(119b) Investigating Changes in Extracellular Matrix Architecture in a 3D Glioblastoma Model

Glioblastoma (GBM) is the most common type of brain cancer. Despite current treatment methods including resection, chemotherapy, and radiotherapy, the median survival time of patients has not been significantly altered in the last 10 years. In GBM tumors, the microenvironment includes extracellular matrix (ECM) components, cancer cells, stromal cells, and various growth factors. The GBM tumor ECM is unique in that it differs from surrounding brain tissue. It has been reported that a GBM tumor can be up to 12-fold stiffer than normal brain tissue. Additionally, the composition of the ECM is altered due to the increased collagen deposition from stromal and cancer cells. Understanding structural changes to the ECM in GBM can aid in understanding how these cells form and maintain the tumor environment.

Methods

We have assembled a 3D model of the perivascular niche in GBM. We assembled hydrogels composed of Type 1 collagen or HyStem-C® (Sigma), a gelin and HA-based biomaterial. A human GBM cell line LN229 (ATCC) was utilized since these cells display a mesenchymal subtype, and are moderately invasive. Human umbilical vein endothelial cells (HUVECs) were used as a vasculature mimic. While the vasculature is comprised of different cell types, endothelial cells are a principal component and are commonly used as an in vitro model.

Layered 3D hydrogels were prepared in glass-bottom tissue culture well plates. First, a thin gel of collagen (either 1.1 mg/mL or 2.2 mg/mL) was deposited to prevent cells from adhering to the glass surface beneath. This was followed by a layer of collagen I or HyStem-C®. After gelation, a fibronectin-coated coverslip of HUVECs was placed atop the stacked gels. For co-cultures, the second cell type was seeded around the coverslip on day 7. For LN229 only cultures, a blank coverslip was utilized to mimic HUVEC co-culture, and the LN229 cells were seeded around the coverslip as in co-cultures.

Results

The storage moduli of the hydrogels were measured to be 0.336 ± 0.073 Pa, 1.593 ± 0.111 Pa, and 241.373 ± 43.299 Pa for 1.1 mg/mL collagen, 2.2 mg/mL collagen, and HyStem-C®, respectively. HUVECs, LN229 cells, and co-cultures were cultured for up to 14 days. Gel contraction was observed in all cultures. Contraction was approximately 14% and 34 % in HUVEC and LN229 monocultures, respectively, at day 14. Gel contraction in co-cultures reached up to 68% in softer gels and 34% for cultures in stiffer gels. Cellular proliferation was also significantly increased in co-cultures by 10 – 16-fold from day 8 to day 14. Monocultures of each cell type showed statistically insignificant increases in proliferation of 1 – 4-fold. Physical colocalization of HUVECs and LN229 cells was also observed. Colocalization was found in up to 30% of total LN229 cells, and increased with higher collagen content. This indicates a role for the chemical composition of a biomaterial scaffold. Both gel contraction and colocalization data indicate that LN229-HUVEC interactions are influenced by the surrounding ECM.

Future and Ongoing Work

Currently we are investigating additional aspects of ECM remodeling, including the endocytosis of the biomaterial scaffold, matrix metalloproteinase (MMP) secretion, and other physical properties such as porosity and changes to collagen architecture. The increased stiffness of the ECM in GBM tumors can result in a barrier to drug diffusion. In the future, we plan to test the administration of chemotherapy treatments to investigate how ECM remodeling can affect drug efficacy in vitro.