Single-cell transcriptomics reveals shared immunosuppressive landscapes of mouse and human neuroblastoma

BACKGROUND High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity

BET and CDK Inhibition Reveal Differences in the Proliferation Control of Sympathetic Ganglion Neuroblasts and Adrenal Chromaffin Cells

Neuroblastoma arising from the adrenal differ from ganglionic neuroblastoma both genetically and clinically, with adrenal tumors being associated with a more severe prognosis. The different tumor properties may be linked to specific tumor founder cells in adrenal and sympathetic ganglia. To address this question, we first set up cultures of mouse sympathetic neuroblasts and adrenal chromaffin cells. These cultures were then treated with various proliferation inhibitors to identify lineage-specific responses. We show that neuroblast and chromaffin cell proliferation was affected by WNT, ALK, IGF1, and PRC2/EZH2 signaling inhibitors to a similar extent. However, differential effects were observed in response to bromodomain and extraterminal (BET) protein inhibitors (JQ1, GSK1324726A) and to the CDK-7 inhibitor THZ1, with BET inhibitors preferentially affecting chromaffin cells, and THZ1 preferentially affecting neuroblasts. The differential dependence of chromaffin cells and neuroblasts on BET and CDK signaling may indicate different mechanisms during tumor initiation in sympathetic ganglia and adrenal

Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma

Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in Alk(F1178L) mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI Alk(R1279Q) and MYCN/KI Alk(F1178L) mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.ISSN:2041-172

Breakpoint Features of Genomic Rearrangements in Neuroblastoma with Unbalanced Translocations and Chromothripsis

<div><p>Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including <i>ALK</i>, <i>NBAS</i>, <i>FHIT</i>, <i>PTPRD</i> and <i>ODZ4</i>. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.</p></div

Inter and large intra-chromosomal rearrangements predicted by SVDetect.

<p>DELETION deletion of a fragment.</p><p>INS_FRAGMT insertion of a short fragment (fragment is known).</p><p>INV_INS_FRAGMT insertion of a short fragment (fragment is known, fragment is inverted).</p><p>INV_FRAGMENT inversion (both ends of the inversion are confirmed by read pairs).</p><p>INVERSION inversion (only one end of the inversion is confirmed by read pairs).</p><p>LARGE_DUPLICATION large duplication (size of the duplicated fragment is greater than average insert size).</p><p>TRANSLOCATION translocation.</p

Molecular characterization of unbalanced translocations in the CLB-Ga cell line.

<p><b>A</b>, Characterization at the base-pair level of an acquired unbalanced translocation der(4)t(4;17). The translocation was initially demonstrated by 24-color karyotyping (left panel) and loss of 4q as well as gain of 17q was consistently observed by read coverage analysis with the FREEC algorithm (middle panels). NGS data identified abnormally mapped reads corresponding to this inter-chromosomal rearrangement. Further PCR analysis confirmed that this rearrangement was somatic as detected in CLB-Ga cell line as well as in a bone marrow sample contaminated with tumor cells but not in the corresponding lymphoblastoid LL-Ga cell line (right panel). <b>B</b>, Characterization at the base-pair level of an acquired unbalanced translocation der(5)t(5;11). The translocation was initially evidenced by 24-color karyotyping (left panel) and loss of 5q as well as gain of 11p was consistently observed by FREEC (middle panels). NGS data identified reads corresponding to an unbalanced translocation between 5q and 11p; however, orientation of the stuck fragments was not compatible with the observed derivative chromosome. NGS data also revealed an intra-chromosomal inverted duplication at 5q close to the rearrangement between 5q and 11p. The FREEC profile indeed revealed a small region of gain preceding the 5q deletion. Further PCR analysis confirmed both rearrangements and indicated that they were somatic as detected in the CLB-Ga cell line as well as in the bone marrow sample contaminated with tumor cells but not in the LL-Ga cell line (right panels). Sequencing of the PCR products indicates that the first breakpoint falls into the first intron of the <i>CDX1</i> gene, which is consequently truncated. An inverted duplication is observed between positions 149 499 753 and 149 551 306 comprising the first exon of the <i>CDX1</i> gene as well as exons 1 to 17 of the <i>PDGFRB</i> gene. The breakpoint at position 41 211 793 on chromosome 11 corresponds to the first intron of the <i>LRRC4C</i> gene. Thus, this junction may result in a chimeric transcript including <i>PDGFRB</i> and <i>LRRC4C</i>.</p

Chromothripsis in two primary NB tumors.

<p>FREEC analysis showing the shattering of: <b>A</b>, chromosome 1 in NB1141; <b>D</b>, chromosome 6 in NB1142. All inter-chromosomal rearrangements and intra-chromosomal SVs encompassing regions longer than 50 kb identified in mate-pair sequencing are visualized using the Circos tool. <b>B</b>, NB1141 tumor; <b>E</b>, NB1142 tumor; <b>C</b>, zoom on chromosome 1 and distal part of chromosome 2p arm (0–35 Mb) in NB1141; <b>F</b>, zoom on chromosomes 6 and 19 in NB1142. Line’s thickness is related to the numbers of pairs identified in each link.</p