Background: Cancers, such as head and neck squamous cell carcinoma (HNSCC), have populations of cancer initiating cells (CICs), which are considered to contribute to cancer recurrence and drug resistance. CICs of solid tumors can form spheroids in vitro when grown in suspension due to contact-independent survival and proliferation. HNSCCs can form spheroids in culture (orospheres). Despite extensive characterization in other cancers, the suitability for orospheres as an in vitro model for HNSCC remains poorly characterized.
Our lab has shown that human beta-defensin 3 (hBD-3), an epithelial cell-derived peptide, is overexpressed at the invasive front in HNSCCs, co-localizes with BMI-1, a well-known CIC biomarker, and promotes a microenvironment favorable for tumor growth. While CIC markers are only identified via biopsy, including BMI-1, hBD-3 can be detected extracellularly. Thus, hBD-3 is a potential biomarker for CIC identification via minimally invasive procedures.
We compared orosphere formation and gene expression in HNSCC cells grown in monolayer or suspension when treated with cetuximab and cisplatin. We hypothesized that the drugs would decrease expression of CIC markers (hBD-3 and BMI-1) and orosphere formation.
Methodology: Two HNSCC cell lines (HSC3 and Scc9) were grown in suspension and as monolayers. Cells were cultured in low-glucose DMEM, 10% fetal bovine serum, and 100 U/mL penicillin–streptomycin at 37C and 5% CO2 and treated with epidermal growth factor (100 ng/mL), cetuximab (200 ng/mL), or cisplatin (2.5 mM). Orosphere formation was microphotographed for seven days and compared to media controls. All cells were analyzed by qPCR for expression of hBD-3 and BMI-1 with GAPDH as the housekeeping control.
Results: Orospheres were successfully grown in suspension from single cell colonies into aggregations of CICs and reformed. When manually dissociated, the single HNSCC cells proliferated and regrew to form orospheres, suggesting self-renewal. Few or no orospheres formed in serum-free media compared to serum-supplemented media. Cetuximab treatment resulted in smaller spheroids and qPCR showed diminished hBD-3 and BMI-1 transcript expression. Cisplatin treatment, however, formed spheroids comparable to positive controls and qPCR did not show diminished hBD-3 or BMI-1 mRNA expression. Interestingly, hBD-3 expression was higher in orospheres than cells that were cultured in monolayer.
Conclusions: We generated spheroids using HNSCC cell lines, which were previously characterized as CIC-containing orospheres (Krishnamurthy and Nör 2012). EGFR-specific inhibition by cetuximab was able to inhibit orosphere formation, block CIC proliferation and growth, and hBD-3 expression. In contrast, cisplatin, a known genotoxic drug, failed to inhibit orosphere formation, growth, and hBD-3 expression. The role of cetuximab will be further validated by analyzing protein levels of hBD-3 and BMI-1 in monolayer and orosphere cultures. Our data suggests that upon further study, hBD-3 has the potential to be a possible prognostic biomarker following therapy.