Noninvasive Estimation of Tumor Interstitial Fluid Pressure from Subharmonic Scattering of Ultrasound Contrast Microbubbles.

The noninvasive estimation of interstitial fluid pressure (IFP) using ultrasound contrast agent (UCA) microbubbles as pressure sensors will provide tumor treatments and efficacy assessments with a promising tool. This study aimed to verify the efficacy of the optimal acoustic pressure in vitro in the prediction of tumor IFPs based on UCA microbubbles' subharmonic scattering. A customized ultrasound scanner was used to generate subharmonic signals from microbubbles' nonlinear oscillations, and the optimal acoustic pressure was determined in vitro when the subharmonic amplitude reached the most sensitive to hydrostatic pressure changes. This optimal acoustic pressure was then applied to predict IFPs in tumor-bearing mouse models, which were further compared with the reference IFPs measured using a standard tissue fluid pressure monitor. An inverse linear relationship and good correlation (r = -0.853, p < 0.001) existed between the subharmonic amplitude and tumor IFPs at the optimal acoustic pressure of 555 kPa, and pressure sensitivity was 1.019 dB/mmHg. No statistical differences were found between the pressures measured by the standard device and those estimated via the subharmonic amplitude, as confirmed by cross-validation (mean absolute errors from 2.00 to 3.09 mmHg, p > 0.05). Our findings demonstrated that in vitro optimized acoustic parameters for UCA microbubbles' subharmonic scattering can be applied for the noninvasive estimation of tumor IFPs.

Mechanosensitive channel MscL induces non-apoptotic cell death and its suppression of tumor growth by ultrasound.

Mechanosensitive channel of large conductance (MscL) is the most thoroughly studied mechanosensitive channel in prokaryotes. Owing to its small molecular weight, clear mechanical gating mechanism, and nanopore forming ability upon opening, accumulating studies are implemented in regulating cell function by activating mechanosensitive channel of large conductance in mammalian cells. This study aimed to investigate the potentials of mechanosensitive channel of large conductance as a nanomedicine and a mechano-inducer in non-small cell lung cancer (NSCLC) A549 cells from the view of molecular pathways and acoustics. The stable cytoplasmic vacuolization model about NSCLC A549 cells was established via the targeted expression of modified mechanosensitive channel of large conductance channels in different subcellular organelles. Subsequent morphological changes in cellular component and expression levels of cell death markers are analyzed by confocal imaging and western blots. The permeability of mitochondrial inner membrane (MIM) exhibited a vital role in cytoplasmic vacuolization formation. Furthermore, mechanosensitive channel of large conductance channel can be activated by low intensity focused ultrasound (LIFU) in A549 cells, and the suppression of A549 tumors in vivo was achieved by LIFU with sound pressure as low as 0.053 MPa. These findings provide insights into the mechanisms underlying non-apoptotic cell death, and validate the nanochannel-based non-invasive ultrasonic strategy for cancer therapy.

Deep learning for the diagnosis of suspicious thyroid nodules based on multimodal ultrasound images.

Objectives: This study aimed to differentially diagnose thyroid nodules (TNs) of Thyroid Imaging Reporting and Data System (TI-RADS) 3-5 categories using a deep learning (DL) model based on multimodal ultrasound (US) images and explore its auxiliary role for radiologists with varying degrees of experience. Methods: Preoperative multimodal US images of 1,138 TNs of TI-RADS 3-5 categories were randomly divided into a training set (n = 728), a validation set (n = 182), and a test set (n = 228) in a 4:1:1.25 ratio. Grayscale US (GSU), color Doppler flow imaging (CDFI), strain elastography (SE), and region of interest mask (Mask) images were acquired in both transverse and longitudinal sections, all of which were confirmed by pathology. In this study, fivefold cross-validation was used to evaluate the performance of the proposed DL model. The diagnostic performance of the mature DL model and radiologists in the test set was compared, and whether DL could assist radiologists in improving diagnostic performance was verified. Specificity, sensitivity, accuracy, positive predictive value, negative predictive value, and area under the receiver operating characteristics curves (AUC) were obtained. Results: The AUCs of DL in the differentiation of TNs were 0.858 based on (GSU + SE), 0.909 based on (GSU + CDFI), 0.906 based on (GSU + CDFI + SE), and 0.881 based (GSU + Mask), which were superior to that of 0.825-based single GSU (p = 0.014, p< 0.001, p< 0.001, and p = 0.002, respectively). The highest AUC of 0.928 was achieved by DL based on (G + C + E + M)US, the highest specificity of 89.5% was achieved by (G + C + E)US, and the highest accuracy of 86.2% and sensitivity of 86.9% were achieved by DL based on (G + C + M)US. With DL assistance, the AUC of junior radiologists increased from 0.720 to 0.796 (p< 0.001), which was slightly higher than that of senior radiologists without DL assistance (0.796 vs. 0.794, p > 0.05). Senior radiologists with DL assistance exhibited higher accuracy and comparable AUC than that of DL based on GSU (83.4% vs. 78.9%, p = 0.041; 0.822 vs. 0.825, p = 0.512). However, the AUC of DL based on multimodal US images was significantly higher than that based on visual diagnosis by radiologists (p< 0.05). Conclusion: The DL models based on multimodal US images showed exceptional performance in the differential diagnosis of suspicious TNs, effectively increased the diagnostic efficacy of TN evaluations by junior radiologists, and provided an objective assessment for the clinical and surgical management phases that follow.

Biomimetic nanoparticles directly remodel immunosuppressive microenvironment for boosting glioblastoma immunotherapy.

Glioblastoma (GBM), as a very aggressive cancer of central nervous system, is very challenging to completely cure by the conventional combination of surgical resection with radiotherapy and chemotherapy. The success of emerging immunotherapy in hot tumors has attracted considerable interest for the treatment of GBM, but the unique tumor immunosuppressive microenvironment (TIME) of GBM leads to the failure of immunotherapy. Here, we show the significant improvement of the immunotherapy efficacy of GBM by modulating the TIME through novel all-in-one biomimetic nanoparticles (i.e. CS-I/J@CM NPs). The nanoparticles consist of utrasmall Cu2-x Se nanoparticles (NPs) with outstanding intrinsic properties (e.g., photo-responsive Fenton-like catalytic property for inducing immunogenic cell death (ICD) and alleviating the hypoxia of tumor), indoximod (IND, an inhibitor of indoleamine-2,3-dioxygenease in tumor), JQ1 (an inhibitor for reducing the expression of PD-L1 by tumor cells), and tumor cell membrane for improving the targeting capability and accumulation of nanoparticles in tumor. We reveal that these smart CS-I/J@CM NPs could drastically activate the immune responses through remodeling TIME of GBM by multiple functions. They could (1) increase M1-phenotype macrophages at tumor site by promoting the polarization of tumor-associated macrophages through the reactive oxygen species (ROS) and oxygen generated from the Fenton-like reaction between nanoparticles and H2O2 within tumor under NIR II irradiation; (2) decrease the infiltration of Tregs cells at tumor site through the release of IND; (3) decrease the expression of PD-L1 on tumor cells through JQ1. The notable increments of anti-tumor CD8+T cells in the tumor and memory T cells (TEM) in the spleen show excellent therapy efficacy and effectively prevent the recurrence of GBM after modulation of the TIME. This work demonstrates the modulation of TIME could be a significant strategy to improve the immunotherapy of GBM and other cold tumors.

Inhibiting autophagy flux and DNA repair of tumor cells to boost radiotherapy of orthotopic glioblastoma.

Radio-resistance of glioblastoma (GBM) remains a leading cause of radiotherapy failure because of the protective autophagy induced by X-Ray irradiation and tumor cells' strong capability of repairing damaged DNA. It is of great importance to overcome the radio-resistance for improving the efficacy of radiotherapy. Herein, we report the novel mechanism of core-shell copper selenide coated gold nanoparticles (Au@Cu2-xSe NPs) inhibiting the protective autophagy and DNA repair of tumor cells to drastically boost the radiotherapy efficacy of glioblastoma. We reveal that the core-shell Au@Cu2-xSe NPs can inhibit the autophagy flux by effectively alkalizing lysosomes. They can increase the SQSTM1/p62 protein levels of tumor cells without influencing their mRNA. We also reveal that Au@Cu2-xSe NPs can increase the ubiquitination of DNA repair protein Rad51, and promote the degradation of Rad51 by proteasomes to prevent the DNA repair. The simultaneous inhibition of protective autophagy and DNA repair significantly suppress the growth of orthotopic GBM by using radiotherapy and our novel Au@Cu2-xSe NPs. Our work provides a new insight and paradigm to significantly improve the efficacy of radiotherapy by rationally designing theranostic nano-agents to simultaneously inhibit protective autophagy and DNA repair of tumor cells.

On-Chip Multicolor Photoacoustic Imaging Flow Cytometry.

On-chip imaging flow cytometry has been widely used in cancer biology, immunology, microbiology, and drug discovery. Pure optical imaging combined with flow cytometry to derive chemical, structural, and morphological features of cells provides systematic insights into biological processes. However, due to the high concentration and strong optical attenuation of red blood cells, preprocessing is necessary for optical flow cytometry while dealing with whole blood. In this study, we develop an on-chip photoacoustic imaging flow cytometry (PAIFC), which combines multicolor high-speed photoacoustic microscopy and microfluidics for cell imaging. The device employs a micro-optical scanner to achieve a miniaturized outer size of 30 × 17 × 24 mm3 and ultrafast cross-sectional imaging at a frame rate of 1758 Hz and provides lateral and axial resolutions of 2.2 and 33 µm, respectively. Using a multicolor strategy, PAIFC is able to differentiate cells labeled by external contrast agents, detect melanoma cells with an endogenous contrast in whole blood, and image melanoma cells in blood samples from tumor-bearing mice. The results suggest that PAIFC has sufficient sensitivity and specificity for future cell-on-chip applications.

Quantitative characterization of the colorectal cancer in a rabbit model using high-frequency endoscopic ultrasound.

PURPOSE: Colonoscopy accompanied with biopsy works as the routine endoscopic strategy for the diagnosis of colorectal cancer (CRC) in clinic; however, the colonoscopy is limited to the tissue surface. During the last decades, enabling technologies are emerging to complement with the colonoscopy for better administration of CRC. The conventional low-frequency (<12 MHz) endoscopic ultrasound (EUS) guided fine-needle aspiration (FNA) has been widely used to assess the lesion penetration. With the high-frequency ultrasound transducer (>20 MHz), EUS allows more precise visualization of the colorectal abnormalities. In order to achieve the accurate detection or in situ characterization of the colorectal lesions, the EUS diagnosis needs more patho-physiological related information in the micro-structural or molecular level. Quantitative ultrasound (QUS) technique, which could extract the micro-structural information from the ultrasound radio-frequency (RF) signal, is promising for the non-invasive tissue characterization. To date, the knowledge of the high-frequency endoscopic QUS for the CRC characterization has not been fully determined. METHODS: In this work, to our best knowledge, it is the first application of the QUS technique based on a customized high-frequency EUS system (30.5 MHz center frequency) to characterize the colorectal malignancies in a VX2 rabbit CRC model. To eliminate the response from the ultrasound electronic system and transducer, the ultrasound signals from colon tissue were calibrated. And, the resulting quasi-liner ultrasound spectra were fit by the linear regression test. As a result, three spectral parameters, including the slope (k), intercept (I) and Midband Fit (M), were obtained from the best-fit line. The three spectral parameters were compared between the malignant tissue regions and adjacent normal tissue regions of the colon tissue specimen ex vivo. The independent t-test was conducted between the three parameters from the normal and malignant group. The statistical method of Fisher Linear Discriminant (FLD) was used to explore the linear combinations of the three parameters, so as to provide more tissue micro-structural features than the single parameter alone. The three FLD values were derived from three different combinations among k, I and M. The threshold was selected from the statistical analysis to optimize the differentiation criteria between the malignant and the normal tissues. The color-coded images were used to display the local FLD values and combined with the EUS B-mode image. RESULTS AND CONCLUSIONS: The mean Midband Fit (M) and intercept (I) showed significant differences between the malignant and normal tissue regions. The statistical analysis showed that there were significant differences in all the mean FLD values of the spectral parameter combinations (kI, kM and IM) (t test, P < 0.05). And, the combined image result from the B-mode image and color-coded image could visually correlate with the histology result. In conclusion, the high-frequency endoscopic QUS technique was potential to be used as a complementary method to distinguish the colorectal malignancies by leveraging its morphological and micro-structural ultrasound information.

Low-intensity pulsed ultrasound ameliorates depression-like behaviors in a rat model of chronic unpredictable stress.

INTRODUCTION: There is an unmet need for better nonpharmaceutical treatments for depression. Low-intensity pulsed ultrasound (LIPUS) is a novel type of neuromodulation that could be helpful for depressed patients. OBJECTIVE: The goal of this study was to investigate the feasibility and potential mechanisms of LIPUS in the treatment of depression. METHODS: Chronic unpredictable stress (CUS) was used to generate rats with depression-like features that were treated with four weeks of LIPUS stimulation of the ventromedial prefrontal cortex. Depression-like behaviors were assessed with the sucrose preference, forced swim, and open field tests. BDNF/mTORC1 signaling was examined by Western blot to investigate this potential molecular mechanism. The safety of LIPUS was evaluated using hematoxylin-eosin and Nissl staining. RESULTS: Four weeks of LIPUS stimulation significantly increased sucrose preference and reduced forced swim immobility time in CUS rats. LIPUS also partially reversed the molecular effects of CUS that included decreased levels of BDNF, phosphorylated tyrosine receptor kinase B (TrkB), extracellular signal-regulated kinase (ERK), mammalian target of rapamycin complex 1 (mTORC1), and S6 kinase (S6K). Moreover, histological staining revealed no gross tissue damage. CONCLUSIONS: Chronic LIPUS stimulation can effectively and safely improve depression-like behaviors in CUS rats. The underlying mechanisms may be related to enhancement of BDNF/ERK/mTORC1 signaling pathways in the prefrontal cortex (PFC). LIPUS is a promising noninvasive neuromodulation tool that merits further study as a potential treatment for depression.

In vivo transrectal imaging of canine prostate with a sensitive and compact handheld transrectal array photoacoustic probe for early diagnosis of prostate cancer.

In this study, we built a novel handheld array photoacoustic probe by integrating multiple optical components with a transrectal ultrasound (TRUS) transducer array. The optical components deliver laser energy and TRUS is used for photoacoustic signal detection. Compared to the previously reported probe, the new photoacoustic probe utilizes an optimized light illumination scheme to enhance the utilization-efficiency of the laser energy, thus improving the imaging sensitivity of the probe. In addition, the new probe is compact and easy to handle for clinicians. We validated the use of this photoacoustic probe for prostate cancer imaging through both phantom studies and in vivo canine model study, which mimics the prostate cancer conditions. The results showed that the probe is suitable for clinical use and can be used in the clinics for several potential clinical applications, including early diagnosis of prostate cancer, targeted image-guided biopsy, and image-guided intra-operative procedure.

Second near-infrared photodynamic therapy and chemotherapy of orthotopic malignant glioblastoma with ultra-small Cu2-xSe nanoparticles.

The treatment of malignant glioblastoma is a huge challenge due to the existence of the blood-brain barrier. Herein, we report the treatment of orthotopic malignant glioblastoma with imaging guided second near-infrared (NIR-II) photodynamic therapy and chemotherapy by using drug-loaded ultra-small Cu2-xSe theranostic nanoparticles (NPs). Ultra-small Cu2-xSe NPs possess a strong absorbance in the NIR-II window, and their absorption at 1064 nm is around 2 times that at 808 nm. Their strong NIR-II absorbance and the deeper-tissue penetration of NIR-II light ensure excellent photodynamic therapy performance under irradiation with a 1064 nm laser. We also demonstrate that ultra-small Cu2-xSe NPs can produce vast amounts of reactive oxygen species via electron transfer (for ˙OH generation) and energy transfer (for 1O2 generation) mechanisms under irradiation. In addition, these NPs can be effectively and locally transported into orthotopic malignant glioblastoma with the assistance of focused ultrasound. The deposited Cu2-xSe NPs can be used for photoacoustic imaging to guide the combined NIR-II photodynamic therapy and chemotherapy. The results show that the tumor growth can be significantly suppressed. This work demonstrates the great potential of drug-loaded ultra-small Cu2-xSe NPs as a promising therapeutic agent for the treatment of orthotopic malignant glioblastoma.

Low-intensity ultrasound suppresses low-Mg2+-induced epileptiform discharges in juvenile mouse hippocampal slices.

OBJECTIVE: It has been shown that low-intensity ultrasound (LIUS) can suppress seizures in some laboratory studies. However, the mechanism of the suppression effect of LIUS remains unclear. The goal of this study is to investigate the modulation effects of focused LIUS on epileptiform discharges in mouse hippocampal slices as well as the underlying mechanism. APPROACH: Epileptiform discharges in hippocampal slices of 8 d-old mice were induced by low-Mg2+ artificial cerebrospinal fluid and recorded by a micro-electrode array in vitro. LIUS was delivered to hippocampal slices to investigate its modulation effects on epileptiform discharges. Pharmacological experiments were conducted to study the mechanism of the modulation effects. MAIN RESULTS: LIUS suppressed the amplitude, rate and duration of ictal discharges. For inter-ictal discharges, LIUS suppressed the amplitude but facilitated the rate. LIUS suppressed the spontaneous spiking activities of pyramidal neurons in CA3, and the suppression effect was eliminated by Kaliotoxin. The suppression effect of LIUS on epileptiform discharges was weakened when the perfusion was mixed with Kaliotoxin. SIGNIFICANCE: Those findings demonstrate that LIUS suppresses the epileptiform discharges in 8 d-old mouse hippocampal slices and that its suppression effect can mainly attributed to the activation of mechanosensitive Kv1.1 channels.

Evaluation of Brain Tumor in Small Animals Using Plane Wave-Based Power Doppler Imaging.

Precisely evaluating the characteristics of a glioma tumor in vivo is challenging when performing surgical resection clinically. The infiltration characteristics of a tumor make precise resection difficult because of uncertainties about the surrounding vasculature and the relationships with functional structures. Magnetic resonance imaging is routinely used to distinguish the area of a glioma, but it cannot resolve details of the vascular network around or inside the tumor. Ultrasound imaging is a real-time imaging modality that has been applied clinically in intra-operative surgery, and the sensitivity of flow measurements in the brain is improved by ultrafast plane wave imaging. This study applies a plane wave-based power Doppler imaging method to visualize the blood flow distribution in glioma models in vivo. This new imaging method makes it possible to delineate the flow structure of a glioma tumor in the brain of a small animal. The tumor can be distinguished from normal brain tissue, and different sections of the tumor contain different flow structures. The normalized blood flow intensities (mean ± standard deviation) within regions of interest were 0.33 ± 0.13, 0.72 ± 0.15, 0.36 ± 0.23 and 0.06 ± 0.07 for the type I normal rat, type I glioma rat, type II normal rat and type II glioma rat, respectively. Quantification analysis verified the feasibility of using this plane wave-based Doppler imaging method to evaluate brain tumors in small animals.

A Delayed-Excitation Data Acquisition Method for High-Frequency Ultrasound Imaging.

High-frequency ultrasound imaging (at >20 MHz) has gained widespread attention due to its high spatial resolution being useful for basic cardiovascular and cancer research involving small animals. The sampling rate of the analog-to-digital converter in a high-frequency ultrasound system usually needs to be higher than 120 MHz in order to satisfy the Nyquist sampling-rate requirement. However, the sampling rate is typically within the range of 40-60 MHz in a traditional ultrasound system, and so we propose a delayed-excitation method for performing high-frequency ultrasound imaging with a traditional data acquisition scheme. In this method, the transmitted pulse is delayed by a certain time period so that the ultrasound echo data are aligned into high-sampling-rate slots. Wire and tissue-mimicking phantoms were imaged to evaluate the performance of the proposed method, whereas a porcine small-intestine specimen and an excised rabbit eyeball were used for in vitro imaging evaluations. The test results demonstrate that the proposed method allows high-frequency ultrasound imaging to be implemented using a traditional ultrasound sampling system.

Simulation Study of an Ultrasound Retinal Prosthesis With a Novel Contact-Lens Array for Noninvasive Retinal Stimulation.

Millions of people around the world suffer from varying degrees of vision loss (including complete blindness) because of retinal degenerative diseases. Artificial retinal prosthesis, which is usually based on electrical neurostimulation, is the most advanced technology for different types of retinal degeneration. However, this technology involves placing a device into the eyeball, and such a highly invasive procedure is inevitably highly risk and expensive. Ultrasound has been demonstrated to be a promising technology for noninvasive neurostimulation, making it possible to stimulate the retina and induce action potentials similar to those elicited by light stimulation. However, the technology of ultrasound retinal stimulation still requires considerable developments before it could be applied clinically. This paper proposes a novel contact-lens array transducer for use in an ultrasound retinal prosthesis (USRP). The transducer was designed in the shape of a contact lens so as to facilitate acoustic coupling with the eye liquid. The key parameters of the ultrasound transducer were simulated, and results are presented that indicate the achievement of 2-D pattern generation and that the proposed contact-lens array is suitable for multiple-focus neurostimulation, and can be used in a USRP.

Computer-assisted assessment of ultrasound real-time elastography: initial experience in 145 breast lesions.

PURPOSE: To develop and evaluate a computer-assisted method of quantifying five-point elasticity scoring system based on ultrasound real-time elastography (RTE), for classifying benign and malignant breast lesions, with pathologic results as the reference standard. MATERIALS AND METHODS: Conventional ultrasonography (US) and RTE images of 145 breast lesions (67 malignant, 78 benign) were performed in this study. Each lesion was automatically contoured on the B-mode image by the level set method and mapped on the RTE image. The relative elasticity value of each pixel was reconstructed and classified into hard or soft by the fuzzy c-means clustering method. According to the hardness degree inside lesion and its surrounding tissue, the elasticity score of the RTE image was computed in an automatic way. Visual assessments of the radiologists were used for comparing the diagnostic performance. Histopathologic examination was used as the reference standard. The Student's t test and receiver operating characteristic (ROC) curve analysis were performed for statistical analysis. RESULTS: Considering score 4 or higher as test positive for malignancy, the diagnostic accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 93.8% (136/145), 92.5% (62/67), 94.9% (74/78), 93.9% (62/66), and 93.7% (74/79) for the computer-assisted scheme, and 89.7% (130/145), 85.1% (57/67), 93.6% (73/78), 92.0% (57/62), and 88.0% (73/83) for manual assessment. Area under ROC curve (Az value) for the proposed method was higher than the Az value for visual assessment (0.96 vs. 0.93). CONCLUSION: Computer-assisted quantification of classical five-point scoring system can significantly eliminate the interobserver variability and thereby improve the diagnostic confidence of classifying the breast lesions to avoid unnecessary biopsy.

Scanning-mode 2D acoustic radiation force impulse (s2D-ARFI) imaging based on GPU acceleration.

Acoustic radiation force impulse (ARFI) technique is a quantitative method for tissue stiffness assessment. It has been proved to be less operator dependent than the quasi-static elastography, and has more simple hardware architecture than the supersonic shearwave imaging (SSI) technique, which make it easier to be miniaturized for some special clinical applications. However, unlike the SSI, ARFI cannot provide real-time 2D images of tissue stiffness distribution mainly due to its data-intensive and time-consuming algorithms. In this study, the algorithms of ARFI were modified and improved to fit for the parallel computation on graphics processing unit (GPU), and the quasi-real-time scanning-mode 2D ARFI images (s2D-ARFI) were implemented on a self-developed compact system. High ratio of the time consumptions between the algorithms using CPU and using GPU has been verified, and it was also proved that there was no distinct difference between the stiffness images obtained by these two methods. The s2D-ARFI provides us an additional choice for quantitatively imaging the tissue stiffness, and has a potential to be miniaturized and used in the emergency treatments in field first-aid and the donor evaluation for organ transplantation.