Conceived and designed the experiments: RS SS. Performed the experiments: TRF FN SJ RS. Analyzed the data: TRF FN SJ RS SS. Wrote the paper: TRF RS SS.
The authors have declared that no competing interests exist.
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. 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.
In 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
Thus, 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.
The CXCR4 chemokine receptor is a plasma membrane receptor that regulates an array of trafficking events during organogenesis, hematopoesis and inflammation
Consequently, much attention has been directed towards the detection and localization of CXCR4 receptors in human primary tumors. Earlier studies have assessed CXCR4 expression using reverse transcription-polymerase chain reaction (RT-PCR)
In the present study, we have extensively characterized the new rabbit monoclonal anti-CXCR4 antibody UMB-2, which is directed against the carboxyl-terminal tail of the receptor. We demonstrate that UMB-2 selectively detects its cognate receptor in fixed cells and tissues. In contrast to currently available monoclonal and polyclonal antibodies, UMB-2 efficiently detects bona fide CXCR4 plasma membrane receptors. Thus, the development of UMB-2 will now allow the establishment of guidelines for routine performance of CXCR4 immunohistochemistry in human tumors.
Rabbit polyclonal anti-CXCR4 antibodies {2144} and {1181} were generated against the following sequence KGKRGGHSSVSTESESSSFHSS, which corresponds to residues 338–359 of the human CXCR4 receptor. This sequence is identical in mouse, rat and human CXCR4 receptors. Anti-CXCR4 antibodies {2144} and {1181} have been extensively characterized previously in mouse and rat tissues
The following tumors were investigated: breast carcinoma (n = 36); ovarian carcinoma (n = 22); cervical carcinoma (n = 16); endometrial carcinoma (n = 4); gastric cancer (n = 13), colorectal adenocarcinoma (n = 23); pancreatic adenocarcinoma (n = 29); prostate cancer (n = 24); carcinoid (n = 18), growth hormone-secreting pituitary adenoma (n = 8); pheochromocytoma (n = 20); glioblastoma multiforme (n = 18); astrocytoma grade II (n = 8); astrocytoma grade III (n = 8). All tissue specimens had been fixed in formalin and were then embedded in paraffin.
Plasmids encoding the human CXCR4 and human CCR7 receptors were obtained from UMR cDNA Resource Center (Rolla, MO). Human embryonic kidney 293 (HEK-293) cells were stably transfected with either CXCR4 or CCR7. Cells were grown on coverslips overnight and either not exposed or exposed to 100 ng/ml SDF-1 or 100 ng/ml MIP-3 (R&D Systems). Cells were then fixed and incubated with anti-CCR7 {1188, 1 µg/ml}, anti-CXCR4 {UMB-2, hybridoma supernatant at a dilution of 1∶100}, anti-CXCR4 {6190, 1–10 µg/ml}, anti-CXCR4 {44716, 5–15 µg/ml} or anti-CXCR4 {12G5, 1–25 µg/ml} antibodies followed by the appropriate cyanine 3.18-conjugated secondary antibodies (Amersham, Braunschweig, Germany). Specimens were mounted and examined using a Leica TCS-NT laser scanning confocal microscope as described
HEK-293 cells stably transfected with CCR7 or CXCR4 as well as brains from CXCR4-deficient mice and their wild-type littermates were lysed in detergent buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.2 mM phenylmethylsulfonylfluoride, 10 mg/ml leupeptin, 1 mg/ml pepstatin A, 1 mg/ml aprotinin and 10 mg/ml bacitracin) and glycoproteins were enriched using wheat germ lectin agarose beads as described
Seven µm paraffin sections were cut and floated onto positively charged slides and immunohistochemically stained as described. For antigen retrieval, sections were microwaved in 10 mM citric acid (pH 6.0) for 20 min at 600 W. Specimens were then incubated with anti-CXCR4 {UMB-2, 1∶10}, anti-CXCR4 {6190, 1–10 µg/ml}, anti-CXCR4 {44716, 5–15 µg/ml} or anti-CXCR4 {12G5, 1–25 µg/ml} antibodies overnight at 4°C. Staining of primary antibody was detected using the appropriate biotinylated secondary antibodies followed by incubation with avidin-biotinylated peroxidase solution. Tissue was then rinsed and stained with diaminobenzidine for 15 min. Cell nuclei were lightly counterstained with hematoxylin. For immunohistochemical controls, the primary antibody was either omitted or adsorbed with homologous or heterologous peptides for 2 h at room temperature.
Specificity of anti-CXCR4 antibody {UMB-2} was monitored using Western blot analysis of extracts prepared from CXCR4-expressing and CCR7-expressing HEK 293 cells. When membrane preparations from these cells were electrophoretically separated and blotted onto nitrocellulose, UMB-2 revealed a single broad band migrating at
Specificity of UMB-2 was then monitored in Western blot experiments using tissues from CXCR4-deficient mice and their wild-type littermates. The sequence used to generate UMB-2 is identical in mice, rats and humans. Using the rabbit polyclonal anti-CXCR4 antibodies {2144} and {1181}, we have previously shown that the CXCR4 receptor is expressed in distinct populations of neurons in the embryonic brain and that CXCR4 expression strongly declines during development
First, UMB-2 was tested on a variety of formalin-fixed, paraffin-embedded human breast cancers, which have previously been reported to express CXCR4
A series of 247 human tissue samples was then immunohistochemically stained with UMB-2. Prominent CXCR4 staining of the tumor cells was observed in a number of mammary, pancreatic, prostate, cervical and ovarian carcinomas (
Tumor type (n) | CXCR4 n (%) | Tumoral blood vessels CXCR4 pos. n (%) |
Mammary carcinoma (36) | 13 (36) | 4 (11) |
Ovarian carcinoma (22) | 8 (36) | 0 |
Cervical carcinoma (16) | 6 (38) | 0 |
Endometrial carcinoma (4) | 1 (25) | 0 |
Gastric carcinoma (13) | 4 (30) | 2 (15) |
Colorectal carcinoma (23) | 5 (22) | 1 (4) |
Pancreatic carcinoma (29) | 16 (55) | 11 (38) |
Prostate carcinoma (24) | 19 (79) | 0 |
Carcinoid (18) | 7 (39) | 1 (6) |
GH-adenoma (8) | 0 | 0 |
Pheochromocytoma (20) | 1 (5) | 2 (10) |
Astrocytoma grade II (8) | 0 | 0 |
Astrocytoma grade III (8) | 0 | 0 |
Glioblastoma multiforme (18) | 0 | 16 (89) |
Finally, the rabbit monoclonal anti-CXCR4 antibody {UMB-2} was compared with mouse monoclonal antibody 12G5 and other frequently used commercially available antibodies. As depicted in Supplemental
In an effort to study the pattern of CXCR4 receptor protein expression in human normal and neoplastic tissues, we extensively characterized the novel rabbit monoclonal anti-CXCR4 antibody {UMB-2}. Our results demonstrate that the cytoplasmic tail of the CXCR4 receptor can serve as an epitope for the generation of monoclonal antibodies that effectively stain formalin-fixed, paraffin-embedded human and rodent tissues. Several lines of evidence indicate that UMB-2 specifically detects its targeted receptor and does not crossreact. First, in Western blots of membranes from CXCR4-transfected cells UMB-2 detected a broad band migrating at
In contrast to currently available anti-CXCR4 antibodies, the novel rabbit monoclonal antibody UMB-2 yielded highly effective plasma membrane staining of tumor cells without any detectable immunostaining of cell nuclei. The plasma membrane localization would be compatible with our understanding of CXCR4 function in cancer cell migration and homing
The rabbit monoclonal antibody UMB-2 will overcome a number of limitations of currently available antibodies, e.g. the most frequently used commercially available anti-CXCR4 antibodies have only been selected for their binding to native receptors on living cells and have not been adequately characterized on fixed cells and tissues
Comparative analysis of UMB-2 and currently available CXCR4 antibodies using transfected cells. A, Western blot analysis of the specificity of anti-CXCR4 antibodies. Membrane preparations from HEK-293 cells stably transfected to express either CCR7 or CXCR4 were separated on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were then incubated with 1 µg/ml anti-CCR7 {1188}, anti-CXCR4 {UMB-2} hybridoma supernatant at a dilution of 1∶100, 10 µg/ml anti-CXCR4 {6190}, 15 µg/ml anti-CXCR4 {44716} or 25 µg/ml anti-CXCR4 {12G5} antibodies. Blots were developed using enhanced chemiluminescence. Note that only UMB-2 detected the appropriate CXCR4 receptor band. Ordinate, migration of protein molecular weight markers (Mr×10−3). B, characterization of CXCR4 antibodies by immunofluorescent staining of transfected cells. B, HEK-293 cells stably transfected to express CCR7 or CXCR4 were either not exposed or exposed to 100 ng/ml MIP-3 or 100 ng/ml SDF-1 for 30 min, and subsequently fixed and immunofluorescently stained with 1 µg/ml anti-CCR7 {1188}, anti-CXCR4 {UMB-2} at a dilution of 1∶100, 10 µg/ml anti-CXCR4 {6190}, 15 µg/ml anti-CXCR4 {44716} or 25 µg/ml anti-CXCR4 {12G5} antibodies. UMB-2 selectively detected plasma membrane immunofluorescence in CXCR4-expressing cells that rapidly translocated into the cytosol after SDF-1 exposure. Note that none of the commercially available antibodies was able to detect CXCR4 in fixed cells or cell lysates under otherwise identical conditions. Representative results from one of two independent experiments are shown. Scale bar, 20 µm.
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Comparative analysis of immunohistochemical staining of human tissues using UMB-2 and currently available CXCR4 antibodies. Adjacent sections of a variety of human tumors were dewaxed, microwaved in citric acid and incubated with anti-CXCR4 {UMB-2} at a dilution of 1∶10, 10 µg/ml anti-CXCR4 {6190}, 15 µg/ml anti-CXCR4 {44716} or 25 µg/ml anti-CXCR4 {12G5} antibodies. Sections were sequentially treated with biotinylated secondary antibodies AB solution. Sections were then developed in diaminobenzidine and lightly counterstained with hematoxylin. Note that UMB-2 immunohistochemistry allowed a clear distinction between CXCR4 positive and CXCR4 negative tumors. Whereas anti-CXCR4 {44716} and anti-CXCR4 {12G5} produced a predominant nuclear staining, anti-CXCR4 {6190} produced no staining under otherwise identical conditions. Scale bar 50 µm.
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We would like to thank Dana Mayer for technical assistance, and Christoph Röcken, Christian Mawrin and Matthias Evert for providing reagents.