Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

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.

Rapid Uptake and Degradation of CXCL12 Depend on CXCR7 Carboxyl-terminal Serine/Threonine Residues

“…Compounds and Antibodies-CXCL12 (PeproTech, Hamburg, Germany), 125 I-CXCL12 (ARI0129, Hartmann Analytic, Braunschweig, Germany), AMD3100 (A5602, Sigma), cycloheximide (C4859, Sigma), -protein phosphatase (P0753, New England Biolabs GmbH, Frankfurt am Main, Germany), CCX733 and 11G8 monoclonal mouse anti-CXCR7 antibody (ChemoCentryx, Mountain View, CA) (22), anti-transferrin receptor antibody (13-6800; Invitrogen), and UMB-2 monoclonal rabbit anti-CXCR4 antibody (23). Polyclonal rabbit antisera against the hemagglutinin (HA) epitope MYPYDVPDYA were generated by injecting the peptide into rabbits 631-633 after coupling to keyhole limpet hemocyanin and emulsification with Freund's adjuvant.…”

“…We then addressed CXCL12-dependent phosphorylation of the CXCR7-4tail mutant using the rabbit monoclonal CXCR4 antibody UMB-2, which was raised against a synthetic peptide corresponding to the 22 C-terminal amino acids of CXCR4 (23). Recently, we established that binding of UMB-2 to CXCR4 is severely impaired after CXCL12 stimulation/phosphorylation of CXCR4 and that dephosphorylation of CXCR4 by -PP reinstates binding of UMB-2 to CXCR4 (Fig.…”

In Vitro Evaluation of Molecular Tumor Targets in Nuclear Medicine: Immunohistochemistry Is One Option, but Under Which Conditions?

“…At first, rather basic information is needed, namely a full description of the antibody characteristics (antibody name, manufacturer, code number, antigen sequence if known, species raised in, monoclonal/polyclonal designation, clone designation if applicable, lot number for polyclonal antibodies). It should further be confirmed that the antibody has been tested successfully in cell lines expressing the receptor, and negative controls in cell lines devoid of the receptor or in wild-type/knockout mice should also be provided (3,4,10,11). Preferably, such data should be available for both fresh frozen and the corresponding formalin-fixed materials.…”

“…By definition, GPCRs are cell surface receptors with 7 transmembrane-spanning domains. Therefore, under normal conditions, we expect to see immunoreactivity localized at the cell membrane (10,11,(13)(14)(15). A typical example of the somatostatin receptor sst 2 expressed in a neuroendocrine tumor is shown in Figure 1A.…”

“…Putting most of the above-mentioned recommendations into practice will likely prevent unnecessary controversies and reduce experimental discrepancies, as recently shown by examples in the field of receptors for somatostatin, chemokines, and glucagonlike peptide 1 (3,4,10,11,23,24).…”