Pedrón, Sara

Abstract Radiation therapy (RT) has long been included in the treatment of glioblastoma (GBM). However, radioresistance in cancer cells as well as toxicity in normal tissues are major obstacles to clinical efficacy. Improved understanding of the mechanisms of tumor microenvironment-induced radioresistance during and after radiation therapy can provide fundamental insights to improve clinical outcomes in GBM. Here, using three-dimensional engineered hydrogel models in vitro, we report the influence of extracellular matrix, hypoxia, and adjacent neuronal cells in radiotherapeutic sensitivity. We find that mechanical cues and oxygen availability regulate cellular response to radiation, with softer matrices allowing for more DNA damage. Hyaluronan fragments from the extracellular matrix also modulate rapid metabolic response to radiation, especially in hypoxic environments. We show that neuronal networks influence tumor metabolic activity and the inflammatory response. Overall, we demonstrate here that alternative radiation strategies, such as low dose rate radiation therapy and microenvironmental regulation, have the potential to be more effective in a specific subset of radiosensitive GBM tumors.

Abstract Despite extensive advances in cancer research, glioblastoma (GBM) still remains a very locally invasive and thus challenging tumor to treat, with a poor median survival. Tumor cells remodel their microenvironment and utilize extracellular matrix to promote invasion and therapeutic resistance. It is aimed here to determine how GBM cells exploit hyaluronan (HA) to maintain proliferation using ligand‐receptor dependent and ligand‐receptor independent signaling. Tissue engineering approaches are used to recreate the 3D tumor microenvironment in vitro, then analyze shifts in metabolism, hyaluronan secretion, HA molecular weight distribution, as well as hyaluronan synthetic enzymes (HAS) and hyaluronidases (HYAL) activity in an array of patient‐derived xenograft GBM cells. It is revealed that endogenous HA plays a role in mitochondrial respiration and cell proliferation in a tumor subtype‐dependent manner. A tumor‐specific combination treatment of HYAL and HAS inhibitors is proposed to disrupt the HA stabilizing role in GBM cells. Taken together, these data shed light on the dual metabolic and ligand – dependent signaling roles of hyaluronan in glioblastoma.

Abstract Biomaterial-based ex vivo models of healthy and diseased brain tissue are among those tools that may help study the complexity of cerebral tissue to better diagnose, prevent, and treat brain diseases. We have established engineered brain tumor biomaterials based on methacrylamide-functionalized gelatin hydrogels and used microfluidic-forming techniques to generate platforms that combine transitions of biophysical and biomolecular properties found in the glioblastoma tumor microenvironment, from the core to the tumor margins (1). Moreover, this biomaterial approach is able to monitor the response of patient-derived xenograft (PDX) cell populations with different molecular signatures; in particular, we have analyzed the amplified and the constitutively activated vIII mutant EGF receptor. We have also characterized PDX response to targeted inhibitors, such as erlotinib, a tyrosine kinase inhibitor that specifically blocks EGFR (2). Patients with glioblastoma (GBM) exhibit poor survival rates, tied both to the significant intra and interpatient heterogeneity of the tumor as well as the complex signaling pathways underlying malignancy. Spatial and temporal gradients regulate cell proliferation, migration, and differentiation during cancer. Therefore, the use of a microfluidic approach to fabricate patterned biomaterials, have the ability to examine transitions between defined environments (e.g., glioma core, periphery and neural tissue). Cells within these structured materials can be fully analyzed by imaging, secretomic and gene expression analyses. Cell morphology evolved to a more spread shape from low to high crosslinking density of gelatin and hypoxic levels depended on cell concentration. Using patient derived GBM tumor samples we are able to generate a miniaturized tumor tissue analog to examine how the heterogeneities within the tumor microenvironment impact glioma malignant phenotype, growth, and therapeutic efficacy. The ability to manipulate tumor extracellular matrix (ECM) can improve therapeutic outcomes and restrict glioma infiltration. Our in vitro tumor model is able to analyze the relationship between HA and the invasive phenotype of GBM tumor cells. We show that cell growth, motility and proteomic responses of GBM cells within our platform were significantly altered by HA molecular weight in response to a tyrosine kinase inhibitor. These results provide additional insights regarding the importance of extracellular microenvironment in the invasive potential of GBM tumors. We have demonstrated that repeated dosing of erlotinib promotes expression of PDGFRb in EGFR vIII mutant cells and that extracellular HA plays a favorable role in the inhibition of EGFR, through STAT3 deactivation (3). (1) Pedron S et al. (2015), Adv. Mater., 27: 1567; (2) Pedron S. et al. (2017), Adv. Healthcare Mater., 6: 1700529; (3) Pedron S, et al. (2019), Biomaterials, 219: 119371. Citation Format: Sara Pedron, Jann N. Sarkaria, Brendan A.C. Harley. Engineered glioblastoma tumor models reveal extracellular matrix signals influence the efficacy of targeted inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO035.

Abstract 3D biomaterial models have potential to explore the influence of the tumor microenvironment on aberrant signaling pathways and compensatory signals using patient‐derived cells. Glioblastoma (GBM) tumors are highly heterogeneous, with both cell composition and extracellular matrix biophysical factors seen as key regulators of malignant phenotype and treatment outcomes. Amplification, overexpression, and mutation of the epidermal growth factor receptor (EGFR) tyrosine kinase have been identified in 50% of GBM patients. Here, hyaluronic acid (HA) decorated methacrylamide‐functionalized gelatin (GelMA) hydrogels are used to examine the synergies between microenvironmental factors and a model EGFR tyrosine kinase inhibitor (TKI) using patient‐derived xenograft cells. The in vitro behavior of 3 patient‐derived xenografts that reflect a clinically relevant range of EGFR variants is characterized: GBM10 (EGFR, wild type), GBM12 (EGFR+), and GBM6 (EGFRvIII). GelMA hydrogels support xenograft culture; cells remain viable, active, respond to matrix‐immobilized HA, and upregulate genes associated with matrix remodeling and tumor growth. Interestingly, matrix‐immobilized HA alters the response of GBM cells to a model tyrosine kinase inhibitor, erlotinib. While constitutively activated EGFRvIII cells are sensitive to TKI in gelatin hydrogels, hyaluronic acid mediated adhesive signaling interacts with EGFRvIII signaling to increase cell metabolic activity, increase soluble hyaluronic acid synthesis, and modify response to erlotinib exposure.

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.