Sand Laurens G L, Buckle Tessa, van Leeuwen Fijs W B, Corver Willem E, Kruisselbrink Alwine B, Jochemsen Aart G, Hogendoorn Pancras C W, Szuhai Károly
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden, The Netherlands.
BMC Cancer. 2017 May 26;17(1):383. doi: 10.1186/s12885-017-3352-z.
Ewing 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.
The 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.
The 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.
The 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.
尤因肉瘤是一种侵袭性强、具有高度转移性的原发性骨与软组织肿瘤，最常发生于青少年的骨骼。患者，尤其是那些被诊断为转移性疾病的患者，总体生存率较低。趋化因子受体CXCR4在尤因肉瘤的肿瘤微环境中具有关键的促肿瘤发生作用，并且被认为与转移倾向增加有关。与CXCR4 RNA表达研究相比，早期关于尤因肉瘤中CXCR4蛋白表达的研究结果相互矛盾。此前，我们证明了使用荧光标记的CXCR4特异性肽MSAP-Ac-TZ14011可在体内检测到CXCR4表达。因此，我们使用MSAP-Ac-TZ14011研究了尤因肉瘤细胞系中膜性CXCR4的表达。
通过流式细胞术使用杂交肽MSAP-Ac-TZ14011研究EWS细胞系中CXCR4膜表达水平，并将其与CXCR4 RNA表达水平相关联。将测量结果与在各种细胞制备条件下使用CXCR4抗体ab2074检测到的水平进行比较。此外，通过共聚焦荧光显微镜随时间分析染色模式。
与抗CXCR4抗体ab2074相比，杂交肽MSAP-Ac-TZ14011水平显示CXCR4膜表达与CXCR4 RNA表达水平之间具有更强且更好的相关性。使用活细胞共聚焦显微镜，通过杂交肽MSAP-Ac-TZ14011检测到CXCR4膜染色和内化，并且信号强度与CXCR4 mRNA表达水平良好相关。
基于荧光标记的CXCR4靶向肽的方法为使用流式细胞术或活细胞荧光显微镜研究活细胞中CXCR4膜表达提供了一种可靠的替代抗体染色的方法。荧光标记的CXCR4靶向肽能够在体内检测尤因肉瘤中CXCR4的表达，这可能有助于对抗CXCR4治疗的病例进行分层。
