Abstract 1592: CXCR4 pathways in CTCs: from bioinformatics to immunophenotype

Abstract Introduction: Bioinformatics’ analysis regarding gene expression in Normal tissue vs cancer tissue, Normal blood vs cancer patients’ blood and Normal blood vs cancer tissue revealed a subgroup of 24 genes which potentially could be overexpressed in CTCs. Among these genes are: CXCR4, a chemokine receptor which is involved in tumor metastasis and, JUNB a transcription factor participating in CXCR4 pathway. The objective of this study was to confirm the results of bioinformatics’ analysis in samples from breast cancer patients and therefore to evaluate the expression of CXCR4 and JUNB in CTCs. Methods: Triple staining immunofluoresence with panytokeratin/CXCR4/JUNB antibodies were performed in SKBR3, MDA-MB231, MCF7 and Hela cell lines. The same experiments were performed in PBMCs from normal (n = 10.) subjects and in PBMCs (n = 55) from untreated metastatic breast cancer patients. Results: Since CXCR4, as a chemokine receptor is also expressed on hematopoietic cells, its expression on CTCs was quantified using the ARIOL system. We used cell lines and both normal vs patients’ PBMCs in order to establish an expression pattern of both molecules. Quantification experiments among cell lines revealed that CXCR4 was overexpressed in SKBR3 following the subsequent hierarchy SKBR3>MCF7>MDA-MB231>Hela. Accordingly, the expression pattern of JUNB in cell lines was SKBR3>Hela>MCF7>MDA-MB231 with higher expression in SKBR3 and lower in MDA-MB 231. Statistical analysis revealed significant differences in the expression of both molecules between healthy donors’ PBMCs and patient's PBMCs. Subsequently, CTCs were detected in 17 out of 55 screened patients (31%). Mean intensity of CTCs for CXCR4 was 34.38 and it was higher than Hela and lower than SKBR3. Patients with CXCR4-positive CTCs (with mean expression higher than 95% of normal PBMCs) were 53%. In addition 84% of the examined CTCs had expression profile higher than normal PBMCs. JUNB expression in CTCs (above 95% of normal PBMCs) was identified in 76.92% of patients. JUNB mean expression in CTCs was 26.1 and it was lower than SKBR3 and higher than MCF7. Furthermore 64.3% of the total CTCs have expression higher than Normal PBMCs. Conclusion: CXCR4 and JUNB are highly expressed in CTCs derived from breast cancer patients, in agreement to bioinformatics’ analysis. Quantification of immunofluoresence potentially delineates a subgroup of patients with high expression of CXCR4 and JUNB that could benefit from target therapies. Citation Format: G. Kallergi, V. Tsintari, S. Sfakianakis, M. Zervakis, D. Mavroudis, V. Georgoulias. CXCR4 pathways in CTCs: from bioinformatics to immunophenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1592. doi:10.1158/1538-7445.AM2015-1592