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Localized drug delivery and uptake can benefit from the combined action of ultrasound and microbubbles at a specific site. Some of the possible mechanisms suggested are vessel poration and/or cell poration, but the exact acoustic parameters that trigger those phenomena remain unknown. Ex vivo machine perfusion of human-sized organs is a technique that provides an ideal environment for pre-clinical investigations with high physiologic relevance not possible with in vitro experiments. In this work, ex vivo machine-perfused pig livers were combined with an image-guided therapy system to investigate microvascular flow changes caused by the interaction of ultrasound-driven microbubbles with the vasculature. The effects of acoustic pressure (1.7-4 MPa peak negative pressures) and number of cycles (1000 or 20 cycles) were examined. Perfusion changes caused by the action of ultrasound on microbubbles in the microcirculation were qualitatively and quantitatively assessed with contrast-enhanced ultrasound and used as a metric of the extent of vessel perforation, thus, extravasation. Areas that were exposed to peak negative pressures above 1.7 MPa underwent a detectable and irreversible perfusion change. Complete devascularization of the area exposed to ultrasound was observed at much larger acoustic pressures ( 4 MPa). Shorter acoustic pulses (20 cycles) produced markedly fewer perfusion changes than longer pulses (1000 cycles) under the same acoustic amplitude exposure. 42 2676 2686 2676-2686

Immunogenic cell death (ICD) is an intriguing concept within cancer immunotherapy. Indeed, by inducing a specific means of cellular demise, a protective immune response can be triggered against the dying cells. As such, the dying cancer cells are—in a way—turned into a vaccine, attracting immune cells to the site of cell death, and activating them to trigger an anticancer immune response. Since the early discovery of ICD, more mechanistic insights into the hallmarks of ICD have been unraveled and several state-of-the-art cancer therapies have been identified as bona fide ICD inducers (1, 2).One of the cancer therapies which could be a potential candidate to induce ICD is high-intensity focused ultrasound (HIFU) (3). HIFU is currently used as a way of debulking solid tumors in a non-invasive, image-controlled and precise way without the use of ionizing radiation. Depending on the ultrasound parameters, tumor tissue can be ablated either in a mechanical (tissue disruption) or thermal way (heating and coagulative necrosis). Interestingly, there are some indications that HIFU may also affect the immune system: experiments in mice and man have shown attraction/activation of immune cells to/in the tumor site (4, 5). Of note: these immunostimulating effects were reported to be more prominent for mechanical HIFU than thermal HIFU. However, neither the mechanisms behind these observations, nor the possible link to the induction of ICD have been investigated. Therefore, the key aim of work is to investigate the potential of HIFU to induce ICD. Using a custom-designed HIFU set-up, we were able to—in a precise, automated and controlled way—expose B16F0 melanoma cells to HIFU in vitro. The HIFU-exposed tumor cells were subsequently evaluated for the occurrence of apoptosis and ICD hallmarks. More specifically, we evaluated the exposure of calreticulin (CRT) on the cell membrane and release of high mobility group box 1 (HMGB-1) and adenosine triphosphate (ATP). Our data show that application of mechanical HIFU protocols (using short, repeated ultrasound bursts) induce a significant fraction of apoptotic cells (as measured by a loss of mitochondrial membrane potential) within 4h after treatment. Importantly, already within the first hour after HIFU exposure, high levels of the ICD hallmark and Toll-like receptor 4 agonist HMGB-1 were found in the supernatant of the HIFU-treated cells, compared to untreated controls. To determine the effect of HIFU on ATP release, a quinacrine assay was performed to stain intracellular ATP-containing vesicles. Using flow cytometry, we observed that HIFU triggered exocytosis of these vesicles, with 2- to 3-fold reductions in quinacrine fluorescence at 4h and 24h after HIFU exposure, respectively. In addition, at 24h after HIFU exposure, >30% of the cells exposed CRT on the outer leaflet of the cell membrane, which was a drastic increase compared to blanks (

Outcomes of beta cell islet transplantation at subcutaneous sites have been disappointing due to hypoxia-induced oxidative stress by poor vascularization. However, subcutaneous engraftment still has advantages like the simple surgical procedure under local anesthesia and the possibility to biopsy when needed1. Therefore, it is important to have non-invasive imaging techniques available to monitor the exact location and fate of transplanted islets. Subcutaneously transplanted islets offer the unique opportunity to perform in vivo optical imaging, which is often limited by the short penetration depth of light when using intraportal transplantations. For validation purposes, 19F MRI using perfluorocarbon based particles can be used, which draws attention in the field of cellular imaging2 thanks to the lack of background signal and possibility for quantification of transplanted cells. These perfluorocarbon particles could easily be tacked with a fluorescent dye to be also detected by fluorescence imaging. To perform BLI, islets/cells need to be transduced by viral vectors. The present work deals with the use of multimodal in vivo imaging (BLI / Fluorescence /19F MRI) to image and monitor the transplanted pancreatic islets in mice. By combining different imaging techniques, we want to overcome the limitations in sensitivity, resolution and specificity of the individual method. Materials and methods Perfluorocarbon (PFCE) particle and lentiviral vector (LV): Cationic PFCE particles were prepared according to previous report3 with 3.5mol% red DiD fluorescent dye (particle concentration: 4.5*109 particles/ml, particle mean size: ±0.513μm). Lentiviral vector carrying the hEFlα promoter to drive triple flag tagged firefly luciferase (3flagFluc) and a puromycin resistance gene (PuroR) was used for cell/islet transduction4. Cell line and pancreatic islets isolation: Tumor insulin producing cells (INS-1E) were used for uptake validation as well as pancreatic islets isolated from Wistar rats (female, 8-10 weeks) using the collagenase digestion method. In-vitro studies: Phantoms containing PFCE labeled cells/pancreatic islets filled with 2% agarose were made for performing 19F MRI. PFCE labeled or LV transfected cells/islets were placed in 24 well plates for either fluorescent imaging or BLI. Animal model: C57Bl6 mice (male, 8-10 weeks) were subcutaneously injection of either 200 PFCE labeled/LV transfected pancreatic islets or one million PFCE labeled/LV transfected cells at one side with another 200μl PBS injected at the other side as negative control. Microscopy and Confocal imaging: OLYMPUS CKX41 Microscope (Tokyo, Japan) was used for cell/pancreatic islets visualization. Confocal images were also acquired using a Nikon A1R Eclipse Ti confocal microscope (Tokyo, Japan). NMR spectroscopy: NMR experiments were carried out using a 400 MHz Bruker Avance II NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) with the following acquisition parameters: 350 ppm spectral width, 8 s relaxation delay, 128/2048 number of acquisitions and 64k data points. Optical imaging: Both fluorescence images and BLI images were acquired with an IVIS Spectrum system (PerkinElmer, Waltham, USA). For fluorescence imaging, epi-fluorescence illumination mode (FOV = 13.4 cm, Excitation peak = 648 nm, Emission peak = 670 nm) was used. D-luciferin (126 mg/kg) was administered intravenously after the cell/islets transplantation for BLI. MRI experiments: Both in vitro and in vivo MRI were performed using 9.4T Biospec small animal MRI system (Bruker, Ettlingen, Germany) equipped with a home-made surface coil tuneable and matchable to both 1H MRI and 19F MRI with same setups. RARE sequence was used for both in vitro and in vivo studies with some parameters modifications (RARE factor= 8/18; FOV= 6.4 cm*6.4 cm/8 cm*4 cm; TE= 15.9 ms; TR= 6 s (1H)/1 s (19F); slice thickness= 0.5 mm(1H)/2.5 mm(19F); Number of average= 2(1H)/1000(19F)). Results The pancreatic islets/INS-1E cells were incubated with PFCE particles/lentiviral vector and then further used for in vitro and in vivo studies. Viability essays confirmed that PFCE particle and lentiviral vector did not affect cells/pancreatic islets’ function and morphology. Microscopy images show both INS-1E cells and pancreatic islets have excellent uptake of PFCE particles after 24 hours’ incubation. Confocal image confirms the internalization of the PFCE particles by some pancreatic cells. NMR results further confirm and quantify the uptake, which is around 4*1012 19F atoms/cell and 1*1015 19F atoms/pancreatic islet. In vitro BLI shows both INS-1E cells and pancreatic islets could be transduced successfully after a 48 hours’ transduction. Phantoms containing labeled cells or pancreatic islets confirm good visualization by 19F MRI (approx. 200 labeled islets) within an hour acquisition. After subcutaneous transplantation, cells/ islets could be detected and monitored in vivo by all three different imaging modalities, resulting in the following detectability thresholds: approx. 100 islets for 19F MRI and 50 islets for both BLI and fluorescent imaging. Initial follow up results of PFCE labeled islets show the fluorescent signal could be tracked for over two weeks, while the 19F MRI signal can be sustained for over 3 weeks. Conclusion and Discussion By using subcutaneous islet transplantation, we were able to cross-validate their location and quantity using a tri-modal in vivo imaging platform. All three different modalities could provide direct or indirect information regarding the number of engrafted pancreatic islets. Future work will focus on the follow up after the transplantation of islets in diabetic animals using all three imaging modalities. The pancreatic islet’s tri-modal imaging has the potential to overcome the limitations in sensitivity, resolution and specificity of the individual methods. References 1.Sakata N et al., Diabetes Metab Res Rev 30(1), 1-10(2014); 2. Ahrens ET et al., Nature Biotechnology 23, 983-987(2005); 3.Dewitte H et al., Journal of Controlled Release 169, 141-149(2013); 4.Wolfs E et al., Plos One 9, issue 4(2014). ispartof: Molecular Imaging And Biology vol:20 issue:6 pages:940-951 ispartof: location:United States status: published

Localized drug delivery and uptake can benefit from the combined action of ultrasound and microbubbles at a specific site. Some of the possible mechanisms suggested are vessel poration and/or cell poration, but the exact acoustic parameters that trigger those phenomena remain unknown. Ex vivo machine perfusion of human-sized organs is a technique that provides an ideal environment for pre-clinical investigations with high physiologic relevance not possible with in vitro experiments. In this work, ex vivo machine-perfused pig livers were combined with an image-guided therapy system to investigate microvascular flow changes caused by the interaction of ultrasound-driven microbubbles with the vasculature. The effects of acoustic pressure (1.7–4 MPa peak negative pressures) and number of cycles (1000 or 20 cycles) were examined. Perfusion changes caused by the action of ultrasound on microbubbles in the microcirculation were qualitatively and quantitatively assessed with contrast-enhanced ultrasound and used as a metric of the extent of vessel perforation, thus, extravasation. Areas that were exposed to peak negative pressures above 1.7 MPa underwent a detectable and irreversible perfusion change. Complete devascularization of the area exposed to ultrasound was observed at much larger acoustic pressures ( 4 MPa). Shorter acoustic pulses (20 cycles) produced markedly fewer perfusion changes than longer pulses (1000 cycles) under the same acoustic amplitude exposure. 42 2676 2686 2676-2686

Targeted drug delivery under image guidance is gaining more interest in the drug-delivery field. The use of microbubbles as contrast agents in diagnostic ultrasound provides new opportunities in noninvasive image-guided drug delivery. In the present study, the imaging and therapeutic properties of novel doxorubicin liposome-loaded microbubbles are evaluated. The results showed that at scanning settings (1.7 MHz and mechanical index 0.2), these microbubbles scatter sufficient signal for nonlinear ultrasound imaging and can thus be imaged in real time and be tracked in vivo. In vitro therapeutic evaluation showed that ultrasound at 1 MHz and pressures up to 600 kPa in combination with the doxorubicin liposomeloaded microbubbles induced 4-fold decrease of cell viability compared with treatment with free doxorubicin or doxorubicin liposome-loaded microbubbles alone. The therapeutic effectiveness is correlated to an ultrasound-triggered release of doxorubicin from the liposomes and an enhanced uptake of the free doxorubicin by glioblastoma cells. The results obtained demonstrate that the combination of ultrasound and the doxorubicin liposome-loaded microbubbles can provide a new method of noninvasive image-guided drug delivery. ABSTRACT FROM AUTHOR] Copyright of IEEE Transactions on Ultrasonics Ferroelectrics & Frequency Control is the property of IEEE and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) 60 78

Cancer is a genetic disease, caused by mutations in the genome of normal cells. Chemical and physical damage to the cellular genome can induce these mutations resulting in the transformation of a healthy cell into a tumor cell. Over the past years, researchers have acquired a basic understanding of tumor onset. Several important proto-oncogenes and proteins involved in angiogenesis have been identified, leading to the development and clinical use of several new anticancer agents. We succeeded in preparing microbubbles which are able to carry genetic (pDNA, siRNA) and chemotherapeutic (doxorubicin) drugs. We proved that these microbubbles can selectively deliver their genetic or chemotherapeutic drugs to melanoma cells upon ultrasound exposure. We also showed that the activity of currently available drug carriers (pDNA or siRNA lipoplexes and doxorubicin-liposomes) was significantly improved by microbubble coupling and ultrasound exposure. This enables us to obtain a space and time controlled drug delivery guided by ultrasound. The fact that ultrasound and microbubbles have already been approved for ultrasound contrast imaging and are currently used in daily clinic, makes it very plausible that the microbubble concept reported in this thesis may find its way as an advanced drug delivery system.

Cancer immunotherapy, where a patient's immune system is harnessed in the battle against a tumor, is currently on the rise. One possible approach comprises the in vitro modulation of dendritic cells with tumor antigens, after which the cells are injected as a therapeutic cancer vaccine. Despite extensive progress and promising (pre)clinical results, such cancer vaccines often remain patient-specific, laborious in production and therefore very expensive. In this thesis, these issues are addressed by developing imageable biomaterials as cancer immunotherapeutics. Firstly, we highlight the use of antigen-loaded perfluorocarbon particles for 19F MRI tracking of dendritic cells is explored. Such imageable cellular vaccines could provide information on the fate and migratory potential of the cells after their injection, and therefore give important initial feedback on the vaccine efficacy. Secondly, we investigated an alternative strategy that makes use of mRNA-loaded microbubbles for ultrasound-guided and ultrasound-triggered immune activation. We demonstrated that this principle could indeed be used to induce antigen expression in dendritic cells in vitro. Moreover, by simultaneously delivering both antigen mRNA and adjuvant mRNA (TriMix), highly immunogenic dendritic cell vaccines could be produced, allowing complete tumor regression in 30% of the vaccinated animals, which were protected against tumor recurrence. Interestingly, these mRNA-loaded microbubbles show potential for the immediate in vivo delivery of mRNA to dendritic cells as they spontaneously migrate to the lymph nodes upon subcutaneous injection. What is more, the image feedback information provided by these contrast agents on the lymphatic anatomy underpins their interesting role as immunotheranostic agents.