Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx

<div> <div> <div> <p>The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale architecture, on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to- mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology. </p> </div> </div> </div>

Download Full-text

Abstract LB-B18: Epithelial to mesenchymal transition in human tumor biopsies: Quantitative, histopathological proof of the existence of EMT in vivo by immunofluorescence microscopy

10.1158/1535-7163.targ-15-lb-b18 ◽

2015 ◽

Author(s):

Tony Navas ◽

Robert J. Kinders ◽

Scott M. Lawrence ◽

Katherine Ferry-Galow ◽

Thomas D. Pfister ◽

...

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Immunofluorescence Microscopy ◽

Human Tumor ◽

Mesenchymal Transition ◽

Tumor Biopsies

Download Full-text

Generation of human T cells directed against an agonist epitope of Brachyury, a transcription factor involved in human tumor cell epithelial to mesenchymal transition (EMT)

Journal for ImmunoTherapy of Cancer ◽

10.1186/2051-1426-1-s1-p202 ◽

2013 ◽

Vol 1 (S1) ◽

Author(s):

Benjamin Boyerinas ◽

Jo A Tucker ◽

Diane J Poole ◽

Caroline Jochéms ◽

Claudia Palena ◽

...

Keyword(s):

T Cells ◽

Transcription Factor ◽

Tumor Cell ◽

Epithelial To Mesenchymal Transition ◽

Human Tumor ◽

Human Tumor Cell ◽

Mesenchymal Transition ◽

Human T Cells

Download Full-text

The T-box transcription factor Brachyury promotes epithelial-mesenchymal transition in human tumor cells

Journal of Clinical Investigation ◽

10.1172/jci38379 ◽

2010 ◽

Vol 120 (2) ◽

pp. 533-544 ◽

Cited By ~ 168

Author(s):

Romaine I. Fernando ◽

Mary Litzinger ◽

Paola Trono ◽

Duane H. Hamilton ◽

Jeffrey Schlom ◽

...

Keyword(s):

Transcription Factor ◽

Tumor Cells ◽

Epithelial Mesenchymal Transition ◽

Human Tumor ◽

Human Tumor Cells ◽

Mesenchymal Transition ◽

T Box

Download Full-text

Cancer-Associated Fibroblasts Modulate Transcriptional Signatures Involved in Proliferation, Differentiation and Metastasis in Head and Neck Squamous Cell Carcinoma

Cancers ◽

10.3390/cancers13133361 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3361

Author(s):

Emilia Wiechec ◽

Mustafa Magan ◽

Natasa Matic ◽

Anna Ansell-Schultz ◽

Matti Kankainen ◽

...

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Epithelial To Mesenchymal Transition ◽

Expression Profiles ◽

Gene Expression Profiles ◽

In Vitro Models ◽

Cancer Associated Fibroblasts ◽

Mesenchymal Transition ◽

2D And 3D

Cancer-associated fibroblasts (CAFs) are known to increase tumor growth and to stimulate invasion and metastasis. Increasing evidence suggests that CAFs mediate response to various treatments. HNSCC cell lines were co-cultured with their patient-matched CAFs in 2D and 3D in vitro models, and the tumor cell gene expression profiles were investigated by cDNA microarray and qRT-PCR. The mRNA expression of eight candidate genes was examined in tumor biopsies from 32 HNSCC patients and in five biopsies from normal oral tissue. Differences in overall survival (OS) were tested with Kaplan–Meier long-rank analysis. Thirteen protein coding genes were found to be differentially expressed in tumor cells co-cultured with CAFs in 2D and 81 in 3D when compared to tumor cells cultured without CAFs. Six of these genes were upregulated both in 2D and 3D (POSTN, GREM1, BGN, COL1A2, COL6A3, and COL1A1). Moreover, two genes upregulated in 3D, MMP9 and FMOD, were significantly associated with the OS. In conclusion, we demonstrated in vitro that CAF-derived signals alter the tumor cell expression of multiple genes, several of which are associated with differentiation, epithelial-to-mesenchymal transition (EMT) phenotype, and metastasis. Moreover, six of the most highly upregulated genes were found to be overexpressed in tumor tissue compared to normal tissue.

Download Full-text

Ultra-Low Colcemid Doses Induce Microtubule Dysfunction as Revealed by Super-Resolution Microscopy

10.1101/2020.08.13.249664 ◽

2020 ◽

Author(s):

Ashley M Rozario ◽

Sam Duwé ◽

Cade Elliott ◽

Riley B Hargreaves ◽

Peter Dedecker ◽

...

Keyword(s):

Side Effects ◽

Single Molecule ◽

Super Resolution ◽

Short Exposure ◽

Low Doses ◽

Anti Cancer ◽

Drug Regimens ◽

20 Nm ◽

Super Resolution Microscopy ◽

Microtubule Function

ABSTRACTMicrotubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt micro-tubules. However, their side effects require the development of safer drug regimens that still retain clinical efficacy. Currently, many questions remain regarding microtubule-interacting drugs at clinically relevant and ultra-low doses. Here, we use super-resolution microscopies (single molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. Short exposure to 30 - 80 nM colcemid results in aberrant microtubule curvature while microtubule fragmentation is detected upon treatment with ≥100 nM colcemid. Remarkably, even ultra-low doses (5 hours at <20 nM) led to subtle but significant microtubule architecture remodeling and suppression of microtubule dynamics. These challenges to microtubule function represent less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs, and therefore offer potential for improved understanding and design of anti-cancer agents.

Download Full-text

Super-resolution microscopy unveils FIP200-scaffolded, cup-shaped organization of mammalian autophagic initiation machinery

10.1101/712828 ◽

2019 ◽

Cited By ~ 2

Author(s):

Samuel J Kenny ◽

Xuyan (Shirley) Chen ◽

Liang Ge ◽

Ke Xu

Keyword(s):

Three Dimensional ◽

Super Resolution ◽

Subcellular Location ◽

Outer Face ◽

Structural Protein ◽

Er Exit Sites ◽

Autophagy Pathway ◽

20 Nm ◽

Yeast Homolog ◽

Super Resolution Microscopy

AbstractAutophagy is an essential physiological process by which eukaryotic cells degrade and recycle cellular materials. Although the biochemical hierarchies of the mammalian autophagy pathway have been identified, questions remain regarding the sequence, subcellular location, and structural requirements of autophagosome formation. Here, we characterize the structural organization of key components of the mammalian autophagic initiation machinery at ∼20 nm spatial resolution via three-color, three-dimensional super-resolution fluorescence microscopy. We thus show that upon cell starvation, FIP200, a large structural protein of the ULK1 complex with no direct yeast homolog, scaffolds the formation of cup-like structures located at SEC12-enriched remodeled ER-exit sites prior to LC3 lipidation. This cup scaffold, then, provides a structural asymmetry to enforce the directional recruitment of downstream components, including the Atg12-Atg5-Atg16 complex, WIPI2, and LC3, to the cup inside. Moreover, we provide evidence that the early autophagic machinery is recruited in its entirety to these cup structures prior to LC3 lipidation, and gradually disperses and dissociates on the outer face of the phagophore membrane during elongation. We thus shed new light on the physical process of mammalian autophagic initiation and development at the nanometer-scale.

Download Full-text

Lactoferrin Reverses Methotrexate Driven Epithelial Barrier Defect by Inhibiting TGF-β Mediated Epithelial to Mesenchymal Transition

10.1101/2019.12.23.878207 ◽

2019 ◽

Author(s):

Thomas E. Wallach ◽

Vasudha Srivastava ◽

Efren Reyes ◽

Ophir D. Klein ◽

Zev J. Gartner

Keyword(s):

Barrier Function ◽

Epithelial To Mesenchymal Transition ◽

Epithelial Mesenchymal Transition ◽

Intestinal Barrier ◽

Human Breast Milk ◽

Barrier Dysfunction ◽

Mesenchymal Transition ◽

Barrier Defect ◽

Β Receptor ◽

20 Nm

ABSTRACTBACKGROUND AND AIMSMethotrexate is an important tool in the arsenal of oncologists, gastroenterologists, and rheumatologists. At low doses it induces intestinal barrier dysfunction that may induce side effects such as gastrointestinal discomfort and liver injury. Previous studies suggest that lactoferrin can improve barrier function in a variety of contexts. This study set out to determine the mechanism of methotrexate induced barrier dysfunction and assess the effect of lactoferrin and other components of human breast milk on this dysfunction.METHODSUsing a murine enteroid model and Caco2 spheroids, we measured flux of basolateral-administered fluorescent dextran into the lumen. Barrier dysfunction was induced using methotrexate (220 nM) or lipopolysaccharide (20 nM). Human lactoferrin was added at 0.8 mg/ml (10 µM). RNAseq was performed on exposed samples.RESULTSLactoferrin blocks methotrexate-induced barrier dysfunction in murine enteroids. Similar results were observed when barrier dysfunction was induced in Caco2 spheroids with methotrexate and LPS, but not ML7. RNAseq revealed activation of TGF-β response genes and epithelial-mesenchymal transition (EMT) by methotrexate, which normalized in the presence of lactoferrin. TGF-β receptor inhibition (RepSox) blocked methotrexate induced barrier dysfunction in Caco2 spheroids. 20 nM TGF-β induced barrier dysfunction in Caco2 spheroids which was also inhibited by lactoferrin.CONCLUSIONSMethotrexate induces barrier dysfunction by activation of an EMT program promoted by TGF-β signaling and inhibited by lactoferrin. Lactoferrin is also protective of barrier function in an LPS-induced model. The likely mechanism of this effect is blockade of EMT programs induced by TGF-β.

Download Full-text

Effect of IMP3 on epithelial-to-mesenchymal transition and the function of colon cancer stem cells.

Journal of Clinical Oncology ◽

10.1200/jco.2015.33.3_suppl.631 ◽

2015 ◽

Vol 33 (3_suppl) ◽

pp. 631-631

Author(s):

Ping Wei ◽

Dawei Li ◽

Ye Xu ◽

Sanjun Cai ◽

Xiang Du

Keyword(s):

Colon Cancer ◽

Epithelial To Mesenchymal Transition ◽

Epithelial Mesenchymal Transition ◽

Clinical Stage ◽

Human Tumor ◽

Disease Free Survival ◽

Underlying Mechanism ◽

Vital Role ◽

Migration Ability ◽

Mesenchymal Transition

631 Background: EMT could promote the acquisition of stem-like properties in cancer cells and cancer stem cell (CSC) has EMT properties. However, the underlying mechanism of the interaction between EMT and CSC still remain unclear. We previously identified Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) plays a vital role in colon cancer carcinogenesis. Here, we aimed to identify the gene function between EMT and CSC in colon cancer. Methods: The expression of IMP3 was analyzed in human tumor tissues. Colonospheres were isolated from colon cancer cell lines and the biological features of IMP3 were investigated in the process of EMT and colonoshpere cells. Results: Increased IMP3 levels were significantly correlated with higher clinical stage, T classification, LNM, presence of distant metastasis. Patients with IMP3-positive localized tumors had lower 5-year disease-free survival (DFS) and overall survival (OS) than those with IMP3-negative tumors. Multivariate survival analysis showed that IMP3 was an independent prognostic marker for DFS and OS. Expanded colonospheres contained cells that expressed high levels of CD133 and had the ability to promote migration and activate the epithelial-mesenchymal transition. Down regulation of IMP3 in HCT116 cells inhibited the invasion, migration ability and epithelial-mesenchymal transition and the properties of colonospheres. Conclusions: These findings suggest that IMP3 plays an important role in colon cancer tumorigenesis and metastasis through transactivation of colon CSC with EMT property, which is helpful to identify critical marker and therapeutic target.

Download Full-text