Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

Single-cell transcriptomics and epigenomics unravel the role of monocytes in neuroblastoma bone marrow metastasis.

Metastasis is the major cause of cancer-related deaths. Neuroblastoma (NB), a childhood tumor has been molecularly defined at the primary cancer site, however, the bone marrow (BM) as the metastatic niche of NB is poorly characterized. Here we perform single-cell transcriptomic and epigenomic profiling of BM aspirates from 11 subjects spanning three major NB subtypes and compare these to five age-matched and metastasis-free BM, followed by in-depth single cell analyses of tissue diversity and cell-cell interactions, as well as functional validation. We show that cellular plasticity of NB tumor cells is conserved upon metastasis and tumor cell type composition is NB subtype-dependent. NB cells signal to the BM microenvironment, rewiring via macrophage mgration inhibitory factor and midkine signaling specifically monocytes, which exhibit M1 and M2 features, are marked by activation of pro- and anti-inflammatory programs, and express tumor-promoting factors, reminiscent of tumor-associated macrophages. The interactions and pathways characterized in our study provide the basis for therapeutic approaches that target tumor-to-microenvironment interactions.

Human repair-related Schwann cells adopt functions of antigen-presenting cells in vitro.

The plastic potential of Schwann cells (SCs) is increasingly recognized to play a role after nerve injury and in diseases of the peripheral nervous system. Reports on the interaction between immune cells and SCs indicate their involvement in inflammatory processes. However, the immunocompetence of human SCs has been primarily deduced from neuropathies, but whether after nerve injury SCs directly regulate an adaptive immune response is unknown. Here, we performed comprehensive analysis of immunomodulatory capacities of human repair-related SCs (hrSCs), which recapitulate SC response to nerve injury in vitro. We used our well-established culture model of primary hrSCs from human peripheral nerves and analyzed the transcriptome, secretome, and cell surface proteins for pathways and markers relevant in innate and adaptive immunity, performed phagocytosis assays, and monitored T-cell subset activation in allogeneic co-cultures. Our findings show that hrSCs are phagocytic, which is in line with high MHCII expression. Furthermore, hrSCs express co-regulatory proteins, such as CD40, CD80, B7H3, CD58, CD86, and HVEM, release a plethora of chemoattractants, matrix remodeling proteins and pro- as well as anti-inflammatory cytokines, and upregulate the T-cell inhibiting PD-L1 molecule upon pro-inflammatory stimulation with IFNγ. In contrast to monocytes, hrSC alone are not sufficient to trigger allogenic CD4+ and CD8+ T-cells, but limit number and activation status of exogenously activated T-cells. This study demonstrates that hrSCs possess features and functions typical for professional antigen-presenting cells in vitro, and suggest a new role of these cells as negative regulators of T-cell immunity during nerve regeneration.

Landscape of Bone Marrow Metastasis in Human Neuroblastoma Unraveled by Transcriptomics and Deep Multiplex Imaging.

While the bone marrow attracts tumor cells in many solid cancers leading to poor outcome in affected patients, comprehensive analyses of bone marrow metastases have not been performed on a single-cell level. We here set out to capture tumor heterogeneity and unravel microenvironmental changes in neuroblastoma, a solid cancer with bone marrow involvement. To this end, we employed a multi-omics data mining approach to define a multiplex imaging panel and developed DeepFLEX, a pipeline for subsequent multiplex image analysis, whereby we constructed a single-cell atlas of over 35,000 disseminated tumor cells (DTCs) and cells of their microenvironment in the metastatic bone marrow niche. Further, we independently profiled the transcriptome of a cohort of 38 patients with and without bone marrow metastasis. Our results revealed vast diversity among DTCs and suggest that FAIM2 can act as a complementary marker to capture DTC heterogeneity. Importantly, we demonstrate that malignant bone marrow infiltration is associated with an inflammatory response and at the same time the presence of immuno-suppressive cell types, most prominently an immature neutrophil/granulocytic myeloid-derived suppressor-like cell type. The presented findings indicate that metastatic tumor cells shape the bone marrow microenvironment, warranting deeper investigations of spatio-temporal dynamics at the single-cell level and their clinical relevance.

Novel Insights into Diagnosis, Biology and Treatment of Primary Diffuse Leptomeningeal Melanomatosis.

Primary diffuse leptomeningeal melanomatosis (PDLMM) is an extremely rare and aggressive cancer type for which best treatment strategies remain to be elucidated. Herein, we present current and prospective diagnostic strategies and treatment management of PDLMM. Against the background of an extensive literature review of published PDLMM cases and currently employed therapeutic strategies, we present an illustrative case of a pediatric patient suffering from PDLMM. We report the first case of a pediatric patient with PDLMM who received combination treatment including trametinib and everolimus, followed by intravenous nivolumab and ipilimumab with concomitant intensive intraventricular chemotherapy, resulting in temporary significant clinical improvement and overall survival of 7 months. Following this clinical experience, we performed a comprehensive literature review, identifying 26 additional cases. By these means, we provide insight into current knowledge on clinical and molecular characteristics of PDLMM. Analysis of these cases revealed that the unspecific clinical presentation, such as unrecognized increased intracranial pressure (present in 67%), is a frequent reason for the delay in diagnosis. Mortality remains substantial despite diverse therapeutic approaches with a median overall survival of 4 months from diagnosis. On the molecular level, to date, the only oncogenic driver reported so far is mutation of NRAS (n = 3), underlining a close biological relation to malignant melanoma and neurocutaneous melanosis. We further show, for the first time, that this somatic mutation can be exploited for cerebrospinal fluid liquid biopsy detection, revealing a novel potential biomarker for diagnosis and monitoring of PDLMM. Last, we use a unique patient derived PDLMM cell model to provide first insights into in vitro drug sensitivities. In summary, we provide future diagnostic and therapeutic guidance for PDLMM and first insights into the use of liquid biopsy and in vitro models for this orphan cancer type.

Schwann cell plasticity regulates neuroblastic tumor cell differentiation via epidermal growth factor-like protein 8.

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development.

An annotated fluorescence image dataset for training nuclear segmentation methods.

Fully-automated nuclear image segmentation is the prerequisite to ensure statistically significant, quantitative analyses of tissue preparations,applied in digital pathology or quantitative microscopy. The design of segmentation methods that work independently of the tissue type or preparation is complex, due to variations in nuclear morphology, staining intensity, cell density and nuclei aggregations. Machine learning-based segmentation methods can overcome these challenges, however high quality expert-annotated images are required for training. Currently, the limited number of annotated fluorescence image datasets publicly available do not cover a broad range of tissues and preparations. We present a comprehensive, annotated dataset including tightly aggregated nuclei of multiple tissues for the training of machine learning-based nuclear segmentation algorithms. The proposed dataset covers sample preparation methods frequently used in quantitative immunofluorescence microscopy. We demonstrate the heterogeneity of the dataset with respect to multiple parameters such as magnification, modality, signal-to-noise ratio and diagnosis. Based on a suggested split into training and test sets and additional single-nuclei expert annotations, machine learning-based image segmentation methods can be trained and evaluated.

Neuroblastoma cells undergo transcriptomic alterations upon dissemination into the bone marrow and subsequent tumor progression.

Neuroblastoma is the most common extracranial solid tumor in childhood. The vast majority of metastatic (M) stage patients present with disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis and relapse. Although these cells represent a major obstacle in the treatment of neuroblastoma patients, insights into their expression profile remained elusive. The present RNA-Seq study of stage 4/M primary tumors, enriched BM-derived diagnostic and relapse DTCs, as well as the corresponding BM-derived mononuclear cells (MNCs) from 53 patients revealed 322 differentially expressed genes in DTCs as compared to the tumors (q < 0.001, |log2 FC|>2). Particularly, the levels of transcripts encoded by mitochondrial DNA were elevated in DTCs, whereas, for example, genes involved in angiogenesis were downregulated. Furthermore, 224 genes were highly expressed in DTCs and only slightly, if at all, in MNCs (q < 8 × 10-75 log2 FC > 6). Interestingly, we found the transcriptome of relapse DTCs largely resembling those of diagnostic DTCs with only 113 differentially expressed genes under relaxed cut-offs (q < 0.01, |log2 FC|>0.5). Notably, relapse DTCs showed a positional enrichment of 31 downregulated genes on chromosome 19, including five tumor suppressor genes: SIRT6, BBC3/PUMA, STK11, CADM4 and GLTSCR2. This first RNA-Seq analysis of neuroblastoma DTCs revealed their unique expression profile in comparison to the tumors and MNCs, and less pronounced differences between diagnostic and relapse DTCs. The latter preferentially affected downregulation of genes encoded by chromosome 19. As these alterations might be associated with treatment failure and disease relapse, further functional studies on DTCs should be considered.

Impact of Disseminated Neuroblastoma Cells on the Identification of the Relapse-Seeding Clone.

Purpose: Tumor relapse is the most frequent cause of death in stage 4 neuroblastomas. Since genomic information on the relapse precursor cells could guide targeted therapy, our aim was to find the most appropriate tissue for identifying relapse-seeding clones.Experimental design: We analyzed 10 geographically and temporally separated samples of a single patient by SNP array and validated the data in 154 stage 4 patients.Results: In the case study, aberrations unique to certain tissues and time points were evident besides concordant aberrations shared by all samples. Diagnostic bone marrow-derived disseminated tumor cells (DTCs) as well as the metastatic tumor and DTCs at relapse displayed a 1q deletion, not detected in any of the seven primary tumor samples. In the validation cohort, the frequency of 1q deletion was 17.8%, 10%, and 27.5% in the diagnostic DTCs, diagnostic tumors, and DTCs at relapse, respectively. This aberration was significantly associated with 19q and ATRX deletions. We observed a significant increased likelihood of an adverse event in the presence of 19q deletion in the diagnostic DTCs.Conclusions: Different frequencies of 1q and 19q deletions in the primary tumors as compared with DTCs, their relatively high frequency at relapse, and their effect on event-free survival (19q deletion) indicate the relevance of analyzing diagnostic DTCs. Our data support the hypothesis of a branched clonal evolution and a parallel progression of primary and metastatic tumor cells. Therefore, searching for biomarkers to identify the relapse-seeding clone should involve diagnostic DTCs alongside the tumor tissue. Clin Cancer Res; 23(15); 4224-32. ©2017 AACR.

Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype.

The remarkable feature of Schwann cells (SCs) to transform into a repair phenotype turned the spotlight on this powerful cell type. SCs provide the regenerative environment for axonal re-growth after peripheral nerve injury (PNI) and play a vital role in differentiation of neuroblastic tumors into a benign subtype of neuroblastoma, a tumor originating from neural crest-derived neuroblasts. Hence, understanding their mode-of-action is of utmost interest for new approaches in regenerative medicine, but also for neuroblastoma therapy. However, literature on human SCs is scarce and it is unknown to which extent human SC cultures reflect the SC repair phenotype developing after PNI in patients. We performed high-resolution proteome profiling and RNA-sequencing on highly enriched human SC and fibroblast cultures, control and ex vivo degenerated nerve explants to identify novel molecules and functional processes active in repair SCs. In fact, we found cultured SCs and degenerated nerves to share a similar repair SC-associated expression signature, including the upregulation of JUN, as well as two prominent functions, i.e., myelin debris clearance and antigen presentation via MHCII. In addition to myelin degradation, cultured SCs were capable of actively taking up cell-extrinsic components in functional phagocytosis and co-cultivation assays. Moreover, in cultured SCs and degenerated nerve tissue MHCII was upregulated at the cellular level along with high expression of chemoattractants and co-inhibitory rather than -stimulatory molecules. These results demonstrate human SC cultures to execute an inherent program of nerve repair and support two novel repair SC functions, debris clearance via phagocytosis-related mechanisms and type II immune-regulation. GLIA 2016;64:2133-2153.

Metronomic topotecan impedes tumor growth of MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy induced senescence.

Poor prognosis and frequent relapses are major challenges for patients with high-risk neuroblastoma (NB), especially when tumors show MYCN amplification. High-dose chemotherapy triggers apoptosis, necrosis and senescence, a cellular stress response leading to permanent proliferative arrest and a typical senescence-associated secretome (SASP). SASP components reinforce growth-arrest and act immune-stimulatory, while others are tumor-promoting. We evaluated whether metronomic, i.e. long-term, repetitive low-dose, drug treatment induces senescence in vitro and in vivo. And importantly, by using the secretome as a discriminator for beneficial versus adverse effects of senescence, drugs with a tumor-inhibiting SASP were identified.We demonstrate that metronomic application of chemotherapeutic drugs induces therapy-induced senescence, characterized by cell cycle arrest, p21(WAF/CIP1) up-regulation and DNA double-strand breaks selectively in MYCN-amplified NB. Low-dose topotecan (TPT) was identified as an inducer of a favorable SASP while lacking NFKB1/p50 activation. In contrast, Bromo-deoxy-uridine induced senescent NB-cells secret a tumor-promoting SASP in a NFKB1/p50-dependent manner. Importantly, TPT-treated senescent tumor cells act growth-inhibitory in a dose-dependent manner on non-senescent tumor cells and MYCN expression is significantly reduced in vitro and in vivo. Furthermore, in a mouse xenotransplant-model for MYCN-amplified NB metronomic TPT leads to senescence selectively in tumor cells, complete or partial remission, prolonged survival and a favorable SASP.This new mode-of-action of metronomic TPT treatment, i.e. promoting a tumor-inhibiting type of senescence in MYCN-amplified tumors, is clinically relevant as metronomic regimens are increasingly implemented in therapy protocols of various cancer entities and are considered as a feasible maintenance treatment option with moderate adverse event profiles.

Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies-A Validation Study.

BACKGROUND: Metastases in the bone marrow (BM) in form of disseminated tumor cells (DTCs) are frequent events at diagnosis and also at relapse in high-risk neuroblastoma patients. The frequently highly diluted occurrence of DTCs requires adequate enrichment strategies to enable their detailed characterization. However, to avoid methodical artifacts we tested whether pre-analytical processing steps-including transport duration, temperature and, importantly, tumor cell enrichment techniques-are confounding factors for gene expression analysis in DTCs. METHODS: LAN-1 neuroblastoma cells were spiked into tumor free BM and/or peripheral blood and: i) kept at room temperature or at 4°C for 24, 48 and 72 hours; ii) frozen down at -80°C and thawed; iii) enriched via magnetic beads. The effect on the gene expression signature of LAN-1 cells was analyzed by qPCR arrays and gene expression microarrays. RESULTS: Neither storage at -80°C in DMSO and subsequent thawing nor enrichment of spiked-in neuroblastoma cells changed the expression of the analyzed genes significantly. Whereas storage at 4°C altered the expression of analyzed genes (14.3%) only at the 72h-timepoint in comparison to the 0h-timepoint, storage at room temperature had a much more profound effect on gene expression by affecting 20% at 24h, 26% at 48h and 43% at 72h of the analyzed genes. CONCLUSION: Using neuroblastoma as a model, we show that tumor cell enrichment by magnetic bead separation has virtually no effect on gene expression in DTCs. However, transport time and temperature can influence the expression profile remarkably. Thus, the expression profile of routinely collected BM samples can be analyzed without concern as long as the transport conditions are monitored.

Bone marrows from neuroblastoma patients: an excellent source for tumor genome analyses.

Neuroblastoma is the most common extra-cranial solid tumor in childhood. Presence of disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis and at relapse is a common event in stage M neuroblastomas. Although the clinical heterogeneity of disseminated neuroblastomas is frequently associated with genomic diversity, so far, only little information exists about the genomic status of DTCs. This lack of knowledge is mainly due to the varying amount of BM infiltrating tumor cells, which is usually below 30% even at diagnosis thereby hampering systematic analyses. Thus, a valuable chance to analyze metastatic and relapse clones is, so far, completely unexploited. In this study, we show that the enrichment of tumor cells in fresh or DMSO frozen BM samples with a minimum of 0.05% or 0.1% infiltration rate, respectively, by applying magnetic bead-based technique increased the DTC content to a sufficient level to allow SNP array analyses in 49 out of 69 samples. In addition, we successfully used non-enriched BM samples with ≥30% DTCs including non-stained and immunostained cytospin and BM smear slides for SNP array analyses in 44 cases. We analyzed the genomic profile of DTCs by an ultra-high density SNP array technique with highest performance detecting all segmental chromosomal aberrations, amplified regions, acquired loss of heterozygosity events and minor aberrations affecting single genes or parts thereof.