Fluorescence microscopy has become a powerful tool to investigate proteins in their natural environment. Well-established techniques like widefield and confocal fluorescence microscopy have commonly ...been used for decades to visualize biomolecules in single cells and tissue sections. Live cell microscopy allows for the investigation of biomolecular trafficking, and other specialized techniques, such as proximity ligation assays (PLA) and fluorescence lifetime imaging microscopy (FLIM), can be used to study interactions between biomolecules of interest. Finally, with the most recent rise of optical super-resolution microscopy, we can investigate target biomolecules in situ with unprecedented detail on the nanometer scale. Here, we discuss various optical microscopy techniques that have successfully been used to image MIF. We highlight applications, advantages, and limitations of each technique. The techniques described here can easily be adapted to investigate other target proteins, their localization, interaction partners, and mechanisms of action.
Chronic stress accelerates metastasis - the main cause of death in cancer patients - through the activation of beta -adrenoceptors ( beta ARs). We have previously shown that beta 2AR signaling in ...MDA-MB-231HM breast cancer cells, facilitates invadopodia formation and invasion in vitro. However, in the tumor microenvironment where many stromal cells also express beta AR, the role of beta 2AR signaling in tumor cells in metastasis is unclear. Therefore, to investigate the contribution of beta 2AR signaling in tumor cells to metastasis in vivo, we used RNA interference to generate MDA-MB-231HM breast cancer cells that are deficient in beta 2AR. beta 2AR knockdown in tumor cells reduced the proportion of cells with a mesenchymal-like morphology and, as expected, reduced tumor cell invasion in vitro. Conversely, overexpression of beta 2AR in low metastatic MCF-7 breast cancer cells induced an invasive phenotype. Importantly, we found that knockdown of beta 2AR in tumor cells significantly reduced the impact of stress on metastasis in vivo. These findings highlight a crucial role for beta 2AR tumor cell signaling in the adverse effects of stress on metastasis, and indicate that it may be necessary to block beta 2AR on tumor cells to fully control metastatic progression.
Two tropomyosin isoforms, human Tm5
NM1 and Tm3, were over-expressed in B35 rat neuro-epithelial cells to examine preferential associations between specific actin and tropomyosin isoforms and to ...determine the role tropomyosin isoforms play in regulating the drug susceptibility of actin filament populations. Immunofluorescence staining and Western blot analysis were used to study the organisation of specific filament populations and their response to treatment with two widely used actin-destabilising drugs, latrunculin A and cytochalasin D. In Tm5
NM1 cells, we observed large stress fibres which showed predominant co-localisation of
β-actin and low-molecular-weight
γ-tropomyosin isoforms. Tm3 cells had an abundance of cellular protrusions which contained both the
β- and
γ-actin isoforms, predominately populated by high-molecular-weight
α- and
β-tropomyosin isoforms. The stress fibres observed in Tm5
NM1 cells were more resistant to both latrunculin A and cytochalasin D than filaments containing the high-molecular-weight tropomyosins observed in Tm3 cells. Knockdown of the over-expressed Tm5
NM1 isoform with a human-specific Tm5
NM1 siRNA reversed the phenotype and caused a reversal in the observed drug resistance. We conclude that there are preferential associations between specific actin and tropomyosin isoforms, which are cell type specific, but it is the tropomyosin composition of a filament population which determines the susceptibility to actin-targeting drugs.
Multiple cell types form specialized protein complexes that are used by the cell to actively degrade the surrounding extracellular matrix. These structures are called podosomes or invadopodia and ...collectively referred to as invadosomes. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner.
Introduction For efficient metastatic dissemination, tumor cells form invadopodia to degrade and move through three-dimensional extracellular matrix. However, little is known about the conditions ...that favor invadopodia formation. Here, we investigated the effect of beta-adrenoceptor signaling - which allows cells to respond to stress neurotransmitters - on the formation of invadopodia and examined the effect on tumor cell invasion. Methods To characterize the molecular and cellular mechanisms of beta-adrenergic signaling on the invasive properties of breast cancer cells, we used functional cellular assays to quantify invadopodia formation and to evaluate cell invasion in two-dimensional and three-dimensional environments. The functional significance of beta-adrenergic regulation of invadopodia was investigated in an orthotopic mouse model of spontaneous breast cancer metastasis. Results beta-adrenoceptor activation increased the frequency of invadopodia-positive tumor cells and the number of invadopodia per cell. The effects were selectively mediated by the beta.sub.2-adrenoceptor subtype, which signaled through the canonical Src pathway to regulate invadopodia formation. Increased invadopodia occurred at the expense of focal adhesion formation, resulting in a switch to increased tumor cell invasion through three-dimensional extracellular matrix. beta.sub.2-adrenoceptor signaling increased invasion of tumor cells from explanted primary tumors through surrounding extracellular matrix, suggesting a possible mechanism for the observed increased spontaneous tumor cell dissemination in vivo. Selective antagonism of beta.sub.2-adrenoceptors blocked invadopodia formation, suggesting a pharmacological strategy to prevent tumor cell dissemination. Conclusion These findings provide insight into conditions that control tumor cell invasion by identifying signaling through beta.sub.2-adrenoceptors as a regulator of invadopodia formation. These findings suggest novel pharmacological strategies for intervention, by using beta-blockers to target beta.sub.2-adrenoceptors to limit tumor cell dissemination and metastasis.
Abstract
Chronic stress accelerates breast cancer metastasis through beta-adrenergic signaling and recent clinical studies found that beta-blockade reduced metastasis in women with breast cancer. ...However, the specific beta-adrenergic receptor subtype(s) through which stress-responsive neurotransmitters mediates effects on cancer remain unknown. To identify the receptor that transmits beta-adrenergic signaling to primary mammary tumors, we used bioluminescence imaging to longitudinally quantify the effect on metastasis of beta-blockers that selectively inhibit beta1 vs. beta 2 adrenergic receptors in an orthotopic mouse model of breast cancer progression. Non-specific beta-blockade with propranolol reduced metastasis by >90% (p = .03), confirming a key role for beta-adrenergic signaling pathways in stress-enhanced metastasis. Treatment with the beta2-selective antagonist ICI118551 similarly reduced stress-enhanced metastasis to control levels. In contrast, beta-1 selective blockade failed to protect against metastasis. Pharmacological intervention studies and gene expression analyses have started to define the downstream signaling pathways of beta-adrenergic regulation of metastasis. These studies may provide mechanistic explanation for recent clinical findings that not all beta-blockers protect against metastasis and provide a framework for selection of cancer patients who may optimally benefit from beta-blockade.
Citation Format: Ming Gene Chai, Caroline P. Le, Sarah J. Creed, Benjamin A L Finnin, Matthew A. Pimentel, David Shackleford, Andreas Moeller, Izhak Haviv, J. Robert Lane, Erica K. Sloan. Beta-blockade of breast cancer metastasis: Receptor regulation and downstream signaling pathways. abstract. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B092.
Genetic disorders and congenital anomalies are the leading causes of infant mortality. Diagnosis of most genetic diseases in neonatal and paediatric intensive care units (NICU and PICU) is not ...sufficiently timely to guide acute clinical management. We used rapid whole-genome sequencing (STATseq) in a level 4 NICU and PICU to assess the rate and types of molecular diagnoses, and the prevalence, types, and effect of diagnoses that are likely to change medical management in critically ill infants.
We did a retrospective comparison of STATseq and standard genetic testing in a case series from the NICU and PICU of a large children's hospital between Nov 11, 2011, and Oct 1, 2014. The participants were families with an infant younger than 4 months with an acute illness of suspected genetic cause. The intervention was STATseq of trios (both parents and their affected infant). The main measures were the diagnostic rate, time to diagnosis, and rate of change in management after standard genetic testing and STATseq.
20 (57%) of 35 infants were diagnosed with a genetic disease by use of STATseq and three (9%) of 32 by use of standard genetic testing (p=0·0002). Median time to genome analysis was 5 days (range 3-153) and median time to STATseq report was 23 days (5-912). 13 (65%) of 20 STATseq diagnoses were associated with de-novo mutations. Acute clinical usefulness was noted in 13 (65%) of 20 infants with a STATseq diagnosis, four (20%) had diagnoses with strongly favourable effects on management, and six (30%) were started on palliative care. 120-day mortality was 57% (12 of 21) in infants with a genetic diagnosis.
In selected acutely ill infants, STATseq had a high rate of diagnosis of genetic disorders. Most diagnoses altered the management of infants in the NICU or PICU. The very high infant mortality rate indicates a substantial need for rapid genomic diagnoses to be allied with a novel framework for precision medicine for infants in NICU and PICU who are diagnosed with genetic diseases to improve outcomes.
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Human Genome Research Institute, and National Center for Advancing Translational Sciences.
Neurodevelopmental disorders (NDDs) affect more than 3% of children and are attributable to single-gene mutations at more than 1000 loci. Traditional methods yield molecular diagnoses in less than ...one-half of children with NDD. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can enable diagnosis of NDD, but their clinical and cost-effectiveness are unknown. One hundred families with 119 children affected by NDD received diagnostic WGS and/or WES of parent-child trios, wherein the sequencing approach was guided by acuity of illness. Forty-five percent received molecular diagnoses. An accelerated sequencing modality, rapid WGS, yielded diagnoses in 73% of families with acutely ill children (11 of 15). Forty percent of families with children with nonacute NDD, followed in ambulatory care clinics (34 of 85), received diagnoses: 33 by WES and 1 by staged WES then WGS. The cost of prior negative tests in the nonacute patients was $19,100 per family, suggesting sequencing to be cost-effective at up to $7640 per family. A change in clinical care or impression of the pathophysiology was reported in 49% of newly diagnosed families. If WES or WGS had been performed at symptom onset, genomic diagnoses may have been made 77 months earlier than occurred in this study. It is suggested that initial diagnostic evaluation of children with NDD should include trio WGS or WES, with extension of accelerated sequencing modalities to high-acuity patients.
PDF
