Methods for treating hypoxia-related cancers

Abstract

The invention provides methods for enhancing the efficacy of a cancer treatment or treating cancer in a subject by administering a composition comprising an anti-cancer agent and a microbubble suspension. The microbubble suspension is characterized by a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subj ect's body weight or a human equivalent dose (HED) range of 2x 108 to 4x 109 bubbles per kg body weight. Ultrasound with a setting of continuous non-thermal pulses with short cycle and sufficient time interval is applied to induce microbubble destruction in a targeted region using continuous non-thermal pulses with short cycles and sufficient time intervals, wherein the mechanical index (MI) value is between 0.4 and 1.2.

Description

METHODS FOR TREATING HYPOXIA-RELATED CANCERSBACKGROUND OF THE INVENTION

[0001] It is known that certain cancers such as pancreatic ductal adenocarcinoma (PDAC) are hypoxia-related and difficult to treat due to late diagnosis, and chemotherapy failure, primarily caused by the harsh hypoxia tumor microenvironment (TME). A harsh hypoxia tumor microenvironment is a tumor microenvironment that is characterized by low levels of oxygen. This can occur due to a number of factors, including the rapid growth of the tumor, which outstrips the ability of the blood vessels to supply it with oxygen, or the presence of tumor cells that secrete factors that inhibit the grow th of new7blood vessels.SUMMARY OF THE INVENTION

[0002] In accordance with the present invention, the present invention provides methods of enhancing efficacy of a cancer treatment in a subject comprising administering to the subject a composition comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2x108to 4xl09bubbles per Kg body w eight; applying ultrasound to induce the microbubble destruction in a targeted region by continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2.

[0003] In another aspect provides methods of treating cancer in a subject comprising administering to the subject a composition comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2xl08to 4xl09bubbles per Kg body weight; applying ultrasound to induce the microbubble destruction in a targeted region by continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2.

[0004] In accordance with the present invention, the present invention provides methods of enhancing efficacy of a cancer treatment in a subj ect, comprising administering to the subj ect a composition comprising an anti-cancer agent and a microbubble suspension, wherein the microbubble suspension has a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subject’s body weight; and applying ultrasound to induce microbubble destruction in a targeted region by continuous non-thermal pulses with short cycle and sufficient time interval, wherein the MI value is 0.4 to 1.2 .

[0005] In yet another aspect provides methods of treating cancer in a subject, comprising administering to the subject a composition comprising an anti-cancer agent and a microbubble suspension, wherein the microbubble suspension has a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subject’s body weight; and applying ultrasound to induce microbubbledestruction in a targeted region by continuous non-thermal pulses with short cycle and sufficient time interval, wherein the MI value is 0.4 to 1.2.INCORPORATION BY REFERENCE

[0006] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The novel features of the invention are set forth with particularity in the appended claims. 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:

[0008] FIG. 1A-B provide study results showing different concentrated microbubbles (i.e.. no bubble, exemplary high concentrated microbubbles, medium concentrated microbubbles and 1 / 20 diluted microbubbles) affecting the tumor hypoxia areas as in images (1 A) and in chart (IB).

[0009] FIG. 2A / B provide study results of adverse effects of mice after the treatments. (2A) Mice body weight variation. Mice's body weight was recorded every two to three days until sacrificing. Control mice without treatments (n=8), MB only group received sonoporation only (n=3). The Dox-only group received doxorubicin (n=l 1), Dox+MB-induced sonoporation group received doxorubicin plus Power Doppler-based sonoporation (n=10). (2B) Table of Blood biochemical tests. Mice's blood was collected 24 hours after treatment for biochemical examination. Values are presented as the average ± SD. P-values using student's t-tests comparing Dox or Dox+MB versus control, *; P<0.05 (No difference between control and MB only; Dox and Dox+MB among all parameters). ALP = Alkaline phosphatase, ALT = alanine aminotransferase, AST = aspartate aminotransferase, TBIL = Total Bilirubin. PHOS= phosphorus levels, CRE = Creatinine, BUN = blood urea nitrogen.

[0010] FIG. 3 shows preliminary results of sonoporation with doxorubicin under different ultrasound modes (CPA and B mode) and mechanical index (MI), demonstrating variation in hypoxic tumor area (PIMO / tumor area %).

[0011] FIG. 4A-D provide study results of the change of UN-KC-6141 tumor microenvironment following Power Doppler-based sonoporation with exemplary microbubbles (e.g., NH002) and doxorubicin. (4A) Tumor hypoxia regions were indicated with Pimonidazole staining (shown in white), and the white dashed line indicated the tumor border. Scale bar = 200 pm. (4B) Quantification of hypoxia (Pimonidazole4) region (fraction of tumor area coverage). N A 3 for each time point and group. (4C) Representative images of the perfused tumor area 3 days following Power Doppler-based sonoporation with exemplary’ microbubbles (e.g., NH002) anddoxorubicin (Dox+MB) vs doxorubicin only (Dox only). Non-perfused area (white dash circle) was discerned under contrast mode as the dark area. Scale bar = 1 cm. (4D) Statistics of tumor perfused area variation compared to the day before the treatment. A two-tailed unpaired t-test was used to compare every two groups. *: P <0.05, **: P <0.01. N>3 in each group.

[0012] FIG. 5A-B provide study results of the effect of doxorubicin treatment and B-mode sonoporation on UN-KC-6141 tumor-bearing mice. ( A) Kaplan-Meier survival curve of UN- KC-6141 tumor-bearing mice, Control mice without any treatments (n=3), Dox only group receive doxorubicin only (n=3), and Dox+MB group receive doxorubicin plus B-mode-based sonoporation (n=3). (5B) Quantification of the largest tumor section area of orthotopic UN-KC- 6141 tumor-bearing mice. A two-tailed unpaired t-test was used to compare the tumor size between the control and dox groups at each time point. *: P < 0.05, **: P < 0.01.

[0013] FIG. 6A-C show study results of administering doxorubicin (Dox) with Power Dopplerbased sonoporation which slows down UN-KC-6141 tumor progression. (6A) Kaplan-Meier survival curve of PD AC-bearing mice. Control mice without any treatments (n=10), MB (microbubbles) only group received sonoporation without Dox (n=3). The Dox-only group received doxorubicin (n=12), Dox+MB group received doxorubicin plus Power Doppler-based sonoporation (n=10). (6B) The largest tumor section area of orthotopic UN-KC-6141 tumor examined at 10, 13, 16, and 21 post-tumor implantation. (6C) The largest secondary tumor section area of orthotopic UN-KC-6141 tumor examined 21 days post tumor implantation. A two-tailed unpaired t-test was used to compare the tumor size at each time point between the Dox only and Dox+MB groups. *: p<0.05, **: p<0.01, ***: p<0.001.

[0014] FIG. 7A-E provide study results of the combination of administering gemcitabine with Power Doppler-based sonoporation, which slows down UN-KC-6141 tumor progression. (7A) Kaplan-Meier survival curve of PDAC, Control mice without any treatments (n=3), GEM-only group received gemcitabine only (n=4), GEM+MB group received gemcitabine plus Power Doppler-based sonoporation (n=4). (7B) The largest tumor section area of orthotopic UN-KC- 6141 tumor-bearing mice examined at 10, 13, 16, and 21 post-tumor implantation. (7C) The largest secondary tumor section area of orthotopic UN-KC-6141 tumor examined 16 days post + tumor implantation. (7D) Representative images of hypoxia (Pimonidazole , shown in gray) area in tumor. Scale bar = 200 pm. (7E) Quantitative data of hypoxia area in the tumor. A two-tailed unpaired t-test w as used to compare the tumor size at each time point between the GEM-only and GEM+MB groups. *: p<0.05, **: p<0.01.

[0015] FIG. 8A-D show study results where Power Doppler-based NH002 sonoporation with Chemotherapy (PDNSC) strengthens the aPDLl treatment and slows down UN-KC-6141 tumor progression (i.e., efficacy enhancement). (8A) Representative images of cytotoxicity T cells inthe tumor area. Cytotoxicity T cells were indicated with CD8 staining (white dots). Scale bar = 100 pm. (8B) Quantification of CD8+T cells in the tumor region (per field). Ni 3 for each time point and group. (8C) Kaplan-Meier survival curve of PDAC. aPDLl group received aPDLl only (n=4), Dox+aPDLl group received doxorubicin plus aPDLl (n=6). The Dox+MB+aPDLl group received doxorubicin plus Power Doppler-based sonoporation and aPDLl (n=7). (8D) The largest tumor section area of orthotopic UN-KC-6141 tumor-bearing mice examined at 10, 13, 16, and 21 post-tumor implantation. A two-tailed unpaired t-test was used to compare the tumor size at each time point between the Dox+aPDLl and Dox+MB+aPDLl groups. *: P <0.05, **: P <0.01, ***: p<0.001, ****: p<0.0001. N>3 in each group.

[0016] FIG. 9A / B illustrate the change of CD4 T cells in tumors following PDNSC. (9 A) Representative images of CD4 T cells in the tumor area. Tumors were collected as depicted in Figure 3 A. CD4 T cells were indicated with CD4 staining (white dots). Scale bar = 100 pm. (9B) Quantification of CD4 T cells in the tumor region (per field). A two-tailed unpaired t-test was used to compare every two groups. *: P <0.05, **: P <0.01. N>3 in each group.

[0017] FIG. 10A-C provide study results of the combination of administering liposomal drug Onivyde™ with Power Doppler-based sonoporation, which slows down UN-KC-6141 tumor progression. (10A) The largest tumor section area of orthotopic UN-KC-6141 tumor-bearing mice examined at 10, 13, 16. 21 and 25 post-tumor implantation. Scale bar = 1 cm. (10B) The largest secondary tumor section area of orthotopic UN-KC-6141 tumor examined 1 days post tumor implantation. (10C) Kaplan-Meier survival curve of PDAC, Control mice without any treatments (n=3), Onivyde only group received Onivyde™ only (n=7), Onivyde+MB group received Onivyde™ plus Power Doppler-based sonoporation (n=5).DETAILED DESCRIPTION OF THE INVENTION

[0018] Peritoneal carcinomatosis refers to the presence of cancerous cells or tumors within the peritoneal cavity, which houses vital abdominal organs such as the stomach, liver, and intestines. Hypoxia, a condition of reduced oxygen levels, is a key feature of the tumor microenvironment in peritoneal carcinomatosis, promoting tumor progression, therapy resistance, and poor clinical outcomes. This phenomenon is particularly relevant in gastrointestinal (GI) tract cancers, including colorectal, gastric, and pancreatic cancers, where hypoxic regions contribute to aggressive tumor behavior and limited treatment efficacy. Additionally, hypoxia is commonly observed in other malignancies associated with peritoneal carcinomatosis, such as ovarian cancer, mesothelioma, appendiceal cancer, as well as cancers of the breast, kidney, lung, liver, bladder, and prostate. The following sections provide a detailed overview of the relationship between hypoxia and its impact on various cancer types.• Ovarian Cancer: Advanced ovarian cancer can spread to the peritoneal cavity and develop areas of hypoxia. Ovarian cancer often involves the formation of ascites (fluid buildup), which can further contribute to a hypoxic environment. (Klemba, Bodnar et al. 2020)• Colorectal Cancer: Colorectal cancer that has spread to the peritoneum can also experience hypoxia. These tumors can arise from the colon or rectum and metastasize to the peritoneal lining. (Mi, Mu et al. 2020)• Gastric (Stomach) Cancer: Peritoneal carcinomatosis can occur in cases of advanced gastric cancer, and hypoxia can develop in the tumor nodules present in the peritoneal cavity. (Taylor 2018)• Pancreatic Cancer: Pancreatic cancer can metastasize to the peritoneal cavity and develop areas of hypoxia, similar to how it can cause hypoxia when it spreads to other locations. (Shah, Sheppard et al. 2020)• Mesothelioma: Peritoneal mesothelioma, a rare cancer originating in the peritoneal lining, can also exhibit hypoxia in the tumor microenvironment. (Taylor 2018)• Appendiceal Cancer: Certain types of appendiceal cancer, such as pseudomyxoma peritonei, can lead to the accumulation of mucinous tumor deposits in the peritoneal cavity, which may be associated with hypoxia. (Taylor 2018)• Breast Cancer: While not exclusive to aggressive forms, hypoxia can be observed in certain subtypes of breast cancer, particularly those that are more advanced or have a poor prognosis. (Tutzauer, Sjbstrdm et al. 2022)• Renal Cell Carcinoma: Kidney cancer, specifically clear cell renal cell carcinoma, often develops hypoxia due to genetic mutations that affect blood vessel formation. (Schodel, Grampp et al. 2016)• Lung Cancer: Both small cell and non-small cell lung cancers can develop hypoxia. Hypoxia-inducible factors (HIFs) play a role in promoting tumor survival in the low- oxygen environment of the lungs. (Ziolkowska-Suchanek 2021)• Liver Cancer: Hepatocellular carcinoma (the most common type of liver cancer) can experience hypoxia due to the rapid grow th and demand for blood supply. (Bao and Wong 2021)• Bladder Cancer: Invasive bladder cancer can develop hypoxia as it grows and spreads. (Peixoto, Fernandes et al. 2016)• Prostate Cancer: While not all prostate cancers exhibit hypoxia, advanced or aggressive forms can develop low-oxygen regions. (Mohamed. Tesen et al. 2023)

[0019] As certain hypoxia-related cancers such as pancreatic cancer (pancreatic ductal adenocarcinoma, “PDAC”) are hard to cure due to late diagnosis, and failure of suitable chemotherapy, a novel therapeutic intervention for these types of cancers, (e.g., PDAC) is needed.

[0020] Ultrasound contrast agents, particularly microbubbles, have been explored as potential enhancers for imaging and therapeutic applications. However, conventional microbubbles often suffer from limitations such as low gas volume, insufficient concentration, and suboptimal size distribution, which may reduce their effectiveness in penetrating tumor tissues and enhancing drug delivery.

[0021] The present invention further provides microbubble formulation featuring a higher concentration (about 5xlOlo / mL), greater gas volume, and a controlled smaller size in contrast with the prior commercially available microbubbles. The gas volume of the formulation ranges from about 190 to 235 pL / mL with smaller sizes ranging in 0.9 to 1.5 pm. These enhanced characteristics enable improved formulation stability, stronger acoustic responsiveness under ultrasound exposure, and more effective therapeutic outcomes. These improvements further optimize microbubble-mediated drug delivery, particularly in challenging tumor microenvironments such as hypoxic and poorly perfused cancers.

[0022] The combination of ultrasound and microbubble, usually called sonoporation, has been a promising approach for diagnosis and enhanced drug deliver}'. However, only few studies address the impact of sonoporation on the tumor microenvironment (TME) of PDAC, especially regarding the ultrasound settings and microbubble properties required for effective treatment. Conventional ultrasound contrast agents, such as SonoVue (Bracco) and Sonazoid (GE Healthcare), are primarily designed for diagnostic imaging and are typically formulated at lower bubble concentrations (2><108and 1.2x l09bubbles / mL, respectively) with gas volumes of 8 pL / mL for both. The highest available in the market, Definity (Lantheus), has a bubble concentration of I x lO10bubbles / mL and a gas volume of about 150 pL / mL (see e.g., the FDA- approved label for Definity®, Lantheus Medical Imaging, U.S. FDA, 2011) but with bigger sizes (1. 1-3.3 pm). Moreover, all these ultrasound contrast agents / microbubble products are approved solely for use in ultrasound imaging. Consequently, they are permitted only at very low injection doses including both the particle number concentration and gas volume per unit of body weight. In contrast, the present invention utilizes a high-concentration microbubble formulation (up to 7x l010bubbles / mL) with a gas volume of about 190 to 235 pL / mL, ensuring a sufficient quantity of microbubbles is available for therapeutic interactions. In some instances, the microbubble concentration and gas volume are configured to correspond to an effective gas dose in the subject blood stream, ensuring sufficient therapeutic cavitation.

[0023] Microbubble gas volume plays a crucial role in ultrasound-mediated biological effects, influencing cavitation activity and drug delivery' efficiency. Studies have shown that gas volume can impact acoustic responsiveness, but its precise role in treating hypoxic cancers remains unclear. The present invention identifies and validates an effective gas volume range for microbubble-mediated drug deliver}' under low mechanical index (MI) conditions to reduce the hypoxic area of tumor.

[0024] As shown in FIG. 1A-B, the exemplary' high-concentration microbubble formulation (i.e., lx bubble) led to a greater reduction in PIMO+ / tumor area %, where PIMO+ regions indicate tumor hypoxia (IB). A lower PIMO+ / tumor area % suggests improved tumor oxygenation and a more favorable tumor microenvironment. As demonstrated in Example 1, an exemplary microbubble formulation in the invention methods was administered via IV (intravenous) injection at ahuman equivalent dose (HED) range of 2xfO8to 4xl09bubbles per kg body weight, corresponding to a gas volume of 1.24 pL / kg to 30.57 pL / kg. The results indicate that while microbubble formulations at the lower end of this range (e g., a 20-fold dilution of the high- concentration formulation) contributed to some reduction in tumor hypoxia, the effect was more pronounced at higher concentrations approaching the upper limit of this range. Table 1 A and IB provide study results quantifying the effect of different microbubble concentrations on tumor hypoxia reduction. Table 1 A presents tumor hypoxic area percentages and their corresponding human equivalent doses (TIED) for different microbubble dosages, highlighting the lower and upper effective limits. Table IB details the gas volume dosing required for these effects, specifying the estimated lower and upper dosing limits.

[0025] Table 1 A. Tumor hypoxic area percentages and the corresponding human equivalent doses (HED) by unit of bubbles per kg body weight

[0026] Table IB. Tumor hypoxic area percentages and the corresponding human equivalent doses (HED) by unit of gas volume.

[0027] In certain embodiments, the microbubble suspension is administered at a human equivalent dose (HED) of 4x l08bubbles per kg body weight (equal to 24.72-30.57 pL / kg gas volume).

[0028] Ultrasound settings are crucial for successful sonoporation. This invention utilized the diagnostic ultrasound system with a broadband linear array transducer for all experiments, making it easily translatable to clinical practice. Typically, one of ordinary skilled person in the art would apply B-mode- based sonoporation (e.g., with a derated MI of 0.2, a 0.3% duty cycle, and a center frequency of 1.9 MHz), referencing a clinical study (see e.g., Dimcevski, G. et al. A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer. Journal of Controlled Release 243, 172-181, doi: 10.1016 / j .jconrel. 2016.10.007 (2016)). However, as shown in FIG. 5A / B. this B-mode approach showed little or limited benefits for tumor inhibition and survival in a comparable murine model. Unexpectedly, it was found that by changing the setting from B-mode to a setting of continuous non-thermal pulses with short cycle and sufficient time interval provides superior benefits for tumor inhibition and survival as shown in the murine model disclosed herein. The term "continuous non-thermal pulses with short cycles and sufficient time interval", refers to a mode of ultrasound delivery where a set of multiple short-duration, i.e. short cycles, acoustic pulses is transmitted repeatedly and continuously as a burst or a pulse train without inducing thermal effects or tissue heating. And, each set of acoustic pulses is transmitted with a sufficient time interval to allow circulating microbubbles (in the bloodstream) to perfuse into the targeted tumor region, continuously replenishing the microbubbles and enhancing the cavitation effect. In contrast, B-mode ultrasound typically transmits a single short-cycle pulse per transmission. It does not deliver repeated pulses at the same spatial location within a short timeframe. The continuous pulsed mode. i.e. continuous non-thermal pulses with short cycles and sufficient time interval, described here delivers denser acoustic energy temporally, with multiple short pulses grouped in each transmission, and longer intervals between transmissions — differentiating it from B-mode in both temporal and spatial pulse characteristics. It may be considered that themicrobubble administration needs to be altered from four bolus injections to continuous infusion, ensuring microbubbles to be perfused into the tumor's vessel network and generated on-site cavitation. With sustained microbubble delivery, reperfusion of microbubbles to the tumor region could be achieved. The adequacy of the transmission interval between each set of acoustic pulses needs to be adjusted based on the area or volume covered by the ultrasound insonation and the administered microbubble dosage. Ideally, the selected transmission interval should allow microbubbles to fully replenish the entire targeted tumor region.

[0029] By comparing different sonoporation modes, the continuous non-thermal pulses with short cycle and sufficient time interval (“CPA” mode, e.g., Power Doppler mode) that provides 15 more pulses in one set of transmission than the traditional B-mode is used, which increases the cavitation doses in a regular diagnostic ultrasound system. Unexpectedly, tt is found that only certain ROI selection used in the Power Doppler provides the desired therapeutic benefit as illustrated in the FIG. 3. Such finding allows physicians or sonographers specifically target the tumor area for sonoporation without affecting nearby organs. The study results in Example 2 demonstrate that the above adjustments are critical to a successful cancer treatment. In accordance with the practice of the present invention, microbubbles used in the invention methods undergo targeted, non-thermal manipulation pulses through continuous ultrasound with precisely modulated pulse lengths.

[0030] It is found that mechanical index (“MI”) values in sonoporation below 0.2 result in linear microbubble oscillation, while values between 0.2 and 0.5 lead to nonlinear oscillation.Microbubble will expand and burst when MI exceeds 0.6. According to the labeling of the marketed ultrasound contrast agents, such as Definity (Lantheus Medical Imaging, MA), in the absence of sufficient safety data, it is generally believed that microbubbles should not be used at mechanical indexes higher than 0.8. Therefore, ensuring microbubbles generate significant oscillations and robust mechanical forces for observable therapeutic effects on surrounding cells or tissues while prioritizing safety is essential. It was found that when applying the intermediate mechanical index of 0.4 and observed no acute compliance issues in mice, as supported by the biochemistry test results (see FIG. 2A-B). Furthermore, as supported by the preliminary results in the study of CPA vs B mode (see FIG. 3), it is realized that the better PIMO+ area / tumor area % is achieved when MI values are between 0.4 to 1.2. Hypoxic probe PIMO was used to track tumor hypoxia region by injection of PIMO one hour before mouse sacrifice. PIMO+ area could be presented using primary and secondary antibody. PIMO+ area / tumor area % was calculated as PIMO+ pixels (transformed to area by scale bar) divided by tumor pixels (transformed to area by scale bar). Tumor areas were circled by the dense area of Hoechst staining.

[0031] In some embodiments, MI value used in the invention method is 0.4 to 1.2. In certain embodiments, MI value is 0.5-1.0. In certain embodiments, MI value is 0.6 to 0.8.

[0032] The highly proliferative tumor cells and inadequate blood supply lead to hypoxia within TME, thus hindering the delivery and therapeutic efficacy of chemical drugs. Hypoxia in tumors becomes more severe after chemotherapy due to the toxicity of chemo-drugs, such as gemcitabine and doxorubicin, on endothelial cells, further compromising blood flow and reducing oxygen delivery'. Moreover, a study demonstrated that the hypoxia tumor microenvironment becomes fibrotic after chemotherapy, potentially affecting oxygen diffusion and increasing the status of tumor hypoxia (see, e.g., Park, J. K. et al. The anti-fibrotic effect of GV 1001 combined with gemcitabine on treatment of pancreatic ductal adenocarcinoma. Oncotarget 7, 75081-75093, doi: 10. 18632 / oncotarget. 12057 (2016)). In the study examples disclosed herein, doxorubicin (Dox), gemcitabine, and Onivyde® (irinotecan liposome injection) were each co-administered with the exemplary- microbubble formulation (e.g., NH002) followed by continuous, non-thermal ultrasound pulses (e.g., Power Doppler mode). In the UN-KC-6141 pancreatic tumor model, tumor hypoxia progressively increased over time in the control group. Tumors treated with Dox alone exhibited persistently high levels of hypoxia throughout the observation period (See, e.g., FIG 4B). In contrast, the combination of Dox or gemcitabine with microbubble-mediated sonoporation significantly reduced tumor hypoxia (see, e.g., FIG. 4B, 7D- E). Furthermore, co-administration of microbubbles with Dox, gemcitabine, or Onivyde® (irinotecan liposome injection), followed by ultrasound exposure, effectively slowed overall tumor progression (including secondary tumors in different area of organs) and extended overall survival in tumor-bearing mice (Example 2. see, e.g., FIG. 6-7, 10). As the invention methods disclosed herein clearly provide treatment benefits to tumors in different area of organs corresponding to ovarian cancer, colorectal cancer, gastric (stomach) cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, and prostate cancer, it is evidenced that the invention methods apply to these cancers.

[0033] These results validate the claimed therapeutic method of treating cancer by administering a composition comprising microbubbles and an anti-cancer agent (e.g., Dox, Gem, Onivyde® (irinotecan liposome injection)) at a human equivalent dose (HED) of 2* 108to 4x109bubbles / kg, followed by applying ultrasound with continuous non-thermal pulses at a mechanical index (MI) of 0.4-1.2. The disclosed invention provides a clinically translatable approach to overcome chemotherapy -induced hypoxia and improve treatment efficacy in cancers such as pancreatic ductal adenocarcinoma.

[0034] Tumor perfusion was also observed to decrease after Dox treatment. However, combining Power Doppler-based NH002 sonoporation with Chemotherapy (PDNSC) such as Doxsignificantly resulted in a nearly two-fold perfusion enhancement, reversing the upraised hypoxia area caused by doxorubicin treatment. Interestingly, the well-perfused status was sustained by combined therapy for at least six days after treatment and subsided after that (See, e.g., FIG 4C- D). These data suggested that PDNSC could provide an optimal therapeutic window for combining with other adjuvant therapies, such as radiation therapy or Immune checkpoint blockade (ICB) therapy, to enhance the efficiency of anti-tumor response.

[0035] ICB therapy has emerged as a viable and promising treatment for cancer by enhancing the function of T lymphocytes with monoclonal antibodies targeting the programmed cell death protein- l(PD-l), cytotoxic T lymphocyte-associated protein-4 (CTLA-4), and programmed death-ligand l(PD-Ll), various types of cancer patients benefited. However, targeted T-cell therapy has been unsuccessful in some hypoxia-related cancer patients such as PDAC patients due to the harsh TME of PDAC, preventing T cells from locating proximal enough to the cancer cells (Hosein, A. N.. Dougan, S. K... Aguirre, A. J. & Maitra, A. Translational advances in pancreatic ductal adenocarcinoma therapy. Nature Cancer 3, 272-286, doi: 10.1038 / s43018-022- 00349-2 (2022)). In accordance with the practice of the present invention, it w as found that PDNSC modulated the hypoxic tumor microenvironment of PDAC by reducing hypoxia, enhancing perfusion, and increasing T-cell infiltration, as demonstrated in FIG. 8A-8B and 9A- 9B. These alterations in the TME are believed to enhance the efficacy of aPDLl therapy. In accordance with embodiments of the present invention, the combination of Doxorubicin, microbubbles, and aPDLl antibody resulted in greater tumor regression and extended survival in PDAC tumor-bearing mice compared to monotherapies or dual combinations (see, e.g., FIG. 8C- D). These findings demonstrate that PDNSC enables enhanced ICB therapy and provide a novel strategy for the treatment of PDAC.

[0036] Various chemotherapy combinations, with or without adjuvant therapy, have been extensively studied for the treatment of PDAC. The present invention provides a specific sonoporation setting using PDNSC that reduces tumor hypoxia and enhances the efficacy of chemotherapeutic agents, including small-molecule drugs such as doxorubicin and gemcitabine, for treating pancreatic tumors. In vivo studies demonstrated that PDNSC improves tumor perfusion and reduces hypoxia (see, e.g., FIG. 4A-D), leading to delayed overall tumor progression including the secondary tumors and extended survival in PDAC -bearing mice treated with either doxorubicin or gemcitabine (see, e.g., FIGs. 6-7). Compared to small-molecule drugs, liposomal formulations exhibit prolonged pharmacokinetic (PK) profiles in vivo, allowing them to more effectively leverage sustained improvements in the tumor microenvironment achieved by the present invention. These improvements include reduced hypoxia, enhanced drug permeability, and increased therapeutic response. In accordance with embodiments of the presentinvention, PDNSC was also applied to liposomal drug formulations. In particular, preclinical studies confirmed that PDNSC significantly enhanced the therapeutic efficacy of Oniv de™. a clinically approved liposomal irinotecan formulation used in PDAC treatment. In preclinical PDAC models, the combination of PDNSC with Onivyde™ resulted in substantial overall tumor size reduction including the secondary tumors and prolonged survival compared to Onivyde™ treatment alone (see, e.g., FIG. 10).These findings demonstrate that the PDNSC-mediated tumor microenvironment remodeling — including increased perfusion, enhanced local drug delivery, and reduced hypoxia — benefits both small-molecule and liposomal chemotherapeutic agents in the treatment of pancreatic cancer. However, other chemotherapeutic regimens commonly used in PDAC patients, such as gemcitabine plus nab-paclitaxel and FOLFIRINOX (5-FU, irinotecan, folinic acid, oxaliplatin), can also be effectively applied in combination with the present invention.

[0037] In some embodiments, the anti-cancer agent comprises liposomal formulated drugs such as Onivyde™ (irinotecan liposome injection), which has demonstrated enhanced tumor reduction and prolonged survival when combined with the disclosed sonoporation method in in vivo studies. The results of the preclinical studies demonstrate the enhanced therapeutic efficacy of small-molecule chemotherapy drugs, such as doxorubicin and gemcitabine, by reducing tumor hypoxia, increasing local perfusion, and improving outcomes. Furthermore, this invention compositions and methods also boost the effectiveness of liposomal chemotherapy drugs, like Onivyde™, leading to significant tumor size reduction and increased survival time.

[0038] It is evidenced by the present invention, the administration of doxorubicin or gemcitabine combined with sonoporation significantly reduced hypoxia further, whereas sonoporation alone did not exhibit such a pronounced effect illustrated in FIG 4B. Thus, it is surprisingly found that the exemplary anti-cancer agents (e.g., doxorubicin and gemcitabine) synergize with CPA mode (e.g., Power Doppler) applied particular microbubbles sonoporation to increase tumor perfusion and reduce the tumor hypoxic region. In some embodiments, the anti-cancer agent is administered simultaneously with sonoporation to achieve the best outcome.

[0039] In some embodiments provide methods of enhancing efficacy of a cancer treatment in a subject comprising administering to the subject a composition comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2x108to 4x109bubbles per Kg body weight; applying ultrasound to induce the microbubble destruction in a targeted region with a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2. The methods enhance ultrasound-mediated cavitation effects and reducing tumor hypoxia. In some embodiments provide a method of enhancing efficacy of a cancer treatment in a subject comprising administering to the subject a compositioncomprising an anti-cancer agent and a microbubble suspension, wherein the microbubble suspension has a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subject’s body weight; and applying ultrasound to induce microbubble destruction in a targeted region by continuous non-thermal pulses with short cycle and sufficient time interval, wherein the MI value is 0.4 to 1.2. The methods enhance ultrasound-mediated cavitation effects and reducing tumor hypoxia.

[0040] In some embodiments provide a pharmaceutical composition for use in enhancing efficacy of a cancer treatment in a subject, comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2x108to 4xl09bubbles per Kg body weight with application of ultrasound to induce the microbubble destruction in a targeted region in a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2. In some embodiments provide a use of pharmaceutical composition comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2x108to 4xl09bubbles per Kg body weight in the manufacture of a medicament, with application of ultrasound in a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2 to induce the microbubble destruction in a targeted region, for enhancing efficacy of a cancer treatment in a subject.

[0041] In some embodiments the subject is a mammal. In certain embodiments, the subject is a human. In some embodiments, the anti-cancer agent comprises doxorubicin, gemcitabine, gemcitabine plus nab-paclitaxel, 5-FU, irinotecan, folinic, oxaliplatin, FOLLFIRINOX (5-FU, irinotecan, folinic, oxaliplatin) or liposome formulated anti-cancer agent such as irinotecan liposome injection (Onivyde™), or the like. In certain embodiments, the anti-cancer agent is doxorubicin, gemcitabine, Onivyde™ or combinations thereof. In some embodiments, the composition in invention methods disclosed herein further comprise anti-PDLl (aPDLl) antibody, or the like. In some embodiments, the cancer is ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, and prostate cancer. In certain embodiments, the cancer is pancreatic cancer such as pancreatic ductal adenocarcinoma.

[0042] In some embodiments provide methods of treating cancer in a subject comprising administering to the subject a composition comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2x108to 4xl09bubbles per Kg body weight; applying ultrasound to induce the microbubble destruction in a targeted region with a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2. In some embodiments provide a pharmaceutical composition for use intreating cancer in a subject, comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2xl08to 4xl09bubbles per Kg body weight with application of ultrasound to induce the microbubble destruction in a targeted region in a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2. In some embodiments provide a use of pharmaceutical composition comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2xl08to 4xl09bubbles per Kg body weight in the manufacture of a medicament, with application of ultrasound in a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2 to induce the microbubble destruction in a targeted region, for treating cancer in a subject.

[0043] In some embodiments the subject is a mammal. In certain embodiments, the subject is a human. In some embodiments, the anti-cancer agent comprises doxorubicin, gemcitabine, gemcitabine plus nab-paclitaxel. 5-FU, irinotecan, folinic, oxaliplatin, FOLLFIRINOX (5-FU. irinotecan, folinic, oxaliplatin) or liposome formulated anti-cancer agent such as irinotecan liposome injection, or the like. In certain embodiments, the anti-cancer agent is doxorubicin and / or gemcitabine. In some embodiments, the composition in invention methods disclosed herein further comprise anti-PDLl (aPDLl) antibody, or the like. In some embodiments, the cancer is ovanan cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, and prostate cancer. In certain embodiments, the cancer is pancreatic cancer such as pancreatic ductal adenocarcinoma.

[0044] In some embodiments, the present invention provides methods of treating cancer in a subject, comprising administering to the subject a composition comprising an anti-cancer agent and a microbubble suspension, wherein the microbubble suspension is characterized by a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subject’s body weight. The method further comprises applying ultrasound to induce microbubble destruction in a targeted region using continuous non-thermal pulses with short cycle and sufficient time interval, wherein the mechanical index (MI) value is 0.4 to 1.2. The method enhances ultrasound-mediated cavitation effects and reducing tumor hypoxia. In some embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In some embodiments, the anti-cancer agent comprises doxorubicin, gemcitabine, gemcitabine plus nab-paclitaxel, 5-FU. irinotecan, folinic acid, oxaliplatin, FOLFIRINOX (5-FU, irinotecan, folinic acid, oxaliplatin), or liposome-formulated anti-cancer agents such as irinotecan liposome injection (Onivyde™), or the like. In certain embodiments, the anti-cancer agent is doxorubicin, gemcitabine, and / or Onivyde™.

[0045] In some embodiments provide a pharmaceutical composition for use in treating cancer in a subject, comprising an anti-cancer agent and microbubbles suspension wherein the microbubble suspension has a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subject’s body weight, with application of ultrasound to induce the microbubble destruction in a targeted region in a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2. In some embodiments provide a use of pharmaceutical composition comprising an anti-cancer agent and microbubbles suspension wherein the microbubble suspension has a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subject’s body weight, in the manufacture of a medicament with application of ultrasound in a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2 to induce the microbubble destruction in a targeted region for treating cancer in a subject. In some embodiments, the composition used in the disclosed invention methods further comprises an anti-PD-Ll (aPD-Ll) antibody or other immunotherapeutic agents. In some embodiments, the cancer is ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, and prostate cancer. In certain embodiments, the cancer is pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC). In certain embodiments, the method further applies to malignant cancer comprising ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma cancer, lung cancer, liver cancer, or bladder cancer.

[0046] All of the various embodiments or options described herein can be combined in any and all variations. The following Examples serve only to illustrate the invention and are not to be construed in any way to limit the invention.Examples Example 1. Feasibility Study on Power Doppler-based Sonoporation: Effects of Microbubble Gas Volume and HEP Dosage on Tumor Hypoxia Reduction.

[0047] Method and materials

[0048] Mice: Eight-week-old C57BL / 6J male mice were purchased from the National Laboratory7Animal Center of Taiwan. All animal performances followed the guidelines of the Institutional Animal Care and Use Committee (IACUC) of National Tsing Hua University, Taiwan (IACUC approval No. 109067).

[0049] Cell line: UN-KC-6141, a murine PDAC cell line (given by Prof. Surinder K. Batra, the University of Nebraska Medical Center, Omaha, Nebraska, USA), was incubated at 37°C, 5% CO2 under humid conditions. Cells were maintained in Dulbecco’s modified Eagles medium (DMEM; Gibco®, 12100046, Grand Island, NY, USA) with 10 % fetal bovine serum (FBS;Gibco®, 16000044), 1% penicillin-streptomycin (PS; Gibco®, 15140122). Before utilizing the cells, Mycoplasma contamination was examined by an EZ-PCR™ Mycoplasma detection kit (Biological Industry, 20-700-20, Beit HaEmek, Israel).

[0050] In vivo pancreatic tumor model

[0051] The orthotopic pancreatic tumor model was generated by implanting UN-KC-6141 cells into the pancreas as described previously with minor modifications. Briefly, mice were anesthetized with a 1: 1 mixture ofZoletil®50 (Virbac, 7J7ZA, Carros, France) and 2% Rompun® (Bayer Healthcare Animal Health, CAPROM-L-003, Germany). A small incision was made on the left abdomen (spleen side). The spleen was dragged out along with pancreas tissue. A cell number 1x104, in the form of a semi-solid gel spheroid, was embedded into the pancreas head. To form the semi-solid spheroid, 2 pl of 1 : 1 mixture with DMEM and Matrix gel (Coming, 356237, Bedford, MA, USA) were used and pumped by an automatic pump (KD Scientific, 311. Holliston, MA. USA). The wound was closely sutured and covered with ointment to avoid infection. The body weight of mice was recorded every other day after tumor inoculation. Tumorbearing mice were euthanized after showing neurologic deficits (lethargy, failure to canter, dyspnea, and back arching).

[0052] Sonoporation treatment protocol

[0053] Treatments were conducted on day 10, when the average tumor size was approximately 30 to 60 mm3, and all subjects also had secondary metastatic tumors (identified via ultrasound images, e.g., in the spleen, abdomen, kidney, or other sites outside pancreas). The secondary7metastatic tumors are corresponding to ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, or prostate cancer. Mice were positioned, and an intravenous (IV) injection of 2.5 mg / kg doxorubicin (Dox; Pfizer, ADRIAMYCIN®, New York, USA) was given together with 6xl07, 1.2xl08orl.2x!09NH002 microbubbles per 25g mice (at the human equivalent dose (HED) about 2xl08, 4x108or 4x109bubbles per kg body weight, respectively). A ring stand and a three-prong clamp held the transducer to ensure precise positioning. The transducer was moved back and forth to cover all the tumor sections during the sonoporation. The Dox-only group went through the same ultrasound scanning procedures without microbubble injection. Control mice were treated with PBS and only imaged with ultrasound to obtain the tumor image with the largest section area.For the Power Doppler-based sonoporation, tumor location was confirmed and circled as the region of interest (ROI) by the Power Doppler mode 10 minutes before sonoporation. Dox (doxorubicin, 2.5 mg / kg) was administered together with microbubble. They were IV delivered with an automatic micropump (KD Scientific. KDS-310, Holliston MA, USA) at an infusion rateof 50 pl / min for 4 minutes. Mice in the microbubble-only group received only microbubbles. Once the infusion started, the B-mode was switched to the already ROI-selected Power Doppler mode, and the mechanical index was set to 0.4. Mice were anesthetized all the time and kept warm during the treatment.

[0054] Tissue immunofluorescence analysis

[0055] The hypoxia bio-marker pimonidazole (PIMO, 160 mg / kg, HPI-100, HPI, Burlington, MA, USA) was IP injected into mice one hour before sacrifice. The tumor tissue was embedded with the Optimal Cutting Temperature (OCT) compound (Sakura, Finetek, Torrance, CA, USA) and immediately kept at -80 C . Frozen tissues were sliced by the cryo-microtome (Leica, CM1850, Heidelberger, Germany) with 10 pm thickness and adhered to the salinized slide (MUTO PURE CHEMICALS. 511614, Toky o, Japan). The frozen sections were fixed with cold methanol and permeabilized with 0.1% Tween-20 (Sigma, St. Louis, MO, USA) and subsequently blocked with 4% FBS, 1% goat serum (Gibco®, 16210-064) in PBS to reduce the non-specific binding for 1 hour at room temperature. First antibody7was stained as follows: mouse anti-Hypoxy biomarker (1 : 100, HPI, HPI-100 kit) overnight at 4°C . Secondary' antibodies conjugated with Alexa Fluor 488 goat anti-mouse (1 :200 Thermo Fisher Scientific, A21121, Waltham MA, USA) was stained for 1 hour at room temperature. Hoechst33342 (2.5 mg / ml, Thermo Fisher Scientific, Hl 399) was stained to visualize the nucleus. For preservation, slices were mounted using an anti-fade mounting medium (VECTOR, H-1000, Burlingame, CA, USA). Images were taken by the AxioCam MCR-5 on Axiovertskop 40 microscope (Carl Zeiss.Axioskop 40 FL, Goettingen, GERMANY) and analyzed by Image-Pro 6.0 software.

[0056] Study Results

[0057] To examine whether sonoporation with different microbubble dosages could alter the tumor microenvironment, particularly its hypoxic status, an in vivo test was conducted using microbubbles at a human equivalent dose (HED) of 4 x 109bubbles / kg. Ten days after tumor inoculation, mice were treated with either doxorubicin (Dox) alone or Dox plus sonoporation under power Doppler mode (i.e., a setting of continuous non-thermal pulses with short cycle and sufficient time interval). The sonoporation treatment was applied using three microbubble concentrations: the full HED dose (4 x 109bubbles / kg). median 10-fold diluted dose (4xl08bubbles / kg) and a 20-fold diluted dose (2 x 108bubbles / kg). One day after treatment, mice were sacrificed, and tumor tissues were analyzed for hypoxic status using the pimonidazole (PIMO) marker. The results showed that in mice receiving Dox alone (without sonoporation), the hypoxic tumor area was approximately 36%. In contrast, tumors treated with sonoporation at the full microbubble concentration showed a significantly reduced hypoxic area of 8%. Interestingly, even at the 20 x diluted concentration, the hypoxic area w as 21% (Figure 1A and IB). Thesefmdings suggest that sonoporation effectively reduces tumor hypoxia, with a stronger effect observed at higher microbubble concentrations. Moreover, at least 2 x 108bubbles / kg is required as the lower concentration threshold for hypoxia reduction. Based on gas volume calculations, the estimated gas dosing ranged from 1.24-1.53 pL / kg (lower limit) to 24.72-30.57 pL / kg (upper limit), as summarized in Table 1A / B.

[0058] In some instances, the present invention provides methods for reducing tumor hypoxia through Power Doppler-based sonoporation with a specific character of microbubbles, thereby enhancing chemotherapy efficacy. In certain instances, the method involves administering certain microbubbles (e.g., NH002) disclosed herewith at various gas volumes and concentrations, in a human equivalent dose (HED) to investigate their effects on tumor oxygenation The findings demonstrate that sonoporation significantly reduces tumor hypoxia in a dose-dependent manner, with a lower threshold concentration required for effective hypoxia reduction. In certain embodiments, the disclosed method establishes gas volume dosing parameters for optimizing sonoporation treatment, providing a valuable strategy for improving drug delivery in hypoxic tumor environments.Example 2, Feasibility Study Using Power Doppler-based Sonoporation via Exemplary Microbubbles with Chemotherapy (PDNSC) and Immune-checkpoint Inhibitor to Treat PDAC Tumor.

[0059] The materials and cell lines are the same as in Example 1, with additional methodological details focusing on the in vivo treatment, as described below.

[0060] Pancreatic tumor size assessed by ultrasound imaging

[0061] After tumor implantation, mice were screened with ultrasound imaging on days 10, 13, 16, 21 or 25. Briefly, mice were anesthetized, and the body hair of the observed area was entirely removed by depilation creme. Imaging was performed using a portable ultrasound system (Philips, CX50, Amsterdam, Holland) with an L12-3 linear broadband ultrasound transducer (Philips). Images were acquired with a depth of 3 cm and a mechanical index (MI) value of 0.8. The stomach and spleen were scanned and marked in the ultrasound image as a reference, followed by the pancreas and the tumor. Once the tumor was located, video (frame rate 31 Hz) was recorded to find the largest section area. Ultrasound videos and images were further processed with RadiAnt DICOM Viewer, and the total area of all tumor sections was circled and calculated using Image-Pro 6.0 software.

[0062] Sonoporation treatment protocol

[0063] Different ultrasound sonoporation treatment settings were applied for separate experiments. For the sonoporation under B-mode, mice were randomly separated into threegroups: Control, Dox only, and Dox+MB (doxorubicin plus microbubble-induced sonoporation) ten days after tumor inoculation. Treatments were conducted on day 10 and day 16 for both treatment groups. Mice were positioned, and an intravenous (IV) injection of 2.5 mg / kg doxorubicin (Dox; Pfizer, ADRIAMYCIN®, New York, USA) was given to the mice 10 minutes before sonoporation. The tumors were screened and located right after the administration of Dox. A 50 pl IV injection of 3x108NH002 microbubble was then given to mice every 5 minutes for four consecutive times, and the MI value of the B-mode imaging was set as 0.4 throughout the treatment. A ring stand and a three-prong clamp held the transducer to ensure precise positioning. The transducer was moved back and forth to cover all the tumor sections during the sonoporation. The Dox-only group went through the same ultrasound scanning procedures without microbubble injection. Control mice were treated with PBS and only imaged with ultrasound to obtain the tumor image with the largest section area.

[0064] For the Power Doppler-based sonoporation, tumor location was confirmed and circled as the region of interest (ROI) by the Power Doppler mode 10 minutes before sonoporation. Dox (2.5 mg / kg) or gemcitabine (30 mg / kg) or Onivyde™(20 mg / kg) was administered together with microbubble. The total doses of Dox and microbubble remained the same as the previous B- mode setting. They were IV delivered with an automatic micropump (KD Scientific, KDS-310, Holliston MA, USA) at an infusion rate of 50 pl / min for 4 minutes. Mice in the microbubble- only group received only microbubbles. Once the infusion started, the B-mode was switched to the already ROI-selected Power Doppler mode, and the mechanical index was set to 0.4. Mice were anesthetized all the time and kept warm during the treatment.

[0065] PDNSC combined Immune checkpoint blockade (ICB) therapy

[0066] Ten days after the tumor inoculation, mice were randomly separated into three groups (n>4 for each group): anti-programmed death-ligand 1 only (aPDLl; Bio X cell, InVivoMAb anti-mouse PD-L1. BE0101, NH, USA), Dox plus aPDLl (Dox+aPDLl), Power Doppler sonoporation plus Dox as well as aPDLl (Dox+aPDLl+MB). The PDNSC treatment protocols were the same as in the settings mentioned above. Mice were intraperitoneal (IP) injected aPDLl (8 mg / kg) on days 10, 13, 16, and 19.

[0067] Tumor perfusion test after PDNSC

[0068] A tumor specimen (20 mm3) was dissected from the UN-KC-6141 tumor-bearing mice, and then the xenograft was implanted into the pancreas of the other healthy mouse. The following surgical protocol was the same as the orthotopic pancreatic tumor injection. After the tumor reached 50 mm2under the ultrasound examination, mice were randomly divided into Dox only (mice receive Dox only) and Dox+MB (Dox plus microbubble-induced sonoporation). Tumor perfusion was examined one day before treatment and days 0, 1, 3, and 6 after. For thetumor perfusion examination, 20 pl of 1.2x108microbubbles were IV injected into mice. Then, the dynamic reperfusion was recorded as a video (frame rate 19Hz) in the contrast mode by the CX50 ultrasound system following bubble flashing. The image in the video was snapped 7 seconds after the bubble flashing to calculate the tumor perfusion area. The percentage of tumor perfusion area was calculated from the bright tumor perfused area ((entire tumor area - nonperfused tumor area) / total tumor area) x 100%.

[0069] Tissue immunofluorescence analysis

[0070] The hypoxia bio-marker pimonidazole (PIMO, 160 mg / kg, HPI-100, HPI, Burlington, MA, USA) was IP injected into mice one hour before sacrifice. The tumor tissue was embedded with the Optimal Cutting Temperature (OCT) compound (Sakura, Finetek, Torrance, CA, USA) and immediately kept at -80cC . Frozen tissues were sliced by the cryo-microtome (Leica, CM1850, Heidelberger, Germany) with 10 pm thickness and adhered to the salinized slide (MUTO PURE CHEMICALS, 511614, Tokyo, Japan). The frozen sections were fixed with cold methanol and permeabilized with 0.1% Tween-20 (Sigma, St. Louis, MO, USA) and subsequently blocked with 4% FBS, 1% goat serum (Gibco®, 16210-064) in PBS to reduce the non-specific binding for 1 hour at room temperature. First antibodies were stained as follows: purified rat anti-mouse CD8 (1 : 100, BD Pharmingen, 550281), purified rat anti-mouse CD4 (1 : 100, BD Pharmingen, 550280), and mouse anti-Hypoxy biomarker (1: 100, HPI, HPI-100 kit) overnight at 4°C . Secondary antibodies conjugated with Alexa Fluor 488 goat anti-mouse (1:200 Thermo Fisher Scientific, A21121, Waltham MA, USA), Alexa Fluor 594 goat anti-rat (1 : 200 Thermo Fisher Scientific, Al 1012), were stained for 1 hour at room temperature. Hoechst33342 (2.5 mg / ml, Thermo Fisher Scientific, H1399) was stained to visualize the nucleus. For preservation, slices were mounted using an anti-fade mounting medium (VECTOR, H-1000, Burlingame, CA. USA). Images were taken by the AxioCam MCR-5 on Axiovertskop 40 microscope (Carl Zeiss, Axioskop 40 FL, Goettingen, GERMANY) and analyzed by Image-Pro 6.0 software.

[0071] H&E staining

[0072] Methanol fixed section slides were air-dried and stained with Hematoxylin (SIG-MA, GHS232-1L) for 1 minute, followed by water rinsing. The slides were treated with 0.25% Ammonia after rinsing. The air-dried slices were subsequently stained with Eosin (SIGMA, HT110116-500ML) for 20 seconds and mounted with VectaMount® Permanent (VECTOR, H- 5000).

[0073] Blood chemistry test

[0074] The blood (200 pl) was collected from tumor-bearing mice 24 hours post-treatment to examine the acute side effects of the treatments. The whole blood was centrifuged at 2000 x g for10 minutes at 4°C. 60 JJ1 of blood serum was collected from the supernatant and tested on the reagent disc (AmiShield, 001-3GYC, Taoyuan, Taiwan) with the biochemical analytical instrument (AmiShield, VCA-TE-300).

[0075] Statistics: Statistical analysis was performed by the Prism software 8.0 with two-tailed Student’s t-tests, and a P-value < 0.05 was recognized as statistical significance.

[0076] Study Results

[0077] To further explore the feasibility of sonoporation using a traditional clinical diagnostic ultrasound system with B-mode combined with chemotherapy for PDAC treatment, the chemodrug Dox was tested in vivo studies. The effect of Dox on tumor control was then examined by injecting Dox (2.5 mg / kg) into tumor-bearing mice on days 10 and 16 after inoculation (FIG. 5A / B). A total amount of 5 mg / kg of Dox was reported to be a safe dose. Sonoporation was performed on the same day as Dox administration on days 10 and 16. The clinical program was modified to inject Dox 10 minutes before sonoporation. and NH002 microbubbles were given every 5 minutes for four consecutive times. The mechanical index was set at 0.4 to ensure sufficient oscillation of microbubbles while preventing bio-effects. The results showed that two doses of Dox treatment could slightly extend the surviving days of mice-bearing UN-KC-6141 tumors from 22.0 to 24.0 days (FIG. 5 A). A significant tumor growth delay was noted on day 13 and continued to day 21 (FIG. 5B). However, despite observing a trend of reduction in tumor size from day 16 to day 21 compared to the Dox treatment group, B-mode-based sonoporation did not improve the therapeutic efficacy7of Dox. The mice survival curve showed a slight increase in median survival (24.0 vs. 26.0 days) but without a significant difference between the Dox-only group and the sonoporation plus Dox group (FIG. 5B). The study results show that applying continuous non-thermal pulses with short cycle and sufficient time interval (Power Doppler mode) based sonoporation enhances the efficacy of Dox-mediated PDAC tumor grow th delay.

[0078] The unsatisfactory treatment outcome might be attributed to the low- penetration and gradually dropping microbubble concentration within the UN-KC-6141 tumor. Additionally, the concentration of Dox in the mouse plasma was reported to decrease within 10 minutes after drug administration. To improve the effect of sonoporation on drug penetration, an automatic pump for continuous infusion w as used to maintain a constant influx of microbubbles into the tumor during the period of sonoporation, and the Dox was delivered simultaneously with microbubbles for sonoporation. A new protocol using the Power Doppler imaging mode was applied to this study' (FIG. 6) to explicitly target the tumor area for sonoporation and provide more pulses to increase the sonoporation effect. The mice's survival result demonstrated that Power Dopplerbased sonoporation significantly enhanced Dox efficacy by increasing the median surviving time from 27.0 to 33.5 days. The additional survival results of the mice from the treatment exhibited athreefold increase, from 3 days (Dox only) to 9.5 days (Dox+MB), compared to the control group with a survival time of 24.0 days. The 30-day survival rate increased from 30% in the Dox-only group to 50% in the Dox+MB group, with one mouse surviving for over six months (FIG. 6A). The ultrasound image showed that Dox+MB therapy could further inhibit tumor progression area than Dox monotherapy, resulting in 27% and 26% tumor area reduction on days 16 and 21, respectively (FIG. 6B). Also, the secondary tumor size further showed 25% reduced in the Dox+MB therapy group compared with Dox only (FIG. 6C). The study results show that applying continuous non-thermal pulses with short cycle and sufficient time interval (Power Doppler mode) based sonoporation enhances the efficacy of Dox-mediated overall PDAC tumor growth delay (including secondary tumors). On the other hand, sonoporation only (MB only) didn’t affect tumor progression, and there was no significant difference in the mice survival compared to the control group (FIG. 6B). After treatment, the body weight of mice in both the Dox and Dox+MB groups experienced a slight 5% decrease, followed by a rapid recovery within three days (FIG. 2A).

[0079] In contrast, the body w eight of mice in the control and MB-only groups remained stable (FIG. 2A). To confirm if there were acute adverse reactions mediated by Dox or MB treatment, blood from tumor-bearing mice was collected and examined for biochemical values. The results revealed that only the Dox-related acute adverse reactions, such as liver inflammation (indicated by a significant increase in Alkaline Phosphatase (ALP) levels compared to the control), were observed in the Dox and Dox+MB groups. Additionally, a slight increase in phosphorus levels (PHOS) was noted after doxorubicin treatment, as compared to the control and MB-only group, though this difference was insignificant (FIG. 2B). There were no notable distinctions between the Dox and Dox+MB groups across all parameters. These data suggested that the Pow er Doppler-based NH002 sonoporation with Chemotherapy (PDNSC) was safe under this protocol and could significantly prolong the mice's survival by enhancing the efficacy of chemotherapy in pancreatic tumors.

[0080] According to the monitoring of tumor size, surprisingly, a significant reduction in tumor size was found at day 16 (the second treatment of Dox and sonoporation) and persisted until day 21 (FIG. 6B), suggesting a postponed effect than the direct enhancement of drug uptake reported in other sonoporation studies. Without binding to any theories, it was hypothesized that the outcome might result from the altered TME after the sonoporation treatment. To examine whether the preclinical sonoporation setting disclosed herein triggered any changes in the TME, tumor tissues were collected on day 1, day 3, and day 6 after the first treatment and examined by immunofluorescence staining. It is known that hypoxia in PDAC is an intrinsic factor causing resistance to chemotherapy. Previously it was reported that the UN-KC-6141 had low MVD anda high percentage of hypoxia. The hypoxia status after treatments was further examined by staining the hypoxia probe, PIMO. The results showed that the Dox treatment significantly increased in the hypoxic area on day 1 and day 3 post-treatment compared to other groups. However, combination therapy led to significant decreases in hypoxia on day 1 (8.5% versus 36.5%) and day 3 (9.5% versus 28.4%) compared to Dox treatment. A slight decrease in the hypoxic region was also found on day 6 (16.1% versus 25.9%). However, it did not reach a statistical difference, indicating that the effect of combined treatment was sustained for at least 3 days and gradually attenuated 6 days after the first treatment (FIG. 4A, B). There was no statistical difference in the tumor hypoxic area between the control and the sonoporation-only groups in the observation window.

[0081] To further explore the effect of the PDNSC, contrast-enhanced ultrasound was employed to continuously monitor variations in tumor perfusion over a week. Here, the xenograft model was used to achieve a better tumor delineation for precise quantification. The results indicated a significant increase in the variation of tumor perfusion area in the combination treatment group on day 1 (133% versus 67%) and day 3 (132% versus 64%) compared to the group that received Dox only (FIG. 4D). The increased tumor perfusion declined from day 3 to day 6, reaching similar levels of tumor perfusion between the two groups (FIG. 4D). These data suggested that sonoporation with Dox significantly relieved the tumor's hypoxic status and increased tumor perfusion. Clearly, it is evidenced that PDNSC altered the PDAC tumor microenvironment in favor of the drug effect.

[0082] Gemcitabine has been the standard frontline drug for PDAC patients since 1997. To assess the compatibility of the sonoporation setting disclosed herein with gemcitabine, an in vivo study (FIG. 7A-E) examining the combined effect of gemcitabine and sonoporation was conducted. Results showed that the combination treatment prolonged the survival of mice compared to those treated with gemcitabine alone (39.0 days versus 33.0 days, FIG. 7A). Ultrasound images revealed additional tumor growth inhibition in the combined treatment group on day 16 (33% tumor reduction) and day 21 (27% tumor reduction), compared to the gemcitabine-only group (FIG. 7B). Also, the secondary tumors in the combined treatment group showed 38% reduction compared to the gemcitabine-only group (FIG. 7C). Moreover, analysis of tumor hypoxia after treatment (FIG. 7D-E) demonstrated that combined sonoporation with gemcitabine significantly reduced the hypoxic area compared to gemcitabine alone (7.6% versus 33.4%). These findings indicate that PDNSC could combine therapeutic drugs, like Dox or gemcitabine, to modify the TME and enhance chemotherapeutic efficacy against pancreatic tumors.

[0083] The median extended survival time and treatment effectiveness improvement rate for each group and drug combination is summarized in Table 2. It is clearly evidenced that the treatment effectiveness improvement rate of Dox+MB+aPDLl group is more than 6 folds when no MB was applied to Dox+aPDLl group.Table 2. Results of the median extended survival time and treatment effectiveness improvement rate for each group in ICB Study

[0084] Onivyde® (irinotecan liposome injection), a nano-liposomal irinotecan formulation, is widely used as a first-line treatment for metastatic PDAC in combination with 5 -fluorouracil, oxaliplatin and leucovorin. To evaluate the effectiveness of sonoporation with Onivyde®, an in vivo study was conducted using the disclosed Power Doppler-based sonoporation (PDNSC) method. The combination of sonoporation and Onivyde® significantly enhanced tumor suppression compared to Onivyde® alone. Mice receiving the combined treatment exhibited a prolonged survival benefit (32 days vs. 28 days. FIG. IOC). Tumor volume measurements demonstrated an additional -27% tumor reduction in the sonoporation + Onivyde® group on days 16 and 21 compared to Onivyde® monotherapy (FIG. 10A). Also, the secondary tumors in the combined treatment group showed 39% reduction compared to the Onivyde®-only group (FIG. 10B). These findings indicate that PDNSC enhances the therapeutic efficacy of Onivyde, leading to greater tumor suppression and prolonged survival in pancreatic tumor models.

[0085] The median extended survival time and treatment effectiveness improvement rate for each group and drug combination is summarized in Table 3. It is clearly shown the superior thetreatment effectiveness improvement rate of Gem+MB group as well as Onivyde®+MB group in comparison with the corresponding groups without MB.

[0086] Table 3. Results of the median extended survival time and treatment effectiveness improvement rate for each group

[0087] Immunotherapy Study Results

[0088] T cells act as a crucial modulator in the host immune system against tumors, extending the tumor eradication of chemotherapy. However, the T-cell infiltrating was strongly hindered by the harsh TME of PDAC. To further test the effect of PDNSC on T-cell infiltration into tumors, the T cells within TME were identified using CD8 and CD4 markers on day 1, day 3. and day 6 following the initial treatment. The results demonstrated that both cytotoxic T cells (CD8+) and helper T cells (CD4+) were rarely found in control, Dox-only, and MB-only groups (FIG. 8A-B, FIG. 9A-B). Combined treatment resulted in a mild increase of CD8+T cell infiltration on day 3 compared to control (P=0.03), Dox- (P=0.08), and MB-only groups (P=0.09), but a profound expansion on day 6 with a 3.9-fold increase compared to any other groups (FIG. 8A-B).Infiltration of CD4+T cells on day 6 with a 2.5-fold increase in the combined treatment group was also observed (FIG. 9A-B). The above results demonstrated that the PDNSC changed the TME of PDAC, decreasing hypoxia, enhancing tumor perfusion, and increasing T-cell infiltrates.

[0089] Immune checkpoint blockade (ICB) therapy has emerged as a pivotal and promising treatment for cancer. However, it has not yet been proven effective for PDAC patients due to the harsh TME. To further assess the potential for enhancing the efficacy of ICB therapy on PDACs through PDNSC, the anti-PDLl (aPDLl) antibody was administered to tumor-bearing mice. Mice were administrated with aPDLl on days 10, 13, 16, and 19, while the Dox andsonoporation treatment followed the previous schedule. The results revealed that triple combination treatment (Dox+MB+aPDLl) significantly prolonged the median survival of mice (46 days) compared to those of Dox+aPDLl treatment (28 days) and aPDLl only (25 days) (FIG. 8C). The triple combination therapy also significantly suppressed tumor growth compared to Dox+aPDLl or aPDLl alone (FIG. 8D). These data demonstrate that PDNSC can enhance the effectiveness of aPDLl treatment and strengthen the combination of chemo-immunotherapy against pancreatic tumors. In some embodiments, the composition in invention methods disclosed herein further comprise anti-PDLl (aPDLl) antibody, or the like.

[0090] The median survival time and extended survival time for each group is summarized in Table 4. It is clearly shown that Dox+MB+aPDLl provides superior results in both median survival time and extended survival time.

[0091] Table 4. Results of median survival time and extended survival time for each group in ICB Study.

[0092] In some embodiments provide improved treatment efficacy of pancreatic ductal adenocarcinoma (PDAC). In certain embodiments provide methods of applying the continuous non-thermal pulses with short cycle and sufficient time interval (Power Doppler mode) based sonoporation to enhance the chemotherapy and Immune checkpoint blockade therapy in a subj ect. The methods are particularly useful to treat tumors that does not well response to Immune checkpoint blockade therapy. In certain embodiments the claimed method illustrates the power of PDNSC to enhance chemotherapy efficacy and improve the effectiveness of Immune checkpoint blockade therapy.Example 3, Feasibility Study Using Power Doppler-based Sonoporation via Exemplary High Concentrated Microbubbles with Chemotherapy to Treat Metastasis Tumors.

[0093] To determine whether the therapeutic method established in Example 2 can be effectively applied to the treatment of metastatic pancreatic ductal adenocarcinoma (PDAC), an orthotopic tumor model exhibiting strong metastatic and invasive capabilities, was used with the same materials, cell lines, and procedures described in Example 2.

[0094] The orthotopic tumor model was established by injecting UN-KC-6141 cells directly into the pancreas of mice. The orthotopic tumor model is characterized by extensive metastasis to abdominal organs, including the spleen, kidney, liver, colon, stomach, lung, bile duct, and diaphragm. The resulting survival times were inversely correlated with the number of cells implanted. Mice receiving I x lO4cells showed a median survival of approximately 23 days, while those receiving Ix lO5and 2x l05cells had shorter median survival times of about 17.5 and 17.0 days, respectively, indicating that higher tumor burden accelerated disease progression.

[0095] Study Results

[0096] Beyond primary tumors, upon euthanasia due to neurological symptoms, all tumorbearing mice exhibited secondary tumors in abdominal organs such as the liver, kidney, spleen, and intestine. The incidence of secondary tumors was independent of the initial injection cell count. Metastatic sites were recorded at sacrifice, with 68, 73 out of 87 mice showing secondary tumors in the abdomen and spleen, respectively (see Table 5). Additionally, 50, 36, and 22 out of the total 87 mice developed kidney, liver, and colon metastases, respectively (Table 5). Real- world data revealed that the most common metastatic sites were the abdomen (peritoneum) and liver, aligning closely with our orthotopic model. Notably, lung metastasis was rare in this murine tumor model despite a reported clinical diagnosis metastatic rate of 10%.

[0097] Table 5. Results of secondary' tumors in metastatic sites.*Metastasis site recorded when mice were sacrificed.

[0098] These findings highlight that all experimental results in the present disclosure were generated using a rigorous and clinically relevant orthotopic PDAC model that naturally recapitulates metastatic dissemination to major abdominal organs. The metastatic burden and tumor progression closely reflect human disease, thereby reinforcing the translational value and clinical relevance of the present invention of sonoporation-based therapeutic strategy described herein. As such, it is clearly evidenced that the invention methods disclosed herein can apply totreatments of ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma cancer, lung cancer, liver cancer, or bladder cancer.

[0099] In certain embodiments, the invention methods disclosed herein further apply to malignant cancer comprising ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma cancer, lung cancer, liver cancer, or bladder cancer.

[0100] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WHAT IS CLAIMED IS:

1. A method of enhancing efficacy of a cancer treatment in a subject comprising administering to the subject a composition comprising an anti-cancer agent and microbubbles suspension in a human equivalent dose (HED) range of 2xl08to 4xl09bubbles per Kg body weight; applying ultrasound to induce the microbubble destruction in a targeted region with a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2.

2. The method of claim 1, wherein the subject is a mammal.

3. The method of claim 1, the subject is a human.

4. The method of claim 1, wherein the anti-cancer agent comprises doxorubicin, gemcitabine, gemcitabine plus nab-paclitaxel, 5-FU, irinotecan, folinic, oxaliplatin, FOLLFIRINOX (5-FU, irinotecan, folinic, oxaliplatin) or liposome formulated anticancer agent such as Onivyde® (irinotecan liposome injection), or the like.

5. The method of claim 4, wherein the anti-cancer agent is doxorubicin, gemcitabine, Onivyde® (irinotecan liposome injection) or combinations thereof.

6. The method of any claims of claim 1-5, wherein the method further comprises an immune checkpoint blockade (ICB) therapy.

7. The method of claim 6, wherein the composition of the methods further comprises anti- PDL1 (aPDLl) antibody, or the like.

8. The method of any claims of claim 1-7, wherein the cancer is ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, or prostate cancer.

9. The method of claim 1, wherein the cancer is pancreatic cancer such as pancreatic ductal adenocarcinoma.

10. The method of claim 9, wherein the method applies to malignant cancer comprising ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma cancer, lung cancer, liver cancer, or bladder cancer.

11. A method of treating cancer in a subject comprising administering to the subject a composition comprising an anti-cancer agent and microbubbles suspension a human equivalent dose (HED) range of 2x108to 4x109bubbles per Kg body weight; applying ultrasound to induce the microbubble destruction in a targeted region with a setting of continuous non-thermal pulses with short cycle and sufficient time interval wherein MI value is 0.4 to 1.2.

12. The method of claim 11, wherein the subject is a mammal.

13. The method of claim 11, the subject is a human.

14. The method of claim 11, wherein the anti-cancer agent comprises doxorubicin, gemcitabine, gemcitabine plus nab-paclitaxel, 5-FU. irinotecan, folinic. oxaliplatin. FOLLFIRINOX (5-FU, irinotecan, folinic, oxaliplatin) or liposome formulated anticancer agent such as Onivyde® (irinotecan liposome injection), or the like.

15. The method of claim 14, wherein the anti-cancer agent is doxorubicin, gemcitabine, Onivyde® (irinotecan liposome injection), or combinations thereof.

16. The method of any claims of claim 1 1-15, wherein the method further comprises an immune checkpoint blockade (ICB) therapy.

17. The method of claim 16, wherein the composition of the methods further comprises anti- PDL1 (aPDLl) antibody, or the like.

18. The method of any claims of claim 11-17, wherein the cancer is ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, or prostate cancer.

19. The method of claim 18, wherein the cancer is pancreatic cancer such as pancreatic ductal adenocarcinoma.

20. The method of claim 11, wherein the method applies to malignant cancer comprising ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma cancer, lung cancer, liver cancer, or bladder cancer.

21. A method of enhancing efficacy of a cancer treatment in a subject comprising administering to the subject a composition comprising an anti-cancer agent and a microbubble suspension, wherein the microbubble suspension has a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subject's body weight; and applying ultrasound to induce microbubble destruction in a targeted region by continuous nonthermal pulses with short cycle and sufficient time interval, wherein the MI value is 0.4 to 1.2.

22. The method of claim 21, wherein the subject is a mammal.

23. The method of claim 21. wherein the subject is a human.

24. The method of claim 21, wherein the anti-cancer agent comprises doxorubicin, gemcitabine, gemcitabine plus nab-paclitaxel, 5-FU, irinotecan, folinic, oxaliplatin, FOLLFIRINOX (5-FU, irinotecan, folinic, oxaliplatin) or liposome formulated anticancer agent such as irinotecan liposome injection, or the like.

25. The method of claim 24, wherein the anti-cancer agent is doxorubicin, gemcitabine, Onivyde® (irinotecan liposome injection), or combinations thereof.

26. The method of any claims of claim 21-25, wherein the method further comprises an immune checkpoint blockade (ICB) therapy.

27. The method of claim 26, wherein the composition of the methods further comprises anti- PDL1 (aPDLl) antibody, or the like.

28. The method of any claims of claim 21-27, wherein the cancer is ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, or prostate cancer.

29. The method of claim 21, wherein the cancer is pancreatic cancer such as pancreatic ductal adenocarcinoma.

30. The method of claim 21, wherein the method applies to malignant cancer comprising ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma cancer, lung cancer, liver cancer, or bladder cancer.

31. A method of treating cancer in a subject comprising administering to the subject a composition comprising an anti-cancer agent and a microbubble suspension, wherein the microbubble suspension has a gas concentration range of 1.24 pL / kg to 30.57 pL / kg of the subject’s body weight; and applying ultrasound to induce microbubble destruction in a targeted region by continuous non- thermal pulses with short cycle and sufficient time interval, wherein the MI value is 0.4 to 1.2.

32. The method of claim 31, wherein the anti-cancer agent is doxorubicin, gemcitabine, Onivyde® (irinotecan liposome injection), or combinations thereof.

33. The method of any claims of claim 31, wherein the method further comprises an immune checkpoint blockade (ICB) therapy.

34. The method of claim 31, wherein the composition of the methods further comprises anti- PDL1 (aPDLl) antibody, or the like.

35. The method of claim 31, wherein the cancer is ovarian cancer, colorectal cancer, gastric (stomach) cancer, pancreatic cancer, mesothelioma, appendiceal cancer, breast cancer, renal cell carcinoma, lung cancer, liver cancer, bladder cancer, or prostate cancer.

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