Inhibition of the membrane repair protein annexin-A2 prevents tumor invasion and metastasis.

Cancer cells are exposed to major compressive and shearing forces during invasion and metastasis, leading to extensive plasma membrane damage. To survive this mechanical stress, they need to repair membrane injury efficiently. Targeting the membrane repair machinery is thus potentially a new way to prevent invasion and metastasis. We show here that annexin-A2 (ANXA2) is required for membrane repair in invasive breast and pancreatic cancer cells. Mechanistically, we show by fluorescence and electron microscopy that cells fail to reseal shear-stress damaged membrane when ANXA2 is silenced or the protein is inhibited with neutralizing antibody. Silencing of ANXA2 has no effect on proliferation in vitro, and may even accelerate migration in wound healing assays, but reduces tumor cell dissemination in both mice and zebrafish. We expect that inhibiting membrane repair will be particularly effective in aggressive, poor prognosis tumors because they rely on the membrane repair machinery to survive membrane damage during tumor invasion and metastasis. This could be achieved either with anti-ANXA2 antibodies, which have been shown to inhibit metastasis of breast and pancreatic cancer cells, or with small molecule drugs.

Radiosensitisation by olaparib through focused ultrasound delivery in a diffuse midline glioma model.

BACKGROUND AND PURPOSE: Diffuse midline glioma H3K27-altered (DMG) is an aggressive, inoperable, predominantly paediatric brain tumour. Treatment strategies are limited, resulting in a median survival of only 11 months. Currently, radiotherapy (RT), often combined with temozolomide, is considered the standard of care but remains palliative, highlighting the urgency for new therapies. Radiosensitisation by olaparib, an inhibitor of PARP1 and subsequently PAR-synthesis, is a promising treatment option. We assessed whether PARP1 inhibition enhances radiosensitivity in vitro and in vivo following focused ultrasound mediated blood-brain barrier opening (FUS-BBBO). METHODS: Effects of PARP1 inhibition were evaluated in vitro using viability, clonogenic, and neurosphere assays. In vivo olaparib extravasation and pharmacokinetic profiling following FUS-BBBO was measured by LC-MS/MS. Survival benefit of FUS-BBBO combined with olaparib and RT was assessed using a patient-derived xenograft (PDX) DMG mouse model. RESULTS: Treatment with olaparib in combination with radiation delayed tumour cell proliferation in vitro through the reduction of PAR. Prolonged exposure of low olaparib concentration was more efficient in delaying cell growth than short exposure of high concentration. FUS-BBBO increased olaparib bioavailability in the pons by 5.36-fold without observable adverse effects. A Cmax of 54.09 µM in blood and 1.39 µM in the pontine region was achieved following administration of 100 mg/kg olaparib. Although RT combined with FUS-BBBO mediated olaparib extravasation delayed local tumour growth, survival benefits were not observed in an in vivo DMG PDX model. CONCLUSIONS: Olaparib effectively radiosensitises DMG cells in vitro and reduces primary tumour growth in vivo when combined with RT. Further studies are needed to investigate the therapeutic benefit of olaparib in suitable preclinical PDX models.

Dynamic Fluorescence Microscopy of Cellular Uptake of Intercalating Model Drugs by Ultrasound-Activated Microbubbles.

PURPOSE: The combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs. PROCEDURES: SYTOX Green, an intercalating fluorescent dye that displays >500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line. RESULTS: Ultrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 × 10-3 to 2.2 × 10-3 s-1 for 1 and 20 µM, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells. CONCLUSIONS: These data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line.

Correction for photobleaching in dynamic fluorescence microscopy: application in the assessment of pharmacokinetic parameters in ultrasound-mediated drug delivery.

We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates. To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally. Photobleaching rates up to 5.0 10(-3) s(-1) were measured when applying power densities up to 0.2 W.cm(-2). By applying the three-compartment model, the model drug uptake rate of 6.0 10(-3) s(-1) was measured independent of the applied laser power. The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization.