Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and human neuroblastomas

Copyright © 2011 Cantilena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel Open Access Publishing Fund.Wnt signalling is an important component of vertebrate development, required for specification of the neural crest. Ten Wnt receptors [Frizzled receptor 1-10 (Fzd1-10)] have been identified so far, some of which are expressed in the developing nervous system and the neural crest. Here we show that expression of one such receptors, Fzd6, predicts poor survival in neuroblastoma patients and marks rare, HIF1/2 α-positive cells in tumour hypoxic areas. Fzd6 positive neuroblastoma cells form neurospheres with high efficiency, are resistant to doxorubicin killing and express high levels of mesenchymal markers such as Twist1 and Notch1. Expression of Fzd6 is required for the expression of genes of the noncanonical Wnt pathway and the spheres forming activity. When transplanted into immunodeficient mice, neuroblastoma cells expressing the Fzd6 marker grow more aggressively than their Fzd6 negative counterparts. We conclude that Fzd6 is a new surface marker of aggressive neuroblastoma cells with stem cell-like features.This work was sponsored by the Wellcome Trust, the RICC cancer fund, SPARKS, the Italian Association for Cancer Research, Regione Liguria and the Italian Ministry of Health

Human γδ T-Cells: from surface receptors to the therapy of high-risk leukemias

γδ T lymphocytes are potent effector cells, capable of efficiently killing tumor and leukemia cells. Their activation is mediated by γδ T-cell receptor (TCR) and by activating receptors shared with NK cells (e.g., NKG2D and DNAM-1). γδ T-cell triggering occurs upon interaction with specific ligands, including phosphoantigens (for Vγ9Vδ2 TCR), MICA-B and UL16 binding protein (for NKG2D), and PVR and Nectin-2 (for DNAM-1). They also respond to cytokines undergoing proliferation and release of cytokines/chemokines. Although at the genomic level γδ T-cells have the potential of an extraordinary TCR diversification, in tissues they display a restricted repertoire. Recent studies have identified various γδ TCR rearrangements following either hematopoietic stem cell transplantation (HSCT) or cytomegalovirus infection, accounting for their "adaptive" potential. In humans, peripheral blood γδ T-cells are primarily composed of Vγ9Vδ2 chains, while a minor proportion express Vδ1. They do not recognize antigens in the context of MHC molecules, thus bypassing tumor escape based on MHC class I downregulation. In view of their potent antileukemia activity and absence of any relevant graft-versus-host disease-inducing effect, γδ T-cells may play an important role in the successful clinical outcome of patients undergoing HLA-haploidentical HSCT depleted of TCR αβ T/CD19+ B lymphocytes to cure high-risk acute leukemias. In this setting, high numbers of both γδ T-cells (Vδ1 and Vδ2) and NK cells are infused together with CD34+ HSC and may contribute to rapid control of infections and leukemia relapse. Notably, zoledronic acid potentiates the cytolytic activity of γδ T-cells in vitro and its infusion in patients strongly promotes γδ T-cell differentiation and cytolytic activity; thus, treatment with this agent may contribute to further improve the patient clinical outcome after HLA-haploidentical HSCT depleted of TCR αβ T/CD19+ B lymphocytes

Normal CMR bi-atrial and biventricular reference values in sickle cell disease patients without heart damage

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): The MIOT project receives "no-profit support" from industrial sponsorships (Chiesi Farmaceutici S.p.A., ApoPharma Inc.). Background. Cardiac function indices in patients with hemoglobinopathies are different from those in healthy population, mainly due to chronic anemia. Normal reference values specific for SCD patients are not available by CMR. Aim. We aimed to define the normal cut-off value in SCD patients for bi-atrial and biventricular cardiac magnetic resonance (CMR) parameters. Methods. We considered forty-eight adult SCD patients with no known risk factors or cardiac disease, normal electrocardiogram, no macroscopic myocardial fibrosis, and all cardiac segments with T2*≥20 ms, consecutively enrolled in the MIOT network (Myocardial iron overload in thalassemia). SCD patients were compared with ninety-six healthy controls and 96 thalassemia major (TM) patients without cardiac damage, both matched for age and gender. Nine pediatric SCD patients were also analysed in comparison with 9 TM patients and 9 healthy subjects matched for age and gender. Cine images were acquired to quantify biventricular function parameters: LV and RV end-diastolic volume (EDV), end-systolic volume (ESV) and stroke volume (SV) were normalized for body surface area (EDVI, ESVI, SVI), as well as biventricular mass and atrial areas. Myocardial iron overload was assessed by segmental T2* technique. Late gadolinium enhancement (LGE) images were acquired for evaluation of macroscopic myocardial fibrosis. Results. In all three groups males showed higher biventricular volumes and mass indexes than females. SCD male patients had significantly higher LVEDVI (p < 0.0001), LVESVI (p = 0.010), LVSVI (p = 0.003), cardiac index (p = 0.002), LV and RV mass index (p = 0.008 and p = 0.001, respectively) and left and right atrial areas (p < 0.001 and p = 0.011) than healthy subjects. No significant differences were found in RVEDVI, EVESVI and biventricular EF. Compared to healthy volunteers, females with SCD showed a larger LVEDVI (p = 0.020), LVSVI (p = 0.039), RV mass index (p = 0.002) and left atrial area (p = 0.008). SCD and TM patients showed comparable values of bi-atrial and biventricular volumes and function. When compared to TM, SCD patients showed a larger LV (p < 0.001) and RV mass index (p = 0.001) in male group and a larger RV mass index (p = 0.001) in female group. Table 1 shows the cut-offs for bi-atrial and biventricular MR parameters for adult SCD patients by gender. No significant differences in MR parameters were found among the pediatric groups. Conclusions. Normal reference ranges of bi-atrial and biventricular MR parameters for adult males and females SCD patients were established. The use of these reference values will prevent possible misdiagnosis of cardiomyopathy in patients with SCD

PD-L1 expression in metastatic neuroblastoma as an additional mechanism for limiting immune surveillance

The prognosis of high-risk neuroblastoma (NB) remains poor, although immunotherapies with anti-GD2 antibodies have been reported to provide some benefit. Immunotherapies can be associated with an IFNγ storm that induces in tumor cells the “adaptive immune resistance” characterized by the de-novo expression of Programmed Death Ligands (PD-Ls). Tumor cells can also constitutively express PD-Ls in response to oncogenic signaling. Here, we analyze the constitutive and the inducible surface expression of PD-Ls in NB cells. We show that virtually all HLA class Ipos NB cell lines constitutively express PD-L1, whereas PD-L2 is rarely detected. IFNγ upregulates or induces PD-L1 both in NB cell lines in vitro and in NB engrafted nude/nude mice. Importantly, after IFNγ stimulation PD-L1 can be acquired by NB cell lines, as well as by metastatic neuroblasts isolated from bone marrow aspirates of high-risk NB patients, characterized by different MYCN amplification status. Interestingly, in one patient NB cells were poorly responsive to IFNγ stimulation, pointing out that responsiveness to IFNγ might represent a further element of heterogeneity in metastatic neuroblasts. Finally, we document the presence of lymphocytes expressing the PD-1 receptor in NB-infiltrated bone marrow of patients. PD-1pos cells are mainly represented by αβ T cells, but also include small populations of γδ T cells and NK cells. Moreover, PD-1pos T cells have a higher expression of activation markers. Overall, our data show that a PD-L1-mediated immune resistance mechanism occurs in metastatic neuroblasts and provide a biological rationale for blocking the PD-1/PD-Ls axis in future combined immunotherapeutic approaches

Grb7 Upregulation Is a Molecular Adaptation to HER2 Signaling Inhibition Due to Removal of Akt-Mediated Gene Repression

The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2+ breast cancer cell line SKBR3, and Grb7 in MCF7 cells. Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs