3D cell culture alters signal transduction and drug response in head and neck squamous cell carcinoma

Abstract

Epidermal growth factor receptor (EGFR) upregulation is a typical characteristic of head and neck squamous cell carcinoma (HNSCC). However, tyrosine kinase inhibitors have not yet been able to achieve enough therapeutic benefit in clinical trials to justify their use in standard therapy regimens. At present, little is known about the reasons for this treatment failure. In the present study, the HNSCC cell lines UM-SCC-11B and UM-SCC-22B were tested for their response to tyrosine kinase inhibitors (TKI) under 2D and 3D cell culture conditions. Absorption and luciferase-based viability assays were used for this, as well as optical evaluation via fluorescence microscopy. In addition, EGFR and HER3 expression as well as the downstream signalling pathways PI3K/AKT/mTOR and RAS/RAF/MEK/ERK were investigated using western blotting. Cell line UM-SCC-11B revealed a strong resistance to lapatinib under 3D cell culture conditions, while a good response to TKI therapy was observed under 2D cell culture conditions. An associated overexpression of phosphorylated HER3 under 3D cell culture conditions offered a plausible explanation for the altered treatment response. The results of the present study represent an idea of how signalling mechanisms of cancer cells can be changed using different cell culture methods. Overall, 3D cell culture could be an important component in the analysis of resistance mechanisms in cancer therapy.

Keywords: HER3 expression; afatinib; epidermal growth factor receptor; lapatinib; spheroid culture.

Figure 1.

Viability analysis of 11B, 22B, 74A and UD01 cells after treatment with TKI for 48 h. Viability was calculated in comparison to the corresponding control group. Empty bars resemble that no viable cells could be detected. The data are presented as mean values ± SD (n=3), *P≤0.05 vs. control.

Figure 2.

(A) Fluorescence Microscopy of 11B and 22B cells in 2D cultures, stained with Calcein AM/Ethidiumhomodimer-1, after treatment with TKI for 48 h (magnification, ×100). Living cells are shown in green; red cells resemble apoptotic/necrotic cells. The white circle highlights an example of the colony formation of 11B cells. (B) Dose/response curves for 11B and 22B cells in 2D culture under treatment with TKI for 48 h. The data are presented as mean values ± SD, n=3.

Figure 3.

(A) Fluorescence microscopy of 11B and 22B cells in 3D cultures, stained with Sytox-Green/ATP-Red, under treatment with TKI for 48 h (magnification, ×100). Living cells are shown in red; green cells resemble apoptotic/necrotic cells. (B) Dose/response curves for 11B and 22B cells in 3D culture under treatment with TKI for 48 h. The data are presented as mean values ± SD, n=3.

Figure 4.

Western blotting analysis of cell lysates acquired from 11B and 22B cells in 2D or 3D cultures after treatment with lapatinib or afatinib for 48 h. EGFR, epidermal growth factor receptor; p-, phosphorylated.