Inherent Interfacial Mechanical Gradients in 3D Hydrogels Influence Tumor Cell Behaviors

Cells sense and respond to the rigidity of their microenvironment by altering their morphology and migration behavior. To examine this response, hydrogels with a range of moduli or mechanical gradients have been developed. Here, we show that edge effects inherent in hydrogels supported on rigid substrates also influence cell behavior. A Matrigel hydrogel was supported on a rigid glass substrate, an interface which computational techniques revealed to yield relative stiffening close to the rigid substrate support. To explore the influence of these gradients in 3D, hydrogels of varying Matrigel content were synthesized and the morphology, spreading, actin organization, and migration of glioblastoma multiforme (GBM) tumor cells were examined at the lowest (<50 µm) and highest (>500 µm) gel positions. GBMs adopted bipolar morphologies, displayed actin stress fiber formation, and evidenced fast, mesenchymal migration close to the substrate, whereas away from the interface, they adopted more rounded or ellipsoid morphologies, displayed poor actin architecture, and evidenced slow migration with some amoeboid characteristics. Mechanical gradients produced via edge effects could be observed with other hydrogels and substrates and permit observation of responses to multiple mechanical environments in a single hydrogel. Thus, hydrogel-support edge effects could be used to explore mechanosensitivity in a single 3D hydrogel system and should be considered in 3D hydrogel cell culture systems

Cloning and characterization of a calcitonin receptor from the guinea pig brain

Calcitonin is a 32 amino acid peptide hormone of neural crest origin secreted by the parafollicular cells of the thyroid. It is believed that calcitonin, through specific and high affinity interactions with the calcitonin receptor, mediates a diverse spectrum of effective such as hypocalcemia, analgesia, anorexia, suppression of gastric acid secretion, and inhibition of prolactin secretion. However, which of these actions represents the true physiologic role for calcitonin has not been clearly defined. To better understand the physiology of calcitonin and calcitonin receptors, a calcitonin receptor was cloned from a guinea pig brain using a degenerate reverse transcription/polymerase chain reaction (RT/PCR) strategy. Sequence analysis of this receptor showed that it contains 478 amino acids and is a member of the seven transmembrane G-protein coupled receptor superfamily. COS 1 cells transfected with the cloned guinea pig calcitonin receptor cDNA and activated by salmon calcitonin produced a 10 fold increase in intracellular cAMP accumulation, EC\sb{50} of 0.1 nM. In this system, human calcitonin was $\u3e250$ fold less potent and the related neuropeptides, rat $\alpha$CGRP and rat amylin, did not activate the guinea pig calcitonin receptor at physiological concentrations. Stimulation of the guinea pig calcitonin receptor in transfected cells by salmon calcitonin resulted in $\u3e2$ fold increase in phosphatidylinositol hydrolysis with an EC\sb{50} of 2.5 nM. A PCR based strategy to detect possible subtypes of the guinea pig calcitonin receptor revealed that guinea pigs express a single calcitonin receptor subtype. Such a finding contrasts previous observations that other calcitonin receptors from different species contain multiple receptor subtypes. This may indicate that the other subtypes of calcitonin receptors may not have a primary role in calcitonin biology but rather represent species specific modifications of the calcitonin receptor. Tissue distribution of the guinea pig calcitonin receptor was determined by a combination of RT/PCR, Northern analysis, and expression in Xenopus oocytes. These studies consistently demonstrated that the guinea pig calcitonin receptors have the highest expression in the diencephalon. The abundance of calcitonin receptors in the guinea pig brain suggests that this receptor may participate in regulating some functions of the central nervous system, such as analgesia and anorexia

Distal Anterior Inferior Cerebellar Artery Aneurysm Masquerading as a Cerebellopontine Angle Tumor: Case Report and Review of Literature

We present the case of a distal anterior inferior cerebellar artery (AICA) aneurysm masquerading as a cerebellopontine angle tumor in a 60-year-old right-handed man with previously undiagnosed polyarteritis nodosa (PAN). The patient presented with a 2-month history of progressive right-sided hearing loss, intermittent severe headache, and sudden onset of complete facial paralysis 3 weeks before admission. Magnetic resonance imaging, including postgadolinium images, showed a 1.2-cm heterogenously enhancing mass that slightly enlarged the right internal auditory canal. A right suboccipital craniotomy was performed, and a partially thrombosed fusiform AICA aneurysm was discovered just anterior to the VII/VIII nerve complex. The aneurysm was trapped and opened, and a thrombectomy was performed. Postoperatively, the patient experienced abdominal pain; liver function tests were abnormal. Investigation revealed a small retroperitoneal hemorrhage and aneurysms of the celiac axis and gastroduodenal arteries. Further investigation revealed an increased erythrocyte sedimentation rate, and a diagnosis of PAN was made. PAN is a well-identified factor in the genesis of peripheral vascular aneurysms. Aneurysms involving the hepatic, renal, coronary, pancreatic, and tibial arteries have been described. PAN is an extremely rare cause of intracranial aneurysm. Patients who present with aneurysms in unusual locations (e.g., distal AICA) should be investigated for vasculopathy and collagen vascular disorders

The Mechanical Fingerprint of a Parallel Polyprotein Dimer

We use the GCN4 oligomerization domain to engineer a covalently linked parallel polyprotein dimer based on the well-studied I27 domain of titin. We use single molecule atomic force microscopy techniques to stretch single polyprotein fibers and verify their mechanical properties. We find that the engineered polyprotein dimers extend in perfect register, doubling the unfolding force and halving the persistence length while keeping the contour length increase unchanged. These experiments directly confirm the mechanical scaling laws proposed for parallel bundles of modular proteins

Mechanical Characterization of Protein L in the Low-Force Regime by Electromagnetic Tweezers/Evanescent Nanometry

Mechanical manipulation at the single molecule level of proteins exhibiting mechanical stability poses a technical challenge that has been almost exclusively approached by atomic force microscopy (AFM) techniques. However, due to mechanical drift limitations, AFM techniques are restricted to experimental recordings that last less than a minute in the high-force regime. Here we demonstrate a novel combination of electromagnetic tweezers and evanescent nanometry that readily captures the forced unfolding trajectories of protein L at pulling forces as low as 10 ∼ 15 pN. Using this approach, we monitor unfolding and refolding cycles of the same polyprotein for a period of time longer than 30 min. From such long-lasting recordings, we obtain ensemble averages of unfolding step sizes and rates that are consistent with single-molecule AFM data obtained at higher stretching forces. The unfolding kinetics of protein L at low stretching forces confirms and extends the observations that the mechanical unfolding rate is exponentially dependent on the pulling force within a wide range of stretching forces spanning from 13 pN up to 120 pN. Our experiments demonstrate a novel approach for the mechanical manipulation of single proteins for extended periods of time in the low-force regime

Glioblastoma Behaviors in Three-Dimensional Collagen-Hyaluronan Composite Hydrogels

Glioblastoma multiforme (GBM) tumors, which arise from glia in the central nervous system (CNS), are one of the most deadly forms of human cancer with a median survival time of ∼1 year. Their high infiltrative capacity makes them extremely difficult to treat, and even with aggressive multimodal clinical therapies, outcomes are dismal. To improve understanding of cell migration in these tumors, three-dimensional (3D) multicomponent composite hydrogels consisting of collagen and hyaluronic acid, or hyaluronan (HA), were developed. Collagen is a component of blood vessels known to be associated with GBM migration; whereas, HA is one of the major components of the native brain extracellular matrix (ECM). We characterized hydrogel microstructural features and utilized these materials to investigate patient tumor-derived, single cell morphology, spreading, and migration in 3D culture. GBM morphology was influenced by collagen type with cells adopting a rounded morphology in collagen-IV versus a spindle-shaped morphology in collagen-I/III. GBM spreading and migration were inversely dependent on HA concentration; with higher concentrations promoting little or no migration. Further, noncancerous astrocytes primarily displayed rounded morphologies at lower concentrations of HA; in contrast to the spindle-shaped (spread) morphologies of GBMs. These results suggest that GBM behaviors are sensitive to ECM mimetic materials in 3D and that these composite hydrogels could be used to develop 3D brain mimetic models for studying migration processes

Quantification of migration speeds (average) of OSU-2 cells at the lowest (<∼50 µm) and highest observation planes (>∼500 µm) investigated.

<p>* indicates statistical significance.</p

miRNA-mediated loss of m6A increases nascent translation in glioblastoma.

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition

Mechanics of the gel-glass interface modeled using FEM.

<p>(A) Stress contour plots of Matrigel with varying height. Axisymmetric elements used. Von Mises stress is an equivalent stress that includes both normal stress (tension/compression) and shear stress contributions. It is calculated from the stress components acting at each location and gives a convenient way of comparing the overall magnitude of stress in different regions. (B) Stress felt at the Matrigel-glass interface as a function of gel height.</p