Expression of osteopontin in cocultures of osteoblastic cells and squamous cell carcinoma cells and its effects on the neoplastic cell phenotype and osteoclastic activation

Author(s)

Lucas Novaes Teixeira

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2015-02-20

Examining board members

Paulo Tambasco de Oliveira; Elizabeth Ferreira Martinez; Sandra Beatriz Chaves Tarquinio; Ricardo Della Coletta; Edgard Graner

• Advisor: Paulo Tambasco de Oliveira

Abstract

The oral squamous cell carcinoma (OSCC) is the most prevalent malignant neoplasm of the oral structures. It may invade bone in up to 56% of the cases and promote osteoclast-mediated bone extracellular matrix (ECM) resorption. Expression of the matricellular protein osteopontin (OPN) in malignant neoplasms, including OSCC, has been positively correlated with aggressive tumor behavior. OPN is the most abundant non collagenous ECM protein in bone, where it preferentially accumulates at interfaces, including cement lines, laminae limitantes and reversal lines, being essential for the adhesion and function of osteoblasts and osteoclasts. Despite the importance attributed to OPN in the tumor microenvironment, indicative of more aggressive neoplastic phenotypes, the effects of osteoblast-derived OPN on OSCC cells and on OSCC-induced osteoclast activity are still not fully understood. The present in vitro study aimed to evaluate temporal expression of OPN in cocultures of human osteoblastic cells and malignant neoplastic epithelial cells and the effects of osteoblast-derived OPN on the neoplastic cell phenotype. Additionally, the effects of cocultures on osteoclastic activity were evaluated. Human OSCC-derived epithelial cells (SCC9 cell line) were plated on Transwell® membranes coated or not by a thin uniform layer of Matrigel and cocultured with human osteoblastic cells (SAOS-2 cell line) during its peak of OPN expression (day 10 of SAOS-2 culture). SCC9 cells exposed to OPN-silenced SAOS-2 cultures by means of interference RNA and SCC9 cells cultured alone were used as controls. At 24 h of coculture, SCC9 cells were quantitatively evaluated for cell adhesion, proliferation, migration and invasion of Matrigel. The impact of coculturing SCC9 and SAOS-2 cells either during the OPN peak expression or under the silencing of OPN was quantitatively evaluated in terms of calcium phosphate resorption by U-937-derived osteoclastic cells and expression of cytokines in the culture medium by ELISA assay. The statistical analyses were carried out using the non-parametric Kruskal-Wallis test (p < 0.05). The results showed a reciprocal induction of SAOS-2 and SCC9 cells in terms of OPN expression over the coculture interval. SAOS-2-secreted OPN altered the SCC9 cell phenotype, leading to enhanced cell adhesion and proliferation and higher Matrigel invasion, which was also enhanced, but to a lesser degree, by SAOS-2 cultures silenced for OPN. Cell migration was not affected. Cocultures with SAOS-2, mainly during the peak expression of OPN, resulted in overexpression of IL 6 and IL 8 by SCC9 cells, which corresponded with an enhanced resorptive capacity of osteoclastic cells. Taken together, the results suggest that osteoblast-derived OPN affects the interactions among malignant neoplastic epithelial cells, osteoblasts and osteoclasts, likely contributing to the progression of bone lesions in OSCC (AU)

• FAPESP's process: 12/08605-6 - Expression of osteopontin in cocultures of osteoblastic cells and squamous cell carcinoma cells and its effects on the neoplastic cell phenotype and osteoclastic activation
• Grantee: Lucas Novaes Teixeira
• Support Opportunities: Scholarships in Brazil - Doctorate