Erlotinib Promotes Ligand-Induced EGFR Degradation in 3D but Not 2D Cultures of Pancreatic Ductal Adenocarcinoma Cells

Abstract

The epithelial growth factor receptor (EGFR) is a tyrosine kinase receptor that participates in many biological processes such as cell proliferation. In addition, EGFR is overexpressed in many epithelial cancers and therefore is a target for cancer therapy. Moreover, EGFR responds to lots of stimuli by internalizing into endosomes from where it can be recycled to the membrane or further sorted into lysosomes where it undergoes degradation. Two-dimensional cell cultures have been classically used to study EGFR trafficking mechanisms in cancer cells. However, it has been widely demonstrated that in 2D cultures cells are exposed to a non-physiological environment as compared to 3D cultures that provide the normal cellular conformation, matrix dimensionality and stiffness, as well as molecular gradients. Therefore, the microenvironment of solid tumors is better recreated in 3D culture models, and this is why they are becoming a more physiological alternative to study cancer physiology. Here, we develop a new model of EGFR internalization and degradation upon erlotinib treatment in pancreatic ductal adenocarcinoma (PDAC) cells cultured in a 3D self-assembling peptide scaffold. In this work, we show that treatment with the tyrosine kinase inhibitor erlotinib promotes EGFR degradation in 3D cultures of PDAC cell lines but not in 2D cultures. We also show that this receptor degradation does not occur in normal fibroblast cells, regardless of culture dimensionality. In conclusion, we demonstrate not only that erlotinib has a distinct effect on tumor and normal cells but also that pancreatic ductal adenocarcinoma cells respond differently to drug treatment when cultured in a 3D microenvironment. This study highlights the importance of culture systems that can more accurately mimic the in vivo tumor physiology.

Keywords: 3D culture; EGFR; PDAC; degradation; drug resistance; pancreatic ductal adenocarcinoma; self-assembling peptides; trafficking.

Figure 1

Cell culture in RAD16-I scaffold. (a)Viability of BxPC-3, PANC-1 and hNDF cultured in 0.15% and 0.5% RAD16-I scaffold measured by MTT assay at day 1 and day 7 of culture. Statistical differences are indicated as * for p < 0.05, ** for p < 0.01, *** for p <0.001 and **** for p < 0.0001, two-way ANOVA, N = 2, n = 3); (b) Z-projection pictures of BxPC-3, PANC-1 and hNDF cells at day 6 of culture stained with Phalloidin (pseudo-colored in yellow) and DAPI (blue). Scale bars represent 20 µm; (c) macroscopic view of hNDF and (d) PANC-1 3D constructs in 0.15% and 0.5% RAD16-I scaffolds at different time points.

Figure 2

EGFR expression in 2D and 3D cultures. (a) EGFR (red) and β1-integrin (green) immunofluorescence counterstained with DAPI (grey) in BxPC-3, PANC-1 and hNDF cells cultured in 2D monolayer and (b) Z-projection of EGFR and β1-integrin immunofluorescence in BxPC-3, PANC-1 and hNDF cells in 3D RAD16-I scaffold at 0.15% peptide concentration. Scale bars represent 10 µm; (c) Western blot bands of EGFR in 2D and 3D cultures; (d) quantification of EGFR in 3D cultures represented as the ratio between soft and stiff cultures. GAPDH was used as loading control. One representative blot is shown. Experiments were repeated three times (N = 3), and statistical differences are indicated as **** for p < 0.0001.

Figure 3

Immunofluorescence analysis of the EGFR in PANC-1 cells incubated with EGF, erlotinib or both in 2D cultures. (a) EGFR (red) and β1-integrin (green) immunofluorescence counterstained with DAPI (grey) in the presence of EGF, erlotinib or both in PANC-1 and close-up sections (gray pictures) of the cell periphery and the perinuclear area labeled with white and empty arrows, respectively. Scale bars represent 10 µm; (b) fluorescence intensity profiles corresponding to the white line in pictures from (a). Different cell regions are indicated as P for cell periphery, N for nucleus and PN for perinuclear area; (c) Manders’ colocalization coefficients. Statistical differences are indicated as * for p < 0.05, ** for p < 0.01 and **** for p < 0.0001.

Figure 4

Immunofluorescence analysis of the EGFR in BxPC-3 cells incubated with EGF, erlotinib or both in 2D cultures. (a) EGFR (red) and β1-integrin (green) immunofluorescence counterstained with DAPI (grey) in the presence of EGF, erlotinib or both and close-up sections (gray pictures). Scale bars represent 10 µm; (b) fluorescence intensity profiles corresponding to the white line in pictures from (a). Different cell regions are indicated as: P for cell periphery, N for nucleus and PN for perinuclear area; (c) Manders’ colocalization coefficients. Statistical differences are indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001.

Figure 5

Immunofluorescence analysis of the EGFR in hNDF cells incubated with EGF, erlotinib or both in 2D cultures. Scale bars represent 10 µm. Insets represent high magnification images of the region indicated by a white square.

Figure 6

EGFR immunofluorescence in PANC-1, BxPC-3and hNDF cells incubated with EGF, erlotinib or both in 0.15% RAD16-I 3D cultures. One representative Z plane is shown. Scale bars represent 20 µm. Insets represent high magnification images of the region indicated by an asterisk (*).

Figure 7

Western blot analysis of the EGFR in BxPC-3, PANC-1 and hNDF cells incubated with EGF or erlotinib or both in 2D cultures and soft and stiff 3D cultures. (a) Western blot bands of EGFR in 2D cultures; (b) Western blot bands of EGFR in 0.15% and 0.5% RAD16-I 3D cultures; (c) densitometry of bands shown in (a) for 2D cultures; (d) densitometry of bands shown in (b) for 3D cultures. GAPDH was used as an internal control. One representative blot is shown. Experiments were repeated three times (N = 3), and statistical differences are indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001.

Figure 8

Western blot analysis of the EGFR in PANC-1 and BxPC-3 cells incubated with EGF and erlotinib in the presence of the proteasome (MG-132) and lysosomes (Bafilomycin A1) inhibitors in 0.15% RAD16-I 3D cultures. (a) Western blot bands of EGFR; (b) densitometry of bands shown in (a). GAPDH was used as an internal control. One representative blot is shown. Experiments were repeated three times (N = 3), and statistical differences are indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001.

Figure 9

Colocalization analysis of the EGFR and EEA1 (early endosomes) in cells incubated with EGF, erlotinib or both in 2D cultures. EGFR (red) and EEA1 (green) immunofluorescence counterstained with DAPI (grey) in (a) PANC-1; (b) BxPC-3 and (c) hNDF cells. Scale bars represent 10 µm; (d) Manders’ colocalization coefficients showing the proportion of EGFR overlapping with EEA1; (e) Manders’ colocalization coefficients showing the proportion of EEA1 overlapping with EGFR. Statistical differences are indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001.

Figure 10

Colocalization analysis of the EGFR and LAMP1 (lysosomes) in cells incubated with EGF, erlotinib or both in 2D cultures. EGFR (red) and LAMP1 (green) immunofluorescence counterstained with DAPI (grey) in (a) PANC-1; (b) BxPC-3 and (c) hNDF cells. Scale bars represent 10 µm. Gray pictures show close-up sections of each marker; (d) Manders’ colocalization coefficients showing the proportion of EGFR overlapping with LAMP1; (e) Manders’ colocalization coefficients showing the proportion of LAMP1 overlapping with EGFR. Statistical differences are indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001.

Figure 11

Immunofluorescence of EGFR and EEA1 in (a) BxPC-3 cells and (b) hNDF incubated with EGF, erlotinib or both in 0.15% RAD16-I 3D cultures. One representative Z plane is shown. Scale bars represent 10 µm. White arrows show EGFR colocalizing with early endosomes.

Figure 12

Immunofluorescence of EGFR and LAMP1 in (a) PANC-1 and (b) BxPC-3 cells incubated with EGF, erlotinib or both in 0.15% RAD16-I 3D cultures. One representative Z plane is shown. Scale bars represent 10 µm. White arrows show EGFR colocalizing with lysosomes.