Sialylation facilitates self-assembly of 3D multicellular prostaspheres by using cycloRGDfK(TPP) peptide

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 cycloRGDfK(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 2 alpha,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.