Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

Abstract

Background: Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro.

Methods: Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations.

Results: EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation.

Conclusion: CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

Figure 1

Sub-culturing of CC cell lines. Medium changes (ch) and time points for cell counting (count) for EGI-1 (A) and TFK-1 (B). Effects of cetuximab on growth of human CC cell lines was determined at the indicated time points. Apoptosis was observed by PI staining at the end of the sub-culture period.

Figure 2

RT-PCR for housekeeping genes and growth factor receptors. A) PCR analysis to ensue the presence and integrity of five housekeeping genes from TFK-1, representative for all cell lines used. B) mRNA expression of growth factor receptors in four CC cell lines (n. d. = not detected).

Figure 3

Western blot for growth factor receptors from different CC cell lines. EGFR, HGFR, IGF2R, VEGFR2 and 3 was detected in EGI-1, HuH28, OZ and TFK-1. IGF1R was visualized in EGI-1 and TFK-1. VEGFR1 was not in the investigated cell lines (n. d. = not determined).

Figure 4

IHC for growth factor receptors on different CC cell lines. Immunostaining of EGFR on EGI-1 (A), HuH28 (C), OZ (E) and TFK-1 (G) with the according isotype staining (B, D, F and H). Immunostaining of HGFR (I), IGF1R (J), IGF2R (K), VEGFR1 (L), VEGFR2 (M) and VEGFR3 (N). IHC revealed expression of the growth factor receptors tested in all human CC cell lines. ABC procedure (A-I, K-M) and APAAP method (J and N). Original magnification × 200.

Figure 5

Effects of cetuximab on growth of human CC cell lines. No growth inhibition was observed for EGI-1 (A). Anti-EGFR antibody decreased growth of TFK-1 (B).

Figure 6

Effects of cetuximab on apoptosis of TFK-1 cells. Single cell gate (A) and PI staining after 24 days of culture (B-G). Cetuximab mediates apoptotic effects in TFK-1 cells at 1000 μg/ml only.