CXCR4 expression in different subsets of CTCS and single (detached) breast cancer cells

Circulating tumor cells (CTCs) mediate tumor dissemination and play the key role in the metastatic cascade [1, 2]. The prognostic value of CTCs in breast cancer has been previously shown. Thus, presence of CTCs in the peripheral blood is associated with the poor prognosis in breast cancer [3, 4]. However, little is known about exact mechanism of migration and trafficking of CTCs in the peripheral blood circulation [2]. Metastasis of cancer cells to distant locations needs intravasation from the primary tumor sites into blood vessels, survival in the circulation, migration to secondary organs, adhesion, and proliferation of cancer cells in targeting organs and tissues [5]. Chemokines might affect the selection of target organs for metastases formation. For example, the CXCR4/ CXCL12 axis makes breast cancer cells move out of the circulation and traffic into organs with high amounts of chemokines, and thus forming metastases [6, 7]. In our previous study, we showed that CTCs were represented by heterogeneous population. Some cancer cells had stem-like or/and EMT (epithelialmesenchymal transition) phenotype, other cancer cells did not have EMT and stemness features [8]. We suggested that CTCs did not always precede metastasis, because they could not have the necessary features, including their CXCR4 expression. So, in present study we investigated the CXCR4 expression in different subsets of CTCs and single (detached) breast cancer cells in primary tumor.

Material and Methods

Patients (n=35) with invasive breast carcinoma of no special type (IC NST) (T1-4N0-2M0) within the range of 29 - 69 years of age (mean age: 49.06 ± 9.78) were treated at the Cancer Research Institute, Tomsk NRMC (Tomsk, Russia) between 2011 and 2017 included (Table 1). IC NST was defined according to the World Health Organization’s recommendations [9]. The venous blood samples were collected in EDTA-treated tubes before surgical intervention and were used for flow cytometry. The formalin-fixed, paraffin-embedded (FFPE) primary tumor samples were used for immunofluorescence analysis. This study was approved by the institutional review board, all patients signed an informed consent for voluntary participation.

Flow Cytometry

Confocal microscopy

Results

As CXCR4 promotes breast cancer metastasis to distant organs where its ligand, SDF-1, is generated in large quantity [10, 11], we examined the expression of these molecules in different subsets of CTCs in the blood of breast cancer patients (Figure 1). The

CXCR4 expression in CTCs without stem-like and EMT phenotype (CK7+CD45-CD44-CD24+/-N-cadherin-CXCR4+) was present in 31.8% (7/22) patients (0.00(0.00-0.95) cell/ml). In 68.2% (15/22) patients, CTCs with CXCR4 exhibited EMT but not stem markers (CK7+CD45-CD44-CD24+/-N-cadherin+CXCR4+) (1.96(0.13-3.59) cell/ml). The stem-like CTCs expressed CXCR4 in 27.3% (6/22) patients. All this cells (0.00(0.00-0.13) cell/ml) didn’t have EMT features (CK7+CD45-CD44+CD24-N-cadherin-CXCR4+). In all patient blood samples (22/22) the CXCR4 expression in CTCs with stem-like and EMT phenotype (CK7+CD45-CD44+CD24-N-cadherin+CXCR4+) was absent (Table 2).

Figure 2. The single (detached) breast cancer cells with CXCR4 expression. The white cursor points to the non stem single (detached) breast cancer cell with EMT features and CXCR4 expression

Figure 1. Flow cytometry analysis of the circulating tumor cells with CXCR4 expression

The non stem-like single (detached) breast cancer cells with EMT features (CK7+CD133-N-cadherin+CXCR4+) expressed CXCR4 in 44.0% (11/25) tumor samples (Figure 2). Our results suggested that only 0.00(0.00-8.00)% of single (detached) CK7+CD133-N-cadherin+ cells exhibited CXCR4 in primary tumor. The single (detached)