Reproduction of the Antitumor Effect of Cisplatin and Cetuximab Using a Three-dimensional Spheroid Model in Oral Cancer

Abstract

Background/Aim: Cancer research has been conducted using cultured cells as part of drug discovery testing, but conventional two-dimensional culture methods are unable to reflect the complex tumor microenvironment. On the other hand, three-dimensional cultures have recently been attracting attention as in vitro models that more closely resemble the in vivo physiological environment. The purpose of this study was to establish a 3D culture method for oral cancer and to verify its practicality. Materials and Methods: Three-dimensional cultures were performed using several oral cancer cell lines. Western blotting was used for protein expression analysis of the collected cell masses (spheroids), and H-E staining was used for structural observation. The cultures were exposed to cisplatin and cetuximab and the morphological changes of spheroids over time and the expression changes of target proteins were compared. Results: Each cell line formed spheroidal cell aggregates and showed enhancement of cell adhesion molecules over time. H-E staining showed tumor tissue-like structures specific to each cell line. Cisplatin showed concentration-dependent antitumor effects due to loss of cell adhesion and spheroid disruption in each cell line, while cetuximab exhibited antitumor effects that correlated with EGFR expression in each cell line. Conclusion: Spheroids made from oral cancer cell lines appeared to have tumor-like characteristics that may reflect their clinical significance. In the future, it may become possible to produce tumor spheroids from tissue samples of oral cancer patients, and then apply them to drug screening and to develop individualized diagnostic and treatment methods.

Keywords: anticancer drug; oral cancer; spheroid; three-dimensional culture.

Figure 1

Morphology of oral cancer cells in 2D and 3D culture. (A) Representative images of SAS and HSC-3 cells in 2D culture. Upper, low magnification images. Bottom, high magnification images. Scale bars, 200 µm. (B) WB showing the levels of E-cadherin and EGFR in SAS and HSC-3 cells. (C) IHC images showing positive staining for E-cadherin and EGFR in SAS and HSC-3 cells. Scale bars, 200 µm. (D) Schematic of the experimental protocol for achieving spheroid recovery over time. (E) Comparison of spheroid diameter changes over time. The experiment was performed in five independent culture wells. (F, G) Morphology and protein expression of oral cancer cells cultured as 3D spheroids in ULA 96-well plates. Scale bars, 200 µm. The WB results show the levels of E-cadherin and EGFR in (F) SAS cells and (G) HSC-3 cells.

Figure 2

Preparation and evaluation of oral cancer spheroid sections. (A) Schematic of the experimental protocol for preparation of spheroid sections. (B) Representative H-E staining images and IHC staining images showing Ki-67 in SAS and HSC-3 spheroids. Scale bar, 100 µm.

Figure 3

Anti-tumor effect of cisplatin on oral cancer spheroids. (A) Schematic diagram from cisplatin addition to spheroid recovery. (B) Schematic of the caspase cascade pathway during cisplatin-induced apoptosis. (C-F) Representative images of cells and changes in expression of E-cadherin and apoptosis-related molecules over 6 days of exposure to different concentrations of cisplatin. Scale bar, 200 µm. (C) SAS cells in 2D culture. (D) SAS cells in 3D culture. (E) HSC-3 cells in 2D culture. (F) HSC-3 cells in 3D culture.

Figure 4

Anti-tumor effect of cetuximab on oral cancer spheroids. (A) Schematic diagram from cetuximab addition to spheroid recovery. (B) Schematic of the inhibition of the EGFR downstream signaling pathway by cetuximab. (C-F) Representative images of cells and changes in expression of E-cadherin, p-EGFR and EGFR over 6 days of exposure to different concentrations of cetuximab. Scale bar, 200 µm. (C) SAS cells in 2D culture. (D) SAS cells in 3D culture. (E) HSC-3 cells in 2D culture. (F) HSC-3 cells in 3D culture.

Figure 5

Spheroids as a tool for determining drug efficacy. (A) Schematic of the experimental protocol for preparation of drug-stimulated spheroid sections. (B) Representative bright field images, H-E staining images and IHC staining images showing cleaved caspase-3 in SAS spheroids with or without cisplatin for 6 days. Scale bar, 200 µm.

Figure 6

Future concepts using 3D cultures in oral cancer treatment.