Impact of human bladder cancer cell architecture on autologous T-lymphocyte activation

To investigate the influence of tumor cell architecture on T-cell activation, we used an autologous human model based on 2 bladder tumor cell lines as targets for cytotoxic tumor-infiltrating lymphocytes (TILs). These tumor cell lines were grown in vitro as either standard 2-dimensional (2D) monolayers or 3-dimensional (3D) spheroids. T-cell activation was determined by measuring the production of three major cytokines (tumor necrosis factor, granulocyte/macrophage colony-stimulating factor and interferon-gamma), known to be secreted by most activated TILs. Changes in the architecture of target cells from 2D to 3D induced a dramatic decrease in their capacity for stimulating TILs. Interestingly, neither TIL infiltration nor MHC class I, B7.1 costimulatory or lymphocyte function-associated factor-3 adhesion molecule downregulation played a major role in this decrease. These findings demonstrate that tumor architecture has a major impact on T-cell activation and might be implicated in the escape of tumor cells from the immune system

Stable tubule only polypeptides (STOP) proteins co-aggregate with spheroid neurofilaments in amyotrophic lateral sclerosis

A major cytopathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of axonal spheroids containing abnormally accumulated neurofilaments. The mechanism of their formation, their contribution to the disease, and the possibility of other co-aggregated components are still enigmatic. Here we analyze the composition of such lesions with special reference to stable tubule only polypeptide (STOP), a protein responsible for microtubule cold stabilization. In normal human brain and spinal cord, the distribution of STOP proteins is uniform between the cytoplasm and neurites of neurons. However, all the neurofilament-rich spheroids present in the tissues of affected patients are intensely labeled with 3 different anti-STOP antibodies. Moreover, when neurofilaments and microtubules are isolated from spinal cord and brain, STOP proteins are systematically co-purified with neurofilaments. By SDS-PAGE analysis, no alteration of the migration profile of STOP proteins is observed in pathological samples. Other microtubular proteins, like tubulin or kinesin, are inconstantly present in spheroids, suggesting that a microtubule destabilizing process may be involved in the pathogenesis of ALS. These results indicate that the selective co-aggregation of neurofilament and STOP proteins represent a new cytopathological marker for spheroids

Comparison of spheroids formed by rat glioma stem cells and neural stem cells reveals differences in glucose metabolism and promising therapeutic applications

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs

Focused Ultrasound Treatment of a Spheroid In Vitro Tumour Model

SIMPLE SUMMARY: Ultrasound waves can be applied for diagnostic and therapeutic purposes. Focused ultrasound is approved for tissue ablation, e.g., in the treatment of uterine fibroids or essential tremors. Besides the non-invasive image-guided surgical intervention at temperatures above 55 °C, FUS is investigated in other fields like blood-brain barrier opening, hyperthermia, and neuromodulation. FUS offers potential as an adjuvant therapy in cancer treatment. Therefore, analysis of FUS effects on cancer cells is necessary. We performed studies on two human cancer cell line spheroids using a newly developed high-throughput in vitro FUS applicator with 32 individual transducers. This study aimed to perform basic experiments with a new in vitro FUS device on a 3D tumour model to acquire insight into the effects of FUS at the cellular level. These experiments may contribute to a better understanding and predictions of cancer treatment efficacy. ABSTRACT: Focused ultrasound (FUS) is a non-invasive technique producing a variety of biological effects by either thermal or mechanical mechanisms of ultrasound interaction with the targeted tissue. FUS could bring benefits, e.g., tumour sensitisation, immune stimulation, and targeted drug delivery, but investigation of FUS effects at the cellular level is still missing. New techniques are commonly tested in vitro on two-dimensional (2D) monolayer cancer cell culture models. The 3D tumour model—spheroid—is mainly utilised to mimic solid tumours from an architectural standpoint. It is a promising method to simulate the characteristics of tumours in vitro and their various responses to therapeutic alternatives. This study aimed to evaluate the effects of FUS on human prostate and glioblastoma cancer tumour spheroids in vitro. The experimental follow-up enclosed the measurements of spheroid integrity and growth kinetics, DNA damage, and cellular metabolic activity by measuring intracellular ATP content in the spheroids. Our results showed that pulsed FUS treatment induced molecular effects in 3D tumour models. With the disruption of the spheroid integrity, we observed an increase in DNA double-strand breaks, leading to damage in the cancer cells depending on the cancer cell type

Scaffold-free bioprinting of mesenchymal stem cells using the Regenova printer: Spheroid characterization and osteogenic differentiation

Limitations in scaffold material properties, such as sub-optimal degradation time, highlight the need for alternative approaches to engineer de novo tissues. One emerging solution for fabricating tissue constructs is scaffold-free tissue engineering. To facilitate this approach, three-dimensional (3D) bioprinting technology (Regenova Bio 3D Printer) has been developed to construct complex geometric shapes from discrete cellular spheroids without exogenous scaffolds. Optimizing spheroid fabrication and characterizing cellular behavior in the spheroid environment are important first steps prior to printing larger constructs. Here, we characterized spheroids of immortalized mouse bone marrow stromal cells (BMSCs) that were differentiated to the osteogenic lineage. Immortalized BMSCs were seeded in low attachment 96-well plates in various numbers to generate self-aggregated spheroids either under the force of gravity or centrifugation. Cells were cultured in control or osteogenic media for up to 28 days. Spheroid diameter, roundness and smoothness were measured. Cell viability, DNA content and alkaline phosphatase activity were assessed at multiple time points. Additionally, expression of osteogenic markers was determined using real time qPCR. Spheroids formed under gravity with 20 K, 30 K and 40 K cells had average diameters of 498.5 ± 8.3 μm, 580.0 ± 32.9 μm and 639.2 ± 54.0 μm, respectively, while those formed under 300G centrifugation with the same numbers of cells had average diameters of 362.3 ± 3.5 μm, 433.1 ± 6.4 μm and 491.2 ± 8.0 μm. Spheroids formed via centrifugation were superior to those formed by gravity, as evidenced by better roundness and smoothness and double the retention of DNA (cellular) content. Cells in spheroids exhibited a robust osteogenic response to the differentiation medium, including higher mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than those cultured in control medium, as well as greater alkaline phosphatase activity. The optimal spheroid fabrication technique from this study was to aggregate 40 K cells under 150–300G centrifugation. In future investigations, these spheroids will be 3D printed into larger tissue constructs

iPSC-Derived Vascular Cell Spheroids as Building Blocks for Scaffold-Free Biofabrication

Recently a protocol is established to obtain large quantities of human induced pluripotent stem cells (iPSC)-derived endothelial progenitors, called endothelial colony forming cells (ECFC), and of candidate smooth-muscle forming cells (SMFC). Here, the suitability for assembling in spheroids, and in larger 3D cell constructs is tested. iPSC-derived ECFC and SMFC are labeled with tdTomato and eGFP, respectively. Spheroids are formed in ultra-low adhesive wells, and their dynamic proprieties are studied by time-lapse microscopy, or by confocal microscopy. Spheroids are also tested for fusion ability either in the wells, or assembled on the Regenova 3D bioprinter which laces them in stainless steel micro-needles (the “Kenzan” method). It is found that both ECFC and SMFC formed spheroids in about 24 h. Fluorescence monitoring indicated a continuous compaction of ECFC spheroids, but stabilization in those prepared from SMFC. In mixed spheroids, the cell distribution changed continuously, with ECFC relocating to the core, and showing pre-vascular organization. All spheroids have the ability of in-well fusion, but only those containing SMFC are robust enough to sustain assembling in tubular structures. In these constructs a layered distribution of alpha smooth muscle actin-positive cells and extracellular matrix deposition is found. In conclusion, iPSC-derived vascular cell spheroids represent a promising new cellular material for scaffold-free biofabrication

Modular and adaptable tumor niche prepared from visible light-initiated thiol-norbornene photopolymerization

Photopolymerized biomimetic hydrogels with adaptable properties have been widely used for cell and tissue engineering applications. As a widely adopted gel crosslinking method, photopolymerization provides experimenters on-demand and spatial-temporal controls in gelation kinetics. Long wavelength ultraviolet (UV) light initiated photopolymerization is among the most popular methods in the fabrication of cell-laden hydrogels owing to its rapid and relatively mild gelation conditions. The use of UV light, however, still causes concerns regarding its potential negative impacts on cells. Alternatively, visible light based photopolymerization can be used to crosslink cell-laden hydrogels. The majority of visible light based gelation schemes involve photoinitiator, co-initiator, and co-monomer. This multi-component initiation system creates added challenges for optimizing hydrogel formulations. Here, we report a co-initiator/co-monomer-free visible light initiated thiol-norbornene photopolymerization scheme to prepare modular biomimetic hydrogels suitable for in situ cell encapsulation. Eosin-Y was used as the sole initiator to initiate modular gelation between synthetic macromers (e.g., thiolated poly(vinyl alcohol) or poly(ethylene glycol)) and functionalized extracellular matrices (ECM), including norbornene-functionalized gelatin (GelNB) and/or thiolated hyaluronic acid (THA). These components are modularly crosslinked to afford bio-inert (i.e., purely synthetic), bioactive (i.e., using gelatin), and biomimetic (i.e., using gelatin and hyaluronic acid) hydrogels. The stiffness of the hydrogels can be easily tuned without affecting the contents of the bioactive components. Furthermore, the use of naturally-derived biomacromolecules (e.g., gelatin and HA) renders these hydrogels susceptible to enzyme-mediated degradation. In addition to demonstrating efficient and tunable visible light mediated gelation, we also utilized this biomimetic modular gelation system to formulate artificial tumor niche and to study the effects of cell density and gel modulus on the formation of pancreatic ductal adenocarcinoma (PDAC) spheroids.

S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage.: S100B is absent in SVZ GFAP expressing cells

International audienceDuring the postnatal development, astrocytic cells in the neocortex progressively lose their neural stem cell (NSC) potential, whereas this peculiar attribute is preserved in the adult subventricular zone (SVZ). To understand this fundamental difference, many reports suggest that adult subventricular GFAP-expressing cells might be maintained in immature developmental stage. Here, we show that S100B, a marker of glial cells, is absent from GFAP-expressing cells of the SVZ and that its onset of expression characterizes a terminal maturation stage of cortical astrocytic cells. Nevertheless, when cultured in vitro, SVZ astrocytic cells developed as S100B expressing cells, as do cortical astrocytic cells, suggesting that SVZ microenvironment represses S100B expression. Using transgenic s100b-EGFP cells, we then demonstrated that S100B expression coincides with the loss of neurosphere forming abilities of GFAP expressing cells. By doing grafting experiments with cells derived from beta-actin-GFP mice, we next found that S100B expression in astrocytic cells is repressed in the SVZ, but not in the striatal parenchyma. Furthermore, we showed that treatment with epidermal growth factor represses S100B expression in GFAP-expressing cells in vitro as well as in vivo. Altogether, our results indicate that the S100B expression defines a late developmental stage after which GFAP-expressing cells lose their NSC potential and suggest that S100B expression is repressed by adult SVZ microenvironment

Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors.

Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson?s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit submicromolar inhibition constants but have different mechanisms of inhibition. The crystal structures of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique glycine residue to achieve their selectivity. These two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states