsy pulses are being delivered; evaluate one or more ultrasound backscatter parameters to predict a degree of cellular destruction within the target tissue volume; and control the histotripsy therapy transducer to stop delivery of the histotripsy pulses based on the evaluation of the ultrasound backscatter parameters.

[0029] A histotripsy therapy system is also provided, comprising: a histotripsy therapy transducer array; a robotic positioning system coupled to the histotripsy therapy transducer array, the robotic positioning system being configured to place a focus of the histotripsy transducer array within the target tissue volume; and an electronic controller configured to: - 5 -SG Docket No. 10860-536.600control the histotripsy therapy transducer to deliver histotripsy pulses to the target tissue volume to generate cavitation within the target tissue volume; receive acoustic cavitation emission (ACE) signals from the cavitation with the histotripsy therapy transducer; calculate a time duration between initiation and collapse of the cavitation from the ACE signals to predict a degree of cellular destruction within the target tissue volume; and control the histotripsy therapy transducer to stop delivery of the histotripsy pulses based on the calculated time duration of the cavitation.DETAILED DESCRIPTION

[0030] Provided herein are systems and methods that provide efficacious non-invasive and minimally invasive therapeutic, diagnostic and research procedures. In particular, provided herein are optimized systems and methods that provide targeted, efficacious histotripsy in a variety of different regions and under a variety of different conditions without causing undesired tissue damage to intervening / non-target tissues or structures.

[0031] Balancing desired tissue destruction in target regions with the avoidance of damage to non-target regions presents a technical challenge. This is particularly the case where time efficient procedures are desired. Conditions that provide fast, efficacious tissue destruction tend to cause undue heating in non-target tissues. Undue heating can be avoided by reducing energy or slower delivery of energy, both of which run contrary to the goals of providing a fast and efficacious destruction of target tissue. Provided herein are a number of technologies that individually and collectively allow for fast, efficacious target treatment without undesired damage to non-target regions.

[0032] The system, methods and devices of the disclosure may be used for the minimally or non-invasive acoustic cavitation and treatment of healthy, diseased and / or injured tissue, including in extracorporeal, percutaneous, endoscopic, laparoscopic, and / or as integrated into a robotically-enabled medical system and procedures. As will be described below, the histotripsy system may include various electrical, mechanical and software sub-systems, including a Cart, Therapy, Integrated Imaging, Robotics, Coupling and Software. The system also may comprise various Other Components, Ancillaries and Accessories, including but not limited to patient surfaces, tables or beds, computers, cables and connectors, networking devices, power supplies, displays, drawers / storage, doors, wheels, illumination and lighting and various simulation and training tools, etc. All systems, methods and means creating / controlling / delivering histotripsy are considered to be a part of this disclosure, including new related inventions disclosed herein.- 6 -SG Docket No. 10860-536.600

[0033] In one embodiment, the histotripsy system is configured as a mobile therapy cart, which further includes a touchscreen display with an integrated control panel with a set of physical controls, a robotic arm, a therapy head positioned on the distal end of the robot, a patient coupling system and software to operate and control the system.

[0034] The mobile therapy cart architecture can comprise internal components, housed in a standard rack mount frame, including a histotripsy therapy generator, high voltage power supply, transformer, power distribution, robot controller, computer, router and modem, and an ultrasound imaging engine. The front system interface panel can comprise input / output locations for connectors, including those specifically for two ultrasound imaging probes (handheld and probe coaxially mounted in the therapy transducer), a histotripsy therapy transducer, AC power and circuit breaker switches, network connections and a foot pedal. The rear panel of the cart can comprise air inlet vents to direct airflow to air exhaust vents located in the side, top and bottom panels. The side panels of the cart include a holster and support mechanism for holding the handheld imaging probe. The base of the cart can be comprised of a cast base interfacing with the rack mounted electronics and providing an interface to the side panels and top cover. The base also includes four recessed casters with a single total locking mechanism. The top cover of the therapy cart can comprise the robot arm base and interface, and a circumferential handle that follows the contour of the cart body. The cart can have inner mounting features that allow technician access to cart components through access panels.

[0035] The touchscreen display and control panel may include user input features including physical controls in the form of six dials, a space mouse and touchpad, an indicator light bar, and an emergency stop, together configured to control imaging and therapy parameters, and the robot. The touchscreen support arm is configured to allow standing and seated positions, and adjustment of the touchscreen orientation and viewing angle. The support arm further can comprise a system level power button and USB and ethemet connectors.

[0036] The robotic arm can be mounted to the mobile therapy cart on arm base of sufficient height to allow reach and ease of use positioning the arm in various drive modes into the patient / procedure work space from set up, through the procedure, and take down. The robotic arm can comprise six degrees of freedom with six rotating joints, a reach of 850 mm and a maximum payload of 5 kg. The arm may be controlled through the histotripsy system software as well as a 12 inch touchscreen polyscope with a graphical user interface. The robot can comprise force sensing and a tool flange, with force (x, y, z) with a range of 50 N, precision of 3.5 N and accuracy of 4.0 N, and torque (x, y, z) with a range of 10.0 Nm, - 7 -SG Docket No. 10860-536.600precision of 0.2 Nm and accuracy of 0.3 Nm. The robot has a pose repeatability of + / - 0.03mm and a typical TCP speed of 1 m / s (39.4 in / s). In one embodiment, the robot control box has multiple I / O ports, including 16 digital in, 16 digital out, 2 analog in, 2 analog out and 4 quadrature digital inputs, and an I / O power supply of 24V / 2A. The control box communication comprises 500 Hz control frequency, Modbus TCP, PROFINET, ethemet / IP and USB 2.0 and 3.0.

[0037] The therapy head can comprise one of a select group of four histotripsy therapy transducers and an ultrasound imaging system / probe, coaxially located in the therapy transducer, with an encoded mechanism to rotate said imaging probe independent of the therapy transducer to known positions, and a handle to allow gross and fine positioning of the therapy head, including user inputs for activating the robot (e.g. for free drive positioning).In some examples, the therapy transducers may vary in size (22 x 17 cm to 28 x 17 cm), focal lengths from 12 - 18 cm, number of elements, ranging from 48 to 64 elements, comprised within 12-16 rings, and all with a frequency of 700 kHz. The therapy head subsystem has an interface to the robotic arm includes a quick release mechanism to allow removing and / or changing the therapy head to allow cleaning, replacement and / or selection of an alternative therapy transducer design (e.g., of different number of elements and geometry), and each therapy transducer is electronically keyed for auto-identification in the system software.

[0038] The patient coupling system can comprise a six degree of freedom, six joint, mechanical arm, configured with a mounting bracket designed to interface to a surgical / interventional table rail. The arm may have a maximum reach of approximately 850 mm and an average diameter of 50 mm. The distal end of the arm can be configured to interface with an ultrasound medium container, including a frame system and an upper and lower boot. The lower boot is configured to support either a patient contacting film, sealed to patient, or an elastic polymer membrane, both designed to contain ultrasound medium (e.g., degassed water or water mixture), either within the frame and boot and in direct contact with the patient, or within the membrane / boot construct. The lower boot provides, in one example, a top and bottom window of approximately 46 cm x 56 cm and 26 cm x 20 cm, respectively, for placing the therapy transducer with the ultrasound medium container and localized on the patient’s abdomen. The upper boot may be configured to allow the distal end of the robot to interface to the therapy head and / or transducer, and to prevent water leakage / spillage. In preferred embodiments, the upper boot is a sealed system. The frame is also configured, in a sealed system, to allow two-way fluid communication between the ultrasound medium container and an ultrasound medium source (e.g., reservoir or fluidics- 8 -SG Docket No. 10860-536.600management system), including, but not limited for filling and draining, as well as air venting for bubble management.

[0039] The system software and work-flow can be configured to allow users to control the system through touchscreen display and the physical controls, including but not limited to, ultrasound imaging parameters and therapy parameters. The graphical user interface of the system comprises a work-flow based flow, with the general procedure steps of 1) registering / selecting a patient, 2) planning, comprising imaging the patient (and target location / anatomy) with the freehand imaging probe, and robot assisted imaging with the transducer head for final gross and fine targeting, including contouring the target with a target and margin contour, of which are typically spherical and ellipsoidal in nature, and running a test protocol (e.g., test pulses) including a bubble cloud calibration step, and a series of predetermined locations in the volume to assess cavitation initiation threshold and other patient / target specific parameters (e.g., treatment depth), that together inform a treatment plan accounting for said target’s location and acoustic pathway, and any related blockage (e.g., tissue interfaces, bone, etc.) that may require varied levels of drive amplitude to initiate and maintain histotripsy. Said parameters, as measured as a part of the test protocol, comprising calibration and multi -location test pulses, are configured in the system to provide input / feedback for updating bubble cloud location in space as needed / desired (e.g., appropriately calibrated to target cross-hairs), as well as determining / interpolating required amplitudes across all bubble cloud treatment locations in the treatment volume to ensure threshold is achieved throughout the volume. Further, said parameters, including but not limited to depth and drive voltage, may be also used as part of an embedded treatability matrix or look up table to determine if additional cooling is required (e.g., off-time in addition to time allocated to robot motions between treatment pattern movements) to ensure robust cavitation and intervening / collateral thermal effects are managed (e.g., staying below t43 curve for any known or calculated combination of sequence, pattern and pathway, and target depth / blockage). The work-flow and procedure steps associated with these facets of planning, as implemented in the system software may be automated, wherein the robot and controls system are configured to run through the test protocol and locations autonomously, or semi -autonomously. Following planning, the next phase of the procedure work-flow, 3) the treatment phase, is initiated following the user accepting the treatment plan and initiating the system for treatment. Following this command, the system is configured to deliver treatment autonomously, running the treatment protocol, until the prescribed volumetric treatment is complete. The status of the treatment (and location of the bubble cloud) is displayed in real-time, adjacent to various treatment parameters, including, but not limited to,- 9 -SG Docket No. 10860-536.600of which may include total treatment time and remaining treatment time, drive voltage, treatment contours (target / margin) and bubble cloud / point locations, current location in treatment pattern (e.g., slice and column), imaging parameters, and other additional contextual data (e.g., optional DICOM data, force torque data from robot, etc.). Following treatment, the user may use the therapy head probe, and subsequently, the freehand ultrasound probe to review and verify treatment, as controlled / viewed through the system user interface. If additional target locations are desired, the user may plan / treat additional targets, or dock the robot to a home position on the cart if no further treatments are planned.

[0040] FIG. 1 A generally illustrates histotripsy system 100 according to the present disclosure, comprising a therapy transducer 102, an imaging system 104, a display and control panel 106, a robotic positioning arm 108, and a cart 110. The system can further include an ultrasound coupling interface and a source of coupling medium, not shown.

[0041] FIG. IB is a bottom view of the therapy transducer 102 and the imaging system 104. As shown, the imaging system can be positioned in the center of the therapy transducer. However, other embodiments can include the imaging system positioned in other locations within the therapy transducer, or even directly integrated into the therapy transducer. In some embodiments, the imaging system is configured to produce real-time imaging at a focal point of the therapy transducer.

[0042] The histotripsy system may comprise one or more of various sub-systems, including a Therapy sub-system that can create, apply, focus and deliver acoustic cavitation / histotripsy through one or more therapy transducers, Integrated Imaging subsystem (or connectivity to) allowing real-time visualization of the treatment site and histotripsy effect through-out the procedure, a Robotics positioning sub-system to mechanically and / or electronically steer the therapy transducer, further enabled to connect / support or interact with a Coupling sub-system to allow acoustic coupling between the therapy transducer and the patient, and Software to communicate, control and interface with the system and computer-based control systems (and other external systems) and various Other Components, Ancillaries and Accessories, including one or more user interfaces and displays, and related guided work-flows, all working in part or together. The system may further comprise various fluidics and fluid management components, including but not limited to, pumps, valve and flow controls, temperature and degassing controls, and irrigation and aspiration capabilities, as well as providing and storing fluids. It may also contain various power supplies and protectors.- 10 -SG Docket No. 10860-536.600CART

[0043] The Cart 110 may be generally configured in a variety of ways and form factors based on the specific uses and procedures. In some cases, systems may comprise multiple Carts, configured with similar or different arrangements. In some embodiments, the cart may be configured and arranged to be used in a radiology environment and in some cases in concert with imaging (e.g., CT, cone beam CT and / or MRI scanning). In other embodiments, it may be arranged for use in an operating room and a sterile environment, or in a robotically enabled operating room, and used alone, or as part of a surgical robotics procedure wherein a surgical robot conducts specific tasks before, during or after use of the system and delivery of acoustic cavitation / histotripsy. As such and depending on the procedure environment based on the aforementioned embodiments, the cart may be positioned to provide sufficient workspace and access to various anatomical locations on the patient (e.g., torso, abdomen, flank, head and neck, etc.), as well as providing work-space for other systems (e.g., anesthesia cart, laparoscopic tower, surgical robot, endoscope tower, etc.).

[0044] The Cart may also work with a patient surface (e.g., table or bed) to allow the patient to be presented and repositioned in a plethora of positions, angles and orientations, including allowing changes to such to be made pre, peri and post-procedurally. It may further comprise the ability to interface and communicate with one or more external imaging or image data management and communication systems, not limited to ultrasound, CT, fluoroscopy, cone beam CT, PET, PET / CT, MRI, optical, ultrasound, and image fusion and or image flow, of one or more modalities, to support the procedures and / or environments of use, including physical / mechanical interoperability (e.g., compatible within cone beam CT work-space for collecting imaging data pre-, peri- and / or post-histotripsy).

[0045] In some embodiments one or more Carts may be configured to work together. As an example, one Cart may comprise a bedside mobile Cart equipped with one or more Robotic arms enabled with a Therapy transducer, and Therapy generator / amplifier, etc., while a companion cart working in concert and at a distance of the patient may comprise Integrated Imaging and a console / display for controlling the Robotic and Therapy facets, analogous to a surgical robot and master / slave configurations.

[0046] In some embodiments, the system may comprise a plurality of Carts, all slave to one master Cart, equipped to conduct acoustic cavitation procedures. In some arrangements and cases, one Cart configuration may allow for storage of specific sub-systems at a distance reducing operating room clutter, while another in concert Cart may comprise essentially bedside sub-systems and componentry (e.g., delivery system and therapy).- 11 -SG Docket No. 10860-536.600

[0047] One can envision a plethora of permutations and configurations of Cart design, and these examples are in no way limiting the scope of the disclosure.HISTOTRIPSY

[0048] Histotripsy comprises short, high amplitude, focused ultrasound pulses to generate a dense, energetic, “bubble cloud”, capable of the targeted fractionation and destruction of tissue. Histotripsy is capable of creating controlled tissue erosion when directed at a tissue interface, including tissue / fluid interfaces, as well as well-demarcated tissue fractionation and destruction, at sub-cellular levels, when it is targeted at bulk tissue. Unlike other forms of ablation, including thermal and radiation-based modalities, histotripsy does not rely on heat or ionizing (high) energy to treat tissue. Instead, histotripsy uses acoustic cavitation generated at the focus to mechanically effect tissue structure, and in some cases liquefy, suspend, solubilize and / or destruct tissue into sub-cellular components.

[0049] Histotripsy can be applied in various forms, including: 1) Intrinsic-Threshold Histotripsy: Delivers pulses with at least a single negative / tensile phase sufficient to cause a cluster of bubble nuclei intrinsic to the medium to undergo inertial cavitation, 2) Shock- Scattering Histotripsy: Delivers typically pulses 3-20 cycles in duration. The amplitude of the tensile phases of the pulses is sufficient to cause bubble nuclei in the medium to undergo inertial cavitation within the focal zone throughout the duration of the pulse. These nuclei scatter the incident shockwaves, which invert and constructively interfere with the incident wave to exceed the threshold for intrinsic nucleation, and 3) Boiling Histotripsy: Employs pulses roughly 1-20 ms in duration. Absorption of the shocked pulse rapidly heats the medium, thereby reducing the threshold for intrinsic nuclei. Once this intrinsic threshold coincides with the peak negative pressure of the incident wave, boiling bubbles form at the focus.

[0050] The large pressure generated at the focus causes a cloud of acoustic cavitation bubbles to form above certain thresholds, which creates localized stress and strain in the tissue and mechanical breakdown without significant heat deposition. At pressure levels where cavitation is not generated, minimal effect is observed on the tissue at the focus. This cavitation effect is observed only at pressure levels significantly greater than those which define the inertial cavitation threshold in water for similar pulse durations, on the order of 10 to 30 MPa peak negative pressure.

[0051] Histotripsy may be performed in multiple ways and under different parameters. It may be performed totally non-invasively by acoustically coupling a focused ultrasound transducer over the skin of a patient and transmitting acoustic pulses transcutaneously through overlying (and intervening) tissue to the focal zone (treatment zone and site). It may - 12 -SG Docket No. 10860-536.600be further targeted, planned, directed and observed under direct visualization, via ultrasound imaging, given the bubble clouds generated by histotripsy may be visible as highly dynamic, echogenic regions on, for example, B Mode ultrasound images, allowing continuous visualization through its use (and related procedures). Likewise, the treated and fractionated tissue shows a dynamic change in echogenicity (typically a reduction), which can be used to evaluate, plan, observe and monitor treatment.

[0052] Generally, in histotripsy treatments, ultrasound pulses with 3 or more acoustic cycles are applied, and the bubble cloud formation relies on the pressure release scattering of the positive shock fronts (sometimes exceeding 100 MPa, P+) from initially initiated, sparsely distributed bubbles (or a single bubble). This is referred to as the “shock scattering mechanism”.

[0053] This mechanism depends on one (or a few sparsely distributed) bubble(s) initiated with the initial negative half cycle(s) of the pulse at the focus of the transducer. A cloud of microbubbles then forms due to the pressure release backscattering of the high peak positive shock fronts from these sparsely initiated bubbles. These back- scattered high-amplitude rarefactional waves exceed the intrinsic threshold thus producing a localized dense bubble cloud. Each of the following acoustic cycles then induces further cavitation by the backscattering from the bubble cloud surface, which grows towards the transducer. As a result, an elongated dense bubble cloud growing along the acoustic axis opposite the ultrasound propagation direction is observed with the shock scattering mechanism. This shock scattering process makes the bubble cloud generation not only dependent on the peak negative pressure, but also the number of acoustic cycles and the amplitudes of the positive shocks. Without at least one intense shock front developed by nonlinear propagation, no dense bubble clouds are generated when the peak negative half-cycles are below the intrinsic threshold.

[0054] When ultrasound pulses less than 2 cycles are applied, shock scattering can be minimized, and the generation of a dense bubble cloud depends on the negative half cycle(s) of the applied ultrasound pulses exceeding an “intrinsic threshold” of the medium. This is referred to as the “intrinsic threshold mechanism”.

[0055] This threshold can be in the range of 26 - 30 MPa for soft tissues with high water content, such as tissues in the human body. In some embodiments, using this intrinsic threshold mechanism, the spatial extent of the lesion may be well-defined and more predictable. With peak negative pressures (P-) not significantly higher than this threshold, sub -wavelength reproducible lesions as small as half of the -6dB beam width of a transducer may be generated.- 13 -SG Docket No. 10860-536.600

[0056] With high-frequency Histotripsy pulses, the size of the smallest reproducible lesion becomes smaller, which is beneficial in applications that require precise lesion generation. However, high-frequency pulses are more susceptible to attenuation and aberration, rendering problematical treatments at a larger penetration depth (e.g., ablation deep in the body) or through a highly aberrative medium (e.g., transcranial procedures, or procedures in which the pulses are transmitted through bone(s)). Histotripsy may further also be applied as a low-frequency “pump” pulse (typically < 2 cycles and having a frequency between 100 kHz and 1 MHz) can be applied together with a high-frequency “probe” pulse (typically < 2 cycles and having a frequency greater than 2 MHz, or ranging between 2 MHz and 10 MHz) wherein the peak negative pressures of the low and high-frequency pulses constructively interfere to exceed the intrinsic threshold in the target tissue or medium. The low-frequency pulse, which is more resistant to attenuation and aberration, can raise the peak negative pressure P- level for a region of interest (ROI), while the high-frequency pulse, which provides more precision, can pinpoint a targeted location within the ROI and raise the peak negative pressure P- above the intrinsic threshold. This approach may be referred to as “dual frequency”, “dual beam histotripsy” or “parametric histotripsy.”CONTROLLED CELLULAR RELEASE BY HISTOTRIPSY

[0057] This disclosure describes controlling histotripsy dose to release cell nuclei by disrupting the cell membrane to release extracellular free-flowing cell nuclei containing tumor antigens, which can stimulate potent immune response.

[0058] Histotripsy is a non-invasive ultrasound ablation technology that mechanically breaks down the cells in the target region with millimeter accuracy. Using microsecondlength ultrasound pulses at very high peak negative pressure, cavitation microbubbles are generated using nanometer gas pockets inside the tissuel. The expansion and collapse of the microbubbles occurring with several hundreds of microseconds induces high local mechanical strain and stress to disrupt the cells in the focal region. Each histotripsy pulse produces a portion of cell disruption and increasing number histotripsy pulses produce increasing cell disruption. The cell membrane is most sensitive to histotripsy damage and is the first cellular structure to be disrupted, while the cell nuclei is the most resistant to mechanical damage and is damaged the last. With a specific number of histotripsy pulses (i.e., histotripsy dose), the membrane of most or all cells in the treatment region can be disrupted to cause effective tumor kill, while abundant cell nuclei are released outside the cells. As histotripsy mechanism is mechanical in nature and does not use heating or ionization to denature the protein, the DNA is expected to be intact and functional within the remaining cell nuclei. However, if too high a histotripsy dose is delivered, the extracellular- 14 -SG Docket No. 10860-536.600cell nuclei will be damaged, and eventually all destroyed. The extracellular free-flowing cell nuclei released by a controlled histotripsy dose along with other acellular debris then enters the blood circulation in days, weeks, or even months post histotripsy, releasing tumor neoantigen in the treated sites as well as off-target sites for immune stimulation.

[0059] The primary invention of disclosure is the control of histotripsy dose to ensure tumor cell kill while maximizing the release of extracellular free-flow tumor cell nuclei and thus release of tumor neoantigen. The histotripsy dose is selected such that most or all the cell membranes are disrupted, and tumor cell nuclei become extracellular and free flowing. At too low a dose, not all tumor cell membranes are disrupted. At too high a dose, the extracellular free flowing tumor cell nuclei are destroyed too. The extracellular tumor cell nuclei enable release of tumor neoantigen locally and systemically, as histotripsy-liquefied acellular lysate is absorbed by the body via blood circulation.

[0060] In addition, in certain cases, the tumor antigen is on the cell membrane, and the histotripsy dose can be controlled to maximize the release the tumor antigen on the cell membrane. The histotripsy dose is selected such that most or all the cell membranes are disrupted, and tumor antigen attached to the cell membrane become extracellular and free flowing. At too low a dose, not all tumor cell membranes are disrupted. At too high a dose, the extracellular cell membrane fragments and attached protein are destroyed too. The free- floating extracellular cell membrane fragments and attached protein enable release of tumor antigen locally and systemically, as histotripsy-liquefied acellular lysate is absorbed by the body via blood circulation. An example is given below using the HER2 breast cancer model, where HER2 is a tumor associated antigen attached to the cell membrane.

[0061] In both cases, the release of tumor antigen in turn stimulates innate and adaptive immune response locally and systemically. By maximizing the extracellular free flowing tumor cell nuclei release, this immunostimulation effect can be maximized to maximize the abscopal effect. This immunostimulation effect can also be maximized to combine with immunotherapy drugs to further enhance the therapeutic effect of immunotherapy or to sensitize tumors resistant to current immunotherapy such that those tumors can be treated by immunotherapy effectively. This invention also includes ultrasound-based feedback technology and biomarker measurement to detect and control such dose delivery.

[0062] Feedback technology for dose control tumors require different doses to completely disrupt the cell membrane to release maximal extracellular free-flowing cell nuclei. Human tumors can also vary significantly across patients even for the same tumor type. To deliver personalized optimal histotripsy dose to patients, this disclosure provides the use of quantitative ultrasound feedback (e.g., with B-mode imaging) of one or more backscatter- 15 -SG Docket No. 10860-536.600parameters to pinpoint when cell membranes within the target tumor are disrupted (e.g., complete tumor kill in the target region). This is expected when number of extracellular free- flowing cell nuclei is the highest; or cell membrane fragments are free-flowing; while the tumor antigens are mostly functional. Additional histotripsy doses are expected to gradually damage or reduce cell nuclei or tumor antigen.

[0063] For example, with increasing cell disruption, the ultrasound B-mode image intensity (backscatter intensity) in the target region will reduce, and the B-mode tissue texture becomes more homogeneous. Thus, the B-mode ultrasound backscatter intensity, texture, and / or isotropy analysis may be quantified to predict the complete cell disruption point. In some aspects, the backscatter ultrasound parameters can be evaluated automatically by the system (e.g., with an automatic analysis of the B-mode ultrasound images / backscatter with the histotripsy system). In addition, cavitation collapse time, analyzed from acoustic cavitation emission signals, increases with increasing cellular disruption and plateaus after complete cellular disruption, which can also be used to predict when the complete tumor cell membrane disruption (tumor kill) and the highest number of extracellular free-flowing cell nuclei are achieved.

[0064] Lastly, ultrasound elastography has been used to measure the elastic modulus decrease over histotripsy treatment. The elastic modulus distribution in the treated region becomes more homogenous with increasing cellular disruption. Thus, the elastic modulus measurement may be used as another metric to predict when the highest number of extracellular free-flowing cell nuclei is achieved. The imaging feedback will allow us to predict and achieve the high number of extracellular free-flowing cell nuclei personalized for each human patient receiving histotripsy tumor treatment, maximizing the immune response.

[0065] Biomarker measurement of tumor antigen release: In addition, biomarkers can be used to evaluate the tumor antigen (contained in the tumor cell nuclei) released to extracellular compartment via blood test or tissue biopsy post histotripsy treatment. Biomarkers can be tumor specific antigen or tumor associated antigen for a specific tumor type.

[0066] Histotripsy dose delivery and release of extracellular free flowing tumor cell nuclei: We have demonstrated the proof-of-concept to release extracellular free-flowing cell nuclei by controlling the histotripsy dose delivery in the mouse tumor models. There is also initial evidence showing the release of free-flow cell nuclei is correlated to increased immunostimulation.

[0067] There is also initial evidence showing the release of free-flow cell nuclei is correlated to increased immunostimulation and improved survival (Example 1-3). Example 2- 16 -SG Docket No. 10860-536.600also shows acoustic feedback can be used to predict the maximal cell nuclei release. The final example shows tumor antigen attached to the tumor cell membrane released by histotripsy, which was quantified by biomarker measurement (Example 4).

[0068] Example 1 - Maximize tumor cell nuclei release by histotripsy dose control in the in vivo mouse melanoma model.

[0069] FIGS. 2A-2E: H&E histology slide of the in vivo mouse B16F10 tumors: 2A) control untreated and treated by histotripsy at 2B) 2, 2C) 8, 2D) 20, 2E) 40, and 2F) 100 ppi. At 2 and 8 ppi, partial cellular disruption is observed. At 20ppl and 40ppl, there are no intact cells with substantial extracellular free-flowing cell nuclei (dark dots; examples pointed by yellow arrows). At 100 ppi, there are reduced extracellular free-flowing cell nuclei, and nuclei look more shrunken (examples pointed by yellow arrows).

[0070] In the first example, increasing histotripsy doses (2, 8, 20, 40, and 100 different pulses per location (ppi)) were used to treat the tumor in the mouse B16F10 melanoma model. Mice were inoculated with B16F10 cells on the flank. Once tumors reached 5-7 mm in the largest dimension, histotripsy was applied using 1-cycle pulses at 30MPa peak negative pressure, 100Hz pulse repetition frequency (PRF) by a 1MHz transducer. Histology shows that compared to untreated control (FIG. 2A), small amount of cellular damage was observed at 2 ppi (FIG. 2B). At 8 ppi, some of cell membrane is disrupted, with observable extracellular free-flowing cell nuclei (FIG. 2C). At 20 and 40 ppi, no intact cell membranes are observed inside the treatment region, while there are substantial number of extracellular free-flowing cell nuclei remain (FIGS. 2D-E). At 100 ppi, extracellular free-flowing cell nuclei are shrunken and reduced (FIG. 2G).

[0071] FIGS. 3 A-3C: 3 A) Tumor growth curve (Tumor volume vs. days post inoculation) for untreated control, 8ppl, 20ppl, 40ppl, and lOOppl histotripsy treatment groups (n=4). The treatment groups had slower tumor growth compared to the control group. The 20ppl and 40ppl histotripsy treatment groups had the slower tumor growth compared to 8ppl and lOOppl . 3B)-C) IHC measurements show that the 40ppl groups produced significantly higher CD45 and CD8 T-cell infiltration in the local treated tumor compared to other groups.

[0072] A survival study was conducted by treating 50-90% of the in vivo B16F10 mouse melanoma tumor, and the 20ppl and 40ppl groups produced better tumor control (FIG. 3 A) compared to the untreated, 8ppl, and lOOppl groups. Immunohistochemistry (IHC) measurements at 7 days post-histotripsy show that the 20 and 40ppl groups (particularly the 40ppl group) produced significantly higher CD45 and CD8 T-cell infiltration in the local treated tumor compared to untreated control and other treatment groups (FIGS. 3B-C). The- 17 -SG Docket No. 10860-536.600better survival and increased immune response at 20 and 40 ppi correspond to the increased extracellular free-flowing tumor cell nuclei at 20 and 40 ppi.

[0073] Example 2 - Maximize tumor cell nuclei release by histotripsy dose control in the in vivo mouse colorectal tumor model; Acoustic feedback predicting optimal histotripsy dose to maximize cell nuclei release and best survival and immune response.

[0074] FIGS. 4A-4E: H&E histology slide of the in vivo mouse MC 38 tumors 4A) control untreated and treated by histotripsy at 4B) 15, 4C) 50, and 4D) 100 ppi. There are extracellular free-floating cell nuclei (dark dots; examples pointed by yellow arrows) in 15ppl and 50ppl slides, but no nuclei are available at lOOppl. 4E) CD8+ T cell measurement at 7 days post histotripsy shows the highest CD8+ T cell concentration for the 15ppl group.

[0075] FIGS. 5A-5B: 5 A) The increase of the cavitation lifespan plateaued at around 15PPL of the histotripsy dose. 5B) Kaplan-Meier survival curves for different histotripsy doses in the murine MC38 CRC model (n = 6-12 per group) showed the best survival results (complete tumor regression in all mice) for the 15PPL group.

[0076] In a second example, histotripsy was applied to the mouse MC38 subcutaneous colorectal tumor model (FIG. 4A). Histotripsy was applied using 2-cycle pulses at 30MPa peak negative pressure by a 1MHz transducer. Three doses were tested: 15ppl at 35Hz PRF, 50ppl at 50Hz RPF, and lOOppl at 190Hz PRF. At 15ppl, there is abundant free-flowing cell nuclei with almost all cell membrane disrupted (FIG. 4B). At 50 ppi, the number of free- flowing cell nuclei is reduced (FIG. 4C). At 100 ppi, almost all cell nuclei are destroyed (FIG. 4D).

[0077] Correspondingly, the immune response measured at 7 days post histotripsy shows the trend of the highest CD8+ T cell concentration for the 15ppl treatment group. Among all treatment groups, the CD8+ T cell concentration for the 15ppl group is the only one that is statistically significantly higher compared to the control untreated group (p=0.002). The CD8+ T cell concentration for the 15ppl group is also significantly higher than the 8ppl (p=0.007) and 100 ppi (p=0.049) group. The sample size is 10 for the control group and 5 for each treatment group.

[0078] This example also shows acoustic feedback received by the histotripsy transducer can be used to predict the optimal histotripsy dose delivery control. In this example, prior to the treatment, following the clinical histotripsy treatment workflow, histotripsy was applied to 7 selected points within the target tumor to check targeting, and the acoustic cavitation emission (ACE) signals were received for each point using the transmit-receive capable histotripsy transducer in an in vivo MC38 colorectal tumor model. The cavitation lifespan was calculated as the time duration between the initiation and collapse of the cavitation- 18 -SG Docket No. 10860-536.600bubbles, during which acoustic shockwave signals are emitted from the bubbles and received by the histotripsy ultrasound transducer elements in the pre-treatment targeting check. The cavitation lifespan increased with the histotripsy dose and the increase plateaued around 15ppl (FIG. 5 A). As shown above, the 15ppl resulted in complete cellular membrane disruption and released the highest number of free-floating cell nuclei to the extracellular space as shown by histology (FIG. 4A). The 15ppl also resulted in the highest CD8+ T-cell activation (FIG. 4B) and the best survival (FIG. 5B). This suggests that the ACE signals received during the pre-treatment targeting check can be used to determine the optimal histotripsy dose for maximal tumor cell nuclei release corresponding to best survival and immune response.

[0079] Example 3 - Maximize tumor cell nuclei release by histotripsy dose control in the in vivo mouse Merkle Cell Carcinoma (MCC) model.

[0080] FIG. 6: H&E histology slide of the in vivo mouse MC 38 tumors control untreated and treated by histotripsy at 10, 25, 50, 100, and 300 ppi. At lOppl and 25ppl, there is partial tumor cellular disruption. At 50ppl, most tumor cells are disrupted in the target region, with free floating tumor cell nuclei released. The free-floating cell nuclei reduced at lOOppl. At 300ppl, there is no observable cellular and subcellular components inside the treated region.

[0081] FIG. 7: Kaplan-Meier survival curves for two cohorts of experiments. Left: The first cohort includes four groups of mice: untreated control and 10, 25, and 50 ppi treatment groups (n=5-6). Right: The second cohort includes four groups of mice: untreated control and 50, 100, and 300 ppi treatment groups (n=5-6).

[0082] In a third example, histotripsy was applied to the in vivo mouse MCC tumor model. Histotripsy was applied using 2-cycle pulses at 30MPa peak negative pressure by a 1.5MHz transducer. Five doses were tested: 10, 25, 50, 100, and 300 ppi. PRF was varied between 25-200 Hz to keep treatment time constant. Acute H&E histology (FIG. 6) shows that at lOppl and 25ppl, there is only partial tumor cellular disruption. At 50ppl, most tumor cells are disrupted in the target region, with substantial free floating tumor cell nuclei released. The free-floating cell nuclei reduced at lOOppl. At 300ppl, there is no observable cellular and subcellular components inside the treated region.

[0083] Two cohorts of survival experiments were performed. The first cohort includes four groups of mice: untreated control and 10, 25, and 50 ppi treatment groups (n=5-6). The second cohort includes four groups of mice: untreated control and 50, 100, and 300 ppi treatment groups (n=5-6). In both cohorts, the 50ppl treatment group resulted in the longest survival (FIG. 7), which corresponds to the most free-floating tumor cell nuclei released and almost complete tumor cell disruption at 50pl. The lowest dose treatment group with partial- 19 -SG Docket No. 10860-536.600tumor ablation (1 Oppl) and the overdose treatment group with no tumor cell nuclei remaining (3OOppl) had the worst survival outcome and showed no benefit compared to the untreated control group (FIG. 7).

[0084] Example 5 - Release of tumor antigen attached to the cell membrane fragment by histotripsy dose control.

[0085] FIGS. 8A-8C: 8A) Released HER2 levels of histotripsy -treated cell suspension in cell free fragments showing higher HER2 release with increasing histotripsy dose. 8B) Released HER2 levels of histotripsy -treated tumors in cell free fragments detected by UPLC at 285 nm showing increased HER2 release with increasing histotripsy dose. 8C) Western blot analysis of histotripsy cell free fragment (C.F.) probed with monoclonal HER2 antibody. Lanes 1 and 2 were ladder (13 pL) and rHER2 (0.012 pg), respectively as references. Lanes 3-6 were the analysis for cell lysis with a 15 pg of protein per lane. Lane 3 was untreated, lanes 4-6 were the low, moderate, and high dose histotripsy treatment, respectively. LH: lOppl; MH: 50ppl; HH: lOOppl.

[0086] HER2+ breast cancer is an aggressive form of breast cancer that is often resistant to existing therapies. Even with newer treatment strategies, approximately 30% of patients relapse, often developing metastatic disease, and the 5-year survival rate for these patients is only approximately 22%. HER2 is expressed 50-100 times more in tumor cells than in normal cells, serving as a tumor antigen and pathological biomarker that distinguishes cancerous from noncancerous breast cells. HER2 is often expressed on the surface of cancer cells.

[0087] Histotripsy treatment was performed on in vitro HER2 breast cancer cells and in vivo HER2 mouse breast tumor model using a custom built, 8-element 1 MHz array transducer using 1-2 cycle pulses at 100Hz pulse repetition frequency (PRF), and an estimated peak negative pressure of ~53 MPa. Samples were targeted via ultrasound imaging using a L40-8 / 12 20 MHz ultrasound probe coaxially aligned with the therapy transducer. The treatment pattern was generated by setting the three-dimensional boundaries and populating an ellipsoid grid with equal spacing between points, which was 1 mm for the in vitro treatment and 0.5 mm for the ex vivo tumor treatment. Cell culture samples were gently vortexed to ensure homogenous suspension prior to treatment, while ex vivo tumors were targeted with a sub-total volume to minimize suspended tissue movement during treatment. Three histotripsy doses were delivered by varying the number of pulses per location (10, 50, 100 ppi).

[0088] In the in vitro HER2 breast cancer cell suspension, we measured the HER2 released from tumor cells by histotripsy disruption of the cell membrane, allowing cell - 20 -SG Docket No. 10860-536.600membrane fragment with HER released into extracellular compartment. This study was performed on in vitro cultured E0771E2 breast cancer cell line expressed HER2 as a tumor associated antigen. E0771E2 cells were created by engineering mouse mammary tumor E0771 cells with human wildtype HER2. While untreated cells displayed very lowed protein levels, all histotripsy treatments released statistically significantly more proteins in the cell free supernatants compared to untreated controls. In the in vitro cell suspension tube, the untreated cells kept moving into the target region with cavitation-induced flow, such that there were always untreated cells in the target region, and overdose seen in vivo due to repeatedly treating the same cells were not observed in this in vitro setting. Thus, increased histotripsy dose resulted in a higher release of extracellular protein and HER2 (FIG. 8). High dose (lOOppI) histotripsy triggered significantly more cell lysis and released more proteins into the cell free fragments than the low dose (1 Oppl) histotripsy (FIG. 8 A). Furthermore, released HER2 levels of HT -treated cells in cell free fragments were analyzed by ultraperformance liquid chromatography (UPLC). The cell-free fragments exhibited a peak with a retention time at ~9.3 minutes similar to rHER2, a positive control, suggesting that the nature of the HER2 released by HT is in line with rHER2. Consistent with histotripsy-mediated release of proteins analyzed by BCA protein assay, this test also showed a dose-dependent triggered release of HER2 in the cell free fragments (FIG. 8B). In addition, Western blot assay with a monoclonal antibody against HER2 was used to check whether the presence of HER2 protein in cell free fragments post histotripsy treatment. rHER2 protein, a positive control, appeared at 110 kDa (FIG. 8C). A protein band of the same size was found in the cell free fragments of E0771E2 cells treated with HT (Lanes 4-6, FIG. 8C) while this specific band was barely detected in the untreated cells (Lanes 3 and 7), demonstrating the histotripsy-mediated release of HER2 from cells. Additionally, the 110 kDa protein band intensity was dose-dependent with a 0.5x,~3.5x, and ~5x fold increase in density for the low (Lane 4. 1 Oppl), moderate (Lane 5, 50ppl), and high (Lane 6. lOOppl) histotripsy doses respectively compared to the untreated sample (Lane 3).

[0089] FIGS. 9A-9D. HER2 detected after in vivo histotripsy treatment of the mouse HER2 breast cancer model. 9A) Representative ultrasound image of the histotripsy cavitation cloud during treatment. Analysis of in vivo histotripsy E0771E2 tumor cell free fragments / supernatants with 9B) UPLC assay, detected at 285 nm; 9C) BCA protein assay. 9D) Western blot assay probed with monoclonal HER2 antibody. Ladder was loaded with 15 pL / lane; Positive control rHER2 was loaded with 0.01 pg / lane; Samples were loaded with 50 pg / lane protein. n = 4. *** p < 0.001.- 21 -SG Docket No. 10860-536.600

[0090] Finally, in in-vivo experiments of E0771E2 tumor with stable and functional HER2 expression was used for histotripsy treatment. C57BL / 6 HER2 transgenic mice were inoculated with E0771E2 cells. Eight transgenic HER2 mice were undergone in vivo experiment, with 4 mice subjected to histotripsy treatment at lOOppl while another 4 mice without histotripsy treatment as a control group. Ultrasound images clearly showed that the HT cavitation cloud could accurately target tumor tissues (FIG. 9A). The results were consistent with in vitro experiments. Both UPLC analysis (FIG. 9B) and BCA assay (FIG. 9C) detected the significant increase of protein level in cell free fragments after histotripsy treatment. UPLC analysis once again showed that the histotripsy treated tumor lysis exhibited a HER2 peak at around 9.2-9.3 minutes, overlapping with the peak of rHER2 (FIG. 9B), indicating that both rHER2 protein and the released HER2 protein had the same size and structure. Western blot specifically displayed the high level of HER2 antigen spots (FIG. 9D), indicating that histotripsy could in vivo trigger the release of tumor antigens that is potentially significant for immunotherapies.

[0091] Additional systems, methods and parameters to deliver optimized histotripsy, using shock scattering, intrinsic threshold, and various parameters enabling frequency compounding and bubble manipulation, are herein included as part of the system and methods disclosed herein, including additional means of controlling said histotripsy effect as pertains to steering and positioning the focus, and concurrently managing tissue effects (e.g., prefocal thermal collateral damage) at the treatment site or within intervening tissue. Further, it is disclosed that the various systems and methods, which may include a plurality of parameters, such as but not limited to, frequency, operating frequency, center frequency, pulse repetition frequency, pulses, bursts, number of pulses, cycles, length of pulses, amplitude of pulses, pulse period, delays, burst repetition frequency, sets of the former, loops of multiple sets, loops of multiple and / or different sets, sets of loops, and various combinations or permutations of, etc., are included as a part of this disclosure, including future envisioned embodiments of such.

[0092] When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being - 22 -SG Docket No. 10860-536.600“directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0093] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”.

[0094] Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0095] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.- 23 -SG Docket No. 10860-536.600

[0096] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0097] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0098] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional - 24 -SG Docket No. 10860-536.600features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0099] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.- 25 -SG Docket No. 10860-536.600

Claims

CLAIMSWhat is claimed is:

1. A method of providing histotripsy therapy, comprising: delivering histotripsy pulses with a histotripsy therapy transducer to a target tissue volume to generate cavitation within the target tissue volume; obtaining B-mode ultrasound images of the target tissue volume while the histotripsy pulses are being delivered; evaluating one or more ultrasound backscatter parameters to predict a degree of cellular destruction within the target tissue volume; and stopping delivery of the histotripsy pulses based on the evaluation of the ultrasound backscatter parameters.

2. The method of claim 1, wherein the one or more ultrasound backscatter parameters comprises backscatter intensity.

3. The method of claim 1, wherein the one or more ultrasound backscatter parameters comprises backscatter texture.

4. The method of claim 1, wherein the one or more ultrasound backscatter parameters comprises backscatter isotropy.

5. A method of providing histotripsy therapy, comprising: delivering histotripsy pulses with a histotripsy therapy transducer to a target tissue volume to generate cavitation within the target tissue volume; receiving acoustic cavitation emission (ACE) signals from the cavitation with the histotripsy therapy transducer; calculating a time duration between initiation and collapse of the cavitation from the ACE signals to predict a degree of cellular destruction within the target tissue volume; and stopping delivery of the histotripsy pulses based on the calculated time duration of the cavitation.

6. The method of claim 5, further comprising evaluating changes in the time duration as additional histotripsy pulses are delivered.- 26 -SG Docket No. 10860-536.6007. The method of claim 6, further comprising identifying a plateau in the time duration of the cavitation.

8. The method of claim 7, further comprising associating complete cellular membrane disruption of the target tissue volume with the plateau in the time duration of the cavitation.

9. The method of any of claims 1 or 5, further comprising adjusting delivery of histotripsy pulses based on the degree of cellular destruction.

10. The method of cany of claims 1 or 5, wherein the target tissue volume comprises a tumor.

11. The method of any of claims 1 or 5, wherein the target tissue volume comprises liver tissue.

12. A histotripsy therapy system, comprising: a B-mode ultrasound imaging system configured to obtain B-mode ultrasound images of the target tissue volume; a histotripsy therapy transducer array; a robotic positioning system coupled to the histotripsy therapy transducer array, the robotic positioning system being configured to place a focus of the histotripsy transducer array within the target tissue volume; and an electronic controller configured to: control the histotripsy therapy transducer to deliver histotripsy pulses to the target tissue volume to generate cavitation within the target tissue volume; control the B-mode ultrasound imaging system to obtain B-mode ultrasound images of the target tissue volume while the histotripsy pulses are being delivered; evaluate one or more ultrasound backscatter parameters to predict a degree of cellular destruction within the target tissue volume; and control the histotripsy therapy transducer to stop delivery of the histotripsy pulses based on the evaluation of the ultrasound backscatter parameters.

13. A histotripsy therapy system, comprising: a histotripsy therapy transducer array;- 27 -SG Docket No. 10860-536.600a robotic positioning system coupled to the histotripsy therapy transducer array, the robotic positioning system being configured to place a focus of the histotripsy transducer array within the target tissue volume; and an electronic controller configured to: control the histotripsy therapy transducer to deliver histotripsy pulses to the target tissue volume to generate cavitation within the target tissue volume; receive acoustic cavitation emission (ACE) signals from the cavitation with the histotripsy therapy transducer; calculate a time duration between initiation and collapse of the cavitation from the ACE signals to predict a degree of cellular destruction within the target tissue volume; and control the histotripsy therapy transducer to stop delivery of the histotripsy pulses based on the calculated time duration of the cavitation.- 28 -SG Docket No. 10860-536.600

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