Harley, Brendan A.C.

Abstract The extracellular matrix (ECM) is increasingly recognized as having a key role in cancer development. We have developed a biomaterial tumor mimic that recapitulates systematically the main characteristics and heterogeneities of glioblastoma ECM. With the use of these 3D in vitro platforms we seek to understand the key features that make this cancer so challenging to treat. We are using this device in three different ways: (1) to further understand how particularities of brain tumor ECM affect cancer cells and assist in the diagnostic of different glioblastoma (GBM) tumor types, (2) provide new models for drug screening to afford more personalized therapies and (3) study the mechanisms of resistance to current therapies in order to identify more effective combinatorial treatments and new therapeutic targets. Hyaluronic acid (HA), is the main component of the brain ECM and GBM is associated with aberrant HA secretion and overexpression of receptors associated with HA, such as CD44 and EGFR. We cultured GBM patient-derived xenograft (PDX) cells within gelatin based hydrogels that contained variable concentrations of HA. Cell proliferation and gene expression analyses demonstrate that biomimetic hydrogels support xenograft culture and cells upregulate matrix remodeling genes and others related to tumor growth in response to matrix biophysical properties. We also used these platforms to evaluate the mechanisms of cell resistance to tyrosine kinase inhibitors. For this purpose, we first focused on the epidermal growth factor receptor (EGFR), which has been identified as a molecular target and associated with worse clinical outcomes. We characterized the 3D in vitro behavior of 3 PDX that represent these EGFR variants: GBM10 (EGFR, wild type), GBM12 (EGFR+) and GBM6 (EGFRvIII). We studied the relationship between the HA contained in the surrounding matrix and response of GBM cells to a tyrosine kinase inhibitor (TKI), erlotinib. Results indicate that while EGFR+ cells are sensitive to TKI in HA hydrogels, HA seems to collaborate with EGFRvIII signaling to stir cell activity. Immunoblots demonstrate that this enhanced cell activity is related to a significant increase in PDGFR concentration. Blockade of the CD44 receptor, in combination with erlotinib treatment, does not affect phosphorylation rates of PI3K or ERK, however, it leads to a significant decrease of STAT3 phosphorylation. This is translated into a lower tumor cell metabolic activity in HA matrices. Finally, we show that, patient-derived xenograft cells resistant to erlotinib in vivo (GBM10), become sensitive when CD44 is blocked in HA-containing matrices, as demonstrated by a decrease of phosphorylated PI3K. In summary, we highlight the importance of extracellular HA in EGFR inhibition efficiency. We demonstrate that this biomaterial tumor model can be used as a valuable tool in the mechanistic studies of tumor development and prediction of tyrosine kinase inhibitors efficacy. Citation Format: Sara Pedron, Gabrielle L. Wolter, Emily Chen, Jann N. Sarkaria, Brendan A. Harley. Glioblastoma tumor model to analyze the mechanisms of resistance to tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5776. doi:10.1158/1538-7445.AM2017-5776

Abstract The extracellular matrix (ECM) is increasingly recognized as having a key role in cancer development. Advances in biomaterials design and preparation have led to highly defined sophisticated platforms that can recapitulate key features of the complex tumor microenvironment. We have developed a biomaterial tumor mimic that recapitulates systematically the main characteristics and heterogeneities of glioblastoma ECM. With the use of these 3D in vitro platforms we seek to understand the key features that make this cancer so challenging to treat. We are using this device in three different ways: (1) to further understand how particularities of brain tumor ECM affect cancer cells and assist in the diagnostic of different GBM tumor types, (2) provide new models for drug screening to afford more personalized therapies and (3) study the mechanisms of resistance to current therapies in order to identify more effective combinatorial treatments and new therapeutic targets. Glioblastoma (GBM) tumors are highly heterogeneous, both cell composition and ECM biophysical properties are key factors in cancer cell malignancy and treatment outcomes. Hyaluronic acid (HA), is the main component of the brain extracellular matrix and GBM is associated with aberrant HA secretion and overexpression of receptors associated with HA, such as CD44 and EGFR. We use a microfluidic system to culture GBM patient-derived cells within gelatin based hydrogels that contained gradually increasing concentrations of HA, in order to recreate the heterogeneity of the tumor mass. Cell proliferation and gene expression analyses demonstrate that biomimetic hydrogels support xenograft culture; cells remain viable, active, and upregulate matrix remodeling genes and others related to tumor growth. More importantly, we found that GBM cells respond to changes in the ECM composition presenting different gene expression profiles (VEGF, IL6, MMP2, MMP9, HAS3, CD44) depending on the particularities of every tumor, such as EGFR overexpression or PTEN suppression. These findings pave the way for new clinical tools that allow a more precise cataloging of GBM tumor types and tailoring treatments to individual patients. We have also used these platforms to evaluate potential therapeutic strategies for different GBM xenograft cells and to study the mechanisms of cell resistance. For this purpose, we first focused on the epidermal growth factor receptor (EGFR), which has been identified as a molecular target and associated with worse clinical outcomes. Amplification, overexpression and mutation of the EGFR tyrosine kinase has been identified in 50% of GBM patients. The genetic aberration of EGFR (EGFR+) often comes with the overexpression of the mutant EGFR variant III (EGFRvIII). We characterized the 3D in vitro behavior of 3 patient-derived xenografts that represent these EGFR variants: GBM10 (EGFR, wild type), GBM12 (EGFR+) and GBM6 (EGFRvIII). We studied the relationship between the HA contained in the surrounding matrix and response of GBM cells to a tyrosine kinase inhibitor (TKI), erlotinib. We demonstrate that an interaction exists between CD44 and EGF receptors. Results indicate that while EGFR+ cells are sensitive to TKI in HA hydrogels, HA seems to collaborate with EGFRvIII signaling to stir cell activity. Our results indicate that a combination of both bound and cell secreted soluble HA may influence GBM sensitivity and resistance to erlotinib. Moreover, immunoblots that analyze PI3K pathway support the hypothesis that matrix-bound HA is a key factor in the negative response of vIII tumors to EGFR inhibitors, and that the benefits of EGFR inhibition in GBMs may be found in combination to other inhibitors of ECM signaling. In summary, we demonstrate that engineered tumor avatar biomaterials can be used to reproduce some of the main characteristics of brain tumor microenvironment and become a valuable tool in the mechanistic studies of tumor development and treatment efficacy. Citation Format: Sara Pedron, Amanda M. Pritchard, Jann N. Sarkaria, Mark A. Schroeder, Brendan A. Harley. Engineered biomaterials as essential tools to determine the mechanisms of resistance to tyrosine kinase inhibitors in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B05.