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.

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.

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.