Patient-derived organoids (PDOs) as a novel in vitro model for neuroblastoma tumours.

BACKGROUND: Neuroblastoma (NB) is a paediatric tumour of the sympathetic nervous system. Half of all cases are defined high-risk with an overall survival less than 40% at 5 years from diagnosis. The lack of in vitro models able to recapitulate the intrinsic heterogeneity of primary NB tumours has hindered progress in understanding disease pathogenesis and therapy response. METHODS: Here we describe the establishment of 6 patient-derived organoids (PDOs) from cells of NB tumour biopsies capable of self-organising in a structure resembling the tissue of origin. RESULTS: PDOs recapitulate the histological architecture typical of the NB tumour. Moreover, PDOs expressed NB specific markers such as neural cell adhesion molecules, NB84 antigen, synaptophysin (SYP), chromogranin A (CHGA) and neural cell adhesion molecule NCAM (CD56). Analyses of whole genome genotyping array revealed that PDOs maintained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Furthermore, the PDOs showed stemness features and retained cellular heterogeneity reflecting the high heterogeneity of NB tumours. CONCLUSIONS: We were able to create a novel preclinical model for NB exhibiting self-renewal property and allowing to obtain a reservoir of NB patients' biological material useful for the study of NB molecular pathogenesis and to test drugs for personalised treatments.

Quasi-3D morphology and modulation of focal adhesions of human adult stem cells through combinatorial concave elastomeric surfaces with varied stiffness.

In vivo cell niches are complex architectures that provide a wide range of biochemical and mechanical stimuli to control cell behavior and fate. With the aim to provide in vitro microenvironments mimicking physiological niches, microstructured substrates have been exploited to support cell adhesion and to control cell shape as well as three dimensional morphology. At variance with previous methods, we propose a simple and rapid protein subtractive soft lithographic method to obtain microstructured polydimethylsiloxane substrates for studying stem cell adhesion and growth. The shape of adult renal stem cells and nuclei is found to depend predominantly on micropatterning of elastomeric surfaces and only weakly on the substrate mechanical properties. Differently, focal adhesions in their shape and density but not in their alignment mainly depend on the elastomer stiffness almost regardless of microscale topography. Local surface topography with concave microgeometry enhancing adhesion drives stem cells in a quasi-three dimensional configuration where stiffness might significantly steer mechanosensing as highlighted by focal adhesion properties.

Highly sticky surfaces made by electrospun polymer nanofibers.

We report on a comprehensive study of the unique adhesive properties of mats of polymethylmethacrylate (PMMA) nanofibers produced by electrospinning. Fibers are deposited on glass, with varying of the diameter and the relative orientation of the polymer filaments (random vs. aligned configuration). While no significant variation is observed in the static contact angle (∼130°) of deposited water drops upon changing the average fiber diameter up to the micrometer scale, fibers are found to exhibit unequalled water adhesion. Placed vertically, they can hold up water drops as large as 60 µL, more than twice the values typically obtained with hairy surfaces prepared by different methods. For aligned fibers with anisotropic wetting behavior, the maximum volume measured in the direction perpendicular to the fibers goes up to 90 µL. This work suggests new routes to tailor the wetting behavior on extended areas by nanofiber coatings, with possible applications in adsorbing and catalytic surfaces, microfluidic devices, and filtration technologies.

Wnt activation promotes neuronal differentiation of glioblastoma.

One of the biggest challenges in tumour research is the possibility to reprogram cancer cells towards less aggressive phenotypes. In this study, we reprogrammed primary Glioblastoma multiforme (GBM)-derived cells towards a more differentiated and less oncogenic phenotype by activating the Wnt pathway in a hypoxic microenvironment. Hypoxia usually correlates with malignant behaviours in cancer cells, but it has been recently involved, together with Wnt signalling, in the differentiation of embryonic and neural stem cells. Here, we demonstrate that treatment with Wnt ligands, or overexpression of ß-catenin, mediate neuronal differentiation and halt proliferation in primary GBM cells. An hypoxic environment cooperates with Wnt-induced differentiation, in line with our finding that hypoxia inducible factor-1α (HIF-1α) is instrumental and required to sustain the expression of ß-catenin transcriptional partners TCF-1 and LEF-1. In addition, we also found that Wnt-induced GBM cell differentiation inhibits Notch signalling, and thus gain of Wnt and loss of Notch cooperate in the activation of a pro-neuronal differentiation program. Intriguingly, the GBM sub-population enriched of cancer stem cells (CD133(+) fraction) is the primary target of the pro-differentiating effects mediated by the crosstalk between HIF-1α, Wnt, and Notch signalling. By using zebrafish transgenics and mutants as model systems to visualize and manipulate in vivo the Wnt pathway, we confirm that Wnt pathway activation is able to promote neuronal differentiation and inhibit Notch signalling of primary human GBM cells also in this in vivo set-up. In conclusion, these findings shed light on an unsuspected crosstalk between hypoxia, Wnt and Notch signalling in GBM, and suggest the potential to manipulate these microenvironmental signals to blunt GBM malignancy.

BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1α stability and MGMT expression.

Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in particular BMP2, are known to promote differentiation and growth inhibition in GBM cells. For this reason, we investigated whether a BMP2-based treatment would increase TMZ response in hypoxic drug-resistant GBM-derived cells. Here we show that BMP2 induced strong differentiation of GBM stem-like cells and subsequent addition of TMZ caused dramatic increase of apoptosis. Importantly, we correlated these effects to a BMP2-induced downregulation of both hypoxia-inducible factor-1α (HIF-1α) and MGMT. We report here a novel mechanism involving the HIF-1α-dependent regulation of MGMT, highlighting the existence of a HIF-1α/MGMT axis supporting GBM resistance to therapy. As confirmed from this evidence, over-stabilization of HIF-1α in TMZ-sensitive GBM cells abolished their responsiveness to it. In conclusion, we describe a HIF-1α-dependent regulation of MGMT and suggest that BMP2, by down-modulating the HIF-1α/MGMT axis, should increase GBM responsiveness to chemotherapy, thus opening the way to the development of future strategies for GBM treatment.

Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

Activation of the Notch pathway occurs commonly in T acute lymphoblastic leukemia (T-ALL) because of mutations in Notch1 or Fbw7 and is involved in the regulation of cell proliferation and survival. Deregulated Notch3 signalling has also been shown to promote leukemogenesis in transgenic mice, but the targets of Notch3 in human T-ALL cells remain poorly characterized. Here, we show that Notch3 controls levels of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1). In a model of T-ALL cell dormancy, both Notch3 activation and MKP-1 expression were upregulated in aggressive compared with dormant tumors, and this inversely correlated with the levels of phosphorylated p38 and extracellular signal-regulated kinase1/2 (ERK1/2) MAPKs, two canonical MKP-1 targets. We demonstrate that MKP-1 protein levels are regulated by Notch3 in T-ALL cell lines because its silencing by RNA interference or treatment with γ-secretase inhibitors induced strong MKP-1 reduction whereas activation of Notch3 signalling had the opposite effect. Furthermore, MKP-1 has an important role in T-ALL cell survival because its attenuation by short hairpin RNA significantly increased cell death under stress conditions. This protective function has a key role in vivo, as MKP-1-deficient cells showed impaired tumorigenicity. These results elucidate a novel mechanism downstream of Notch3 that controls the survival of T-ALL cells.

Hybrid planar microresonators with organic and InGaAs active media.

The authors report on the fabrication of hybrid planar micro-resonators based on InGaAs microdisks with an evaporated organic material. Samples of InGaAs grown on InP(100) substrates are obtained by Chemical Beam Epitaxy, and microdisks of InGaAs with different diameters are fabricated by focused ion beam. The hybrid disks are obtained by the subsequent evaporation of 8-hydroxyquinoline aluminium doped with 4-Dicyanomethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran on the InGaAs microdisks. The devices, characterized by micro- and confocal photoluminescence imaging and spectroscopy, exhibit emission around 650 nm, from the organic material for disks with different radius. Finally, simultaneous emission in the visible and at whispering gallery resonant modes in the 1350-1450 nm range are observed due to excitation transfer to InGaAs. These devices open the possibility to combine the flexibility of organics with the high gain of III-V compounds for wavelength down conversion and telecom applications.

Low-loss and highly polarized emission from planar polymer waveguides.

The waveguiding properties and amplified spontaneous emission (ASE) of a blend of light-emitting gain-conjugated polymers were investigated. ASE-induced line narrowing occurs for excitation fluences larger than 100 microJ cm(-2), with a maximum optical-gain coefficient of 8 cm(-1). Energy transfer between the host and guest polymers, significantly reducing the self-absorption, leads to a loss coefficient of the waveguide as low as 0.3 cm(-1), which is believed to be the lowest value reported for active organic gain slabs and a highly polarized emission, with a polarization contrast up to 0.65. These results indicate that gain-conjugated polymer blends are state-of-the-art organic materials for lasing devices.

Gene therapy of ovarian cancer with IFN-alpha-producing fibroblasts: comparison of constitutive and inducible vectors.

Ovarian cancer represents a malignancy suitable for cell and gene therapy approaches owing to its containment within the peritoneal cavity, even at advanced tumor stages. As regulation of transgene expression would be preferable for conducting clinical trials for reasons of safety, we investigated whether intraperitoneal (i.p.) administration of retroviral vector-transduced fibroblasts encoding murine interferon-alpha (IFN-alpha) could have therapeutic activity, and compared its effect with the antitumor effects of fibroblasts producing IFN-alpha under a rapamycin analogue (AP21967)-inducible promoter. Human and murine fibroblasts were recruited into the solid component of transplantable ovarian cancer-grown i.p. in severe combined immunodeficiency mice. Multiple administrations of fibroblasts producing IFN-alpha in a constitutive manner showed therapeutic efficacy, leading to significant prolongation of survival in the majority of animals, associated with inhibition of tumor angiogenesis. Compared to cells transduced by the constitutive vector, fibroblasts transduced by the inducible vector released twofold higher IFN-alpha levels in vitro, following induction by AP21967, and production of the cytokine was under pharmacologic control both in vitro and in vivo. However, these cells elicited only modest therapeutic effects in vivo. Overall, these findings indicate that intracavitary IFN-alpha gene therapy using engineered fibroblasts requires sustained production of IFN-alpha to achieve durable antitumor effects.

Making silicon hydrophobic: wettability control by two-lengthscale simultaneous patterning with femtosecond laser irradiation.

We report on the wettability properties of silicon surfaces, simultaneously structured on the micrometre-scale and the nanometre-scale by femtosecond (fs) laser irradiation to render silicon hydrophobic. By varying the laser fluence, it was possible to control the wetting properties of a silicon surface through a systematic and reproducible variation of the surface roughness. In particular, the silicon-water contact angle could be increased from 66° to more than 130°. Such behaviour is described by incomplete liquid penetration within the silicon features, still leaving partially trapped air inside. We also show how controllable design and tailoring of the surface microstructures by wettability gradients can drive the motion of the drop's centre of mass towards a desired direction (even upwards).