Evaluation of CD44 variant expression in oral, head and neck squamous cell carcinomas using a triple approach and its clinical significance.

Cancer stem cells possess the qualities of self-renewal, tumorigenesis and the ability to recapitulate a heterogeneous tumor. Our group was the first to isolate head and neck squamous cell carcinoma (HNSCC) stem cells using the cell surface marker CD44. CD44 is a trans-membrane glycoprotein with a multitude of key-functions that regulate cancer cell proliferation and metastasis. The variety of CD44 functions is due to tissue-specific patterns of glycosylation of the extracellular portion, and to the multiple protein isoforms (CD44 variants, CD44v) generated by alternative splicing. This study investigates the expression pattern of CD44 variants in HNSCC. Ten cell lines from the most common HNSCC locations and representative of various clinical outcomes were assayed by quantitative realtime PCR, flow cytometry and immunofluorescence comparatively with normal oral keratinocytes. The CD44 v4 and v6 were exclusively abundant in HNSCC while the isoform v1,2 was expressed in normal oral keratinocytes. Of interest, the highest level of CD44v6 expression was detected in advanced metastatic HNSCC, suggesting a link between CD44v6 expression and HNSCC metastasis, while the highest CD44v4 was detected in a stage IV HNSCC refractory to chemotherapy which developed recurrence. Oral-derived HNSCC expressed the highest CD44v4 and v6, and levels corresponded with staging, showing also an increasing tendency with recurrence and metastasis. CD44v were detected predominantly in smaller cells (a characteristic that has been associated with stem cell properties) or cells with mesenchymal morphology (a characteristic that has been associated with the migratory and invasive potential of epithelial tumor cells), suggesting that CD44v differential expression in HNSCC may be representative of the morphological changes inherent during tumor progression towards a more aggressive potential, and thus contributing to the individual tumor biology. The mechanism of CD44 variant involvement in HNSCC progression and metastasis is under investigation.

Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.

For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.