Sialylation transmogrifies human breast and pancreatic cancer cells into 3D multicellular tumor spheroids using cyclic RGD-peptide induced self-assembly.

Roman Akasov,Myron R Szewczuk,Fiona Haxho,Ronald J Neufeld,Sergey V Burov,Vanessa Samuel,Sabah Haq,Elena Markvicheva

doi:10.18632/oncotarget.11868

Abstract

Multicellular tumor spheroids (MTS) have been at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is highly influenced by aberrant cell surface-specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate MTS formation are not well defined. Matrix-free spheroids from breast MCF-7 and pancreatic PANC1 cancer cell lines and their respective tamoxifen (TMX) and gemcitabine (Gem) resistant variants were generated using the RGD platform of cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK (TPP)). MCF-7 and MCF-7 TMX cells formed tight spheroids both in the classical agarose-and RGD-based platforms while all PANC1 cells formed loose aggregates. Using lectin histochemistry staining, sialidase assay, neuraminidase (Vibrio cholerae) and oseltamivir phosphate (OP) neuraminidase inhibitor treatments, MCF-7 and PANC1 cells and their drug-resistant variants expressed different sialic acid (SA) content on their cell surfaces. α-2,3- and α-2,6-sialic acid surface residues facilitated spheroid formation under cyclo-RGDfK(TPP)-induced self-assembly. Pretreatment with α-2,3- SA specific Maackia amurensis (MAL-II) lectin, α-2,6-SA specific Sambucus nigra (SNA) lectin, and exogenous α-2,6-SA specific neuraminidase (Vibrio cholerae) dose-dependently reduced spheroid volume. OP enhanced cell aggregation and compaction forming spheroids. PANC1 and MDA-MB231 xenograft tumors from untreated and OP-treated RAGxCγ double mutant mice expressed significantly higher levels of α-2,3- SA over α-2,6-SA. MCF-7 spheroids also expressed a high α-2,3-SA to α-2,6-SA ratio. These results suggest that the relative levels of specific sialoglycan structures on the cell surface correlate with the ability of cancer cells to form avascular multicellular tumor spheroids and in vivo xenograft tumors.

Highlights

The multicellular tumor spheroid (MTS) is a promising 3D model platform that enables the study of tumor cell development, morphology, cellular motility and drug resistance in vitro [1,2,3,4]
These results suggest that the relative levels of specific sialoglycan structures on the cell surface correlate with the ability of cancer cells to form avascular multicellular tumor spheroids and in vivo xenograft tumors
Sialylation of cell surface glycoproteins in multicellular tumor spheroid formation Since Multicellular tumor spheroids (MTS) and CD24- and CD44-overexpressing cancer stem-like PANC1 cells show high expression of fucosylated glycans using specific lectins binding to www.impactjournals.com/oncotarget the spheroid cells [32], we examined prior to spheroid formation the cell surface sialylation of monolayer MCF7, MCF-7 TMX cells (Figure 3), and PANC1 and PANC1GemR cells (Figure 4)

Summary

Introduction

The multicellular tumor spheroid (MTS) is a promising 3D model platform that enables the study of tumor cell development, morphology, cellular motility and drug resistance in vitro [1,2,3,4]. Since spheroids resemble the 3D architecture of avascular tumors, including multicellular www.impactjournals.com/oncotarget arrangement and extracellular matrix deposition typically found in vivo, spheroid cells demonstrate enhanced resistance to chemotherapy [9]. Novel MTS formations, under matrix-free conditions, are being developed to study the 3D architecture of avascular tumor models [1, 9, 11,12,13], especially in relation to metastasis, invasion and therapeutic drug screening [13, 14]. The molecular development of MTS formation by cancer cells may involve (a) cell surface proteins binding fibronectin which induces 3D cohesion [15], (b) under conditions of random positioning machine (RPM) simulating microgravity, the expression of 28 genes aside from β -tubulin is mutually controlled by a key cytokine interleukin-8 (IL-8 or CXCL8) gene within the framework of 6 extracellular, 6 membrane, 15 cytoplasmic and 2 nuclear proteins [16], and/or (c) the integrins’ interactions with the extracellular matrices (ECM) and intracellular components within the cellular cytoskeleton in particular response to mechanical stimulation [16, 17]

Methods

Results

Conclusion