How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Dynamic phenotypic heterogeneity and the evolution of multiple RNA subtypes in Hepatocellular Carcinoma: the PLANET study

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for Hepatocellular Carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across TNM stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types

Anti-angiogenic alternatives to VEGF blockade

Angiogenesis is a major requirement for tumour formation and development. Anti-angiogenic treatments aim to starve the tumour of nutrients and oxygen and also guard against metastasis. The main anti-angiogenic agents to date have focused on blocking the pro-angiogenic vascular endothelial growth factors (VEGFs). While this approach has seen some success and has provided a proof of principle that such anti-angiogenic agents can be used as treatment, the overall outcome of VEGF blockade has been somewhat disappointing. There is a current need for new strategies in inhibiting tumour angiogenesis; this article will review current and historical examples in blocking various membrane receptors and components of the extracellular matrix important in angiogenesis. Targeting these newly discovered pro-angiogenic proteins could provide novel strategies for cancer therapy

Single-site sonoporation disrupts actin cytoskeleton organization

Sonoporation is based upon an ultrasound-microbubble cavitation routine that physically punctures the plasma membrane on a transient basis. Over such process, the actin cytoskeleton may be disrupted in tandem since this network of subcellular filaments is physically interconnected with the plasma membrane. Here, by performing confocal fluorescence imaging of single-site sonoporation episodes induced by ultrasound-triggered collapse of a single targeted microbubble, we directly observed immediate rupturing of filamentary actin (F-actin) at the sonoporation site (cell type: ZR-75-30; ultrasound frequency: 1 MHz; peak negative pressure: 0.45 MPa; pulse duration: 30 cycles; bubble diameter: 2-4 m). Also, through conducting a structure tensor analysis, we observed further disassembly of the F-actin network over the next 60 min after the onset of sonoporation. The extent of F-actin disruption was found to be more substantial in cells with higher uptake of sonoporation tracer. Commensurate with this process, cytoplasmic accumulation of globular actin (G-actin) was evident in sonoporated cells, and in turn the G:F-actin ratio was increased in a trend similar to drug-induced (cytochalasin D) actin depolymerization. These results demonstrate that sonoporation is not solely a membrane-level phenomenon: organization of the actin cytoskeleton is concomitantly perturbed

Cellular and subcellular impact of low-intensity ultrasound: stimulus or stress

The therapeutic applicability of ultrasound is perhaps well demonstrated with the advent of High-Intensity Focused Ultrasound (HIFU) that works by rapidly heating tissues to induce ablation. Ultrasound also holds tremendous therapeutic potential at low intensities that are near or below the clinical diagnosis limit (720 mW/cm2 spatial-peak time-averaged intensity, as set forth by FDA), presumably acting through a mechanical interaction pathway. However, unlike HIFU where the biological outcome is typically instant cell death, low-intensity ultrasound may induce numerous short-term and long-term outcomes that may be suppressive or proliferative depending on the acoustic exposure parameters. In order to unravel various therapeutic effects that may be induced by low-intensity ultrasound, it is essential to pursue cellular-level investigations across different cell types and under various acoustic settings. In this talk, we shall present the latest findings from a series of low-intensity ultrasound biophysics studies conducted by our research group. First to be discussed is our work on using ultrasound as a repulsive cue for modulating neuronal development dynamics in-vitro. By monitoring neuronal cell morphology in real-time during pulsed ultrasound exposure, we observed that they would undergo neurite retraction and cell body shrinkage. Such an ultrasound-neuron interaction was found to be mediated by a mechanotransduction mechanism, as determined from our ion-channel blockage experiments and confocal microscopy observations. Similar morphological findings were also obtained for other cell types including fibroblasts and stem cells. Interestingly, we discovered that, after the end of ultrasound exposure, proliferation of the treated cells was significantly enhanced. This suggests that transient low-intensity ultrasound exposure is after all non-destructive and may stimulate cellular growth in the long run. The wave-matter interactions of low-intensity ultrasound become more complex (yet more intriguing) when microbubbles are introduced as agents to induce acoustic cavitation. Temporary membrane perforation may be readily achieved in this case (often referred to as sonoporation), and it has been tipped as an emerging paradigm for drug/gene delivery. However, we discovered that this way of permeating the cellular membrane would inadvertently pose stress to living cells even after membrane resealing has occurred. In particular, sonoporated cells were found to exhibit membrane shrinkage and intracellular lipid accumulation. Also, as compared to normal cells, their DNA synthesis kinetics was found to be significantly lengthened, and the onset of cell-cycle arrest was evident. In some instances, programmed cell death (i.e. apoptosis) may even take place. This prompts the need to refine and optimize sonoporation for drug/gene delivery purposes in order to maintain cellular viability. On the other hand, this may represent another way of inducing cell death in contrast to HIFU-based thermal ablation.link_to_OA_fulltex