Cell biomechanics and metastatic spreading: a study on human breast cancer cells

2010/2011Despite the intensive research of the past decades in oncology, cancer invasion and metastasis still represent the most important problem for treatment and the most common cause of death in cancer patients. Metastasis refers to the spread of malignant cells from a primary tumour to distant sites of the body and the adaptation of these cancer cells to a new and different tissue microenvironment. Usually, millions of cells can be released by a tumour into the circulation every day, but only a tiny minority of these cells are able to reach and colonize a distant organs: the utter inefficiency of the metastatic process implies that cells might strongly need biomechanical alterations that allow them to invade and colonize different tissues. The hypothesis that cellular biomechanics may play a significant role in tumour genesis and cancer invasion, gains every day more and more support: therefore characterizing these properties in connection with the membrane and cytoskeleton organization could be very important for understanding better the migration mechanisms and to develop new diagnostics and therapeutics tools. The goal of our study was the mechanical characterization of cell lines chosen as model of cancer progression using different biophysical techniques and the correlation of the mechanical properties with possible alterations of the cytoskeleton structure and plasma membrane composition. We used a custom built Optical Tweezers to extract the local viscoelastic properties of the cell plasma membrane, an Atomic Force Microscopy (AFM) to locally measure cell elasticity of cells, and a Microfluidic Optical Stretcher to measure the deformability of cells as whole bodies. We investigated then the actin organization of the cytoskeleton by STimulated Depletion and Emission (STED) and confocal microscopy. The lipid composition of cells was analysed by MALDI-mass spectroscopy in order to correlate the mechanical alterations of cells with alteration at the cytoskeleton and plasma membrane level. The cell lines analyzed derive from breast tissue and represent a model of human epithelial cells towards malignancy. In particular, two cell lines -MDA-MB-231 and MCF-7- provided by American Type Culture Collection (ATCC) were originally derived from breast cancers patients with different level of cancer aggressiveness. Cells were chosen according to the nowadays accepted classification of breast cancer based on gene expression pattern and proteomic expression, which divide breast cancers in subtypes that differ in terms of risk factor, distribution, prognosis, therapeutic treatment responsiveness, clinical outcomes and survival. The third cell line, HBL-100, is an immortalized but non-neoplastic cell line derived from the milk of a nursing mother with no evidence of breast lesions, representing a earlier stage of the cell transformation. A pulling membrane tether approach by means of Optical Tweezers has been chosen since it allows an accurate quantitative characterization of local viscoelastic properties of plasma membranes. Bovine Serum Albumine (BSA) coated silica beads were used to bind the plasma membrane and grab membrane tethers of several microns measuring the force exerted on the bead. By fitting with the Kelvin body model our force-elongation curves obtained by experimental data we extracted the parameters of interest: tether stiffness, membrane bending rigidity, and tether viscosity. We observed that lower values of tether stiffness and membrane bending rigidity corresponded to cells associated to a higher aggressive behaviour, while viscosity showed an inverse tendency. We also probed elasticity of the cells using by indentation experiments with AFM. We used a bead probe attached to the cantilever and measured the Young Modulus. The results obtained could not clearly discriminate the three cell types in terms of elasticity. Cell deformability was further investigated by means of Microfluidic Optical Stretcher. Cells in suspension were trapped by two counter propagating laser beams of low intensity from two optical fibers. Adjusting the intensity of the laser light, the forces acting on the cell surface increased, leading to a measurable elongation of the cell body along the laser beam axis. With MOS we were able to discriminate between cancer and control cells lines, while differences between the two cancer cell lines were not significant. However a trend could be observed: lower aggressive tumour cells were more resistant to deformation compared to the higher aggressive tumour cells. We investigated the cells cytoskeleton structure by STED and confocal microscopy confirming that malignancy involves cytoskeleton structure alterations. Differences in the organization. of the actin filaments and in the presence of actin drifts were observed. We peformed also a preliminary analysis of the cell lipid composition by MALDI MASS spectroscopy. We could observe that highly aggressive cells with softer membranes presented alterations at the level of Phosphatidylethanolamines (PEs) and Phosphatidylinositoles (PIs). The work of this thesis is partially published in the article “Custom Built Optical Tweezers for locally probing the viscoelastic properties of cancer cells” in the International Journal of Optomechatronics (June 2011). A second article including the comparative results of the biomechanical analysis on the breast cell lines is in preparation.XXIV Cicl

Well-Being in Alpine Space: How Subjective Determinants Affect Urban and Rural Areas. A Case Study Analysis in South Tyrol, Italy

The paper analyzes urban-rural difference on the individual psychological well-being of residents living in the Autonomous Province of Alto Adige, region on the border between Italy and Austria. Data comes from a cross-sectional survey undertaken in 2010 on a statistical representative sample, based on the PGWBI, an instrument specifically used to measure individual subjective well-being. The study examines the influence of socio-demographic factors, as well as cultural determinants, on the PGWBI. Urban inhabitants were found to perceive higher level of psychological well-being compared to rural ones, while the determinants affecting individual subjective had a greater impact on the rural one

Optical delivery of liposome encapsulated chemical stimuli to neuronal cells

Spatially confined and precise time delivery of neuroactive molecules is an important issue in neurophysiology. In this work we developed a technique for delivering chemical stimuli to cultured neurons consisting in encapsulating the molecules of interest in liposomes. These vectors were then loaded in reservoirs consisting of glass capillaries. The reservoirs were placed in the recording chamber and single liposomes were trapped and transported out by optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses, breaking the liposome membrane. The efficiency of encapsulation and release were first evaluated by loading the liposomes with fluorescein. In order to test the effect of the UV-induced release, liposomes with diameter ranging from 1 to 10 μm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca(2+) dye, were monitored by imaging intracellular Ca(2+). An efficient release from the liposomes was demonstrated by detectable calcium signals, indicating stimulated depolarization of the neuronal cells by KCl. The present technique represents an alternative method for focal chemical stimulation of cultured cells that circumvents some of the limitations of microejection and photorelease of caged compounds

Results of a prospective observational study of autologous peripheral blood mononuclear cell therapy for no-option critical limb-threatening ischemia and severe diabetic foot ulcers

Cell therapy with autologous peripheral blood mononuclear cells (PB-MNCs) may help restore limb perfusion in patients with diabetes mellitus and critical limb-threatening ischemia (CLTI) deemed not eligible for revascularization procedures and consequently at risk for major amputation (no-option). Fundamental is to establish its clinical value and to identify candidates with a greater benefit over time. Assessing the frequency of PB circulating angiogenic cells and extracellular vesicles (EVs) may help in guiding candidate selection

Association between polymorphisms of TAS2R16 and susceptibility to colorectal cancer

Background: genetics plays an important role in the susceptibility to sporadic colorectal cancer (CRC). In the last 10 years genome-wide association studies (GWAS) have identified over 40 independent low penetrance polymorphic variants. However, these loci only explain around 1‑4% of CRC heritability, highlighting the dire need of identifying novel risk loci. In this study, we focused our attention on the genetic variability of the TAS2R16 gene, encoding for one of the bitter taste receptors that selectively binds to salicin, a natural antipyretic that resembles aspirin. Given the importance of inflammation in CRC, we tested whether polymorphic variants in this gene could affect the risk of developing this neoplasia hypothesizing a role of TAS2R16 in modulating chronic inflammation within the gut. Methods: we performed an association study using 6 tagging SNPs, (rs860170, rs978739, rs1357949, rs1525489, rs6466849, rs10268496) that cover all TAS2R16 genetic variability. The study was carried out on 1902 CRC cases and 1532 control individuals from four European countries. Results: we did not find any statistically significant association between risk of developing CRC and selected SNPs. However, after stratification by histology (colon vs. rectum) we found that rs1525489 was associated with increased risk of rectal cancer with a (Ptrend of = 0.0071). Conclusions: our data suggest that polymorphisms within TAS2R16 gene do not have a strong influence on colon cancer susceptibility, but a possible role in rectal cancer should be further evaluated in larger cohorts

mRNA PGC-1α levels in blood samples reliably correlates with its myocardial expression: study in patients undergoing cardiac surgery

et al.[Objective]: Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a transcriptional coactivator that has been proposed to play a protective role in mouse models of cardiac ischemia and heart failure, suggesting that PGC-1α could be relevant as a prognostic marker. Our previous studies showed that the estimation of peripheral mRNA PGC-1α expression was feasible and that its induction correlated with the extent of myocardial necrosis and left ventricular remodeling in patients with myocardial infarction. In this study, we sought to determine if the myocardial and peripheral expressions of PGC-1α are well correlated and to analyze the variability of PGC-1α expression depending on the prevalence of some metabolic disorders. [Methods]: This was a cohort of 35 consecutive stable heart failure patients with severe aortic stenosis who underwent an elective aortic valve replacement surgery. mRNA PGC-1α expression was simultaneously determined from myocardial biopsy specimens and blood samples obtained during surgery by quantitative PCR, and a correlation between samples was made using the Kappa index. Patients were divided into two groups according to the detection of baseline expression levels of PGC-1α in blood samples, and comparisons between both groups were made by chi-square test or unpaired Student’s t-test as appropriate. [Results]: Based on myocardial biopsies, we found that mRNA PGC-1α expression in blood samples showed a statistically significant correlation with myocardial expression (Kappa index 0.66, p<0.001). The presence of higher systemic PGC-1α expression was associated with a greater expression of some target genes such as silent information regulator 2 homolog-1 (x-fold expression in blood samples: 4.43±5.22 vs. 1.09±0.14, p=0.044) and better antioxidant status in these patients (concentration of Trolox: 0.40±0.05 vs. 0.34±0.65, p=0.006). [Conclusions]: Most patients with higher peripheral expression also had increased myocardial expression, so we conclude that the non-invasive estimation of mRNA PGC-1α expression from blood samples provides a good approach of the constitutive status of the mitochondrial protection system regulated by PGC-1α and that this could be used as prognostic indicator in cardiovascular disease.Grant from Sociedad Valenciana de Cardiología, 2013 to Óscar Fabregat-Andrés.Peer Reviewe

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores

During metaphase, forces on kinetochores are exerted by k fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser- cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non- kinetochore microtubules, which we term ‘bridging fibre’, bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape

Prolonged higher dose methylprednisolone vs. conventional dexamethasone in COVID-19 pneumonia: a randomised controlled trial (MEDEAS)

Dysregulated systemic inflammation is the primary driver of mortality in severe COVID-19 pneumonia. Current guidelines favor a 7-10-day course of any glucocorticoid equivalent to dexamethasone 6 mg·day-1. A comparative RCT with a higher dose and a longer duration of intervention was lacking

Polygenic and multifactorial scores for pancreatic ductal adenocarcinoma risk prediction

Most cases of pancreatic ductal adenocarcinoma (PDAC) are asymptomatic in early stages, and the disease is typically diagnosed in advanced phases, resulting in very high mortality. Tools to identify individuals at high risk of developing PDAC would be useful to improve chances of early detection