Single Tissue Samples from Head and Neck Squamous Cell Carcinomas are Representative Regarding the Entire Tumor’s Chemosensitivity to Cisplatin and Docetaxel

Abstract

Background: In multimodal therapy concepts for advanced head and neck squamous cell carcinoma (HNSCC), a valid predictive assay for the quick detection of efficient chemotherapeutic agents is desirable. Questionable so far was whether tissue samples of about 100 mg correctly reflect the chemoresponse of a whole HNSCC. This was proven using an ex-vivo colony-forming assay. Materials and Methods: Of 14 HNSCC, 3 biopsies each were taken from separate sites, minced, and collagenase digested. HNSCC digests were added to microtiter plates containing serial dilutions of chemotherapeutic agents or medium as control. After 72-h incubation, wells were washed and cultures methanol-fixed before Giemsa-staining. Epithelial colonies were counted. Results: 11/14 HNSCC (78.6%) showed sufficient colony formation allowing reliable cut-off detection. Cut-off concentrations (complete chemotherapeutically suppressed colony formation) between 3.3 μM and >50 μM cisplatin, and 0.55 μM and 17.6 μM docetaxel were detected. Inhibition of colony formation to 50% of colonies detected in controls (IC50) was found between 0.2 μM and 17.9 μM cisplatin or 1.5 μM and 13.7 μM docetaxel. Cut-off concentrations and IC50 of the HNSCC fragments showed a strong correlation (docetaxel: r > 0.80, p < 0.005; cisplatin: r > 0.67, p < 0.044), while being only insignificantly different in the t-test for paired samples (docetaxel: p > 0.163; cisplatin: p > 0.167). Conclusion: In most cases, tissue samples of about 100 mg allow a representative assessment of chemoresponse of HNSCC.

Heterogeneity of epithelial and stromal cells of head and neck squamous cell carcinomas in ex vivo chemoresponse

“…For quality control purposes and examination of reproducible test conditions along to each HNSCC colony formation assay, a KB cell chemoresponse assay was carried out simultaneously as previously described [17]. Briefly, KB cells from stock cultures adapted to RPMI 1640 free of phenol red and containing only 20 nM of riboflavin were harvested and adjusted to give a final number of 10 4 KB-cells/well.…”

“…Adherent cells and cell colonies were fixed with 90% (v/v) methanol and air-dried. Giemsa staining allowing for differentiation of epithelial and stromal cell colonies was performed as described [17].…”

“…Indeed, more efficient solutions to overcome critical limitations of conventional in-vitro assays by utilizing flavinprotecting conditions [15] to eliminate flavin-mediated photochemical artifacts [16] are available. Advantageously, they are quality-controlled, and allow also for earlier availability (4 days) of data respective to dosages required to achieve the intended response of the individual HNSCC [17,18].…”

“…We already used this predictive short-time ex-vivo colony-forming assay to confirm that the chemoresponse of epithelial cells within a biopsy in principal is representative for a given HNSCC [17]. But within that study the ex-vivo chemoresponse of stromal cells was not analyzed.…”

“…But within that study the ex-vivo chemoresponse of stromal cells was not analyzed. Moreover, it might be mistakenly concluded from the data shown in [17] that any biopsy of a tumour allows for sufficient statements about the chemoresponse of all cell types present within the tumour. Thus the aim of this consecutive study was to analyze potential differences in the chemoresponse of epithelial and stromal cells in individual HNSCC.…”