Itay Barnea

The chemokine CCL5 as a potential prognostic factor predicting disease progression in stage II breast cancer patients.

Purpose: The aim of this study was to determine the prognostic value of the chemokine CCL5, considered as a promalignancy factor in breast cancer, in predicting breast cancer progression and to evaluate its ability to strengthen the prognostic significance of other biomarkers. Experimental Design: The expression of CCL5, alone and in conjunction with estrogen receptor (ER)-α, ER-β, progesterone receptor (PR), and HER-2/neu (ErbB2), was determined in breast tumor cells by immunohistochemistry. The study included 142 breast cancer patients, including individuals in whom disease has progressed. Results: Using Cox proportional hazard models, univariate analysis suggested that, in stage I breast cancer patients, CCL5 was not a significant predictor of disease progression. In contrast, in stage II patients, the expression of CCL5 (CCL5+), the absence of ER-α (ER-α−), and the lack of PR expression (PR−) increased significantly the risk for disease progression (P = 0.0045, 0.0041, and 0.0107, respectively). The prognostic strength of CCL5, as well as of ER-α−, improved by combining them together (CCL5+/ER-α−: P = 0.0001), being highly evident in the stage IIA subgroup [CCL5+/ER-α− (P = 0.0003); ER-α− (P = 0.0315)]. In the stage II group as a whole, the combinations of CCL5−/ER-α+ and CCL5−/PR+ were highly correlated with an improved prognosis. Multivariate analysis indicated that, in stage II patients, ER-α and CCL5 were independent predictors of disease progression. Conclusions: CCL5 could be considered as a biomarker for disease progression in stage II breast cancer patients, with the CCL5+/ER-α− combination providing improved prediction of disease progression, primarily in the stage IIA subgroup.

MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERα) in regulation of the MUC1 gene expression

BackgroundThe MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter.ResultsUsing TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERα, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed.ConclusionAnalysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERα in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy.

Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation

A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label‐free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive‐index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive‐index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label‐free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label‐free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids.

Localized measurements of physical parameters within human sperm cells obtained with wide-field interferometry

We developed a new method to identify the separate cellular compartments in the optical path delay (OPD) maps of un-labeled spermatozoa. This was conducted by comparing OPD maps of fixed, un-labeled spermatozoa to bright field images of the same cells following labeling. The labeling enabled us to identify the acrosomal and nuclear compartments in the corresponding OPD maps of the cells. We then extracted the refractive index maps of fixed cells by dividing the OPD maps of spermatozoa by the corresponding thickness maps of the same cells, obtained with AFM. Finally, the dry mass of the head, nucleus and acrosome of un-labeled immobile spermatozoa, was measured. This method provides the ability to quantitatively measure the dry mass of cellular compartments within human spermatozoa. We expect that these measurements will assist label-free selection of sperm cells for fertilization.

Automated analysis of individual sperm cells using stain-free interferometric phase microscopy and machine learning

Currently, the delicate process of selecting sperm cells to be used for in vitro fertilization (IVF) is still based on the subjective, qualitative analysis of experienced clinicians using non‐quantitative optical microscopy techniques. In this work, a method was developed for the automated analysis of sperm cells based on the quantitative phase maps acquired through use of interferometric phase microscopy (IPM). Over 1,400 human sperm cells from 8 donors were imaged using IPM, and an algorithm was designed to digitally isolate sperm cell heads from the quantitative phase maps while taking into consideration both the cell 3D morphology and contents, as well as acquire features describing sperm head morphology. A subset of these features was used to train a support vector machine (SVM) classifier to automatically classify sperm of good and bad morphology. The SVM achieves an area under the receiver operating characteristic curve of 88.59% and an area under the precision‐recall curve of 88.67%, as well as precisions of 90% or higher. We believe that our automatic analysis can become the basis for objective and automatic sperm cell selection in IVF.

Individual sperm selection by microfluidics integrated with interferometric phase microscopy

The selection of sperm cells possessing normal morphology and motility is crucial for many assisted reproductive technologies (ART), especially for intracytoplasmic sperm injection (ICSI), as sperm quality directly affects the probability of inducing healthy pregnancy. We present a novel platform for real-time quantitative analysis and selection of individual sperm cells without staining. Towards this end, we developed an integrated approach, combining interferometric phase microscopy (IPM), for stain-free sperm imaging and real-time automatic analysis based on the sperm cell 3D morphology and contents, with a disposable microfluidic device, for sperm selection and enrichment. On testing the capabilities of the microfluidic device, we obtained successful selection of sperm cells with a selectivity of 89.5±3.5%, with no negative-decision sperm cells being inadvertently selected. In addition, we demonstrate the accuracy of sperm cell analysis using IPM by comparing the quantitative analysis produced by our IPM-based algorithm to the qualitative visual analysis performed independently by an experienced embryologist, which resulted in precision and specificity of 100%. We believe that the presented integrated approach has the potential to dramatically change the way sperm cells are selected for ICSI and other ART procedures, making the selection process more objective, quantitative and automatic, and thereby increasing success rates.

Label-Free Quantitative Imaging of Sperm for In-Vitro Fertilization Using Interferometric Phase Microscopy

Accurate morphological evaluation via imaging of sperm cells is a critical step in both routine sperm analysis and sperm cell selection for in-vitro fertilization. In this paper, we review the significant advantages of interferometric phase microscopy (IPM), also called digital holographic microscopy, for label-free sperm imaging in fertility clinics. We first review the current state of the art of label-free sperm cell imaging for in-vitro fertilization, including bright-field, Zernike phase contrast, differential interference contrast (DIC), Hoffman modulation contrast and polarized light microscopy, as well as intracytoplasmic morphologically selected sperm injection (IMSI). Using experimental demonstrations, we compare the limitations of these techniques for label-free sperm imaging. IPM and its advantages are then discussed in detail. These include the ability to receive quantitative data regarding the 3D structure of sperm cells, as well as data concerning sperm biomass and morphology without labeling. The current gap to clinical use is discussed, as well as the possible solution of employing compact and simple holographic modules, which may allow holographic imaging to reach the clinic in the near future.

Simultaneous three-wavelength unwrapping using external digital holographic multiplexing module

We present an external interferometric setup that is able to simultaneously acquire three wavelengths of the same sample instance without scanning or multiple exposures. This setup projects onto the monochrome digital camera three off-axis holograms with rotated fringe orientations, each from a different wavelength channel, without overlap in the spatial-frequency domain, and thus allows the full reconstruction of the three complex wavefronts from the three wavelength channels. We use this new setup for three-wavelength phase unwrapping, allowing phase imaging of thicker objects than possible with a single wavelength, but without the increased level of noise. We demonstrate the proposed technique for micro-channel profiling and label-free cell imaging.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

Stain-free interferometric phase microscopy correlation with DNA fragmentation stain in human spermatozoa

Acridine orange (AO) staining is used to diagnose DNA fragmentation status in sperm cells. Interferometric phase microscopy (IPM) is an optical imaging method based on digital holographic microscopy that provides quantitative morphological and refractive index imaging of cells in vitro without the need for staining. We have imaged sperm cells using stain-free IPM in order to estimate different cellular parameters, such as acrosome dry mass and size, in addition to an embryologist evaluation according to the World Health Organization (WHO)-2010 criteria. Following this, the same sperm cells were stained by AO, imaged using a fluorescence confocal microscope and assessed by the AO-emitted color, forming five DNA fragmentation groups. These DNA fragmentation groups were correlated with the embryologist-based classification and the IPM-based morphological parameters. Our results indicate on significant differences in the IPM-based parameters between groups with different fragmentation levels. Based on the validation with AO, we conclude that stain-free IPM images analyzed digitally may assist in selecting sperm cells with intact DNA prior to intracytoplasmic sperm injection. This information may potentially increase percentage of successful pregnancies.