ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma.

BACKGROUND: Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. METHODS: Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor growth in vivo. Two-sided student t-test was used. RESULTS: We detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2α expression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. In coherence, RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2α protein knockdown resulted in profound downregulation of target genes. CONCLUSIONS: The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2α expression at normoxia suggest that HIF-2α have additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2α has transcriptional and non-transcriptional roles in neuroblastoma.

Inflammatory macrophage derived TNFα downregulates estrogen receptor α via FOXO3a inactivation in human breast cancer cells.

Patients with estrogen receptor α positive (ERα+) breast cancer can respond to endocrine therapy, but treatment resistance is common and associated with downregulation of ERα expression in the dormant residual cells. Here we show, using long-term NSG xenograft models of human breast cancer and primary human monocytes, in vitro primary cell cultures and tumors from breast cancer patients, that macrophage derived tumor necrosis factor alpha (TNFα) downregulates ERα in breast cancer cells via inactivation of the transcription factor Forkhead box O transcription factor 3a (FOXO3a). Moreover, presence of tumor associated macrophages in the primary tumor of breast cancer patients, was associated with ERα negativity, and with worse prognosis in patients with ERα+ tumors. We propose that pro-inflammatory macrophages, despite being tumoricidal, may have direct effects on tumor progression and endocrine resistance in breast cancer patients. Our findings suggest that TNFα antagonists should be evaluated for treatment of ERα+ breast cancer.

The Iroquois homeobox proteins IRX3 and IRX5 have distinct roles in Wilms tumour development and human nephrogenesis.

Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock-out Irx3- /Irx5- mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3-/- cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/ß-catenin-signalling. In contrast, IRX5-/- cells displayed activation of Hippo and non-canonical WNT-signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Discovery of epi-Enprioline as a Novel Drug for the Treatment of Vincristine Resistant Neuroblastoma.

Neuroblastoma is a childhood solid tumour originating from undifferentiated neural progenitor cells of the sympathetic nervous system. Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. In the present study, we aimed to identify novel drugs that can inhibit the growth and survival of chemoresistant neuroblastoma. High-throughput screening identified a small molecule, epi-enprioline that was able to induce apoptosis of vincristine-resistant neuroblastoma cells via the mitochondrial apoptotic pathway. Epi-enprioline reduced tumour growth in multiple preclinical models, including an orthotopic neuroblastoma patient-derived xenograft model in vivo. In summary, our data suggest that epi-enprioline can be considered as a lead compound for the treatment of vincristine-resistant neuroblastoma uncovering a novel strategy, which can be further explored as a treatment for drug-resistant neuroblastoma.

Patient-derived xenografts as preclinical neuroblastoma models.

The prognosis for children with high-risk neuroblastoma is often poor and survivors can suffer from severe side effects. Predictive preclinical models and novel therapeutic strategies for high-risk disease are therefore a clinical imperative. However, conventional cancer cell line-derived xenografts can deviate substantially from patient tumors in terms of their molecular and phenotypic features. Patient-derived xenografts (PDXs) recapitulate many biologically and clinically relevant features of human cancers. Importantly, PDXs can closely parallel clinical features and outcome and serve as excellent models for biomarker and preclinical drug development. Here, we review progress in and applications of neuroblastoma PDX models. Neuroblastoma orthotopic PDXs share the molecular characteristics, neuroblastoma markers, invasive properties and tumor stroma of aggressive patient tumors and retain spontaneous metastatic capacity to distant organs including bone marrow. The recent identification of genomic changes in relapsed neuroblastomas opens up opportunities to target treatment-resistant tumors in well-characterized neuroblastoma PDXs. We highlight and discuss the features and various sources of neuroblastoma PDXs, methodological considerations when establishing neuroblastoma PDXs, in vitro 3D models, current limitations of PDX models and their application to preclinical drug testing.

Neuroblastoma patient-derived orthotopic xenografts retain metastatic patterns and geno- and phenotypes of patient tumours.

Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease often have a poor outcome. Here we describe the establishment of neuroblastoma patient-derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high risk neuroblastoma into immunodeficient mice. In vivo tumour growth was monitored by magnetic resonance imaging and fluorodeoxyglucose-positron emission tomography. Neuroblastoma PDXs retained the undifferentiated histology and proliferative capacity of their corresponding patient tumours. The PDXs expressed neuroblastoma markers neural cell adhesion molecule, chromogranin A, synaptophysin and tyrosine hydroxylase. Whole genome genotyping array analyses demonstrated that PDXs retained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Thus, neuroblastoma PDXs recapitulate the hallmarks of high-risk neuroblastoma in patients. PDX-derived cells were cultured in serum-free medium where they formed free-floating neurospheres, expressed neuroblastoma gene markers MYCN, CHGA, TH, SYP and NPY, and retained tumour-initiating and metastatic capacity in vivo. PDXs showed much higher degree of infiltrative growth and distant metastasis as compared to neuroblastoma SK-N-BE(2)c cell line-derived orthotopic tumours. Importantly, the PDXs presented with bone marrow involvement, a clinical feature of aggressive neuroblastoma. Thus, neuroblastoma PDXs serve as clinically relevant models for studying and targeting high-risk metastatic neuroblastoma.

Orthotopic Wilms tumor xenografts derived from cell lines reflect limited aspects of tumor morphology and clinical characteristics.

BACKGROUND: Wilms tumor (WT) is a pediatric tumor of the kidney, the treatment of which includes heavy chemotherapy. Affected children would likely benefit from more targeted therapies with limited side effects. Establishment of relevant orthotopic WT xenografts is important to better understand mechanisms of WT growth and for preclinical drug testing. PROCEDURE: Here we established and characterized orthotopic xenografts from WT cell lines WiT49, CCG-99-11, and WT-CLS1 to ascertain in what aspects each of them recapitulated WT histology, immunophenotype, invasion, and metastatic spread. RESULTS: WiT49 xenografts recapitulated near triphasic WTs with clear WT1 staining and anaplastic features, but with tumor restricted to the kidney. On the contrary both CCG-99-11 and WT-CLS1 xenografts conveyed metastatic disease. CCG-99-11 showed a blastemal phenotype whereas WT-CLS1 xenografts did not properly reflect any specific WT subtype. CONCLUSIONS: From the three tested cell lines, orthotopic WiT49 xenografts best reflect the triphasic pattern of classical WT.

Early evolutionary branching across spatial domains predisposes to clonal replacement under chemotherapy in neuroblastoma.

Neuroblastoma (NB) is one of the most lethal childhood cancers due to its propensity to become treatment resistant. By spatial mapping of subclone geographies before and after chemotherapy across 89 tumor regions from 12 NBs, we find that densely packed territories of closely related subclones present at diagnosis are replaced under effective treatment by islands of distantly related survivor subclones, originating from a different most recent ancestor compared to lineages dominating before treatment. Conversely, in tumors that progressed under treatment, ancestors of subclones dominating later in disease are present already at diagnosis. Chemotherapy treated xenografts and cell culture models replicate these two contrasting scenarios and show branching evolution to be a constant feature of proliferating NB cells. Phylogenies based on whole genome sequencing of 505 individual NB cells indicate that a rich repertoire of parallel subclones emerges already with the first oncogenic mutations and lays the foundation for clonal replacement under treatment.

Breast cancer associated CD169+ macrophages possess broad immunosuppressive functions but enhance antibody secretion by activated B cells.

CD169+ resident macrophages in lymph nodes of breast cancer patients are for unknown reasons associated with a beneficial prognosis. This contrasts CD169+ macrophages present in primary breast tumors (CD169+ TAMs), that correlate with a worse prognosis. We recently showed that these CD169+ TAMs were associated with tertiary lymphoid structures (TLSs) and Tregs in breast cancer. Here, we show that CD169+ TAMs can be monocyte-derived and express a unique mediator profile characterized by type I IFNs, CXCL10, PGE2 and inhibitory co-receptor expression pattern. The CD169+ monocyte-derived macrophages (CD169+ Mo-M) possessed an immunosuppressive function in vitro inhibiting NK, T and B cell proliferation, but enhanced antibody and IL6 secretion in activated B cells. Our findings indicate that CD169+ Mo-M in the primary breast tumor microenvironment are linked to both immunosuppression and TLS functions, with implications for future targeted Mo-M therapy.

Macrophage infiltration promotes regrowth in MYCN-amplified neuroblastoma after chemotherapy.

Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment.

Patient-derived models: Advanced tools for precision medicine in neuroblastoma.

Neuroblastoma is a childhood cancer derived from the sympathetic nervous system. High-risk neuroblastoma patients have a poor overall survival and account for ~15% of childhood cancer deaths. There is thus a need for clinically relevant and authentic models of neuroblastoma that closely resemble the human disease to further interrogate underlying mechanisms and to develop novel therapeutic strategies. Here we review recent developments in patient-derived neuroblastoma xenograft models and in vitro cultures. These models can be used to decipher mechanisms of metastasis and treatment resistance, for drug screening, and preclinical drug testing. Patient-derived neuroblastoma models may also provide useful information about clonal evolution, phenotypic plasticity, and cell states in relation to neuroblastoma progression. We summarize current opportunities for, but also barriers to, future model development and application. Integration of patient-derived models with patient data holds promise for the development of precision medicine treatment strategies for children with high-risk neuroblastoma.

Clinically relevant treatment of PDX models reveals patterns of neuroblastoma chemoresistance.

Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB). Here, we developed a clinically relevant in vivo treatment protocol mimicking the first-line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN-amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal NB chemoresistance mechanisms. Intrinsic resistance was associated with high genetic diversity and an embryonic phenotype. Relapsed NB with acquired resistance showed a decreased adrenergic phenotype and an enhanced immature mesenchymal-like phenotype, resembling multipotent Schwann cell precursors. NBs with a favorable treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts. Novel integrated phenotypic gene signatures reflected treatment response and patient prognosis. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states. This work sheds light on the mechanisms of NB chemotherapy response and emphasizes the importance of transcriptional cell states in chemoresistance.

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis.

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.

Toward brain tumor gene therapy using multipotent mesenchymal stromal cell vectors.

Gene therapy of solid cancers has been severely restricted by the limited distribution of vectors within tumors. However, cellular vectors have emerged as an effective migratory system for gene delivery to invasive cancers. Implanted and injected multipotent mesenchymal stromal cells (MSCs) have shown tropism for several types of primary tumors and metastases. This capacity of MSCs forms the basis for their use as a gene vector system in neoplasms. Here, we review the tumor-directed migratory potential of MSCs, mechanisms of the migration, and the choice of therapeutic transgenes, with a focus on malignant gliomas as a model system for invasive and highly vascularized tumors. We examine recent findings demonstrating that MSCs share many characteristics with pericytes and that implanted MSCs localize primarily to perivascular niches within tumors, which might have therapeutic implications. The use of MSC vectors in cancer gene therapy raises concerns, however, including a possible MSC contribution to tumor stroma and vasculature, MSC-mediated antitumor immune suppression, and the potential malignant transformation of cultured MSCs. Nonetheless, we highlight the novel prospects of MSC-based tumor therapy, which appears to be a promising approach.

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis.

BACKGROUND: Graft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products. METHODS: Neuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture. RESULTS: PDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft. CONCLUSION: These results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

Anti-tumor effects of rigosertib in high-risk neuroblastoma.

High-risk neuroblastoma has a poor prognosis despite intense treatment, demonstrating the need for new therapeutic strategies. Here we evaluated the effects of rigosertib (ON-01910.Na) in preclinical models of high-risk neuroblastoma. Among several hundred cancer cell lines representing 24 tumor types, neuroblastoma was the most sensitive to rigosertib. Treatment of MYCN-amplified neuroblastoma organoids resulted in organoid disintegration, decreased cell viability, and increased apoptotic cell death. Neuroblastoma response to rigosertib involved G2M cell cycle arrest and decreased phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Rigosertib delayed tumor growth and prolonged survival of mice carrying neuroblastoma MYCN-amplified PDX tumors (median survival: 31 days, treated; 22 days, vehicle) accompanied with increased apoptosis in treated tumors. We further identified vincristine and rigosertib as a potential promising drug combination treatment. Our results show that rigosertib might be a useful therapeutic agent for MYCN-amplified neuroblastomas, especially in combination with existing agents.

Inhibition of fatty acid synthesis induces differentiation and reduces tumor burden in childhood neuroblastoma.

Many metabolic pathways, including lipid metabolism, are rewired in tumors to support energy and biomass production and to allow adaptation to stressful environments. Neuroblastoma is the second deadliest solid tumor in children. Genetic aberrations, as the amplification of the MYCN-oncogene, correlate strongly with disease progression. Yet, there are only a few molecular targets successfully exploited in the clinic. Here we show that inhibition of fatty acid synthesis led to increased neural differentiation and reduced tumor burden in neuroblastoma xenograft experiments independently of MYCN-status. This was accompanied by reduced levels of the MYCN or c-MYC oncoproteins and activation of ERK signaling. Importantly, the expression levels of genes involved in de novo fatty acid synthesis showed prognostic value for neuroblastoma patients. Our findings demonstrate that inhibition of de novo fatty acid synthesis is a promising pharmacological intervention strategy for the treatment of neuroblastoma independently of MYCN-status.

Bone marrow multipotent mesenchymal stroma cells act as pericyte-like migratory vehicles in experimental gliomas.

Bone marrow-derived multipotent mesenchymal stroma cells (MSCs) have emerged as cellular vectors for gene therapy of solid cancers. We implanted enhanced green fluorescent protein-expressing rat MSCs directly into rat malignant gliomas to address their migratory capacity, phenotype, and effects on tumor neovascularization and animal survival. A single intratumoral injection of MSCs infiltrated the majority of invasive glioma extensions (72 +/- 14%) and a substantial fraction of distant tumor microsatellites (32 +/- 6%). MSC migration was highly specific for tumor tissue. Grafted MSCs integrated into tumor vessel walls and expressed pericyte markers alpha-smooth muscle actin, neuron-glia 2, and platelet-derived growth factor receptor-beta but not endothelial cell markers. The pericyte marker expression profile and perivascular location of grafted MSCs indicate that these cells act as pericytes within tumors. MSC grafting did not influence tumor microvessel density or survival of tumor-bearing animals. The antiangiogenic drug Sunitinib markedly reduced the numbers of grafted MSCs migrating within tumors. We found no MSCs within gliomas following intravenous (i.v.) injections. Thus, MSCs should be administered by intratumoral implantations rather than by i.v. injections. Intratumorally grafted pericyte-like MSCs might represent a particularly well-suited vector system for delivering molecules to affect tumor angiogenesis and for targeting cancer stem cells within the perivascular niche.

Therapeutic targeting of KSP in preclinical models of high-risk neuroblastoma.

Neuroblastoma is a childhood malignancy with often dismal prognosis; relapse is common despite intense treatment. Here, we used human tumor organoids representing multiple MYCN-amplified high-risk neuroblastomas to perform a high-throughput drug screen with approved or emerging oncology drugs. Tumor-selective effects were calculated using drug sensitivity scores. Several drugs with previously unreported anti-neuroblastoma effects were identified by stringent selection criteria. ARRY-520, an inhibitor of kinesin spindle protein (KSP), was among those causing reduced viability. High expression of the KSP-encoding gene KIF11 was associated with poor outcome in neuroblastoma. Genome-scale loss-of-function screens in hundreds of human cancer cell lines across 22 tumor types revealed that KIF11 is particularly important for neuroblastoma cell viability. KSP inhibition in neuroblastoma patient-derived xenograft (PDX) cells resulted in the formation of abnormal monoastral spindles, mitotic arrest, up-regulation of mitosis-associated genes, and apoptosis. In vivo, KSP inhibition caused regression of MYCN-amplified neuroblastoma PDX tumors. Furthermore, treatment of mice harboring orthotopic neuroblastoma PDX tumors resulted in increased survival. Our results suggested that KSP inhibition could be a promising treatment strategy in children with high-risk neuroblastoma.

Integrative discovery of treatments for high-risk neuroblastoma.

Despite advances in the molecular exploration of paediatric cancers, approximately 50% of children with high-risk neuroblastoma lack effective treatment. To identify therapeutic options for this group of high-risk patients, we combine predictive data mining with experimental evaluation in patient-derived xenograft cells. Our proposed algorithm, TargetTranslator, integrates data from tumour biobanks, pharmacological databases, and cellular networks to predict how targeted interventions affect mRNA signatures associated with high patient risk or disease processes. We find more than 80 targets to be associated with neuroblastoma risk and differentiation signatures. Selected targets are evaluated in cell lines derived from high-risk patients to demonstrate reversal of risk signatures and malignant phenotypes. Using neuroblastoma xenograft models, we establish CNR2 and MAPK8 as promising candidates for the treatment of high-risk neuroblastoma. We expect that our method, available as a public tool (targettranslator.org), will enhance and expedite the discovery of risk-associated targets for paediatric and adult cancers.