New molecular tools and optical technologies to dissect CXCR4 function in breast cancer

Background

The objective was to study the relationship between CXCR4 expression (total and conformational subsets) and disease outcome in malignant breast disease. Initially, a retrospective study evaluating the clinical significance of CXCR4 expression (as determined by immunohistochemistry and immunofluroscence) with histopathological grade and clinical outcome of breast cancer patients were evaluated.

Methods

Tumour specimens from breast cancer patients treated at the Breast Unit at Guy's Hospital London, with prospectively acquired long-term follow-up (25 years) were used in this study.

Using tissue microarrays (TMAs), of primary breast tumour specimens from a series of 252 invasive ductal and lobular carcinomas were immunolabelled for CXCR4. Polyclonal antibodies to human CXCR4 (Anti-Human CXCR4 ARP4016){CXCR4 peptide ARP 7039 N-terminal extracellular domain (1–38)} and two further anti-human CXCR4 cytoplasmic antibodies against two distinct peptides based on the membrane proximal sequence (GAKFKTSAQHALTSVSRG) and distal cytoplasmic sequence (VSTESESSSFHSS) of huCXCR4 cytoplasmic domain, were used to detect CXCR4.

The immunohistochemical detection of CXCR4 expression, was assessed by 2 independent pathologists (with consensus agreement). Both the proportion and intensity of expression was recorded for the total and subpopulations of CXCR4 recognised by ARP4016 and the two cytoplasmic antibodies, respectively.

For immunofluroscence the average fluorescence intensity/unit area of cells stained with the respective antibodies were plotted and quantified.

Results

The proportion and intensity of invasive cells expressing CXCR4 was significantly less in Grade III infiltrating ductal carcinoma compared with Grades I, II and lobular types (P < 0.0001 by Kruskal-Wallis). There is a complex relationship between survival and total CXCR4 expression, with a subset of high CXCR4 expressing, Grade III tumours showing a trend towards poor prognosis. This association will be further elucidated by results of the CXCR4 cytoplasmic antibody staining.

Conclusion

CXCR4 was expressed uniformly across a spectrum of normal, and a panel of invasive breast tumour cells but only a subset of Grade III tumours expressing high CXCR4 correlated with poor prognosis. It may be that only highly invasive cells that are metastatic and very poorly differentiated express functional CXCR4 receptors. CXCR4 function is subject to complex and potentially tightly controlled regulation in breast cancer cells via differential G protein receptor complex formation and this regulation may play a role in the transition from non metastatic to malignant transformation [1]. The application of new antibody tools and optical technologies to these pathological samples will assist the discovery of new biomarkers that will report on the function of CXCR4 in situ.