As the frequency of melanoma increases, current treatment strategies are struggling to significantly impact patient survival. One of the critical issues in designing efficient therapies is understanding the composition of heterogeneous melanoma tumors in order to target cancer stem cells (CSCs) and drug resistant subpopulations. In this review, we summarize recent findings pertinent to the reemergence of the embryonic Nodal signaling pathway in melanoma and its significance as a prognostic biomarker and therapeutic target. In addition, we offer a novel molecular approach to studying the functional relevance of Nodal-expressing subpopulations and their CSC phenotype.

The green fluorescent protein (GFP) is used extensively to monitor gene expression and protein localization in living cells, particularly in developing embryos from a variety of species. Several GFP mutations have been characterized that improve protein expression and alter the emission spectra to produce proteins that emit green, blue, cyan, and yellow wavelengths. DsRed and its variants encode proteins that emit in the orange to red wavelengths. Many of these commercially available fluorescent proteins have been "codon optimized" for maximal levels of expression in mammalian cells. We have generated several fluorescent protein color variants that have been codon optimized for maximal expression in the ascidian Ciona intestinalis. By analyzing quantitative time-lapse recordings of transgenic embryos, we demonstrate that, in general, our Ciona optimized variants are detected and expressed at higher levels than commercially available fluorescent proteins. We show that three of these proteins, expressed simultaneously in different spatial domains within the same transgenic embryo are easily detectable using optimized fluorescent filter sets for epifluorescent microscopy. Coupled with recently developed quantitative imaging techniques, our GFP variants should provide useful reagents for monitoring the simultaneous expression of multiple genes in transgenic ascidian embryos. Developmental Dynamics 235:456 -467, 2006.

Understanding the immune response can be aided by analyzing the genes that immune cells express in response to stimuli. Since all cells from the same orgainism have the same genetic components it is the selection of which genes are expressed that gives each cell its unique characteristics. Once a cell senses cues from its environment it begins to respond by transcribing its DNA into RNA which is later translated into protein. In order to monitor responses at the earliest possible point the RNA levels of a given gene can be assessed giving the researcher insight to how the cell has chosen to react before the protein is translated. Detecting gene expression has traditionally been limited to technologies that examine RNA in lysed or fixed cell populations. The ability to detect gene expression in live cells would allow for more physiologically relevant information. We have developed a novel RNA detection technology capable of detecting specific mRNA and miRNA in live, intact cells. It allows for carrier-free cellular uptake of the reagent, followed by detection of target RNA, with the ability to perform downstream analysis in the same sample.

The study of CD4+ T-helper (TH) cell differentiation is an important area of research that will aid in the understanding of inflammation and autoimmunity. CD4 + T cells can give rise to many subtypes depending on type of immune response. This study focuses on the TH1, TH2 and TH17 CD4+ T cell subtypes. Each subtype expresses a signature cytokine that directs the type of immune response needed. In order to analyze cytokine expression in TH cultures by flow cytometry, we employed a fixable viability dye to gate out dead cells that can accumulate in long-term differentiation cultures. In addition, flow cytometric analysis provides cell-specific information not obtained by ELISA analysis. Using standard TH culture protocols we differentiated naïve CD4+ T cells into TH1, TH2, and TH17 subtypes. We first stained the cells with the viability dye and then stained with antibodies against the signature cytokine of interest in less than 4 hours. Our data shows that we can easily obtain viability information while simultaneously evaluating cytokine production within our TH culture system. Using the fixable viability dye, we can exclude false positive cytokine staining and therefore obtain more accurate and reproducible intracellular expression data from cultured CD4+ T cells.

One of the most common methods for understanding gene function within a live cell is modulating the gene through knockdown or over‐expression systems. These experiments provide information regarding the role a particular gene plays within a given biological process. Unfortunately, determining the modulation in gene expression is currently performed at the population level as in the case of qPCR and or is ultimately an endpoint determination as in the case of RNA FISH. We propose a new method for determining the RNA expression levels of target genes using a nanoparticle technology which can be used in live cells without the need for transfection reagents. Live cell gene expression detection affords the ability to monitor changes in expression levels over time. More data can be collected over the course of a single experiment at the cellular level while monitoring gene expression in a dynamic manner as opposed to studying many samples at varied time points in the case of RT‐PCR or FISH. Here we present data illustrating the benefits of detecting gene expression in live cells while modulating both Survivin and ER alpha genes. U2OS cells which have an ER alpha knock‐in under the control of an inducible promoter were monitored using the nanoparticle detection method prior to and during the induction process enabling the monitoring of the gene modulation. Knockdown experiments were also performed using Survivin as a target gene to illustrate down regulation of genes and the ability to monitor the loss of function as well. Monitoring the modulation of genes within live cells can assist in determination of gene function by allowing for the collection of many more data points within a single experiment without the need for duplicate samples at various time points. Furthermore, as this method is non toxic the live cells can be further characterized or used in subsequent experiments providing even more data from the same cells.

No abstract

Circulating tumor cells (CTCs), released into the bloodstream from primary and metastatic cancers, are valuable tools for understanding tumor biology. However, since they are rare, their detection is hampered by low efficiency and a lack of standardization in current technologies. To overcome these problems, we used Imaging Flow Cytometery along with fluorescent RNA detection probes to collect imagery from large number of cells to assess the number of CTCs. In this study, we spiked peripheral blood mononucleated cells (PBMCs) with SKBR-3 human breast cancer cells and added probes for relevant RNA targets such as EPCAM and Her-2. Taking advantage of the probes’ ability to detect RNA in live cells, combined with the capacity to acquire multi-spectral images of large numbers of cells, we demonstrate image based parameters that can be used to assess the frequency of CTCs in an objective and statistically significant manner. Citation Format: Shobana Vaidyanathan, Don Weldon, David Basiji, Philip Morrissey. Detection and enumeration of circulating tumor cells using imaging flow cytometery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3074. doi:10.1158/1538-7445.AM2014-3074

Detecting gene expression in immune cells has traditionally been limited to technologies that utilize a reporter construct for a gene of interest or within lysed or fixed cell populations as is the case for RT-PCR. Here we show the ability to detect gene expression in live monocytes and macrophages which allows for more physiologically relevant information based on the cell’s response to given stimuli. Determining which genes were up or down regulated in those cells provides insight into complex gene regulatory networks and immune cell function. Here we present a novel RNA expression detection technology capable of detecting specific mRNA and miRNA in live, intact cells. This technology allows for detection of target RNA, with the ability to perform downstream assays such as Fluorescence-Activated Cell Sorting (FACS) to enrich for a specific subpopulation of live cells based solely on their RNA expression profile. Following FACS sorting based on RNA detection the cells remain viable and fully functional for use in downstream assays allowing researchers the ability to further characterize or determine functionality of the enriched population. Within a population of cells there is inherent homogeneity which can skew results when only looking at the population level as opposed to understanding the single cell resolution of gene expression. Looking at RNA levels in a more dynamic manner proves to be much more informative than looking at duplicate samples over time.