A Tissue Engineered 3D Model of Cancer Cell Invasion for Human Head and Neck Squamous-Cell Carcinoma

Abstract

Head and neck squamous-cell carcinoma (HNSCC) is associated with aggressive local invasiveness, being a main reason for its poor prognosis. The exact mechanisms underlying the strong invasive abilities of HNSCC remain to be elucidated. Therefore, there is a need for in vitro models to study the interplay between cancer cells and normal adjacent tissue at the invasive tumor front. To generate oral mucosa tissue models (OMM), primary keratinocytes and fibroblasts from human oral mucosa were isolated and seeded onto a biological scaffold derived from porcine small intestinal submucosa with preserved mucosa. Thereafter, we tested different methods (single tumor cells, tumor cell spots, spheroids) to integrate the human cancer cell line FaDu to generate an invasive three-dimensional model of HNSCC. All models were subjected to morphological analysis by histology and immunohistochemistry. We successfully built OMM tissue models with high in vivo-in vitro correlation. The integration of FaDu cell spots and spheroids into the OMM failed. However, with the integration of single FaDu cells into the OMM, invasive tumor cell clusters developed. Between segments of regular epithelial differentiation of the OMM, these clusters showed a basal membrane penetration and lamina propria infiltration. Primary human fibroblasts and keratinocytes seeded onto a porcine carrier structure are suitable to build an OMM. The HNSCC model with integrated FaDu cells could enable subsequent investigations into cancer cell invasiveness.

Keywords: 3D tissue model; head and neck squamous-cell carcinoma; oral mucosa; tissue engineering.

Figure 1

(a) Experimental design used to generate an oral mucosal model (OMM) and (b–d) different methods of integrating FaDu tumor cells into the OMM. (b) Integration of multicellular tumor cell spots. (c) Integration of multicellular tumor spheroids. (d) Integration of single tumor cells. Oral mucosal model (OMM), head and neck squamous-cell carcinoma (HNSCC).

Figure 2

Generation of a 3D bioequivalent oral mucosa model (OMM). (a) Comparison of human oral mucosa with the OMM by H&E staining. The red dotted line separates the stratified squamous epithelium from the underlying lamina propria. The black dotted line separates the lamina propria from residual SIS/MUC. (b) Comparison of human oral mucosa with the OMM by immunohistochemistry. Scale bars represent 100 μm. Oral mucosa model (OMM), cytokeratin (CK), control (ctrl).

Figure 3

Failed integration of (a) FaDu tumor cell spots (b) and tumor spheroids into the oral mucosa model (OMM). FaDu cells were transduced lentivirally to constitutively express red fluorescent protein (RFP). Analysis by H&E staining and immunohistochemistry showed a keratinized stratified squamous epithelium, similar to the OMM. While, in (a), no tumor cells could be detected, in (b), RFP-positive tumor cells appeared on the stratum corneum as remnants of the spheroid. The arrowhead indicates tumor cells atop the stratum corneum, which are strongly eosinophilic with fragmented nuclei. Scale bars represent 100 μm. Head and neck squamous-cell carcinoma (HNSCC), cytokeratin (CK), red fluorescent protein (RFP).

Figure 4

Integration of single tumor cells as a mixed suspension consisting of FaDu cells and keratinocytes. Depending on the mixing ratio, sections of normal stratification could still be detected at ratios up to 1:33. This was no longer the case at a ratio of 1:25. From a ratio of 1:33, there were clear signs of invasive tumor growth with basement membrane penetration and invasion into the lamina propria (arrowhead and arrows). Horn beads (asterisk), as another sign of malignancy, could be seen at ratios of 1:25 and 1:10. Scale bars represent 100 μm. Head and neck squamous-cell carcinoma (HNSCC), cytokeratin (CK).