Fluorescent CXCR4 targeting peptide as alternative for antibody staining in Ewing sarcoma

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3352-z) contains supplementary material, which is available to authorized users.

Introducing fluorescence guided surgery into orthopedic oncology: A systematic review of candidate protein targets for Ewing sarcoma

“…A surfaceome study of ES by Town et al showed no expression of CXCR4 in MSC, which makes CXCR4 an attractive target for targeted imaging. Sand et al studied membranous CXCR4 expression in ES cell lines using a fluorescently labeled CXCR4 targeting peptide (MSAP‐Ac‐TZ14011), indicating the feasibility of this agent for FGS. The first in human experience with a radiolabeled version of a similar peptide (Pentixafor) in a small and heterogeneous patient cohort did not completely fulfill the expectations in comparison with standard (18)F‐FDG PET …”

“…CXCR4/SDF‐1 signaling plays a role in chemotaxis of hematopoietic cells and in neuron generation during embryogenesis and adult life. It is absent in most healthy tissues but upregulated in the tumor microenvironment of many tumor types, where it is associated with metastasis, angiogenesis, and tumor growth . Also in ES CXCR4 is associated with tumor progression and metastasis .…”

“…Also in ES CXCR4 is associated with tumor progression and metastasis . Overall 82% of ES tumors are positive for CXCR4 (range 64% to 100%), with cytoplasmic and membranous staining varying from weak to strong . A surfaceome study of ES by Town et al showed no expression of CXCR4 in MSC, which makes CXCR4 an attractive target for targeted imaging.…”

“…Finding a good target might be challenging because of its uniqueness and the limited similarities with other tumor types. Sand et al studied membranous CXCR4 expression on ES cell lines using a fluorescently labeled CXCR4 targeting peptide. They showed that the fluorescently tagged CXCR4 targeting peptide was able to detect CXCR4 on living ES cells.…”

Cyanine Conjugate‐Based Biomedical Imaging Probes

“…[114] Besides, a subset of SPECT/NIRF dual-modal probes for targeted imaging were assembled based on a lysine scaffold: -NH 2 for cyanine fluorophores, -NH 2 for 111 In-DTPA and -COOH for various targeting peptides including cRGD (78), [115] antimicrobial peptide UBI 29−41 (79), [116] and CXCR4 targeting peptide Ac-TZ14011 (80). [117] Unlike most short-lived PET radionuclides, 111 In is suitable for the development of antibody-guided probes owing to its better compatibility with mAbs pharmacokinetics. A case in point is the CAIX-targeting 111 In-girentuximab-IRDye800CW (81), which has shown its feasibility and safety in a phase I study in clear cell renal cell carcinoma patients (NCT02497599) (Figure 14b).…”