A study combining microbubble-mediated focused ultrasound and radiation therapy in the healthy rat brain and a F98 glioma model.

Focused Ultrasound (FUS) has been shown to sensitize tumors outside the brain to Radiotherapy (RT) through increased ceramide-mediated apoptosis. This study investigated the effects of FUS + RT in healthy rodent brains and F98 gliomas. Tumors, or striata in healthy rats, were targeted with microbubble-mediated, pulsed FUS (220 kHz, 102-444 kPa), followed by RT (4, 8, 15 Gy). FUS + RT (8, 15 Gy) resulted in ablative lesions, not observed with FUS or RT only, in healthy tissue. Lesions were visible using Magnetic Resonance Imaging (MRI) within 72 h and persisted until 21 days post-treatment, indicating potential applications in ablative neurosurgery. In F98 tumors, at 8 and 15 Gy, where RT only had significant effects, FUS + RT offered limited improvements. At 4 Gy, where RT had limited effects compared with untreated controls, FUS + RT reduced tumor volumes observed on MRI by 45-57%. However, survival benefits were minimal (controls: 27 days, RT: 27 days, FUS + RT: 28 days). Histological analyses of tumors 72 h after FUS + RT (4 Gy) showed 93% and 396% increases in apoptosis, and 320% and 336% increases in vessel-associated ceramide, compared to FUS and RT only. Preliminary evidence shows that FUS + RT may improve treatment of glioma, but additional studies are required to optimize effect size.

Correlation Between the Peripheral Neuropathy and Levels of hs-CRP, IL-1ß and IL-6 in Senile Parkinson's Disease Patients.

This study was carried out to investigate the correlation between the onset of peripheral neuropathy and levels of hypersensitive C-reactive protein (hs-CRP), interleukin 1ß (IL-1ß) and IL-6 in senile Parkinson's disease (PD) patients. For this purpose, a total of 60 PD patients and 60 age-matched healthy subjects were enrolled in this study and received the assessment for peripheral nerves by using the quantified method. Besides, levels of hs-CRP, IL-1ß and IL-6 in serum were determined to analyze the correlation between the clinical features, including the severity of PD and cognitive decline, and the levels of hs-CRP, IL-1ß and IL-6. Results showed that PD patients had more cases of peripheral neuropathy than those in the healthy control group. Levels of hs-CRP, IL-1ß and IL-6 in the serum of PD patients were much higher than those in the healthy control (P<0.05). Besides, PD patients had lower scores of MMSE and MoCA but higher CNPI scores when compared to the healthy control group. As a result, we found that the severity of peripheral neuropathy was in a positive correlation with the levels of hs-CRP, IL-1ß and IL-6. It was concluded that PD patients generally have peripheral neuropathy that may correlate with the increases in the levels of hs-CRP, IL-1ß and IL-6, and early intervention may mitigate the development and progression of peripheral neuropathy.

Ultrasound-mediated delivery of flexibility-tunable polymer drug conjugates for treating glioblastoma.

Effective chemotherapy delivery for glioblastoma multiforme (GBM) is limited by drug transport across the blood-brain barrier and poor efficacy of single agents. Polymer-drug conjugates can be used to deliver drug combinations with a ratiometric dosing. However, the behaviors and effectiveness of this system have never been well investigated in GBM models. Here, we report flexible conjugates of hyaluronic acid (HA) with camptothecin (CPT) and doxorubicin (DOX) delivered into the brain using focused ultrasound (FUS). In vitro toxicity assays reveal that DOX-CPT exhibited synergistic action against GBM in a ratio-dependent manner when delivered as HA conjugates. FUS is employed to improve penetration of DOX-HA-CPT conjugates into the brain in vivo in a murine GBM model. Small-angle x-ray scattering characterizations of the conjugates show that the DOX:CPT ratio affects the polymer chain flexibility. Conjugates with the highest flexibility yield the highest efficacy in treating mouse GBM in vivo. Our results demonstrate the association of FUS-enhanced delivery of combination chemotherapy and the drug-ratio-dependent flexibility of the HA conjugates. Drug ratio in the polymer nanocomplex may thus be employed as a key factor to modulate FUS drug delivery efficiency via controlling the polymer flexibility. Our characterizations also highlight the significance of understanding the flexibility of drug carriers in ultrasound-mediated drug delivery systems.

Effect of ofloxacin levels on growth, photosynthesis and chlorophyll fluorescence kinetics in tomato.

Antibiotic pollution has become a global environmental pollution problem. Chlorophyll fluorescence is one of the most important indicators reflecting the degree to which plants are influenced by the environment. Ofloxacin (OFL) is a highly toxic antibiotic pollutant, and there are few reports on the effects of changes in OFL levels on tomato chlorophyll fluorescence parameters. In this study, we investigated the responses of tomato growth, photosynthetic activity and chlorophyll fluorescence kinetics to exogenous OFL exposure (as the concentrations of 0, 2.5, 5, 10 and 20 mg L-1). The results showed that lower concentrations of OFL (2.5 mg L-1) had little impact on tomato growth, while plant growth was inhibited with the OFL concentration increasing. At higher OFL concentrations (5, 10 and 20 mg L-1), chloroplasts ruptured, and chlorophyll became degraded, resulting in leaf etiolation. Furthermore, the photosynthetic and photochemical efficiency and electron transfer rate were significantly inhibited by OFL. Moreover, damage to the oxygen-evolving complex on the donor side of PSⅡ prevented electron transfer from QA to QB and led to photoinhibition. In conclusion, higher OFL concentration reduced photosynthesis by destroying the photosynthetic mechanism in tomato, resulting in tomato leaf etiolation and plant growth inhibition.

[Retracted] MicroRNA­488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that several of the panels showing cell invasion assay data in Fig. 3A were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to International Journal of Oncology, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Oncology 53: 1493­1504, 2018; DOI: 10.3892/ijo.2018.4483].

Activation of RARα Receptor Attenuates Neuroinflammation After SAH via Promoting M1-to-M2 Phenotypic Polarization of Microglia and Regulating Mafb/Msr1/PI3K-Akt/NF-κB Pathway.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is a life-threatening subtype of stroke with high rates of mortality. In the early stages of SAH, neuroinflammation is one of the important mechanisms leading to brain injury after SAH. In various central nervous system diseases, activation of RARα receptor has been proven to demonstrate neuroprotective effects. This study aimed to investigate the anti-inflammatory effects of RARα receptor activation after SAH. METHODS: Internal carotid artery puncture method used to established SAH model in Sprague-Dawley rats. The RARα specific agonist Am80 was injected intraperitoneally 1 hour after SAH. AGN196996 (specific RARα inhibitor), Msr1 siRNA and LY294002 (PI3K-Akt inhibitor) were administered via the lateral ventricle before SAH. Evaluation SAH grade, neurological function score, blood-brain barrier permeability. BV2 cells and SH-SY5Y cells were co-cultured and stimulated by oxyhemoglobin to establish an in vitro model of SAH. RT-PCR, Western blotting, and immunofluorescence staining were used to investigate pathway-related proteins, microglia activation and inflammatory response. Results: The expression of RARα, Mafb, and Msr1 increased in rat brain tissue after SAH. Activation of the RARα receptor with Am80 improved neurological deficits and attenuated brain edema, blood brain barrier permeability. Am80 increased the expression of Mafb and Msr1, and reduced neuroinflammation by enhancing the phosphorylation of Akt and by inhibiting the phosphorylation of NF-κB. AGN196996, Msr1 siRNA, and LY294002 reversed the therapeutic effects of Am80 by reducing the expression of Msr1 and the phosphorylation of Akt. In vitro model of SAH, Am80 promoted M1-to-M2 phenotypic polarization in microglia and suppressed the nuclear transcription of NF-κB. CONCLUSION: Activation of the RARα receptor attenuated neuroinflammation by promoting M1-to-M2 phenotypic polarization in microglia and regulating the Mafb/Msr1/PI3K-Akt/NF-κB pathway. RARα might serve as a potential target for SAH therapy.

ACEA Attenuates Oxidative Stress by Promoting Mitophagy via CB1R/Nrf1/PINK1 Pathway after Subarachnoid Hemorrhage in Rats.

METHOD: Endovascular perforation was performed to establish a SAH model of rats. ACEA was administered intraperitoneally 1 h after SAH. The CB1R antagonist AM251 was injected intraperitoneally 1 h before SAH induction. Adenoassociated virus- (AAV-) Nrf1 shRNA was infused into the lateral ventricle 3 weeks before SAH induction. Neurological tests, immunofluorescence, DHE, TUNEL, Nissl staining, transmission electron microscopy (TEM), and Western blot were performed. RESULTS: The expression of CB1R, Nrf1, PINK1, Parkin, and LC3II increased and peaked at 24 h after SAH. ACEA treatment exhibited the antioxidative stress and antiapoptosis effects after SAH. In addition, ACEA treatment increased the expression of Nrf1, PINK1, Parkin, LC3II, and Bcl-xl but repressed the expression of Romo-1, Bax, and cleaved caspase-3. Moreover, the TEM results demonstrated that ACEA promoted the formation of mitophagosome and maintained the normal mitochondrial morphology of neurons. The protective effect of ACEA was reversed by AM251 and Nrf1 shRNA, respectively. CONCLUSIONS: This study demonstrated that ACEA alleviated oxidative stress and neurological dysfunction by promoting mitophagy after SAH, at least in part via the CB1R/Nrf1/PINK1 signaling pathway.

Metformin attenuates early brain injury after subarachnoid hemorrhage in rats via AMPK-dependent mitophagy.

Metformin is the most widely used drug to treat type 2 diabetes and its mitochondrial activity is through activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK plays a dual regulatory role in mito-morphosis, controlling the phosphorylation and activation of dynamin-related protein 1 (DRP1) and mitofusin 2 (MFN2). The aim of this study was to investigate whether metformin could reduce early brain injury (EBI) after subarachnoid hemorrhage (SAH) by activating mitophagy and improving mitochondrial morphology through AMPK. This study used 308 male Sprague-Dawley rats. First, different metformin doses were injected intraperitoneally 30 min post-SAH. The dose that did not significantly alter blood glucose in the rats was selected for subsequent experiments. Before or after sacrificing rats, neurological function, brain water content, and blood-brain barrier (BBB) permeability were measured in each group. Transmission electron microscopy was used to observe the level of mitophagy and mito-morphology in each group. The expression of mitophagic and apoptotic proteins were investigated by immunofluorescence and western blot. Metformin at 20 mg/kg improved neurological function and attenuated brain edema and the disruption of BBB permeability 24 h after SAH. Metformin treatment after SAH promoted mitophagy in an AMPK-dependent manner. In addition to the effects on mitophagy, we also found that metformin alleviated oxidative stress and apoptosis after SAH in an AMPK-dependent manner. Lastly, metformin restored homeostasis between mitochondrial fusion and fission. Metformin attenuated EBI after SAH in rats through AMPK-dependent signaling. These protective effects might be achieved by regulating mitochondrial morphology and promoting mitophagy.

Observed Effects of Whole-Brain Radiation Therapy on Focused Ultrasound Blood-Brain Barrier Disruption.

As focused ultrasound for blood-brain barrier disruption (FUS-BBBD) has progressed to human application, it has become necessary to consider the potential effects of prior irradiation treatments. Using a murine model, we examined the effects of whole-brain irradiation on FUS-BBBD. We first subjected half of the experimental cohort to daily 3-Gy whole-brain irradiation for 10 consecutive days. Then, microbubble-assisted FUS-BBBD was performed unilaterally while the contralateral sides served as unsonicated controls. FUS-BBBD, as evident by measuring the fluorescence yield of extravasated trypan blue dye, was identified at all sites with minimal or no apparent pathology. The peak fluorescence intensity caused by extravasated dye in the sonicated region was 17.5 ± 12.1% higher after radiation and FUS-BBBD than after FUS-BBBD alone, suggesting that prior radiation of the brain may be a sensitizing factor for FUS-BBBD. Radiation alone-without FUS-BBBD-resulted in mild BBB disruption. Hemorrhagic petechiae were observed in 9 of 12 radiated brains, with 77% of them clearly located outside the sonicated area; no petechiae were found in non-irradiated animals. This radiation protocol did not appear to increase the risk for vascular damage associated with FUS-BBBD.

Blood-brain barrier disruption and delivery of irinotecan in a rat model using a clinical transcranial MRI-guided focused ultrasound system.

We investigated controlled blood-brain barrier (BBB) disruption using a low-frequency clinical transcranial MRI-guided focused ultrasound (TcMRgFUS) device and evaluated enhanced delivery of irinotecan chemotherapy to the brain and a rat glioma model. Animals received three weekly sessions of FUS, FUS and 10 mg/kg irinotecan, or irinotecan alone. In each session, four volumetric sonications targeted 36 locations in one hemisphere. With feedback control based on recordings of acoustic emissions, 98% of the sonication targets (1045/1071) reached a pre-defined level of acoustic emission, while the probability of wideband emission (a signature for inertial cavitation) was than 1%. BBB disruption, evaluated by mapping the R1 relaxation rate after administration of an MRI contrast agent, was significantly higher in the sonicated hemisphere (P < 0.01). Histological evaluation found minimal tissue effects. Irinotecan concentrations in the brain were significantly higher (P < 0.001) with BBB disruption, but SN-38 was only detected in <50% of the samples and only with an excessive irinotecan dose. Irinotecan with BBB disruption did not impede tumor growth or increase survival. Overall these results demonstrate safe and controlled BBB disruption with a low-frequency clinical TcMRgFUS device. While irinotecan delivery to the brain was not neurotoxic, it did not improve outcomes in the F98 glioma model.

Identification of functional lncRNAs based on competing endogenous RNA network in osteoblast differentiation.

Adult human mesenchymal stem cells have the potential to differentiate into osteoblast, which plays crucial roles in bone regeneration and repair. Some transcriptional factors (TFs), such as BMP-2 and RUNX2, have been demonstrated to control the differentiation processes. It is important to discover more key regulators in osteoblast differentiation. Recently, some studies found long noncoding RNAs (lncRNAs) participating in osteoblast differentiation, such as MALAT1, DANCR, and ANCR. In this study, we performed a network-based computational analysis to investigate the lncRNA-messenger RNA (mRNA) crosstalks via integrating microRNA (miRNA)-RNA interactions, gene coexpression, and protein-protein interactions. First, multiple topology analyses were performed to osteoblast-differentiation-related lncRNA-mRNA network (ODLMN). Several lncRNAs with central topology structures were identified as key regulators. Results showed that these lncRNAs participated in osteoblast differentiation via phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase, and Ras signals. Previous studies have demonstrated that lncRNAs exert functions by involving in close modules. Second, after performing module searching in ODLMN, two functional modules were identified, which played crucial roles through involving in PI3K/protein kinase B, cyclic adenosine 3',5'-monophosphate, and hypoxia-inducible factor 1 pathways. Third, a subset of core lncRNA-TF crosstalks that might form feedback loops to control the biological processes in osteoblast differentiation was identified. These core lncRNA-TF feedback loops showed more TF binding affinity than other lncRNAs. All these results can help us to uncover the molecular mechanism and provide new targets for bone regeneration and repair.

Acoustic feedback enables safe and reliable carboplatin delivery across the blood-brain barrier with a clinical focused ultrasound system and improves survival in a rat glioma model.

The blood-brain barrier (BBB) restricts delivery of most chemotherapy agents to brain tumors. Here, we investigated a clinical focused ultrasound (FUS) device to disrupt the BBB in rats and enhance carboplatin delivery to the brain using the F98 glioma model. Methods: In each rat, 2-3 volumetric sonications (5 ms bursts at 1.1 Hz for 75s) targeted 18-27 locations in one hemisphere. Sonication was combined with Definity microbubbles (10 µl/kg) and followed by intravenous carboplatin (50 mg/kg). Closed-loop feedback control was performed based on acoustic emissions analysis. Results: Safety and reliability were established in healthy rats after three sessions with carboplatin; BBB disruption was induced in every target without significant damage evident in MRI or histology. In tumor-bearing rats, concentrations of MRI contrast agent (Gadavist) were 1.7 and 3.3 times higher in the tumor center and margin, respectively, than non-sonicated tumors (P<0.001). Tissue-to-plasma ratios of intact carboplatin concentrations were increased by 7.3 and 2.9 times in brain and tumor respectively, at one hour after FUS and 4.2 and 2.4 times at four hours. Tumor volume doubling time in rats receiving FUS and carboplatin increased by 96% and 126% compared to rats that received carboplatin alone and non-sonicated controls, respectively (P<0.05); corresponding increases in median survival were 48% and 66% (P<0.01). Conclusion: Overall, this work demonstrates that actively-controlled BBB disruption with a clinical device can enhance carboplatin delivery without neurotoxicity at level that reduces tumor growth and improves survival in an aggressive and infiltrative rat glioma model.

Intracranial Non-thermal Ablation Mediated by Transcranial Focused Ultrasound and Phase-Shift Nanoemulsions.

High intensity focused ultrasound (HIFU) mechanical ablation is an emerging technique for non-invasive transcranial surgery. Lesions are created by driving inertial cavitation in tissue, which requires significantly less peak pressure and time-averaged power compared with traditional thermal ablation. The utility of mechanical ablation could be extended to the brain provided the pressure threshold for inertial cavitation can be reduced. In this study, the utility of perfluorobutane (PFB)-based phase-shift nanoemulsions (PSNEs) for lowering the inertial cavitation threshold and enabling focal mechanical ablation in the brain was investigated. We successfully achieved vaporization of PFB-based PSNEs at 1.8 MPa with a 740 kHz focused transducer with a pulsed sonication protocol (duty cycle = 1.5%, 10 min sonication) within intact CD-1 mice brains. Evidence is provided showing that a single bolus injection of PSNEs could be used to initiate and sustain inertial cavitation in cerebrovasculature for at least 10 min. Histologic analysis of brain slices after HIFU exposure revealed ischemic and hemorrhagic lesions with dimensions that were comparable to the focal zone of the transducer. These results suggest that PFB-based PSNEs may be used to significantly reduce the inertial cavitation threshold in the cerebrovasculature and, when combined with transcranial focused ultrasound, enable focal intracranial mechanical ablation.

Simultaneous Passive Acoustic Mapping and Magnetic Resonance Thermometry for Monitoring of Cavitation-Enhanced Tumor Ablation in Rabbits Using Focused Ultrasound and Phase-Shift Nanoemulsions.

Thermal ablation of solid tumors via focused ultrasound (FUS) is a non-invasive image-guided alternative to conventional surgical resection. However, the usefulness of the technique is limited in vascularized organs because of convection of heat, resulting in long sonication times and unpredictable thermal lesion formation. Acoustic cavitation has been found to enhance heating but requires use of exogenous nuclei and sufficient acoustic monitoring. In this study, we employed phase-shift nanoemulsions (PSNEs) to promote cavitation and incorporated passive acoustic mapping (PAM) alongside conventional magnetic resonance imaging (MRI) thermometry within the bore of a clinical MRI scanner. Simultaneous PAM and MRI thermometry were performed in an in vivo rabbit tumor model, with and without PSNE to promote cavitation. Vaporization and cavitation of the nanoemulsion could be detected using PAM, which led to accelerated heating, monitored with MRI thermometry. The maximum heating assessed from MRI was well correlated with the integrated acoustic emissions, illustrating cavitation-enhanced heating. Examination of tissue revealed thermal lesions that were larger in the presence of PSNE, in agreement with the thermometry data. Using fixed exposure conditions over 94 sonications in multiple animals revealed an increase in the mean amplitude of acoustic emissions and resulting temperature rise, but with significant variability between sonications, further illustrating the need for real-time monitoring. The results indicate the utility of combined PAM and MRI for monitoring of tumor ablation and provide further evidence for the ability of PSNEs to promote cavitation-enhanced lesioning.

MicroRNA-488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3.

It has been reported that aquaporin 3 (AQP3) expression is associated with the progression of numerous types of cancer and microRNA (miRNA/miR) processing. However, the effects and precise mechanisms of AQP3 in osteosarcoma (OS) have not been fully elucidated. The present study aimed to investigate the interaction between AQP3 and miR­488 in OS. The reverse transcription­quantitative polymerase chain reaction (RT­qPCR) assay was performed to detect the levels of AQP3 and miR­488 in OS tissues and cell lines, respectively. Cell proliferation, invasion and epithelial-mesenchymal transition (EMT) were detected to analyze the biological functions of miR­488 and AQP3 in OS cells. Furthermore, mRNA and protein levels of AQP3 was measured by RT­qPCR and western blot analysis. Furthermore, AQP3 was validated as an miR­488 target using luciferase assays in OS cells. The present study revealed that the miR­488 level was significantly downregulated in OS tissues and cell lines, and that the expression of AQP3 was markedly increased. Notable, the low miR­488 expression level was associated with upregulated AQP3 expression in OS tissues. Furthermore, introduction of miR­488 markedly suppressed the proliferation, invasion and EMT of OS cells. However, miR­488-knockdown increased the proliferation, invasion and EMT of OS cells. The present study demonstrated that miR­488 could directly target AQP3 using bioinformatics analysis and luciferase reporter assays. In addition, AQP3-silencing had similar effects to miR­488 overexpression on OS cells. Overexpression of AQP3 in OS cells partially reversed the inhibitory effects of miR­488 mimic. miR­488 inhibited the proliferation, invasion and EMT of OS cells by directly downregulating AQP3 expression, and miR­488 targeting AQP3 was responsible for inhibition of the proliferation, invasion and EMT of OS cells.

Closed-loop control of targeted ultrasound drug delivery across the blood-brain/tumor barriers in a rat glioma model.

Cavitation-facilitated microbubble-mediated focused ultrasound therapy is a promising method of drug delivery across the blood-brain barrier (BBB) for treating many neurological disorders. Unlike ultrasound thermal therapies, during which magnetic resonance thermometry can serve as a reliable treatment control modality, real-time control of modulated BBB disruption with undetectable vascular damage remains a challenge. Here a closed-loop cavitation controlling paradigm that sustains stable cavitation while suppressing inertial cavitation behavior was designed and validated using a dual-transducer system operating at the clinically relevant ultrasound frequency of 274.3 kHz. Tests in the normal brain and in the F98 glioma model in vivo demonstrated that this controller enables reliable and damage-free delivery of a predetermined amount of the chemotherapeutic drug (liposomal doxorubicin) into the brain. The maximum concentration level of delivered doxorubicin exceeded levels previously shown (using uncontrolled sonication) to induce tumor regression and improve survival in rat glioma. These results confirmed the ability of the controller to modulate the drug delivery dosage within a therapeutically effective range, while improving safety control. It can be readily implemented clinically and potentially applied to other cavitation-enhanced ultrasound therapies.

Effects on P-Glycoprotein Expression after Blood-Brain Barrier Disruption Using Focused Ultrasound and Microbubbles.

Many blood-borne substances attempting to pass through the luminal membrane of brain endothelial cells are acted upon by a variety of metabolizing enzymes or are actively expelled back into the capillary lumen by embedded efflux transporters, such as Permeability-glycoprotein (Pgp). Overexpression of this protein has also been linked to multidrug resistance in cancer cells. Previous studies have shown that focused ultrasound (FUS), when combined with a microbubble agent, has ability to temporarily disrupt blood-brain barrier (BBBD). In this work, we investigated whether modulation of Pgp expression is part of the FUS-induced effects. We found that ultrasound can temporarily suppress Pgp expression. When BBBD was produced at 0.55 MPa, Pgp was suppressed up to 48 hours and restored by 72 hours. At 0.81 MPa, suppression can last 72 hours or longer. These findings support the idea that microbubble-enhanced FUS disrupts the functional components of the BBB through suppression of drug efflux.

Evaluation of permeability, doxorubicin delivery, and drug retention in a rat brain tumor model after ultrasound-induced blood-tumor barrier disruption.

Drug delivery in brain tumors is challenging because of the presence of blood-brain barrier (BBB) and the blood-tumor barrier (BTB). Focused ultrasound (FUS) combined with microbubbles can enhance the permeability of the BTB in brain tumors, as well as disrupting the BBB in the surrounding tissue. In this study, dynamic contrast-enhanced Magnetic Resonance Imaging (DCE-MRI) was used to characterize FUS-induced permeability changes in a rat glioma model and in the normal brain and to investigate the relationship between these changes and the resulting concentration of the chemotherapy agent doxorubicin (DOX). 9L gliosarcoma cells were implanted in both hemispheres in male rats. At day 10-12 after implantation, FUS-induced BTB disruption using 690kHz ultrasound and Definity microbubbles was performed in one of the tumors and in a normal brain region in each animal. After FUS, DOX was administered at a dose of 5.67mg/kg. The resulting DOX concentration was measured via fluorometry at 1 or 24h after FUS. The transfer coefficient Ktrans describing extravasation of the MRI contrast agent Gd-DTPA was significantly increased in both the sonicated tumors and in the normal brain tissue (P<0.001) between the two DCE-MRI acquisitions obtained before and after FUS, while no significant difference was found in the controls (non-sonicated tumor/normal brain tissue). DOX concentrations were also significantly larger than controls in both the sonicated tumors and in the normal tissue volumes at 1 and 24h after sonication. The DOX concentrations were significantly larger (P<0.01) in the control tumors harvested 1h after FUS than in those harvested at 24h, when the tumor concentrations were not significantly different than in the non-sonicated normal brain. In contrast, there was no significant difference in the DOX concentrations between the tumors harvested at 1 and 24h after FUS or in the concentrations measured in the brain at these time points. The transfer coefficient Ktrans for Gd-DTPA and the drug concentrations showed a good linear correlation (R2=0.56). Overall, these data suggest that FUS and microbubbles can not only increase DOX delivery across the BBB and BTB, but that it is retained in the tissue at significantly enhanced levels for at least 24h. Such enhanced retention may increase the potency of this chemotherapy agent and allow for reduced systemic doses. Furthermore, MRI-based estimates of Gd-DTPA transport across these barriers might be useful to estimate local DOX concentrations in the tumor and in the surrounding normal tissue.

Growth inhibition in a brain metastasis model by antibody delivery using focused ultrasound-mediated blood-brain barrier disruption.

HER2-targeting antibodies (i.e. trastuzumab and pertuzumab) prolong survival in HER2-positive breast cancer patients with extracranial metastases. However, the response of brain metastases to these drugs is poor, and it is hypothesized that the blood-brain barrier (BBB) limits drug delivery to the brain. We investigated whether we could improve the response by temporary disruption of the BBB using focused ultrasound in combination with microbubbles. To study this, we inoculated 30 nude rats with HER2-positive cells derived from a brain metastasis of a breast cancer patient (MDA-MB-361). The animals were divided into three groups: a control-group that received no treatment; an antibody-only group that received six weekly treatments of trastuzumab and pertuzumab; and an ultrasound+antibody group that received trastuzumab and pertuzumab in combination with six weekly sessions of BBB disruption using focused ultrasound. In two animals, the leakiness of the tumors before disruption was evaluated using contrast-enhanced T1-weighted magnetic resonance imaging and found that the tumors were not leaky. The same technique was used to evaluate the effectiveness of BBB disruption, which was successful in all sessions. The tumor in the control animals grew exponentially with a growth constant of 0.042±0.011mm(3)/day. None of the antibody-only animals responded to the treatment and the growth constant was 0.033±0.009mm(3)/day during the treatment period. Four of the ten animals in the ultrasound+antibody-group showed a response to the treatment with an average growth constant of 0.010±0.007mm(3)/day, compared to a growth constant 0.043±0.013mm(3)/day for the six non-responders. After the treatment period, the tumors in all groups grew at similar rates. As the tumors were not leaky before BBB disruption and there were no responders in the antibody-only group, these results show that at least in some cases disruption of the BBB is necessary for a response to the antibodies in these brain metastases. Interestingly, only some of the rats responded to the treatment. We did not observe a difference in tumor volume at the start of the treatment, nor in HER2 expression or in contrast-enhancement on MRI between the responders and non-responders to explain this. Better understanding of why certain animals respond is needed and will help in translating this technique to the clinic. In conclusion, we demonstrate that BBB disruption using focused ultrasound in combination with antibody therapy can inhibit growth of breast cancer brain metastasis.

Transcriptome Analysis of WHV/c-myc Transgenic Mice Implicates Cytochrome P450 Enzyme 17A1 as a Promising Biomarker for Hepatocellular Carcinoma.

Early detection of hepatocellular carcinoma (HCC) is critical for successful treatment and favorable prognosis. To identify novel HCC biomarkers, we used the WHV/c-myc transgenic (Tg) mice, an animal model of hepatocarcinogenesis. By analyzing their gene expression profiling, we investigated differentially expressed genes in livers of wild-type and Tg mice. The cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1), a hepatic P450 enzyme, was revealed to be overexpressed in the liver tissues of Tg mice at both preneoplastic and neoplastic stages. Mouse-to-human validation demonstrated that CYP17A1 mRNA and protein were also significantly increased in human HCC tissues compared with paired nontumor tissues (P = 0.00041 and 0.00011, respectively). Immunohistochemical studies showed that CYP17A1 was overexpressed in 67% (58 of 87) of HCC, and strong staining of CYP17A1 was observed in well-differentiated HCCs. Consistent with this, the median serum levels of CYP17A1 were also significantly higher in patients with HCC (140.2 ng/mL, n = 776) compared with healthy controls (31.4 ng/mL, n = 366) and to those with hepatitis B virus (57.5 ng/mL, n = 160), cirrhosis (46.1 ng/mL, n = 147), lung cancer (27.4 ng/mL, n = 109), and prostate cancer (42.1 ng/mL, n = 130; all P < 0.001). Notably, the elevations were seen in most AFP-negative HCC cases. Altogether, through mouse-to-human search and validation, we found that CYP17A1 is overexpressed in HCCs and it has great potentiality as a noninvasive marker for HCC detection. These results provide a rationale for the future development and clinical application of CYP17A1 measurement to diagnose HCC more precisely. Cancer Prev Res; 9(9); 739-49. ©2016 AACR.