An Effective Primary Head and Neck Squamous Cell Carcinoma In Vitro Model

Abstract

Head and neck squamous cell carcinoma is a highly malignant disease and research is needed to find new therapeutic approaches. Faithful experimental models are required for this purpose. Here, we describe the specific cell culture conditions enabling the efficient establishment of primary cell culture models. Whereas a classical 10% serum-containing medium resulted in the growth of fibroblast-like cells that outcompeted epithelial cells, we found that the use of specific culture conditions enabled the growth of epithelial tumor cells from HPV+ and HPV- head and neck cancer tissue applicable for research. EpCAM and high Thy-1 positivity on the cell surface were mutually exclusive and distinguished epithelial and fibroblast-like subpopulations in all primary cultures examined and thus can be used to monitor stromal contamination and epithelial cell content. Interestingly, cells of an individual patient developed tumor spheroids in suspension without the use of ultra-low attachment plates, whereas all other samples exclusively formed adherent cell layers. Spheroid cells were highly positive for ALDH1A1 and hence displayed a phenotype reminiscent of tumor stem cells. Altogether, we present a system to establish valuable primary cell culture models from head and neck cancer tissue at high efficiency that might be applicable in other tumor entities as well.

Keywords: HPV; head and neck cancer; primary cell culture; stroma; tumor spheroids.

Figure 1

HNSCC cell culture establishment in culture medium containing 10% FBS. (A) Fresh patient-derived cultures of S3, S4, and S5 show mixed populations of epithelial and mesenchymal cells. By passaging, epithelial cells were gradually lost in the established cultures and expanding cells displayed fibroblast-like morphology; scale bars = 100 µm. (B) Epithelial cells developed scattered patches of cornifying cells; scale bars = 100 µm. (C) qRT-PCR analysis of passages 1 and 2 (P1 and P2) of S4 revealed decreasing expression of epithelial markers and an increase of mesenchymal markers.

Figure 2

HNSCC cell culture establishment in serum-free CSC-medium. (A) Fresh primary cells of S12 and S15 display large epithelial cell compartment. (B) Growth curves show cell proliferation of S12 cells until passage 1 in CSC-medium followed by a decline in cell numbers after splitting into three different media. S15 cells expanded in DMEM and PNEU-medium, but showed poor proliferation in CSC-medium. (C) Upon initial passaging, cells were split into DMEM, CSC, and PNEU-medium, which resulted in fibroblast-like cell expansion in DMEM; no or neglectable expansion of epithelial cells in CSC-medium; epithelial cells expanded in PNEU-medium until passage 2 of S12 and S15. Then, PNEU cultures displayed growth of fibroblast-like cell types, while epithelial cells frequently acquired a phenotype reminiscent of cornification (arrows); scale bars = 100 µm.

Figure 3

Primary HNSCC cell cultures in PNEU-medium. (A) Initial cultures in passage 0 (P0) and critical events observed in later passages: adherent S18 cells spontaneously transitioned into spheroids in passage 4; epithelial S22 cells expanded until passage 6 and then showed signs of reduced viability and were overgrown by fibroblast-like cells; epithelial cells derived from S23 expanded initially, but in passage 1 these cells exhibited death accompanied by proliferation of fibroblast-like cells; S24-derived cultures contained epithelial cells that expanded continuously until the experiment was terminated in passage 6; S25 cells with epithelial morphology expanded continuously until the experiment was terminated in passage 3; scale bars = 100 µm. (B) Growth curves of primary HNSCC cell cultures reveal varying proliferation rates; AD = adherent; SPH = spheroid.

Figure 4

Serum treatment of primary HNSCC cell cultures. (A) In DMEM, fibroblast-like cell types replaced epithelial cells in adherent cultures of S22 and S24; scale bars = 100 µm. (B) The outgrowth of fibroblasts in DMEM-cultured S22 and S24 cells shown in (A) is accompanied by increased Thy-1 expression and loss of EpCAM expression over six culture passages. Adherent (PNEU AD) and spheroid (PNEU SPH) cells of S18 show constant expression of both markers and inconsistent changes in DMEM + 10% serum; INI = initial tumor tissue; P1 = passage 1.

Figure 5

ALDH1A1 expression in primary HNSCC cell cultures in the initial tumor tissue and within the first three culture passages. (A) Relative expression (%) normalized by patient to compare ALDH1A1 levels relative to other cell entities of the same patient sample show higher ALDH1A1 levels in S18 cultures than in the initial S18 tumor. All other cultures showed lower or similar ALDH1A1 expression in comparison to the original tumor tissue. (B) Passage normalized data to compare ALDH1A1 levels between distinct patient samples within each passage and the initial tumors. Tumor sample S24 shows highest ALDH1A1 expression, whereas in cell culture cells of S18 overtop S24 cells form passage 1 on. (C) Data normalized to compare all samples of all passages and the initial tumor to each other reveals stark gradual enrichment of ALDH1A1 expressing cells in S18 cultures; P1 = passage 1; initial = original tumor tissue.

Figure 6

EpCAM and Thy-1 expression in mixed cell cultures composed of epithelial and mesenchymal populations visualized by indirect immunofluorescence. Cells with epithelial morphology stained highly positive for EpCAM, whereas cells with fibroblast-like morphology display high expression of Thy-1. Both markers are mutually exclusive in cell cultures derived from patients S4, S15, and S24. EpCAM positive cells of S18 and S22 showed weak Thy-1 cell membrane localization. Actin was stained using phalloidin to visualize cell morphology; DNA was stained using Hoechst33342; scale bars: 50 µm.