(616e) Investigating Intracellular Adhesion and Intercellular Signaling in Glioblastoma Models

Glioblastoma (GBM) is the deadliest and most common form of brain cancer. Despite therapeutic intervention, the median survival time of patients is only 15 months. It has been reported in soft-tissue cancers that cells align along with the biomaterial scaffold prior to collective migration. Investigating the intracellular adhesions in GBM cells may help further the understanding of how these cells migrate, as well as elucidate interactions with the matrix. In addition to cell-cell and cell-matrix physical interactions, cytokine and chemokine secretions including TGF-β have been correlated to GBM tumor malignancy and growth. By combining intracellular adhesions and intercellular signaling pathways, it is possible to obtain a clearer picture of interactions between cells in GBM.

Methods

We assembled hydrogels composed of Type 1 collagen or HyStem-C® (Sigma), a gelin and hyaluronic acid (HA)-based biomaterial. A human GBM cell line LN229 (ATCC) was utilized since these cells display a mesenchymal subtype, and are moderately invasive. 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®. LN229 cells were then seeded atop the stacked gels. Co-cultures were also assembled utilizing human umbilical vein endothelial cells (HUVECs) as a mimic of vasculature. The HUVECs were cultured for 7 days to allow the formation of networks before co-culture.

Results

Within 24h in culture, LN229 cells formed bands that were aligned and extended along the z-axis in all biomaterial scaffolds. The adhesion molecule N-cadherin has previously been reported to be expressed by GBM cancer cells in intracellular interactions. Immunostaining revealed LN229 cells expressed N-cadherin within 24 h in culture. The expression of N-cadherin was similar in both mono- and co-cultures as well as across all three biomaterials. In addition to N-cadherin staining, the secretion of TGF-β was also measured, with LN229 monocultures displaying an increase of 9.7 – 22.3-fold in all biomaterials over 7 days of culture. TGF-β secretion in co-cultures increased up to 2.5-fold. TGF-β expression at day 8 was 5.7 – 12.9-fold higher in co-cultures than LN229 monocultures; however, secretion was similar at day 14. Furthermore, increased HUVEC seeding density resulted in a 1.1 – 1.3-fold decrease in TGF-β expression. This suggests that LN229 cells are the primary contributor of TGF-β in co-culture. The expression of chemokine CXCL12 was also measured, with an increase of 2 – 2.6-fold observed from Day 8 to Day 14 in all co-cultures. Increased HUVEC seeding density resulted in up to 1.4-fold increase in CXCL12 secretion. This suggests that HUVECs are in fact the primary source of this chemokine in co-culture. These results indicate that signaling molecules are differentially expressed in co-culture than monoculture.

Ongoing and Future Work

Currently, we are in the process of investigating early and continued expression of N-cadherin. We also plan to examine the expression of additional cell adhesion molecules such as integrins α₅ and β₁, as well as measure the secretion of other cytokines, such as IL10, that could impact intracellular interactions. EC intracellular adhesion will also be investigated through vascular endothelial (VE)-cadherin expression. Investigating how cell adhesion markers and secreted chemokines change as a function of co-culture can lead to a better understanding of intercellular interactions in GBM.