GLUT1 (MSVA-401R)

Glucose transporter 1 (or GLUT1), also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1) protein is coded by the SLC2A1 gene on chromosome 1p34.2. GLUT1 facilitates the transport of glucose across the plasma membranes of cells. Glut1 is also a receptor for vitamin C uptake and the human T-cell leukemia virus (HTLV) I and II. GLUT1 expression occurs in almost all tissues. The level of GLUT1 expression parallels the rate of cellular glucose metabolism. It is particularly high in erythrocytes and in endothelial cells of the blood–brain barrier. GLUT1 is often overexpressed in cancer because many tumors exert a metabolic switch from oxidative phosphorylation to glycolysis which requires an elevated uptake of glucose. In cancer GLUT1 represents a potential therapeutic target for GLUT1 inhibitors such as Bay-876.

Summary: In normal tissues, the strongest GLUT1 immunostaining is seen in amnion, chorion and trophoblast cells of the placenta. GLUT1 staining is also strong in all erythrocytes and their precursor cells. GLUT1 staining of endothelial cells is dependent on the location and tissue type. It is strongest in the brain. An at least weak to moderate staining is also seen in squamous epithelium and urothelium as well as in dendritic cells of germinal centres.

Mesenchymal tissues: GLUT1 staining is absent in muscle, fibrous tissue, and fat. Erythrocytes are strongly GLUT1 positive in all organs. GLUT1 staining also occurs in the perineurium of at least a fraction of peripheral nerves. 

Squamous epithelium: A weak to moderate membranous and cytoplasmic staining is preferentially seen in the lower third of squamous epithelium of various sites including skin and tonsil crypts. 

Gastrointestinal tract: A weak membranous and cytoplasmic, preferentially basolateral GLUT1 staining can be seen in the surface epithelium of the stomach. In the intestine, epithelial cell staining is largely absent but can focally be observed (mostly weak). Epithelial cell staining of variable intensity can be focally seen in the gallbladder. Liver and pancreas are mostly GLUT1 negative but a focal membranous staining of acinar epithelial cells can be seen in the pancreas. Salivary glands and Brunner glands are mostly negative.

Genitourinary tract: A weak to moderate membranous staining occurs in the urothelium with decreasing intensity from the basal to the superficial cell layers. The kidney is usually GLUT1 negative, but few tubuli or collecting ducts may show weak to moderate staining. GLUT1 is largely lacking in testis and epididymis. The prostate is usually negative but a fraction of samples shows a faint GLUT1 staining in basal cells. Seminal vesicles show a predominantly basolateral membranous positivity in a fraction of epithelial cells. 

Female organs: Acinar cells of the breast show a weak cytoplasmic staining. In the endometrium a usually weak staining of both epithelial and stromal cells is usually seen. A weak to moderate cytoplasmic staining of epithelial cells also occurs in the fallopian tube. In the placenta, a predominantly membranous GLUT1 staining is very intense in amnion, chorion and trophoblast cells and also endothelial cells show a particularly strong staining. GLUT1 staining is also moderate to strong in decidua cells. In the ovary, GLUT1 staining is largely limited to blood vessels but absent in stroma, corpus luteum, theca and granulosa cells. 

Respiratory tract: A weak, predominantly cytoplasmic GLUT1 staining occurs in (non-basal) cells of the respiratory epithelium. In the lung, staining is largely limited to erythrocytes.

Endocrine organs: GLUT1 staining is lacking in epithelial cells of the adrenal gland, thyroid, parathyroid gland, and the adenohypophysis

Lymphatic tissues and bone marrow: Moderate to strong GLUT1 staining of follicular dendritic cells in germinal centres while staining is markedly weaker in a fraction of lymphocytic and monocytic interfollicular cells. In the bone marrow, erythrocytes and their precursor cells stain positive.

Brain: An very intense positivity occurs in endothelial cells while all glia cells and associated fibres as well as neurons and Purkinje are GLUT1 negative.

These findings are largely consistent with the RNA and protein data described in the Human Protein Atlas (Tissue expression GLUT1) 

Positive control = Brain: A strong GLUT1 staining of endothelial cells should be seen.

Negative control = Brain: GLUT1 staining should be absent in all cells/structures except blood vessels.

Normal tissue gallery

A positive GLUT1 immunostaining is preferentially seen in squamous cell carcinomas irrespective of their origin but at least a small fraction of positive cases may occur in a broad range of tumor entities.

The TCGA findings on GLUT1 RNA expression in different tumor categories have been summarized in the Human Protein Atlas.

Cancer tissue gallery

No data available at the moment

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply MSVA-401R at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

Agilent / Dako – Autostainer Link 48

Pretreatment in PT-Link for 30 minutes at 95°C (pH high); FLEX peroxidase blocking for 5 minutes (room temperature), MSVA-401R 1:150 for 20 minutes (room temperature), FLEX+ mouse/rabbit (LINKER) for 15 minutes (room temperature), horseradish peroxidase (HRP) for 20 minutes (room temperature), FLEX DAB+Sub-Chromo for 10 minutes (room temperature), FLEX hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, and a longer incubation time of FLEX+LINKER result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

Leica – BOND RX

Dewax at 72°C for 30 seconds; Pretreatment in Bond Epitope Retrieval Solution (ER2 – EDTA pH9) for 20 minutes at 100°C; Peroxidase blocking for 5 minutes (room temperature), MSVA-401R 1:200 for 15 minutes (room temperature), Post primary (rabbit anti mouse) for 8 minutes (room temperature), Polymer (goat anti rabbit) for 8 minutes (room temperature), mixed DAB refine for 10 minutes (room temperature), hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of Post primary and or the Polymer result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

Roche – Ventana Discovery ULTRA

Pretreatment for 64 minutes at 100°C (pH 8,4); CM peroxidase blocking for 12 minutes (room temperature), MSVA-401R 1:150 for 20 minutes at 36°C, secondary antibody (anti-rabbit HQ) for 12 minutes at 36°C, anti-HQ HRP for 12 minutes at room temperature, DAB at room temperature, hematoxylin II at room temperature for 8 minutes, bluing reagent at room temperature for 4 minutes.

These images depict staining results obtained by the protocol described above. It is of note, that the Ventana machines generally require higher antibody concentrations than other commonly used autostainers because the antibodies are automatically diluted during the procedure. Various other protocols can result in an identical result as shown above. A longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of secondary antibody and or the anti-HQ HRP result in stronger staining, potentially at the cost of more background staining.

• GLUT1 inhibitors, such as Bay-876 have shown efficiency in blocking tumor cell growth. It is currently unclear whether the effect of such drugs depends on the cellular GLUT1 expression levels.
• The clinical significance of GLUT1 expression in cancer is unclear.

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

Orthogonal validation: For the antibody MSVA-401R specificity is supported by a general concordance of the immunostaining data with data from three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, which are all summarized in the Human Protein Atlas (Tissue expression GLUT1)). In agreement with MSVA-401R findings, GLUT1 RNA expression has been described to preferentially occur in the placenta, bone marrow and tissues covered by squamous epithelium or urothelium while GLUT1 RNA was largely lacking in tissues without significant GLUT1 immunostaining such as the gastrointestinal tract, endocrine organs, lung, muscle tissues or the liver.

Comparison of antibodies: By using MSVA-401R occasional staining was seen in organs/tissues for which high level RNA expression has not been described. The specificity of such occasional GLUT1 stainings such as seen in stomach, rectum, gallbladder, kidney, prostate, seminal vesicle, respiratory epithelium, endometrium, fallopian tube, decidua cells, placenta, lymph node, and germinal centres of the tonsil is confirmed by similar findings obtained by a second independent commercial antibody termed “validation antibody”. Images comparing MSVA-401R and the independent “validation antibody” in the respective tissues are shown below.

Antibody Comparison: MSVA-401R vs another commercially available Progesterone Receptor antibody called “Validation Antibody”

Normal tissue gallery