Poster T10 - Understanding the TRP Mediated-Calcium Signaling and Epithelial to Mesenchymal Transition of Glioblastoma Multiforme on a Microfluidic Model

Introduction: Glioblastoma multiforme (GBM) is among one of the most deadly, aggressive, and invasive types of cancer. Currently, there is no cure for patients diagnosed with GBM. Due to the cancer’s high rates of motility and invasion, even with treatment, patients are often given a survival time of 12-15 months after diagnosis. This makes GBM of particular interest to scientists who intend to improve outcomes of GBM patients. Hence, multiple studies have been conducted to further our understanding of GBM tumor biology and the mechanisms underlying its malignancy. Our research focuses on how the tumor’s microenvironment can promote migration as GBM interacts with components in the brain's extracellular matrix, triggering processes such as the calcium signaling pathway and the epithelial to mesenchymal transition (EMT). Both have caught the attention of numerous researchers because of its implications in GBM migratory and invasive capabilities.

Materials and

Methods: Using the non-adherent method and microfluidic technology, we have generated a model of three dimensional GBM spheroids that was introduced to a PDMS device with microfluidic channels and matrigel to simulate the physical confinements and the extracellular matrix in the brain tissue, respectively. This procedure has established two phenotypes of GBM, an invasive phenotype where the spheroid is touching the confinements of the channels and sprouts, and a non-invasive one, where the spheroid is not in contact with the confinements.
First, our studies compare calcium levels between the two established phenotypes in our 3D models through the use of FLUO-8AM, a dye that turns fluorescent green upon binding to intracellular calcium. Using fluorescent imaging, we examined the relationship between phenotypes and calcium signaling activity. Second, we investigated the specific effect of TGFβ inhibitor, SB-431542, on the EMT as well as other aberrant outcomes. Using our established models, the TGFβ inhibitor was introduced and the resulting sprouting and migrating behaviors of the invasive cells were quantified.

Results, Conclusions, and Discussions: Our calcium signaling trials showed an increase in fluorescent intensity for the invasive phenotype in comparison to the non-invasive phenotype of the cancer, which could possibly indicate a more active calcium signaling pathway. For the change in calcium concentration over a period of time, our results were consistent, with the invasive phenotype demonstrating more variable concentrations and therefore more calcium exchange. With our EMT investigation, our results showed that the invasive untreated group had a much larger sprouting area compared to the treated invasive group. Additionally there was a significant difference between the treated and untreated invasive groups. In the future, we plan to isolate the cells for transcriptomic analysis, particularly on the mRNA expression of EMT-related genes. The findings of these studies will broaden our knowledge of the complexity of EMT, calcium signaling pathways, and their role in GBM cancer development and metastasis.

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