Abstract
BackgroundThe CXCR4 chemokine receptor regulates migration and homing of cancer cells to specific metastatic sites. Determination of the CXCR4 receptor status will provide predictive information for disease prognosis and possible therapeutic intervention. However, previous attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have produced predominant nuclear and occasional cytoplasmic staining but did not result in the identification of bona fide cell surface receptors.Methodology/Principal FindingsIn the present study, we extensively characterized the novel rabbit monoclonal anti-CXCR4 antibody (clone UMB-2) using transfected cells and tissues from CXCR4-deficient mice. Specificity of UMB-2 was demonstrated by cell surface staining of CXCR4-transfected cells; translocation of CXCR4 immunostaining after agonist exposure; detection of a broad band migrating at M r 38,000–43,000 in Western blots of homogenates from CXCR4-expressing cells; selective detection of the receptor in tissues from CXCR4+/+ but not from CXCR4−/− mice; and abolition of tissue immunostaining by preadsorption of UMB-2 with its immunizing peptide. In formalin-fixed, paraffin-embedded human tumor tissues, UMB-2 yielded highly effective plasma membrane staining of a subpopulation of tumor cells, which were often heterogeneously distributed throughout the tumor. A comparative analysis of the mouse monoclonal antibody 12G5 and other frequently used commercially available antibodies revealed that none of these was able to detect CXCR4 under otherwise identical conditions.Conclusions/SignificanceThus, the rabbit monoclonal antibody UMB-2 may prove of great value in the assessment of the CXCR4 receptor status in a variety of human tumors during routine histopathological examination.
Highlights
The CXCR4 chemokine receptor is a plasma membrane receptor that regulates an array of trafficking events during organogenesis, hematopoesis and inflammation [1]
Recent studies indicate that CXCR4 is one of the critical factors for metastasis homing on specific organ sites in that SDF-1 released in target organs may attract nearby or distant CXCR4-expressing cancer cells [2]
UMB-2 detected prominent immunofluorescence at the level of the plasma membrane only in CXCR4- but not in CCR7-expressing cells, and that SDF-1 exposure induced a rapid translocation of CXCR4 receptor immunostaining from the plasma membrane into the cytosol
Summary
The CXCR4 chemokine receptor is a plasma membrane receptor that regulates an array of trafficking events during organogenesis, hematopoesis and inflammation [1]. The specific ligand to CXCR4, stromal cell-derived factor 1 (SDF-1, CXCL12), is expressed at high levels in lymph nodes, lungs, bone marrow and liver. Recent studies indicate that CXCR4 is one of the critical factors for metastasis homing on specific organ sites in that SDF-1 released in target organs may attract nearby or distant CXCR4-expressing cancer cells [2]. An accurate assessment of the CXCR4 status of a given tumor specimen would provide valuable predictive information for disease prognosis and possible therapeutic intervention. The CXCR4 chemokine receptor regulates migration and homing of cancer cells to specific metastatic sites. Determination of the CXCR4 receptor status will provide predictive information for disease prognosis and possible therapeutic intervention. Previous attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have produced predominant nuclear and occasional cytoplasmic staining but did not result in the identification of bona fide cell surface receptors
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