Associate Professor The University of Texas at Austin, United States
Introduction:: Glioblastoma (GBM) cancer is the most malignant form of brain cancer with a prognosis of ~5% chance of survival 5 years after the diagnosis.1 Following the typical cancer treatment regimen involving surgical removal, chemotherapy and radiation therapy, the remaining cancerous cells become resistant to treatment and increase in malignancy, thus, lowering these patients’ prognosis.2 To combat the revolving door of tumor recurrence, better in vitro platforms are needed to explore and characterize GBM and its plasticity in order to develop more efficient therapies. My project will target this problem by characterizing changes in GBM cells as they interact with an ex vivo, brain-mimetic peri-tumoral microenvironment (TME). Current in vitro models of GBM lack key features to recapitulate this in vivo TME, including a brain-mimetic ECM with stromal cells such as endothelial cells (ECs), immune cells, and other neural cell types. While a researchers have incorporated ECM-mimetic peptides into hydrogel-based, in vitro culture systems, they include collagen, fibrin and/or matrigel because they are widely used in other three dimensional (3D) models, however they are not relatively expressed in the brain ECM compared to other areas of the body.3 Hyaluronic acid (HA) is the primary glycosaminoglycan in the brain ECM. Thus, we are developing an HA-based, 3D scaffolding in which to culture GBM cells and ECs and study their cross-talk. By highlighting mechanisms of communication between these cells, more precise therapeutics can be made to target the cancer to have better efficiency at killing the cells after removal.3
Materials and Methods:: Scaffolds of macroporous annealed particles (MAP), seeded with ECs,4,5 and UV-activated, nanoporous HA hydrogels (photogel), embedded with GBM cells,6 will be used to create spatially defined co-cultures. (Fig. 1) In nanoporous scaffolds, EC proliferation and tube formation is typically dependent on the type of ECs and scaffold composition,7 meaning their ability and likelihood to form tube-like structures varies across experiments. This motivates the use of MAP scaffolds, which induce ECs to form intricate tubular networks templated by the space around each microsphere and microsphere curvature. MAPs are made using an emulsification process followed by a sterilization step to prepare them for cell culture. This scaffold contains a relatively uniform pore size, ultimately controlled by the flow rate of the solutions during particle microfluidic-based fabrication, which standardizes the microenvironment that seeded ECsexperience. GBM cells will be cultured in a photogel incorporating ECM-mimetic peptides, which recapitulate specific cell-ECM interactions in GBM tumors in vivo.8 To explore the dynamic connections between aberrant vascularization and GBM malignancy, I have used this co culture system and observed increased migration towards the ECs cultured in the MAP gel. (figure 2) This preliminary data suggests a connection between the two cell types as expected. Next, I plan to 1) Characterize the relationship between ECs and GBM cells using this platform to explore mechanisms for how they communicate through RNAseq and Luminex assays and then 2) Assess how GBM-EC interaction changes in response to radiation treatment.
Results, Conclusions, and Discussions:: Through time-iterated RNAseq of the separated crosstalk experiments I expect to see gradual changes in expression of key proteins used to communicate with the opposite cell type in response to the other’s secretome. Based on current findings in Ivy Glioblastoma Atlas, VEGF, EMR2, and IBSP etc. should be upregulated due to the lack of nutrients present in the media. These elevated genes will further be reduced to find the important factors used between the two cells using Luminex assays assessing the secreted factors only. With these results we can develop a line of communication outside of the known VEGF importance between these cells. To support this data, chemotactic studies to track GBM migration in response to EC secretome will observe a significant preference in migration and validate the communication between these cell types through secreted factors. In the case where no significant accumulation of factors or migration is observed, this could be due to the lack of physical interaction between the cells or the important factors are no longer highly present in the media due to fast degradation of the molecules after secretion. Following preliminary studies, I will coculture these cells using the design in Figure 1. These experiments will be repeated to assess any differences compared to baseline experiments after radiation. Significant differences are expected in migration and factor secretion implying that co culture systems are more recapitulative of their in vivo environment and these 3D systems are needed to form effective therapies. These results will also provide a starting point for following knockdown/out studies working towards developing therapeutic drugs efficiently targeting GBM-EC interaction. To address my second aim I will irradiate these cells on the first day of the experiment and evaluate how this treatment changes their factor secretion, and GBM migration. I expect these cells to overall increase their dependency of each other as a result of the DNA damage that is induced by the treatment. This data will also highlight dynamic interactions between ECs and GBM cells that facilitate their survival post-radiation, hinting towards in vivo mechanisms of survival GBM cells utilize leading to recurrence.
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