PLoS One. 2013;8(2):e56836.

Insulin-like growth factor receptor I (IGF-IR) and vascular endothelial growth factor receptor 2 (VEGFR-2) are expressed on the circulating epithelial tumor cells of breast cancer patients.

Pizon M, Zimon DS, Pachmann U, Pachmann K.

Transfusion Center Bayreuth, Bayreuth, Germany.



BACKGROUND: Circulating epithelial tumor cell (CETC) analysis is a promising diagnostic field for estimating the risk for metastatic relapse and progression in patients with malignant disease. CETCs characterization can be used as a liquid biopsy for prognostic and predictive purposes in breast and other cancers. IGF-IR and VEGFR-2 play an important role in tumor growth and the progression of cancer disease. The purpose of the current study was therefore to investigate their expression on CETCs.

METHODS: CETCs were determined from the blood of 50 patients suffering from breast cancer. The number of vital CETCs and the expression of IGF-IR and VEGFR-2 were investigated using the maintrac® method.

RESULTS: IGF-IR and VEGFR-2 expression on the surface of CETCs were detected in 84% of patients. A statistically high correlation was found between IGF-IR and VEGFR-2 (r = 0.745 and p<0.001) on the CETCs. The co-expression of both receptors was confirmed in some experiments and ranged between 70% and 100%. Statistically significant correlations were observed between the number of CETCs and IGF-IR (r = 0.315 and p<0.05) and VEGFR-2 (r = 0.310 and p<0.05) expression. The presence of CETCs and the level of IGF-IR and VEGFR-2 expression were not associated with tumor stage, hormone receptor status or nodal/distant metastasis.

SUMMARY: In this study, a parallel and co-expression of IGF-IR and VEGFR-2 was examined on the surface of CETCs in breast cancer patients for the first time. Characterization of CETCs may be a promising approach for the rational design of targeted anticancer therapies.

PMID: 23418605




Breast cancer is one of the most frequent cancers among women in the Western world. Despite improvements in early diagnosis and clinical management, breast cancer kills more than 520,000 people worldwide each year [1]. Most breast cancer deaths are due to metastatic disease [1].

The origin of distant metastases must be cells that have been shed by both primary and metastatic cancers into the blood. Thus circulating tumor cells mediate the hematogenous spread of cancer to distant sites [2]. In many studies, the detection of tumor cells in blood in early and metastatic disease has been shown to correlate with an unfavourable clinical outcome [3]. Using maintrac®, a non-dissipative approach, the enumeration and monitoring the behaviour of circulating epithelial cells (CETCs) in patients with many types of cancer contribute to supervise therapy [4]. They directly reflect the patient´s response or lack of response to therapy allowing for changing and optimizing the current strategy of therapy. Moreover, further characterization of CETCs can be used as a liquid biopsy for repeated follow-up examinations in a variety of human cancers [5].

It has been shown that primary breast tumors as well as CETCs can vary phenotypically between one another and intrinsically. The analysis of these variations could provide a very important tool for the development of new therapeutic strategies [6] and to improve the understanding of molecular events and critical pathways involved in breast cancer. Targeted therapies require the identification of novel targets that have high specificity for the molecules involved in cell growth, survival, migration, invasion, metastasis, apoptosis, cell-cycle progression and angiogenesis [7].

Two receptors, IGF-IR and VEGFR-2, have been shown to play an important role in the growth of the primary tumor and metastasis formation.

IGF-IR belongs to the family of transmembrane receptor tyrosine kinases and is expressed on the cell surface of most tissues. Physiologically, this receptor and its ligands play a key role in the regulation of growth and metabolism. It has recently been demonstrated that IGF-IR is also a key player in cancer development and progression [8]. IGF-IR plays a major role in cancer cell proliferation and survival, and confers resistance to cytotoxic, hormonal and targeted therapies in breast cancer. The signalling through the IGF-IR has been implicated in the resistance to anti-cancer agents, including inhibitors of the HER family of receptors [9].

VEGFR-2 is also a transmembrane receptor that has an important role in endothelial cell development [10]. The majority of VEGFR-2 actions are related to angiogenesis, which is a critical event in tumor progression and metastasis [11]. In addition to its function in angiogenesis, VEGF signalling has been implicated in the ability of breast cancer to proliferate, evade apoptosis and migrate. The VEGFR-2 receptors are widely expressed in breast cancer and also in other tumors including lung, colon, uterus and ovarian cancers [12].

In the present study the number of CETCs was determined per ml of peripheral blood and the parallel expression of these two markers investigated on these cells. Typical such cells are shown in Figure 1. There was no statistically significant difference in CETC numbers according to tumor size, lymph node status and presence of metastasis or clinical pathology. CETCs with additional expression of IGF-IR or VEGFR-2 on their surface were observed in 84% of patients (Fig. 2). The median percentage of CETCs expressing IGF-IR was 32.9% (range 0-83.3%) and expressing VEGFR-2 was 50% (range 0-100%) (Fig. 3). We could show that there was a statistically significant difference between the median of IGF-IR and VEGFR-2 expressing CETCs. The expression of IGF-IR and VEGFR-2 was highly correlated (Fig. 4). However, the expression of either IGF-IR or VEGFR-2 on CETCs did not correlate with ER/PR and Her2/neu status of the primary tumor. The co-expression of both receptors was confirmed in 40 patients and ranged between 70% and 100% (Fig. 5).

Taken together our current study demonstrates that IGF-IR and VEGFR-2 are frequently expressed on the CETCs of patients with breast cancer, independent of stage of disease. IGF-IR and VEGFR-2 may represent important components of growth factor signalling in breast cancer. Using CETCs as a liquid biopsy could aid in selecting appropriate patients for targeted and personalized treatment strategies against cancer.


Legends for figures:

Figure 1Figure 1: Immunostaining of CETCs with anti-EpCAM-FITC- and anti-IgG1-PE-antibody. There is no unspecific staining with anti-IgG1(mouse)-PE-antibody. a) Labeling of the whole cell membrane with an additional cap; b),c) differently intense labelling of the cell membrane because EpCAM-fluorescence varies strongly between the individual cells; d) only exclusively surface located cap is stained.


Figure 2Figure 2: Immunostaining of CETC with anti-EpCAM-FITC and anti-IGF-IR-PE antibodies. a) Typical epithelial antigen-positive cell with green fluorescence. b), c) positive EpCAM CETC, which also has a red surface staining for IGF-IR.


Figure 3Figure 3: Median of percentage IGF-IR and VEGFR-2 expression on the CETCs in 50 breast cancer patients.


Figure 4Figure 4: Correlation between the expression of IGF-IR and VEGFR-2 on the surface of CETCs in 50 breast cancer patients.


Figure 5Figure 5: Fluorescence co-localization of EpCAM, IGF-IR and VEGFR-2 expression on the CETCs. a) Typical CETC with EpCAM green staining, b) the same CETC with IGF-IR red staining, c) CETC with VEGFR-2 blue staining, d) merge of EpCAM and IGF-IR expression, e) merging of EpCAM and VEGFR-2 expression and f) merging of EpCAM, IGF-IR and VEGFR-2 expression on the same CETC.



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