Chemotherapy resistance to glioblastoma (GBM) remains an obstacle that is difficult to overcome, leading to poor prognosis of GBM patients. Many previous studies have focused on resistance mechanisms intrinsic to cancer cells; the microenvironment surrounding tumor cells has been found more recently to have significant impacts on the response to chemotherapeutic agents. Extracellular matrix (ECM) proteins may confer cell adhesion-mediated drug resistance (CAMDR). Here, expression of the ECM proteins laminin, vitronectin, and fibronectin was assessed in clinical GBM tumors using immunohistochemistry. Then, patient-derived GBM cells grown in monolayers on precoated laminin, vitronectin, or fibronectin substrates were treated with cilengitide, an integrin inhibitor, and/or carmustine, an alkylating chemotherapy. Cell adhesion and viability were quantified. Transcription factor (TF) activities were assessed over time using a bioluminescent assay in which GBM cells were transduced with lentiviruses containing consensus binding sites for specific TFs linked to expression a firefly luciferase reporter. Apoptosis, mediated by p53, was analyzed by Western blotting and immunocytofluorescence. Integrin α v activation of the FAK/paxillin/AKT signaling pathway and effects on expression of the proliferative marker Ki67 were investigated. To assess effects of integrin α v activation of AKT and ERK pathways, which are typically deregulated in GBM, and expression of epidermal growth factor receptor (EGFR), which is amplified and/or mutated in many GBM tumors, shRNA knockdown was used. Laminin, vitronectin, and fibronectin were abundant in clinical GBM tumors and promoted CAMDR in GBM cells cultured on precoated substrates. Cilengitide treatment induced cell detachment, which was most pronounced for cells cultured on vitronectin. Cilengitide treatment increased cytotoxicity of carmustine, reversing CAMDR. ECM adhesion increased activity of NFκB and decreased that of p53, leading to suppression of p53-mediated apoptosis and upregulation of multidrug resistance gene 1 (MDR1; also known as ABCB1 or P-glycoprotein). Expression of Ki67 was correlative with activation of the integrin α v -mediated FAK/paxillin/AKT signaling pathway. EGFR expression increased with integrin α v knockdown GBM cells and may represent a compensatory survival mechanism. These results indicate that ECM proteins confer CAMDR through integrin α v in GBM cells.
Increased secretion of hyaluronic acid (HA), a glycosaminoglycan abundant in the brain extracellular matrix (ECM), correlates with worse clinical outcomes for glioblastoma (GBM) patients. GBM cells aggressively invade the brain parenchyma while encountering spatiotemporal changes in their local ECM, including HA concentration. To investigate how varying HA concentrations affect GBM invasion, patient-derived GBM cells are cultured within a soft, 3D matrix in which HA concentration is precisely varied and cell migration observed. Data demonstrate that HA concentration can determine the invasive activity of patient-derived GBM cells in a biphasic and highly sensitive manner, where the absolute concentration of HA at which cell migration peaked is specific to each patient-derived line. Furthermore, evidence that this response relies on phosphorylated ezrin, which interacts with the intracellular domain of HA-engaged CD44 to effectively link the actin cytoskeleton to the local ECM is provided. Overall, this study highlights CD44-HA binding as a major mediator of GBM cell migration that acts independently of integrins and focal adhesion complexes and suggests that targeting HA-CD44-ezrin interactions represents a promising therapeutic strategy to prevent tumor cell invasion in the brain.
Abstract Introduction: This study investigated how the mechanical microenvironment of glioblastoma (GBM) tumors may affect morphology and phenotype of patient-derived GBM and brain endothelial cells (ECs)– both critical components in the perivascular invasive niche. Materials and Methods: Hydrogels were fabricated from thiolated hyaluronic acid (HA-SH) (0.5 w/v%) and 4-arm PEG-SH (1.5 w/v%), 8-arm PEG-norbornene (1.125 w/v%). Gelation occurred upon exposure to UV light (365 nm, 3-5.75 mw/cm2) for 15 s in the presence of a cytocompatible photoinitiator LAP (Lithium Pehnyl (2, 4, 6-trimethylbenzoyl) phosphinate, 0.025 w/v%). Patient-derived gliomaspheres or single dissociated ECs were mixed with hydrogel precursors prior to gelation. Phase contrast images were acquired every 3 days to monitor cell migration. After 9 days, 3D cultures were fixed, and cells visualized with CellMaskTM Green (ThermoFisher) and Hoescht (nuclei). Cell survival following encapsulation was evaluated using a Live/Dead Assay (Life Technologies). A Leica LSP5 confocal microscope was used to image 3D cultures. Storage moduli (G') of hydrogels were measured using shear rheometer (TA DHR-2) with an 8-mm flat plate geometry. Results and Discussion: Hydrogel stiffness (G') was controlled by varying UV intensity to achieve a range of 150-1500 Pa, which is representative of the normal brain and tumor microenvironments, respectively. Gliomaspheres and ECs cultured showed comparable survival 7 days post-encapsulation in both soft and stiff gels. Gliomaspheres cultured in stiff hydrogels remained did not migrate away from spheroids, while cells encapsulated in soft hydrogels exhibited robust migration. Our previous results showed that ECs residing in GBM perivasculature differentially express high amount of integrin-binding sialoprotein (IBSP). Survival of ECs in hydrogels baring different biophysical properties is an appropriate platform to study the effect of stiffness on ECs protein expression. Conclusion: Patient-derived GBM and ECs were successfully cultured in 3D HA hydrogels mimicking biophysical properties of brain or GBM tumor tissue. GBM cells cultured in stiffer environments were unable to migrate, but upon culture in soft environments were found to migrate up to 200 µm away from seeded spheroids within 9 days of cultures. Acknowledgment: This project is funded by NIH 1R21NS0931099-01A1 grant and University of California Cancer Research Coordinating Committee Research Award. NIH Training Grant in Genomic Analysis and Interpretation T32HG002536. Citation Format: Alireza Sohrabi, Jesse Liang, Deepthi Muthukrishnan, Itay Solomon, Carolyn Kim, Amelia Lao, Harley Kornblum, Stephanie Seidlits. Biomimetic, 3D hydrogels to investigate effects of microenvironment biophysical properties on patient-derived glioblastoma (GBM) and endothelial cells (ECs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 181.
Abstract Hyaluronic acid (HA) is a highly abundant glycosaminoglycan within the central nervous system. In glioblastoma (GBM), interactions with HA mediate tumor cell invasion. An improved knowledge of the biological underpinnings of HA-dependent GBM cell migration can facilitate the development of efficacious therapeutics. To better study how HA in the tumor matrix affects invasion, we have developed a tunable, 3D culture system in which to study gliomaspheres (GSs) of patient-derived tumor cells. Photopolymerization (365 nm for 15 seconds) of GSs suspended in a solution 0.025 w/v% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), thiolated 4-arm poly-ethylene glycol (20 kDa, Laysan Bio), 8-arm poly-ethylene glycol-norbornene (20 kDa, Laysan Bio), thiolated peptides containing an ‘RGD’ amino acid motif, and either 0.10, 0.25, 0.50, or 0.75 w/v% of thiolated HA (700 kDa average molecular weight, Lifecore Biomedical) yielded 3D hydrogels of varying HA concentrations and similar mechanical properties. Relative invasive capacity, quantified by shape factor (deviation from circularity) and migration length (maximum extension length of processes from the GS periphery), differed across patient lines and was independent of their Cancer Genome Atlas (TCGA) classification as either mesenchymal or proneural. Hydrogels with higher amounts of HA (>0.25% w/v) generally enhanced invasion as compared to low HA (0.10 w/v%) hydrogels. In certain cell lines, a biphasic relationship between HA concentration and migration was observed, in line with previous reports of CD44 expression and migration. Moreover, inhibition of the CD44-Ezrin-Actin axis, using NSC668394, reduced migratory activity in hydrogels with higher HA (>0.25% w/v), while slightly increasing invasion in hydrogels with low HA (0.10 w/v%). In sum, results demonstrate the use of a matrix-mimetic, 3D hydrogel system in which to study GBM invasion through the local extracellular matrix. Citation Format: Gevick Safarians, Itay Solomon, Alireza Sohrabi, Stephanie Seidlits. Patient derived gliomaspheres exhibit HA concentration dependent invasiveness in 3D hydrogels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3459.
<p>Supplementary Methods describing additional information for procedures in manuscript, including hydrogel fabrication, hydrogel sample cryopreservation, lentivirus preparation, mechanical characterization, diffusion, western blotting, and statistics.</p>
The urban sprawl, the growing population and issues such as visual and environmental maladies, traffic, air pollution, instability and insecurity, and the failure to provide urban services provide urban management with the task of adopting effective solutions such as sustainable development. Considering that the main part of the income of the municipality has been through the sale of congestion, which is one of the unsustainable incomes, in order to achieve sustainable development, perhaps after human being, which in the opinion of a citizen and without human beings, sustainable development of the city has no conceptual meaning. The most important factor is sustainable urban resources. Sources of sustainable income are the missing element and missing element that badly affects the performance of urban management. If municipalities are not able to earn sufficient and sustainable income, they will not be able to establish and operate necessary facilities in the city. Although municipal resources are available in various forms, they do not all have the characteristics of sustainable earnings. Sustainability in revenues requires that these items be of relative continuity and, secondly, the acquisition of these revenues does not endanger the quality of the city and threaten and destroy it. Accordingly, the main objective of the present research is the necessity of realization of resistance economy policies in the field of urban management, with sustainable earnings approach in Tehran municipality using descriptive-analytical method. In order to achieve this goal, firstly, the sentences of sustainable sources of income were extracted in the second five-year plan of the Tehran municipality, and then examined and identified sustainable and unsustainable revenues of the Tehran municipality. Finally, strategies for the provision of sustainable financial resources were presented in the municipality of Tehran. The results of the research show that most of the major revenues of municipalities are not consistent with sustainable concepts and they do not have the continuity and desirability. Therefore, it is imperative that municipalities, by studying the income-based and resilient economy, are better equipped to provide sustainable and sustainable sources of income in order to provide citizens with decent services.
Glioblastoma (GBM) is the most lethal cancer originating in the brain. Its high mortality rate has been attributed to therapeutic resistance and rapid, diffuse invasion – both of which are strongly influenced by the unique microenvironment. Thus, there is a need to develop new models that mimic individual microenvironmental features and are able to provide clinically relevant data. Current understanding of the effects of the microenvironment on GBM progression, established experimental models of GBM and recent developments using bioengineered microenvironments as ex vivo experimental platforms that mimic the biochemical and physical properties of GBM tumors are discussed.
<p>Supplementary figures S1-S12 providing necessary data on additional animal xenograft experiments, essential information characterizing cell lines used in manuscript, and controls for immunostaining and western blots. This supplementary file also provides data for experiment on additional cell lines.</p>
SummaryGlioblastoma (GBM) is characterized by extensive microvascular hyperproliferation. In addition to supplying blood to the tumor, GBM vessels also provide trophic support to glioma cells and serve as conduits for migration into the surrounding brain promoting recurrence. Here, we enriched CD31-expressing glioma vascular cells (GVC) and A2B5-expressing glioma tumor cells (GTC) from primary GBM and utilized RNA sequencing to create a comprehensive interaction map of the secreted and extracellular factors elaborated by GVC that can interact with receptors and membrane molecules on GTC. To validate our findings, we utilized functional assays, including a novel hydrogel-based migration assay and in vivo mouse models to demonstrate that one identified factor, the little-studied integrin binding sialoprotein (IBSP) enhances tumor growth and promotes the migration of GTC along the vasculature. This perivascular niche interactome will serve a resource to the research community in defining the potential functions of the GBM vasculature.
Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.
