Sialylation facilitates self-assembly of 3D multicellular prostaspheres by using cyclo-RGDfK

Background: Prostaspheres-based three dimensional (3D) culture models have provided insight into prostate cancer (PCa) biology, highlighting the importance of cell--cell interactions and the extracellular matrix (EMC) in the tumor microenvironment. Although these 3D classical spheroid platforms provide a significant advance over 2D models mimicking in vivo tumors, the limitations involve no control of assembly and structure with only limited spatial or glandular organization. Here, matrix-free prostaspheres from human metastatic prostate carcinoma PC3 and DU145 cell lines and their respective gemcitabine resistant (GemR) variants were generated by using cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)). Materials and methods: Microscopic imaging, immunocytochemistry (ICC), flow cytometry, sialidase, and WST-1 cell viability assays were used to evaluate the formation of multicellular tumor spheroid (MCTS), cell survival, morphologic changes, and expression levels of [alpha]2,6 and [alpha]2,3 sialic acid (SA) and E- and N-cadherin in DU145, PC3, and their GemR variants. Results: By using the cyclo-RGDfK(TPP) peptide platform in a dose- and time-dependent manner, both DU145 and DU145GemR cells formed small MCTS. In contrast, PC3 and PC3GemR cells formed irregular multicellular aggregates at all concentrations of cyclo-RGDfK(TPP) peptide, even after 6 days of incubation. ICC and flow cytometry results revealed that DU145 cells expressed higher amounts of E-cadherin but lower N-cadherin compared with PC3 cells. By using Maackia amurensis ([alpha]2,3-SA-specific MAL-II) and Sambucus nigra ([alpha]2,6-SA specific SNA) lectin-based cytochemistry staining and flow cytometry, it was found that DU145 and DU145GemR cells expressed 5 times more [alpha]2,6-SA than [alpha]2,3-SA on the cell surface. PC3 cells expressed 4 times more [alpha]2,3-SA than [alpha]2,6-SA, and the PC3GemR cells showed 1.4 times higher [alpha]2,6-SA than [alpha]2,3-SA. MCTS volume was dose-dependently reduced following pretreatment with [alpha]2,6-SA-specific neuraminidase (Vibrio cholerae). Oseltamivir phosphate enhanced cell aggregation and compaction of 3D MCTS formed with PC3 cells. Conclusion: The relative levels of specific sialoglycan structures on the cell surface correlate with the ability of PCa cells to form avascular multicellular prostaspheres. Keywords: PC3 and DU145 cell lines, cadherin, oseltamivir phosphate, gemcitab