Selective inhibition of HDAC6 promotes bladder cancer radiosensitization and mitigates the radiation-induced CXCL1 signalling.

BACKGROUND: Although trimodality therapy resecting tumours followed by chemoradiotherapy is emerged for muscle-invasive bladder cancer (MIBC), chemotherapy produces toxicities. Histone deacetylase inhibitors have been identified as an effective strategy to enhance cancer radiotherapy (RT). METHODS: We examined the role of HDAC6 and specific inhibition of HDAC6 on BC radiosensitivity by performing transcriptomic analysis and mechanism study. RESULTS: HDAC6 knockdown or HDAC6 inhibitor (HDAC6i) tubacin exerted a radiosensitizing effect, including decreased clonogenic survival, increased H3K9ac and α-tubulin acetylation, and accumulated γH2AX, which are similar to the effect of panobinostat, a pan-HDACi, on irradiated BC cells. Transcriptomics of shHDAC6-transduced T24 under irradiation showed that shHDAC6 counteracted RT-induced mRNA expression of CXCL1, SERPINE1, SDC1 and SDC2, which are linked to cell migration, angiogenesis and metastasis. Moreover, tubacin significantly suppressed RT-induced CXCL1 and radiation-enhanced invasion/migration, whereas panobinostat elevated RT-induced CXCL1 expression and invasion/migration abilities. This phenotype was significantly abrogated by anti-CXCL1 antibody, indicating the key regulator of CXCL1 contributing to BC malignancy. Immunohistochemical evaluation of tumours from urothelial carcinoma patients supported the correlation between high CXCL1 expression and reduced survival. CONCLUSION: Unlike pan-HDACi, the selective HDAC6i can enhance BC radiosensitization and effectively inhibit RT-induced oncogenic CXCL1-Snail-signalling, thus further advancing its therapeutic potential with RT.

Ultrasound-guided therapeutic modulation of hepatocellular carcinoma using complementary microRNAs.

Treatment options for patients with hepatocellular carcinoma (HCC) are limited, in particular in advanced and drug resistant HCC. MicroRNAs (miRNA) are non-coding small RNAs that are emerging as novel drugs for the treatment of cancer. The aim of this study was to assess treatment effects of two complementary miRNAs (sense miRNA-122, and antisense antimiR-21) encapsulated in biodegradable poly (lactic-co-glycolic acid) nanoparticles (PLGA-NP), administered by an ultrasound-guided and microbubble-enhanced delivery approach in doxorubicin-resistant and non-resistant human HCC xenografts. Proliferation and invasiveness of human HCC cells after miRNA-122/antimiR-21 and doxorubicin treatment were assessed in vitro. Confocal microscopy and qRT-PCR were used to visualize and quantitate successful intracellular miRNA-loaded PLGA-NP delivery. Up and down-regulation of miRNA downstream targets and multidrug resistance proteins and extent of apoptosis were assessed in vivo in treated human HCC xenografts in mice. Compared to single miRNA therapy, combination therapy with the two complementary miRNAs resulted in significantly (P<0.05) stronger decrease in cell proliferation, invasion, and migration of HCC cells as well as higher resensitization to doxorubicin. Ultrasound-guided delivery significantly increased in vivo miRNA-loaded PLGA-NP delivery in human HCC xenografts compared to control conditions by 5-9 fold (P<0.001). miRNA-loaded PLGA-NP were internalized in HCC cells and anti-apoptotic proteins were down regulated with apoptosis in ~27% of the tumor volume of doxorubicin-resistant human HCC after a single treatment with complementary miRNAs and doxorubicin. Thus, ultrasound-guided delivery of complementary miRNAs is highly efficient in the treatment of doxorubicin- resistant and non-resistant HCC. Further development of this new treatment approach could aid in better treatment of patients with HCC.

Sonoporation: Applications for Cancer Therapy.

Therapeutic efficacy of both traditional chemotherapy and gene therapy in cancer is highly dependent on the ability to deliver drugs across natural barriers, such as the vessel wall or tumor cell membranes. In this regard, sonoporation induced by ultrasound-guided microbubble (USMB) destruction has been widely investigated in the enhancement of therapeutic drug delivery given it can help overcome these natural barriers, thereby increasing drug delivery into cancer. In this chapter we discuss challenges in current cancer therapy and how some of these challenges could be overcome using USMB-mediated drug delivery. We particularly focus on recent advances in delivery approaches that have been developed to further improve therapeutic efficiency and specificity of various cancer treatments. An example of clinical translation of USMB-mediated drug delivery is also shown.

Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer.

Ultrasound induced microbubble cavitation can cause enhanced permeability across natural barriers of tumors such as vessel walls or cellular membranes, allowing for enhanced therapeutic delivery into the target tissues. While enhanced delivery of small (<1nm) molecules has been shown at acoustic pressures below 1MPa both in vitro and in vivo, the delivery efficiency of larger (>100nm) therapeutic carriers into cancer remains unclear and may require a higher pressure for sufficient delivery. Enhanced delivery of larger therapeutic carriers such as FDA approved pegylated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NP) has significant clinical value because these nanoparticles have been shown to protect encapsulated drugs from degradation in the blood circulation and allow for slow and prolonged release of encapsulated drugs at the target location. In this study, various acoustic parameters were investigated to facilitate the successful delivery of two nanocarriers, a fluorescent semiconducting polymer model drug nanoparticle as well as PLGA-PEG-NP into human colon cancer xenografts in mice. We first measured the cavitation dose produced by various acoustic parameters (pressure, pulse length, and pulse repetition frequency) and microbubble concentration in a tissue mimicking phantom. Next, in vivo studies were performed to evaluate the penetration depth of nanocarriers using various acoustic pressures, ranging between 1.7 and 6.9MPa. Finally, a therapeutic microRNA, miR-122, was loaded into PLGA-PEG-NP and the amount of delivered miR-122 was assessed using quantitative RT-PCR. Our results show that acoustic pressures had the strongest effect on cavitation. An increase of the pressure from 0.8 to 6.9MPa resulted in a nearly 50-fold increase in cavitation in phantom experiments. In vivo, as the pressures increased from 1.7 to 6.9MPa, the amount of nanoparticles deposited in cancer xenografts was increased from 4- to 14-fold, and the median penetration depth of extravasated nanoparticles was increased from 1.3-fold to 3-fold, compared to control conditions without ultrasound, as examined on 3D confocal microscopy. When delivering miR-122 loaded PLGA-PEG-NP using optimal acoustic settings with minimum tissue damage, miR-122 delivery into tumors with ultrasound and microbubbles was 7.9-fold higher compared to treatment without ultrasound. This study demonstrates that ultrasound induced microbubble cavitation can be a useful tool for delivery of therapeutic miR loaded nanocarriers into cancer in vivo.

Imaging feedback for histotripsy by characterizing dynamics of acoustic radiation force impulse (ARFI)-induced shear waves excited in a treated volume.

Our previous study indicated that shear waves decay and propagate at a lower speed as they propagate into a tissue volume mechanically fractionated by histotripsy. In this paper, we hypothesize that the change in the shear dynamics is related to the degree of tissue fractionation, and can be used to predict histotripsy treatment outcomes. To test this hypothesis, lesions with different degrees of tissue fractionation were created in agar-graphite tissue phantoms and ex vivo kidneys with increasing numbers of therapy pulses, from 0 to 2000 pulses per treatment location. The therapy pulses were 3-cycle 750-kHz focused ultrasound delivered at a peak negative/positive pressure of 17/108 MPa and a repetition rate of 50 Hz. The shear waves were excited by acoustic radiation force impulse (ARFI) focused at the center of the lesion. The spatial and temporal behavior of the propagating shear waves was measured with ultrasound plane wave imaging. The temporal displacement profile at a lateral location 10 mm offset to the shear excitation region was detected with M-mode imaging. The decay and delay of the shear waves were quantitatively characterized on the temporal displacement profile. Results showed significant changes in two characteristics on the temporal displacement profile: the peak-to-peak displacement decayed exponentially with increasing numbers of therapy pulses; the relative time-to-peak displacement increased with increasing numbers of therapy pulses, and appeared to saturate at higher numbers of pulses. Correspondingly, the degree of tissues fractionation, as indicated by the percentage of structurally intact cell nuclei, decreased exponentially with increasing numbers of therapy pulses. Strong linear correlations were found between the two characteristics and the degree of tissue fractionation. These results suggest that the characteristics of the shear temporal displacement profile may provide useful feedback information regarding the treatment outcomes.

Histotripsy beyond the intrinsic cavitation threshold using very short ultrasound pulses: microtripsy.

Histotripsy produces tissue fractionation through dense energetic bubble clouds generated by short, high-pressure, ultrasound pulses. Conventional histotripsy treatments have used longer pulses from 3 to 10 cycles, wherein the lesion-producing bubble cloud generation depends on the pressure-release scattering of very high peak positive shock fronts from previously initiated, sparsely distributed bubbles (the shock-scattering mechanism). In our recent work, the peak negative pressure (P-) for generation of dense bubble clouds directly by a single negative half cycle, the intrinsic threshold, was measured. In this paper, the dense bubble clouds and resulting lesions (in red blood cell phantoms and canine tissues) generated by these supra-intrinsic threshold pulses were studied. A 32-element, PZT-8, 500-kHz therapy transducer was used to generate very short (<2 cycles) histotripsy pulses at a pulse repetition frequency (PRF) of 1 Hz and P- from 24.5 to 80.7 MPa. The results showed that the spatial extent of the histotripsy-induced lesions increased as the applied P- increased, and the sizes of these lesions corresponded well to the estimates of the focal regions above the intrinsic cavitation threshold, at least in the lower pressure regime (P- = 26 to 35 MPa). The average sizes for the smallest reproducible lesions were approximately 0.9 × 1.7 mm (lateral × axial), significantly smaller than the -6-dB beamwidth of the transducer (1.8 × 4.0 mm). These results suggest that, using the intrinsic threshold mechanism, well-confined and microscopic lesions can be precisely generated and their spatial extent can be estimated based on the fraction of the focal region exceeding the intrinsic cavitation threshold. Because the supra-threshold portion of the negative half cycle can be precisely controlled, lesions considerably less than a wavelength are easily produced, hence the term microtripsy.

Acoustic and photoacoustic molecular imaging of cancer.

Ultrasound and combined optical and ultrasonic (photoacoustic) molecular imaging have shown great promise in the visualization and monitoring of cancer through imaging of vascular and extravascular molecular targets. Contrast-enhanced ultrasound with molecularly targeted microbubbles can detect early-stage cancer through the visualization of targets expressed on the angiogenic vasculature of tumors. Ultrasonic molecular imaging can be extended to the imaging of extravascular targets through use of nanoscale, phase-change droplets and photoacoustic imaging, which provides further molecular information on cancer given by the chemical composition of tissues and by targeted nanoparticles that can interact with extravascular tissues at the receptor level. A new generation of targeted contrast agents goes beyond merely increasing imaging signal at the site of target expression but shows activatable and differential contrast depending on their interactions with the tumor microenvironment. These innovations may further improve our ability to detect and characterize tumors. In this review, recent developments in acoustic and photoacoustic molecular imaging of cancer are discussed.

Imaging feedback of histotripsy treatments using ultrasound shear wave elastography.

Histotripsy is a cavitation-based ultrasound therapy that mechanically fractionates soft solid tissues into fluid-like homogenates. This paper investigates the feasibility of imaging the tissue elasticity change during the histotripsy process as a tool to provide feedback for the treatments. The treatments were performed on agar tissue phantoms and ex vivo kidneys using 3-cycle ultrasound pulses delivered by a 750-kHz therapeutic array at peak negative/positive pressure of 17/108 MPa and a repetition rate of 50 Hz. Lesions with different degrees of damage were created with increasing numbers of therapy pulses from 0 to 2000 pulses per treatment location. The elasticity of the lesions was measured with ultrasound shear wave elastography, in which a quasi-planar shear wave was induced by acoustic radiation force generated by the therapeutic array, and tracked with ultrasound imaging at 3000 frames per second. Based on the shear wave velocity calculated from the sequentially captured frames, the Young's modulus was reconstructed. Results showed that the lesions were more easily identified on the shear wave velocity images than on B-mode images. As the number of therapy pulses increased from 0 to 2000 pulses/location, the Young's modulus decreased exponentially from 22.1 ± 2.7 to 2.1 ± 1.1 kPa in the tissue phantoms (R2 = 0.99, N = 9 each), and from 33.0 ± 7.1 to 4.0 ± 2.5 kPa in the ex vivo kidneys (R2 = 0.99, N = 8 each). Correspondingly, the tissues transformed from completely intact to completely fractionated as examined via histology. A good correlation existed between the lesions' Young's modulus and the degree of tissue fractionation as examined with the percentage of remaining structurally intact cell nuclei (R2 = 0.91, N = 8 each). These results indicate that lesions produced by histotripsy can be detected with high sensitivity using shear wave elastography. Because the decrease in the tissue elasticity corresponded well with the morphological and histological change, this study provides a basis for predicting the local treatment outcomes from tissue elasticity change.

Activation of the Notch1/STAT3/Twist signaling axis promotes gastric cancer progression.

Gastric carcinoma is one of the most common malignancies and a lethal cancer in the world. Notch signaling and transcription factors STAT3 (signal transducer and activator of transcription 3) and Twist regulate tumor development and are critical regulators of gastric cancer progression. Herein, the relationship among Notch, STAT3 and Twist pathways in the control of gastric cancer progression was studied. We found that Twist and phosphorylated STAT3 levels were promoted by the activated Notch1 receptor in human stomach adenocarcinoma SC-M1, embryonic kidney HEK293 and erythroleukemia K562 cells. Notch1 signaling dramatically induced Twist promoter activity through a C promoter binding factor-1-independent manner and STAT3 phosphorylation. Overexpression of Notch1 receptor intracellular domain (N1IC) enhanced the interaction between nuclear STAT3 and Twist promoter in cells. Gastric cancer progression of SC-M1 cells was promoted by N1IC through STAT3 phosphorylation and Twist expression including colony formation, migration and invasion. STAT3 regulated gastric cancer progression of SC-M1 cells via Twist. N1IC also elevated the progression of other gastric cancer cells such as AGS and KATO III cells through STAT3 and Twist. The N1IC-promoted tumor growth and lung metastasis of SC-M1 cells in mice were suppressed by the STAT3 inhibitor JSI-124 and Twist knockdown. Furthermore, Notch1 and Notch ligand Jagged1 expressions were significantly associated with phosphorylated STAT3 and Twist levels in gastric cancer tissues of patients. Taken together, these results suggest that Notch1/STAT3/Twist signaling axis is involved in progression of human gastric cancer and modulation of this cascade has potential for the targeted combination therapy.

An efficient treatment strategy for histotripsy by removing cavitation memory.

Cavitation memory effects occur when remnants of cavitation bubbles (nuclei) persist in the host medium and act as seeds for subsequent events. In pulsed cavitational ultrasound therapy, or histotripsy, this effect may cause cavitation to repeatedly occur at these seeded locations within a target volume, producing inhomogeneous tissue fractionation or requiring an excess number of pulses to completely homogenize the target volume. We hypothesized that by removing the cavitation memory, i.e., the persistent nuclei, the cavitation bubbles could be induced at random locations in response to each pulse; therefore, complete disruption of a tissue volume may be achieved with fewer pulses. To test the hypothesis, the cavitation memory was passively removed by increasing the intervals between successive pulses, ∆t, from 2, 10, 20, 50 and 100, to 200 ms. Histotripsy treatments were performed in red blood cell tissue phantoms and ex vivo livers using 1-MHz ultrasound pulses of 10 cycles at P-/P+ pressure of 21/59 MPa. The phantom study allowed for direct visualization of the cavitation patterns and the lesion development process in real time using high-speed photography; the ex vivo tissue study provided validation of the memory effect in real tissues. Results of the phantom study showed an exponential decrease in the correlation coefficient between cavitation patterns in successive pulses from 0.5 ± 0.1 to 0.1 ± 0.1 as ∆t increased from 2-200 ms; correspondingly, the lesion was completely fractionated with significantly fewer pulses for longer ∆ts. In the tissue study, given the same number of therapy pulses, complete and homogeneous tissue fractionation with well-defined lesion boundaries was achieved only for ∆t ≥ 100 ms. These results indicated that the removal of the cavitation memory resulted in more efficient treatments and homogeneous lesions.

Lesion generation through ribs using histotripsy therapy without aberration correction.

This study investigates the feasibility of using high-intensity pulsed therapeutic ultrasound, or histotripsy, to non-invasively generate lesions through the ribs. Histotripsy therapy mechanically ablates tissue through the generation of a cavitation bubble cloud, which occurs when the focal pressure exceeds a certain threshold. We hypothesize that histotripsy can generate precise lesions through the ribs without aberration correction if the main lobe retains its shape and exceeds the cavitation initiation threshold and the secondary lobes remain below the threshold. To test this hypothesis, a 750-kHz focused transducer was used to generate lesions in tissue-mimicking phantoms with and without the presence of rib aberrators. In all cases, 8000 pulses with 16 to 18 MPa peak rarefactional pressure at a repetition frequency of 100 Hz were applied without aberration correction. Despite the high secondary lobes introduced by the aberrators, high-speed imaging showed that bubble clouds were generated exclusively at the focus, resulting in well-confined lesions with comparable dimensions. Collateral damage from secondary lobes was negligible, caused by single bubbles that failed to form a cloud. These results support our hypothesis, suggesting that histotripsy has a high tolerance for aberrated fields and can generate confined focal lesions through rib obstacles without aberration correction.

Cavitation clouds created by shock scattering from bubbles during histotripsy.

Histotripsy is a therapy that focuses short-duration, high-amplitude pulses of ultrasound to incite a localized cavitation cloud that mechanically breaks down tissue. To investigate the mechanism of cloud formation, high-speed photography was used to observe clouds generated during single histotripsy pulses. Pulses of 5-20 cycles duration were applied to a transparent tissue phantom by a 1-MHz spherically focused transducer. Clouds initiated from single cavitation bubbles that formed during the initial cycles of the pulse, and grew along the acoustic axis opposite the propagation direction. Based on these observations, we hypothesized that clouds form as a result of large negative pressure generated by the backscattering of shockwaves from a single bubble. The positive-pressure phase of the wave inverts upon scattering and superimposes on the incident negative-pressure phase to create this negative pressure and cavitation. The process repeats with each cycle of the incident wave, and the bubble cloud elongates toward the transducer. Finite-amplitude propagation distorts the incident wave such that the peak-positive pressure is much greater than the peak-negative pressure, which exaggerates the effect. The hypothesis was tested with two modified incident waves that maintained negative pressure but reduced the positive pressure amplitude. These waves suppressed cloud formation which supported the hypothesis.

Active focal zone sharpening for high-precision treatment using histotripsy.

The goal of this study is to develop a focal zone sharpening strategy that produces more precise lesions for pulsed cavitational ultrasound therapy, or histotripsy. Precise and well-confined lesions were produced by locally suppressing cavitation in the periphery of the treatment focus without affecting cavitation in the center. The local suppression of cavitation was achieved using cavitation nuclei preconditioning pulses to actively control cavitation in the periphery of the focus. A 1-MHz 513-element therapeutic array was used to generate both the therapy and the nuclei preconditioning pulses. For therapy, 10-cycle bursts at 100-Hz pulse repetition frequency with P-/P+ pressure of 21/76 MPa were delivered to the geometric focus of the therapeutic array. For nuclei preconditioning, a different pulse was delivered to an annular region immediately surrounding the focus before each therapy pulse. A parametric study on the effective pressure, pulse duration, and delivery time of the preconditioning pulse was conducted in red blood cell-gel phantoms, where cavitational damage was indicated by the color change resulting from local cell lysis. Results showed that a short-duration (20 µs) preconditioning pulse at a medium pressure (P-/P+ pressure of 7.2/13.6 MPa) delivered shortly before (30 µs) the therapy pulse substantially suppressed the peripheral damage by 77 ± 13% while complete fractionation in the focal center was maintained. High-speed imaging of the bubble cloud showed a substantial decrease in the maximum width of the bubble cloud by 48 ± 24% using focal zone sharpening. Experiments in ex vivo livers confirmed that highly confined lesions were produced in real tissues as well as in the phantoms. This study demonstrated the feasibility of active focal zone sharpening using cavitation nuclei preconditioning, allowing for increased treatment precision compared with the natural focal width of the therapy transducer.

Examining and analyzing subcellular morphology of renal tissue treated by histotripsy.

Our recent studies have shown that high-intensity pulsed ultrasound can achieve mechanical tissue fragmentation, a process we call histotripsy. Histotripsy has many medical applications where noninvasive tissue removal or significant tissue disruption is needed (e.g., cancer therapy). The primary aim of this study is to investigate tissue regions treated by histotripsy and to characterize the boundary between the treated and untreated zones using transmission electron microscopy (TEM). The nature of the tissue disruption suggests many clinical applications and provides insights on the physical mechanism of histotripsy. Fresh ex vivo porcine kidney tissues were treated using histotripsy. A 1 MHz 100 mm diameter focused transducer was used to deliver 15 cycle histotripsy pulses at a peak negative pressure of 17 MPa and a pulse repetition frequency (PRF) of 100 Hz. Each lesion was produced by a 3 × 3 (lateral) × 4 (axial) grid with 2 mm between adjacent lateral and 3 mm between axial exposure points using mechanical scanning. Two thousand pulses were applied to each exposure point to achieve tissue fragmentation. After treatment, the tissue was processed and examined using TEM. Extensive fragmentation of the tissues treated with histotripsy was achieved. TEM micrographs of the tissue treated by histotripsy, showing no recognizable cellular features and little recognizable subcellular structures, demonstrates the efficacy of this technique in ablating the targeted tissue regions. A boundary, or transition zone, of a few microns separated the affected and unaffected areas, demonstrating the precision of histotripsy tissue targeting. TEM micrographs of the tissue treated by histotripsy showed no discernable cellular structure within the treated region. Histotripsy can minimize fragmentation of the adjoining nontargeted tissues because, as a nonlinear threshold phenomenon, damage can be highly localized. The potential for high lesion precision is evident in the TEM micrographs.

A tissue phantom for visualization and measurement of ultrasound-induced cavitation damage.

Many ultrasound studies involve the use of tissue-mimicking materials to research phenomena in vitro and predict in vivo bioeffects. We have developed a tissue phantom to study cavitation-induced damage to tissue. The phantom consists of red blood cells suspended in an agarose hydrogel. The acoustic and mechanical properties of the gel phantom were found to be similar to soft tissue properties. The phantom's response to cavitation was evaluated using histotripsy. Histotripsy causes breakdown of tissue structures by the generation of controlled cavitation using short, focused, high-intensity ultrasound pulses. Histotripsy lesions were generated in the phantom and kidney tissue using a spherically focused 1-MHz transducer generating 15 cycle pulses, at a pulse repetition frequency of 100 Hz with a peak negative pressure of 14 MPa. Damage appeared clearly as increased optical transparency of the phantom due to rupture of individual red blood cells. The morphology of lesions generated in the phantom was very similar to that generated in kidney tissue at both macroscopic and cellular levels. Additionally, lesions in the phantom could be visualized as hypoechoic regions on a B-mode ultrasound image, similar to histotripsy lesions in tissue. High-speed imaging of the optically transparent phantom was used to show that damage coincides with the presence of cavitation. These results indicate that the phantom can accurately mimic the response of soft tissue to cavitation and provide a useful tool for studying damage induced by acoustic cavitation.

Quantitative ultrasound backscatter for pulsed cavitational ultrasound therapy- histotripsy.

Histotripsy is a well-controlled ultrasonic tissue ablation technology that mechanically and progressively fractionates tissue structures using cavitation. The fractionated tissue volume can be monitored with ultrasound imaging because a significant ultrasound backscatter reduction occurs.This paper correlates the ultrasound backscatter reduction with the degree of tissue fractionation characterized by the percentage of remaining normal-appearing cell nuclei on histology.Different degrees of tissue fractionation were generated in vitro in freshly excised porcine kidneys by varying the number of therapeutic ultrasound pulses from 100 to 2000 pulses per treatment location. All ultrasound pulses were 15 cycles at 1 MHz delivered at 100 Hz pulse repetition frequency and 19 MPa peak negative pressure. The results showed that the normalized backscatter intensity decreased exponentially with increasing number of pulses. Correspondingly, the percentage of normal appearing nuclei in the treated area decreased exponentially as well. A linear correlation existed between the normalized backscatter intensity and the percentage of normal appearing cell nuclei in the treated region. This suggests that the normalized backscatter intensity may be a potential quantitative real-time feedback parameter for histotripsy-induced tissue fractionation. This quantitative feedback may allow the prediction of local clinical outcomes, i.e., when a tissue volume has been sufficiently treated.