In this study we have demonstrated that a CXCR4 targeting fluorescent T140 analogue, MSAP-Ac-TZ14011 tracer peptide, can be used as an alternative for antibodies to determine the CXCR4 cell membrane expression levels in EWS cell lines and that the binding of MSAP-Ac-TZ14011 is not significantly influenced by the used cell dissociation method. The measured levels of cell bound MSAP-Ac-TZ14011, and thereby indirectly the measured CXCR4 levels, were correlated to
CXCR4 mRNA expression and compared with CXCR4 cell membrane levels detected by antibody staining. As the CXCR4 signaling pathway have a stimulating role in the main processes of the TME in many tumor types, CXCR4 could be a candidate biomarker and a potential therapeutic target. [
5,
7]. Moreover, treatment with CXCR4 antagonist T140 and analogues like Ac-TZ14011 inhibited tumor growth [
33]. In EWS, however, although RNA expression has been reported, protein expression in metastases was absent in paraffin embedded material using immunohistochemistry [
8,
9]. CXCR4 consists of multiple isoforms with varying N-terminal ends of which one, CXCR4–2, is dominantly expressed [
34]. Both the N- and C-terminal ends of CXCR4 contain many potential post-translational modification sites and changes at these sites may influence antibody recognition, potentially explaining the various staining patterns observed in earlier studies [
35‐
37]. In addition, CXCR4 protein expression analysis by Western blotting with N-terminal and C-terminal specific antibodies revealed inconclusive staining patterns (Additional file
6: Fig. S3). As the T140 binding site does not contain any reported post-translational modifications and
in situ modeling suggests MSAP-Ac-TZ14011 binds at the same site, this detection could be a better alternative to detect all forms of the receptor [
18,
23,
35,
38]. The detected MSAP-Ac-TZ14011 signals correlated linearly with the earlier obtained RNA expression levels in EWS cell lines and co-localize with CXCR4-GFP expressed in MDA-MB-231 X4 (Figs.
1b and
2a, b). In addition, the detected MSAP-Ac-TZ14011 levels using flow cytometry corresponded to the observations during live cell imaging of the cells (Figs.
1a and
2c, e). Limitations of this method are that cells should be stained alive with MSAP-Ac-TZ14011 and not fixed with methanol prior to staining.
The observed internalization of the MSAP-Ac-TZ14011 in TC32 and MDA-MB-231 X4 confirms previously reported internalization of Ac-TZ14011-FITC [
22]. The CXCR4-GFP overlap with MSAP-Ac-TZ14011 during CXCR4 internalization in MDA-MB-231 X4 and the overlap of MSAP-Ac-TZ14011 with the lysotracker DND-26 in TC32 support the suggestion that upon CXCR4 binding the peptide-CXCR4 complex is internalized. This peptide therefore can be used to detect intracellular located CXCR4 in cells when incubated over a longer period at standard culture conditions. In addition, Ac-TZ14011-based analogues have been studied in in vivo breast-tumor models based on immune uncompromised mice, and the efficacy of this approach has been shown in multiple publications [
20,
24,
39,
40]. One CXCR4-targeting imaging agent has even been studied in a recent clinical trial, indicating the suitability of it to identify tumor cells in patients [
41]. In the preclinical setting the in vivo biodistribution of Ac-TZ14011 analogues was shown to be identical to the distribution in immune-deficient nude mice bearing low CXCR expressing control tumors, with exemption of the uptake levels in the tumor. Evaluation of immune cells in the CXCR4 positive tumors revealed the presence of minor amounts of immune cells in the tumor (1.0–1.3%), which did not influence the visualization of the tumor [
24]. This indicates that effective tumor detection would still be possible, even when a CXCR4 positive stromal cell infiltrates are present. Based on this, we propose that the in vivo detection of CXCR4 expressing tumors in EWS patients might well be possible.
CXCR4 is involved in metastasis and increased CXCR4 RNA expression levels were measured in both metastasis derived cell lines compared to non-metastasis derived cell lines and metastases compared to localized tumors [
8]. In addition, factors inhibiting CXCR4 activation can be used to identify high risk patients [
10]. The detected MSAP-Ac-TZ14011 levels in EWS cell lines positively correlated with the CXCR4 RNA expression levels and the MSAP-Ac-TZ14011 signal overlapped with the CXCR4-GFP membrane signal. When assuming the MSAP-Ac-TZ14011 fluorescence level is correlated to the CXCR4 cell membrane level, metastasis might have a higher CXCR4 cell membrane expression than localized tumors. Such a positive correlation between the migration/invasiveness of a cell line and the CXCR4 cell membrane expression has been observed both in EWS and breast cancer cell lines [
24,
32]. However, clinical data on the cell membrane expression of CXCR4 and its relation with tumor invasiveness is still lacking. Using this method might enable determination of CXCR4 cell membrane expression in patients. This would help to stratify patients for alternative therapies, like anti-CXCR4 therapy and might serve as prognostic marker for EWS patients [
9].