Brain-Mimetic 3D Culture Platforms Allow Investigation of Cooperative Effects of Extracellular Matrix Features on Therapeutic Resistance in Glioblastoma

Abstract

Glioblastoma (GBM) tumors exhibit potentially actionable genetic alterations against which targeted therapies have been effective in treatment of other cancers. However, these therapies have largely failed in GBM patients. A notable example is kinase inhibitors of EGFR, which display poor clinical efficacy despite overexpression and/or mutation of EGFR in >50% of GBM. In addressing this issue, preclinical models may be limited by the inability to accurately replicate pathophysiologic interactions of GBM cells with unique aspects of the brain extracellular matrix (ECM), which is relatively enriched in hyaluronic acid (HA) and flexible. In this study, we present a brain-mimetic biomaterial ECM platform for 3D culturing of patient-derived GBM cells, with improved pathophysiologic properties as an experimental model. Compared with orthotopic xenograft assays, the novel biomaterial cultures we developed better preserved the physiology and kinetics of acquired resistance to the EGFR inhibition than gliomasphere cultures. Orthogonal modulation of both HA content and mechanical properties of biomaterial scaffolds was required to achieve this result. Overall, our findings show how specific interactions between GBM cell receptors and scaffold components contribute significantly to resistance to the cytotoxic effects of EGFR inhibition. Three-dimensional culture scaffolds of glioblastoma provide a better physiological representation over current methods of patient-derived cell culture and xenograft models. .

Peptide‐modified, hyaluronic acid‐based hydrogels as a 3D culture platform for neural stem/progenitor cell engineering

“…Hydrogels were formed by Michael‐type addition between 4‐arm polyethylene glycol‐maleimide (PEG‐Mal, 20 kDa, Laysan Bio) and thiolated HA (700 kDa HA, Lifecore Biomedical) . In brief, carbodiimide chemistry ( N ‐hydroxysuccinimide, NHS; 1‐ethyl‐3‐(3‐dimethylaminopropyl), EDC) was used to add a thiol from cystamine (Sigma‐Aldrich) to the carboxylate group on the glucuronic acid moieties of HA, creating thiolated HA (HA‐SH).…”

“…Prior to gelation and NS/PC encapsulation, PEG‐Mal was dissolved in PBS and sterile‐filtered. Peptides (Genescript, Supplementary Table 1) containing thiols (i.e., cysteines) near the N ‐termini were conjugated via Michael‐type addition to an average of one arm of PEG‐Mal prior to hydrogel formation for a total peptide concentration of 270 μ M in the final hydrogels . PEG‐Mal/peptide solutions were adjusted to 10 mg/mL with PBS and kept on ice.…”

“…Hyaluronic acid preparation and hydrogel formation Hydrogels were formed by Michael-type addition between 4-arm polyethylene glycol-maleimide (PEG-Mal, 20 kDa, Laysan Bio) and thiolated HA (700 kDa HA, Lifecore Biomedical). 64 In brief, carbodiimide chemistry (N-hydroxysuccinimide, NHS; 1-ethyl-3-(3-dimethylaminopropyl), EDC) was used to add a thiol from cystamine (Sigma-Aldrich) to the carboxylate group on the glucuronic acid moieties of HA, creating thiolated HA (HA-SH). Disulfide bridges were broken with dithiothreitol and newly synthesized HA-SH purified via dialysis (12-14 kDa molecular weight cut-off cellulose membrane, Spectrum Labs) in deionized water, adjusted to pH 4 with hydrochloric acid and changed at least twice daily for 3 days.…”

Glioblastoma’s Next Top Model: Novel Culture Systems for Brain Cancer Radiotherapy Research

“…Other groups have generated matrices using hyaluronic acid and chitosan (another key brain ECM component); these matrices more effectively maintain tumor growth patterns and expression of key GSC markers than 2D cultures 92 . These scaffolds have enabled researchers to more fully delineate the effect of the brain ECM on GBM behavior, including invasion 107,108 , growth 109 , and sensitivity to novel therapies 110,111 .…”

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, ).…”