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Immunohistochemical detection of the CXCR4 expression in tumor tissue using the fluorescent peptide antagonist Ac-TZ14011-FITC.

ABSTRACT: Pathology is fundamental in grading, staging, and treatment planning of malignancies. One relatively novel biomarker that may become more important in therapy and diagnostics is the chemokine receptor 4 (CXCR4). Ac-TZ14011 peptide derivatives, functionalized with a radiolabel, can be used for molecular imaging of tumors. Direct fluorescent labeling of the small peptide Ac-TZ14011 with the fluorescent dye fluorescein isothiocyanate (FITC), however, provides an alternative for the detection of CXCR4 expression levels in cells and tumor tissue. In this study, Ac-TZ14011-FITC was validated for CXCR4 staining in human breast cancer cell lines MDAMB231 and MDAMB231(CXCR4+) during flow cytometric analysis. Its efficacy was compared to commercially available antibodies. Competition experiments validated the staining specificity. Confocal imaging revealed that CXCR4 staining was predominantly found on the cell membrane and/or in vesicles formed after endocytosis. Next to being able to differentiate "high" and "low" CXCR4-expressing tumor cells, the fluorescent peptide demonstrates potential in fluorescent immunohistochemistry of tumor tissue. Ac-TZ14011-FITC was able to differentiate MDAMB231 from MDAMB231(CXCR4+) tumor cells and tissue, proving its applicability in the detection of differences in CXCR4 expression levels.

SUBMITTER: van den Berg NS

PROVIDER: S-EPMC3140011 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Immunohistochemical detection of the CXCR4 expression in tumor tissue using the fluorescent peptide antagonist Ac-TZ14011-FITC.

van den Berg Nynke S NS Buckle Tessa T Kuil Joeri J Wesseling Jelle J van Leeuwen Fijs W B FW

Translational oncology 20110801 4

Pathology is fundamental in grading, staging, and treatment planning of malignancies. One relatively novel biomarker that may become more important in therapy and diagnostics is the chemokine receptor 4 (CXCR4). Ac-TZ14011 peptide derivatives, functionalized with a radiolabel, can be used for molecular imaging of tumors. Direct fluorescent labeling of the small peptide Ac-TZ14011 with the fluorescent dye fluorescein isothiocyanate (FITC), however, provides an alternative for the detection of CXC ...[more]

PMID: 21804919

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Project description:This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.

Project description:BackgroundEwing sarcoma is an aggressive, highly metastatic primary bone and soft tissue tumor most frequently occurring in the bone of young adolescents. Patients, especially those diagnosed with a metastatic disease, have a poor overall survival. Chemokine receptor CXCR4 has a key pro-tumorigenic role in the tumor microenvironment of Ewing sarcoma and has been suggested to be involved in the increased metastatic propensity. Earlier studies on CXCR4 protein expression in Ewing sarcoma yielded contradictory results when compared to CXCR4 RNA expression studies. Previously, we demonstrated that CXCR4 expression could be detected in vivo using the fluorescently tagged CXCR4-specific peptide MSAP-Ac-TZ14011. Therefore, we studied the membranous CXCR4 expression in Ewing sarcoma cell lines using MSAP-Ac-TZ14011.MethodsThe CXCR4 membrane expression levels were studied in EWS cell lines by flow cytometry using the hybrid peptide MSAP-Ac-TZ14011 and were correlated to CXCR4 RNA expression levels. The measurements were compared to levels detected using the CXCR4 antibody ab2074 under various cell preparation conditions. In addition, the staining patterns were analyzed by confocal fluorescence microscopy over time.ResultsThe hybrid peptide MSAP-Ac-TZ14011 levels showed a strong and better correlation of CXCR4 membrane expression with the CXCR4 RNA expression levels than observed with the anti-CXCR4 antibody ab2074. With the hybrid peptide MSAP-Ac-TZ14011 using live cell confocal microscopy CXCR4 membrane staining and internalization was detected and the signal intensity correlated well with CXCR4 mRNA expression levels.ConclusionsThe fluorescently labeled CXCR4 targeting peptide-based method provides a reliable alternative to antibody staining to study the CXCR4 membrane expression in live cells using either flow cytometry or live cell fluorescence microscopy. The fluorescently tagged CXCR4 targeting peptide could enable in vivo detection of CXCR4 expression in Ewing sarcoma which may help to stratify cases for anti-CXCR4 therapy.

Dataset Information

Immunohistochemical detection of the CXCR4 expression in tumor tissue using the fluorescent peptide antagonist Ac-TZ14011-FITC.

Publications

Immunohistochemical detection of the CXCR4 expression in tumor tissue using the fluorescent peptide antagonist Ac-TZ14011-FITC.

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