Podocalyxin / PODXL (MSVA-645M)

Podocalyxin (PODXL) is a CD34-related transmembrane sialomucin protein which is coded by the PODXL gene located on chromosome 7q32.2. PODXL is primarily expressed in podocytes of the kidneys, endothelial cells, mesothelial cells, and a few selected epithelial cell types. PODXL knock-out mice die within the first 24 hours of life from renal failure because podocyte foot processes do not form, and intercellular junctions between adjacent podocyte foot processes are abnormal. Germline mutations in PODXL are associated with congenital nephrotic syndrome. Overall PODXL has an important role in tissue development and for optimal glomerular function. PODXL also plays an important role in cancer as it can be mutated and aberrantly expressed in several cancer types. Many cancers with high PODXL expression are derived from PODXL negative normal epithelial cells. PODXL neo-expression was found to be associated with aggressive cancer, drug resistance, and immune evasion. In cancer cells, PODXL can interact with several proteins and downstream signaling pathways that are critical for promoting cancer metastasis and subsequent invasion. Because of its membranous location, PODXL is being evaluated for a role as a therapeutic target protein.

PODXL is most prominently expressed in podocytes of the kidney and also occurs rather abundantly in endothelial cells, mesothelial cells, and a few selected epithelial cell types.

Images describing the PODXL staining pattern in normal tissues obtained by the antibody MSVA-645M are shown in our “Normal Tissue Gallery”.

These findings are largely consistent with the RNA data described in the Human Protein Atlas (Tissue expression PODXL) where a rather ubiquitous expression is described (endothelial cells occur in all tissues).

Positive control = Kidney: A strong PODXL must be seen in glomerular podocytes while at least a weak to moderate PODXL staining must be seen in endothelial cells of small intertubular vessels.

Negative control = Kidney: Tubuli and collecting ducts must be PODXL negative.

Normal tissue gallery

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-645M 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.

• How can PODXL-targeted therapies be effectively developed and optimized for different tumor types?
• What specific molecular pathways are affected by PODXL in cancer?
• What is the prevalence and clinical significance of PODXL expression across tumor types?
• Is there any diagnostic/prognostic/predictive utility of PODXL IHC?

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-645M specificity is in line with RNA expression data from normal tissues which were collected in 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, and which are summarized in the Human Protein Atlas (Tissue expression PODXL). Although PODXL RNA expression was ubiquitous, due to its presence in capillaries of all organs, the highest level of PODXL RNA expression in the kidney and the absence of its expression in the bone marrow are well in agreement with our MSVA-645M IHC data.

Comparison of antibodies: True expression of PODXL in all cell types with PODXL positivity by MSVA-645M is corroborated by an identical staining obtained by a commercially available independent second antibody (termed “validation antibody”). An additional staining of squamous epithelia by the validation antibody which was not seen by MSVA-645M was considered an antibody specific cross-reactivity of our validation antibody.

Normal tissue gallery