Culture on 3D Chitosan‐Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells

Abstract

The lack of in vitro models that support the growth of glioblastoma (GBM) cancer stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly and frequently unsuccessful endeavor. To address these limitations, we synthesize a three-dimensional porous scaffold composed of chitosan and hyaluronic acid (CHA), and compared growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments.

3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs

“…For instance,Todaro et al (2007) produced spheroids with cells obtained from human colon adenocarcinoma tissue fragments and confirmed the expression of a CSC marker, that is, CD133. Other studies showed that the spheroids produced with CSC obtained from cancer cell lines (e.g., BT-474, GBM6, H1650, and MDA-MB-231) demonstrated a higher resistance toward different therapeutics (e.g., 5-FU, camptothecin, carmustine, cisplatin, gemcitabine, lomustine, paclitaxel, temozolomide, and trastuzumab) than 2D cell cultures(Godugu et al, 2013;Reynolds et al, 2017;Rodríguez et al, 2018;K. For example, the 100 µM of oxaliplatin reduced the CD133+ cell viability in only almost equal to 10%, whereas the same treatment reduced the CD133− cell viability to almost equal to 70%(Todaro et al, 2007).Optionally, spheroids can be produced with CSC obtained from cancer cell lines (Figure 3).…”

Biomaterials‐Based Approaches to Tumor Spheroid and Organoid Modeling

“…Glioblastoma (GBM6) cultured in CH scaffold showed enhanced stem cell marker expression resisted treatment of alkylating agents. [100] …”

Three‐dimensional biomimetic hyaluronic acid hydrogels to investigate glioblastoma stem cell behaviors

“…However, very few studies have employed 3D model systems to specifically study GSC behaviors (Nakod et al, ). Of these, majority of studies have utilized HA as an additive in conjunction with polycaprolactone (PCL; Martinez‐Ramos & Lebourg, ); chitosan (Florczyk et al, ; Wang et al, ); chitosan–alginate (Kievit et al, ); or collagen (Herrera‐Perez, Voytik‐Harbin, & Rickus, ); or as the base matrix incorporating synthetic polymers such as poly(ethylene glycol) (PEG; Xiao et al, ; Xiao et al, ). In addition, all of these studies, with the exception of a few (Herrera‐Perez et al, ; Xiao et al, ; Xiao et al, ), have utilized tumor cells grown in the presence of serum, which does not typically favor a stem‐like phenotype (Lee et al, ).…”