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.

Figure 1

Ac-TZ14011-FITC distribution in live and formalin-fixed MDAMB231 and MDAMB231^CXCR4+ tumor cells. (A) Live MDAMB231 and MDAMB231^CXCR4+ cells were incubated with Ac-TZ12011-FITC for 1 hour on ice. (B) Formalin-fixed cells were incubated for 1 hour with Ac-TZ12011-FITC on ice. (C) MDAMB231^CXCR4+ cells were incubated for 1 hour with Ac-TZ12011-FITC on ice, and confocal images were taken (t = 0 minute). Over time, Ac-TZ12011-FITC-CXCR4 receptor complexes internalized via vesicles (t = 0–60 minutes). Original magnification, x630.

Figure 2

Predominant cytoplasmatic CXCR4 expression on formalin-fixed paraffin-embedded and freshly isolated, formalin-fixed, breast cancer tissue. (A) Formalin-fixed paraffin-embedded slides were incubated with the primary anti-CXCR4 antibody clone 12G5, (B) primary anti-CXCR4 antibody clone 2B11 (original magnification, x400; n = 5–10), or (C) incubated with Ac-TZ12011-FITC (original magnification, x630; n = 3). (D) Freshly isolated, formalin-fixed tumor tissue incubated with Ac-TZ12011-FITC showed a different staining pattern. Original magnification, x630; n = 3.