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Comparative Study
. 2008;10(5):R80.
doi: 10.1186/bcr2149. Epub 2008 Sep 29.

Phosphorylated EGFR and PI3K/Akt signaling kinases are expressed in circulating tumor cells of breast cancer patients

Galatea Kallergi  1 , Sofia Agelaki, Antonia Kalykaki, Christos Stournaras, Dimitris Mavroudis, Vassilis Georgoulias
Affiliations
Comparative Study

Phosphorylated EGFR and PI3K/Akt signaling kinases are expressed in circulating tumor cells of breast cancer patients

Galatea Kallergi et al. Breast Cancer Res. 2008.

Abstract

Introduction: The phosphoinositide-3 kinase (PI3K)/Akt pathway, operating downstream of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER)2, is implicated in cell migration and survival. EGFR and HER2 are expressed in circulating tumor cells, but the activation status of downstream signaling molecules has not yet been reported.

Methods: To investigate expression levels of EGFR, HER2, PI3K, and Akt in circulating tumor cells, we used peripheral blood mononuclear cells from 32 cytokeratin-19 mRNA-positive patients with early (n = 16) and metastatic (n = 16) breast cancer.Peripheral blood mononuclear cell cytospins were double stained with cytokeratin antibody along with one of the following: EGFR, phospho-EGFR, HER2, phospho-PI3K, or phospho-Akt antibodies.

Results: EGFR and HER2 were expressed in circulating tumor cells of 38% and 50% patients with early and 44% and 63% patients with metastatic disease, respectively. Interestingly, phospho-PI3K and phospho-Akt expression levels were similar at 88% (14 out of 16) and 81% (13 out of 16), respectively, in circulating tumor cells of patients with early and metastatic disease. Phospho-EGFR was observed in circulating tumor cells of two (33%) early and six (86%) metastatic EGFR-positive patients. Immunomagnetic separation of peripheral blood mononuclear cells, using EpCAM antibody, and subsequent double-staining experiments of circulating tumor cells showed that EGFR was co-expressed with HER2, phospho-Akt and phospho-PI3K kinases, indicating activation of the corresponding survival signaling pathway.

Conclusions: Our findings demonstrate that circulating tumor cells express receptors and activated signaling kinases of the EGFR/HER2/PI3K/Akt pathway, which could be used as targets for their effective elimination.

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Figures

Figure 1
Figure 1
EGFR and HER2 expression in CTCs from breast cancer patients. (a) Representative images of confocal laser scanning microscopic sections of PBMC cytospins stained with pancytokeratins A45 B/B3 (green) and EGFR anti-rabbit (red) antibodies, showing consecutive scanning sections from the upper cytoplasmic region toward the basal attachment site in step sizes of 0.8 μm (magnification: 600×). (b) Representative images of a cytospin, double stained with monoclonal pancytokeratin (A45-B/B3; green) and polyclonal EGFR anti-rabbit (red) antibodies (magnification: 600×). Also shown is quantification of EGFR and CK co-expression in adjuvant and metastatic CTCs. (c) Representative image of cytospin, double stained with polyclonal pancytokeratin and monoclonal HER2 (green) antibodies (magnification: 600×). Quantification of HER2 and CK co-expression in adjuvant and metastatic CTCs. CK, cytokeratin; CTC, circulating tumor cell; EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; PBMC, peripheral blood mononuclear cell.
Figure 2
Figure 2
EGFR, PI3K, and Akt activation in CTCs from breast cancer patients. (a) Representative micrograph of cytospin, double-stained with monoclonal pancytokeratin (A45-B/B3; green) and polyclonal pEGFR anti-rabbit (red) antibodies (magnification: 600×). Also shown is quantification of pEGFR and CK co-expression in adjuvant and metastatic CTCs. (b) Representative image of a cytospin, double stained with monoclonal pancytokeratin (A45-B/B3; green) and polyclonal pPI3K (red) antibodies (magnification: 400×). Also shown is quantification of pPI3K and CK co-expression in adjuvant and metastatic CTCs. (c) Representative micrograph of cytospin, double stained with monoclonal pancytokeratin (A45-B/B3; green) and polyclonal pAkt (red) antibodies (magnification: 600 ×). Also shown is quantification of pAkt and CK co-expression in adjuvant and metastatic CTCs. CK, cytokeratin; CTC, circulating tumor cell; EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; PI3K, phosphoinositide-3 kinase.
Figure 3
Figure 3
Immumomagnetic separation of CTCs. Representative confocal laser scanning micrographs of cytospins after immunomagnetic separation of CTCs with EpCAM antibody. Cell cytospins were double stained with EGFR antibody and one of the following: HER2, pEGFR, pPI3K, or pAkt antibody. Cytospin were also double stained with HER2 antibody and either pPI3K or pAkt antibodies (magnification: 400×). EGFR is co-expressed with HER2, pEGFR, phospho-PI3K, and pAkt kinase. CTC, circulating tumor cell; EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; PBMC, peripheral blood mononuclear cell; PI3K, phosphoinositide-3 kinase.
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