Maria Valeria Corrias

Natural Killer Cell-Mediated Killing of Freshly Isolated Neuroblastoma Cells Critical Role of DNAX Accessory Molecule-1–Poliovirus Receptor Interaction

In the present study, we assessed the susceptibility of freshly isolated neuroblastoma cells to killing mediated by normal human natural killer (NK) cells and analyzed the receptor–ligand interactions that regulate this event. We show that killing of freshly isolated neuroblasts, similar to neuroblastoma cell lines, involves NKp46 and NKp30 (natural cytotoxicity receptors). However, freshly isolated neuroblasts were generally more resistant to NK-mediated lysis than conventional neuroblastoma cell lines. Moreover, a significant heterogeneity in susceptibility to lysis existed among neuroblastomas derived from different patients. Remarkably, susceptibility to lysis directly correlated with the surface expression, on neuroblasts, of poliovirus receptor [PVR (CD155)], a ligand for the DNAX accessory molecule-1 [DNAM-1 (CD226)] triggering receptor expressed by NK cells. Indeed, PVR-expressing neuroblastomas were efficiently killed by NK cells. Moreover, monoclonal antibody-mediated masking of either DNAM-1 (on NK cells) or PVR (on neuroblasts) resulted in strong inhibition of tumor cell lysis. Thus, assessment of the PVR surface levels may represent a novel useful criterion to predict the susceptibility/resistance of neuroblastomas to NK-mediated killing.

Tumor Origin of Endothelial Cells in Human Neuroblastoma

PURPOSE Malignant cells are genetically unstable and prone to develop chemotherapy resistance, whereas tumor vasculature is usually of host origin and genetically stable. Tumor endothelial microvessels attract interest as therapeutic targets, but their genetic instability would curtail such approach. Here, we have investigated the tumor origin of endothelial microvessels in human neuroblastoma (NB). MATERIALS AND METHODS Paraffin-embedded tissue sections from 10 MYCN-amplified tumors (six stage 4, three stage 3, and one stage 1) were studied. Endothelial cells (ECs) were detected by immunofluorescent staining for CD31 or CD105, and MYCN amplification was detected using fluorescence in situ hybridization (FISH). In xenografts of the HTLA-230 human NB cell line, human ECs were detected by CD31 staining, mouse ECs were detected by CD34 staining, and MYCN amplification and murine DNA were detected using FISH. RESULTS MYCN-amplified ECs formed approximately 70% of tumor endothelial microvessels in two stage 4 tumors and 20% in one stage 3 tumor. Similar results were obtained after EC labeling with CD31 or CD105. Staining for alpha-smooth muscle actin in combination with MYCN FISH demonstrated that tumor-derived ECs were coated with pericytes. These vessels were functional because they contained RBCs. Approximately 70% of endothelial vessels from HTLA-230 xenografts stained for human CD31, but not murine CD34, and displayed MYCN amplification, thus proving their tumor origin. CONCLUSION NB-associated endothelial microvessels can originate from tumor cells, and this finding challenges the tenet that tumor vasculature is genetically stable. The possibility that NB-derived ECs are chemotherapy resistant warrants further investigation.

Expression of MAGE-1, MAGE-3 and MART-1 genes in neuroblastoma.

The human MAGE‐1, MAGE‐3 and MART‐1 genes code for antigens that are specifically recognized by cytolytic T lymphocytes in a MHC‐restricted manner. The MAGE‐1 and MAGE‐3 genes are expressed in tumors of different histotypes but not in normal adult tissues (with the exception of testis), while the MART‐1 gene appears to be selectively expressed in melanoma. MAGE‐1, MAGE‐3 and MART‐1 antigens may therefore constitute useful targets for specific anti‐tumor immunization of cancer patients. Here we have investigated the expression of MAGE‐1, MAGE‐3 and MART‐1 in 11 neuroblastoma (NB) cell lines and 73 NB tumor masses. MAGE‐1 and MAGE‐3 transcripts were detected simultaneously in 36% of the cell lines and in 16% of tumor samples. The MAGE‐1 gene was never expressed alone except in one tumor. In contrast, MAGE‐3 mRNA was found in approximately 40% of the NB tumor samples in the absence of MAGE‐1 mRNA. No expression of the MART‐1 gene was observed in any cell line or tumor sample. No correlation was found between MAGE gene expression and clinical stage, event‐free survival and presence or absence of N‐myc amplification.

CXCL12 Does Not Attract CXCR4 + Human Metastatic Neuroblastoma Cells: Clinical Implications

Purpose: The role of CXCR4 in bone marrow localization of neuroblastoma cells has been recently proposed. The aim of this study was to investigate the expression and chemotactic functionality of CXCR4 in human metastatic neuroblastoma cells isolated from the bone marrow and, for comparison, in a panel of neuroblastoma cell lines. Experimental Design: CXCR4 expression and chemotactic functionality were investigated in metastatic neuroblastoma cells isolated from patient bone marrow and in neuroblastoma cell lines. The former cells were isolated as CD45− or GD2+ cells by immunomagnetic bead manipulation. Chemotactic assays were done in a transwell system. Regulator of G protein signaling expression was investigated by reverse transcription-PCR. Results: Metastatic neuroblastoma cells consistently expressed CXCR4, which was also detected in 5 of 10 neuroblastoma cell lines. CXCL12 did not stimulate the chemotaxis of primary tumor cells or cell lines in either normoxia or hypoxia, irrespective of CXCR4 up-regulation detected under the latter condition. Accordingly, neuroblastoma cells failed to modulate filamentous actin and to activate mitogen-activated protein kinase upon treatment with CXCL12. RGS16 mRNA was consistently expressed in primary tumor cells and cell lines, but its down-regulation by RNA interference did not restore CXCR4 chemotactic functionality. Conclusions: These results show unambiguously that CXCR4 expressed in human metastatic neuroblastoma cells is not functional and do not support the clinical use of CXCR4 antagonists to prevent neuroblastoma metastasis.

Neuroblastoma-Derived TGF-β1 Modulates the Chemokine Receptor Repertoire of Human Resting NK Cells

In this study, we show that neuroblastoma (NB) cell conditioning affects the chemokine receptor repertoire of human resting NK cells. In particular, NB cells upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated CX3CR1 in the CD56dim subset. On the contrary, the expression of CXCR1 and CCR7 remained unaltered. The phenomenon was dependent on the release by NB cells of TGF-β1, and rTGF-β1 induced a chemokine receptor repertoire identical to that of NB-conditioned NK cells. The immune modulatory role of TGF-β1 appears to be dose dependent because low amounts of the cytokine were sufficient to modulate CXCR4 and CX3CR1 expression, intermediate amounts modified that of CXCR3, and high amounts were necessary to downregulate the expression of the NKp30 activating receptor. Notably, a similar receptor modulation was observed in rTGF-β2–conditioned NK cells. Finally, the analysis of NK cells from patients with stage 4 NB suggests that NB conditioning could exert in vivo an immune modulatory effect resembling that emerged from in vitro experiments. Altogether our data propose a novel tumor escape-mechanism based on the modulation of chemokine receptors that play pivotal roles in NK cells bone marrow homing, egress, or recruitment into peripheral tissues.

Human NK cell infusions prolong survival of metastatic human neuroblastoma-bearing NOD/scid mice

AimSeveral lines of evidence suggest that NK cell immunotherapy may represent a successful approach in neuroblastoma (NB) patients refractory to conventional therapy. However, homing properties, safety and therapeutic efficacy of NK cell infusions need to be evaluated in a suitable preclinical murine NB model.Materials and methodsHere, the therapeutic efficacy of NK cell infusions in the presence or absence of NK-activating cytokines have been evaluated in a NB metastatic model set up in NOD/scid mice, that display reduced functional activity of endogenous NK cells.ResultsIn NOD/scid mice the injected NB cells rapidly reached all the typical sites of metastatization, including bone marrow. Infusion of polyclonal IL2-activated NK cells was followed by dissemination of these cells into various tissues including those colonized by metastatic NB cells. The early repeated injection of IL2-activated NK cells in NB-bearing NOD/scid mice significantly increased the mean survival time, which was associated with a reduced bone marrow infiltration. The therapeutic effect was further enhanced by low doses of human recombinant IL2 or IL15.ConclusionOur results indicate that NK-based adoptive immunotherapy can represent a valuable adjuvant in the treatment of properly selected NB patients presenting with metastatic disease, if performed in a minimal residual disease setting.

Neuroblastoma mRNAs Predict Outcome in Children With Stage 4 Neuroblastoma: A European HR-NBL1/SIOPEN Study

PURPOSE To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from children with stage 4 neuroblastoma are clinically useful biomarkers of risk. METHODS RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy. RESULTS High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value. CONCLUSION High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.

Immunogenicity of human neuroblastoma

Abstract: Neuroblastoma (NB) is a neuroectodermal tumor that affects children in the first years of life. Half of NB cases present with metastatic disease at diagnosis and have a poor prognosis, in spite of the most advanced chemotherapeutic protocols combined with autologous hematopoietic stem cell transplantation. Among the new avenues for NB treatment that are being explored, immunotherapy has attracted much interest. Emphasis has been placed on monoclonal antibodies directed to tumor‐associated antigens—in particular the disialoganglioside GD2—that have been tested in the clinical setting with promising results. In addition, stimulation of cell‐mediated antitumor effector mechanisms have been attempted—for example, by recombinant interleukin (IL)‐2 administration. Nonetheless, the issue of the immunogenicity of human NB cells has never been thoroughly addressed. Here we shall review the work carried out in our lab in recent years and show that NB cells express tumor‐associated antigens, such as MAGE‐3, but lack constitutive expression of costimulatory molecules and surface HLA class I and II molecules. As such, NB cells are likely to be ignored by the host T cell compartment, since expression of HLA and costimulatory molecules on antigen presenting cells are sine qua non conditions for efficient peptide presentation to T cells and for the subsequent activation and clonal expansion of the latter cells. Notably, in vitro experiments with NB cell lines demonstrated that surface HLA class I molecules and the CD40 costimulatory molecule were upregulated following cell incubation with recombinant interferon‐γ. Interaction of CD40 with recombinant CD40 ligand induced apoptosis of NB cells through a caspase 8‐dependent mechanism. Collectively, these results indicate that the immunogenicity of human NB cells is very low but suggest that manipulation by cytokine administration or gene transfer can increase their immunogenic potential. On the other hand, NB cells represent an excellent target for natural killer cells, the potential role of which in immunotherapy of NB is now being investigated.

Chemokines in neuroectodermal tumour progression and metastasis

Chemokines and their receptors have emerged as pivotal regulators of tumour growth, progression, and metastasis. Here we review the current knowledge on chemokines and receptors likely involved in the development of metastasis of neuroectodermal tumours, with emphasis on neuroblastoma. In this respect, we discuss the controversial role of the CXCR4/CXCL12 axis in bone marrow localization of neuroblastoma cells. In addition, we focus on the ability of neuroblastoma-derived chemokines such as CCL2 and CX3CL1 to attract lymphoid cells to the tumour site. Finally, chemokine receptor and function in other neuroectodermal tumours of adulthood (i.e. melanoma and small cell lung cancer) are discussed.

Peripheral Blood Stem Cell Tumor Cell Contamination and Survival of Neuroblastoma Patients

Purpose: Contribution of peripheral blood stem cell (PBSC) contaminating tumor cells to subsequent relapse and overall survival of neuroblastoma patients remains controversial. Experimental Design: Neuroblastoma cell contamination of 27 PBSC harvests from stage IV neuroblastoma patients was assessed by quantitative RT-PCR for both tyrosine hydroxylase (TH) and GD2 synthase (GD2-s). The effect of PBSC contamination on survival was then analyzed. Results: Seven PBSC tested negative for both markers; 19 were positive for GD2-s, 6 for TH, with 5 positive for both. Survival of the 20 patients with positive PBSC did not differ from that of the patients with negative PBSC (log-rank test, P = 0.134 and 0.218 for event-free survival and overall survival, respectively). By considering the TH and GD2-s results independently, a borderline (P = 0.053) negative effect on event-free survival was observed in patients reinfused with GD2-s-positive PBSC. When the status at transplant was taken into account, only the event-free survival of the patients rescued when in complete remission with GD2-s-negative PBSC was better, although not significantly, than that of patients infused with GD2-s-positive PBSC. Conclusions: Our results obtained in a small cohort of homogeneously treated stage IV patients suggest that patient survival is not affected by PBSC contamination with the exception of a borderline negative effect on event-free survival in patients rescued when in complete remission.