TAMI-21. TUMOR-ASSOCIATED MICROGLIA GUIDE GBM INFILTRATION VIA PLEXIN-B2

Glioblastoma (GBM) is the most common malignant primary brain tumor. The nature of invasiveness of GBM makes complete surgical resection difficult. However, how GBM cells achieve wide infiltration in the brain is poorly understood. Microglia, the resident immune cells in the brain can support GBM growth and invasion, but the underlying mechanisms remain elusive. Here, we show that microglia are activated in a wide field away from tumor boundaries, ahead of tumor cell infiltration. Invading GBM cells are in close contact with microglia, progressively aligned with one another in the direction of tumor invasion. Moreover, ECM is also aligned with the infiltrating tumor and microglia, which may serve as invasion tracks in the brain. Mechanistically, we demonstrate that microglia direct cellular alignment and ECM remodeling in the invasion tracks through an axon guidance receptor Plexin-B2. Myeloid-specific ablation of Plexin-B2 perturbs microglia and tumor cell alignment, microglia migration, ECM organization, and GBM invasiveness. Together, our data reveal a hitherto under-appreciated role of microglia in providing directional cues for GBM invasion through physical interaction and alignment of ECM and tumor cells, thus providing new insights and novel molecular targets in curbing GBM invasion.

Uniaxially fixed mechanical boundary condition elicits cellular alignment in collagen matrix with induction of osteogenesis

Osteocytes differentiated from osteoblasts play significant roles as mechanosensors in modulating the bone remodeling process. While the well-aligned osteocyte network along the trabeculae with slender cell processes perpendicular to the trabeculae surface is known to facilitate the sensing of mechanical stimuli by cells and the intracellular communication in the bone matrix, the mechanisms underlying osteocyte network formation remains unclear. Here, we developed a novel in vitro collagen matrix system exerting a uniaxially-fixed mechanical boundary condition on which mouse osteoblast-like MC3T3-E1 cells were subcultured, evoking cellular alignment along the uniaxial boundary condition. Using a myosin II inhibitor, blebbistatin, we showed that the intracellular tension via contraction of actin fibers contributed to the cellular alignment under the influence of isometric matrix condition along the uniaxially-fixed mechanical boundary condition. Furthermore, the cells actively migrated inside the collagen matrix and promoted the expression of osteoblast and osteocyte genes with their orientations aligned along the uniaxially-fixed boundary condition. Collectively, our results suggest that the intracellular tension of osteoblasts under a uniaxially-fixed mechanical boundary condition is one of the factors that determines the osteocyte alignment inside the bone matrix.

Histological interpretation of differentiated vulvar intraepithelial neoplasia (dVIN) remains challenging—observations from a bi-national ring-study

Differentiated vulvar intraepithelial neoplasia (dVIN) is a premalignant lesion that is known to progress rapidly to invasive carcinoma. Accurate histological diagnosis is therefore crucial to allow appropriate treatment. To identify reliable diagnostic features, we evaluated the inter-observer agreement in the histological assessment of dVIN, among a bi-national, multi-institutional group of pathologists. Two investigators from Erasmus MC selected 36 hematoxylin-eosin-stained glass slides of dVIN and no-dysplasia, and prepared a list of 15 histological features of dVIN. Nine participating pathologists (i) diagnosed each slide as dVIN or no-dysplasia, (ii) indicated which features they used for the diagnosis, and (iii) rated these features in terms of their diagnostic usefulness. Diagnoses rendered by > 50% participants were taken as the consensus (gold standard). p53-immunohistochemistry (IHC) was performed for all cases, and the expression patterns were correlated with the consensus diagnoses. Kappa (ĸ)-statistics were computed to measure inter-observer agreements, and concordance of the p53-IHC patterns with the consensus diagnoses. For the diagnosis of dVIN, overall agreement was moderate (ĸ = 0.42), and pair-wise agreements ranged from slight (ĸ = 0.10) to substantial (ĸ = 0.73). Based on the levels of agreement and ratings of usefulness, the most helpful diagnostic features were parakeratosis, cobblestone appearance, chromatin abnormality, angulated nuclei, atypia discernable under × 100, and altered cellular alignment. p53-IHC patterns showed substantial concordance (ĸ = 0.67) with the consensus diagnoses. Histological interpretation of dVIN remains challenging with suboptimal inter-observer agreement. We identified the histological features that may facilitate the diagnosis of dVIN. For cases with a histological suspicion of dVIN, consensus-based pathological evaluation may improve the reliability of the diagnosis.

A Novel Bioreactor for the Mechanical Stimulation of Clinically Relevant Scaffolds for Muscle Tissue Engineering Purposes

Muscular tissue regeneration may be enhanced in vitro by means of mechanical stimulation, inducing cellular alignment and the growth of functional fibers. In this work, a novel bioreactor is designed for the radial stimulation of porcine-derived diaphragmatic scaffolds aiming at the development of clinically relevant tissue patches. A Finite Element (FE) model of the bioreactor membrane is developed, considering two different methods for gripping muscular tissue patch during the stimulation, i.e., suturing and clamping with pliers. Tensile tests are carried out on fresh and decellularized samples of porcine diaphragmatic tissue, and a fiber-reinforced hyperelastic constitutive model is assumed to describe the mechanical behavior of tissue patches. Numerical analyses are carried out by applying pressure to the bioreactor membrane and evaluating tissue strain during the stimulation phase. The bioreactor designed in this work allows one to mechanically stimulate tissue patches in a radial direction by uniformly applying up to 30% strain. This can be achieved by adopting pliers for tissue clamping. Contrarily, the use of sutures is not advisable, since high strain levels are reached in suturing points, exceeding the physiological strain range and possibly leading to tissue laceration. FE analysis allows the optimization of the bioreactor configuration in order to ensure an efficient transduction of mechanical stimuli while preventing tissue damage.

In Vitro Bioactivity and Biocompatibility of Bio-Inspired Ti-6Al-4V Alloy Surfaces Modified by Combined Laser Micro/Nano Structuring

The bioactivity and biocompatibility play key roles in the success of dental and orthopaedic implants. Although most commercial implant systems use various surface microstructures, the ideal multi-scale topographies capable of controlling osteointegration have not yielded conclusive results. Inspired by both the isotropic adhesion of the skin structures in tree frog toe pads and the anisotropic adhesion of the corrugated ridges on the scales of Morpho butterfly wings, composite micro/nano-structures, including the array of micro-hexagons and oriented nano-ripples on titanium alloy implants, were respectively fabricated by microsecond laser direct writing and femtosecond laser-induced periodic surface structures, to improve cell adherence, alignment and proliferation on implants. The main differences in both the bioactivity in simulated body fluid and the biocompatibility in osteoblastic cell MC3T3 proliferation were measured and analyzed among Ti-6Al-4V samples with smooth surface, micro-hexagons and composite micro/nano-structures, respectively. Of note, bioinspired micro/nano-structures displayed the best bioactivity and biocompatibility after in vitro experiments, and meanwhile, the nano-ripples were able to induce cellular alignment within the micro-hexagons. The reasons for these differences were found in the topographical cues. An innovative functionalization strategy of controlling the osteointegration on titanium alloy implants is proposed using the composite micro/nano-structures, which is meaningful in various regenerative medicine applications and implant fields.