Glioma cell invasion is significantly enhanced in composite hydrogel matrices composed of chondroitin 4- and 4,6-sulfated glycosaminoglycans

Abstract

Glioblastoma multiforme (GBM) is the most aggressive form of astrocytoma accounting for a majority of primary malignant brain tumors in the United States. Chondroitin sulfate proteoglycans (CSPGs) and their glycosaminoglycan (GAG) side chains are key constituents of the brain extracellular matrix (ECM) implicated in promoting tumor invasion. However, the mechanisms by which sulfated CS-GAGs promote brain tumor invasion are currently unknown. We hypothesize that glioma cell invasion is triggered by the altered sulfation of CS-GAGs in the tumor extracellular environment, and that this is potentially mediated by independent mechanisms involving CXCL12/CXCR4 and LAR signaling respectively. This was tested in vitro by encapsulating the human glioma cell line U87MG-EGFP into monosulfated (4-sulfated; CS-A), composite (4 and 4,6-sulfated; CS-A/E), unsulfated hyaluronic acid (HA), and unsulfated agarose (AG; polysaccharide) hydrogels within microfluidics-based choice assays. Our results demonstrated the enhanced preferential cell invasion into composite hydrogels, when compared to other hydrogel matrices (p<0.05). Haptotaxis assays demonstrated the significantly (p<0.05) faster migration of U87MG-EGFP cells in CXCL12 containing CS-GAG hydrogels when compared to other hydrogel matrices containing the same chemokine concentration. This is likely due to the significantly (p<0.05) greater affinity of composite CS-GAGs to CXCL12 over other hydrogel matrices. Results from qRT-PCR assays further demonstrated the significant (p<0.05) upregulation of the chemokine receptor CXCR4, and the CSPG receptor LAR in glioma cells within CS-GAG hydrogels compared to control hydrogels. Western blot analysis of cell lysates derived from glioma cells encapsulated in different hydrogel matrices further corroborate qRT-PCR results, and indicate the presence of a potential variant of LAR that is selectively expressed only in glioma cells encapsulated in CS-GAG hydrogels. These results suggest that sulfated CS-GAGs may directly induce enhanced invasion and haptotaxis of glioma cells associated with aggressive brain tumors via distinct mechanisms.

Chondroitin Sulfate Glycosaminoglycan Scaffolds for Cell and Recombinant Protein-Based Bone Regeneration

“…Briefly, hydrogels were flash frozen in liquid nitrogen and lyophilized. We have not observed any discernible difference in structure and porosity of lyophilized snap frozen CS‐GAG hydrogels reported in this study to hydrogels frozen overnight at −80°C as reported by us previously . They were then mounted on 10 mm SEM stubs and sputter coated (Structure Probe Inc., West Chester, PA) with gold for 30 seconds and imaged under an accelerated voltage of 5 kV.…”

“…Cells were imaged using Fluorescein isothiocyanate (FITC) and Tetramethylrhodamine (TRITC) fluorescent filters 3 hours after seeding. Viability of transduced cells was determined by quantifying the degree of fluorescence overlap of RFP and calcein using the Mander's overlap coefficient parameters in Volocity (PerkinElmer, Waltham, MA) as described previously .…”

Expression and function of chondroitin 4‐sulfate and chondroitin 6‐sulfate in human glioma

“…[32][33][34] CSs have been shown to promote U-87 MG cell invasion by upregulating the expression of the chemokine receptor CXCR4 and CSPG receptor LAR. 35 Of note, the sulfation pattern of GAGs is decisive for the proliferation and adhesion of C6 glioma cells and relates to the invasive behavior of tumor cells by degrading the extracellular matrix. 36,37 C6S may also affect tumor metastasis by remodeling the collagen network in the tumor microenvironment.…”

Sustained Release of Transforming Growth Factor-β1 from Platelet-Rich Chondroitin Sulfate Glycosaminoglycan Gels

“…The sulfate groups on the chondroitin sulfate have a high affinity for positively charged anabolic growth factors and previous work has shown that the use of these gels resulted in sustained release of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) over a period of 15 days. 41,42 Although FGF-2 and BDNF are not anabolic growth factors typically associated with PRP or with cartilage repair, the relevant growth factors in PRP are also positively charged and might interact similarly with CS-GAG hydrogels. In turn, the concept of using biocompatible negatively charged hydrogels for providing sustained release of anabolic growth factors from PRP could be applicable to augmentation of cartilage repair in the knee.…”