Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma

Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management

Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq

Tumor subclasses differ according to the genotypes and phenotypes of malignant cells as well as the composition of the tumor microenvironment (TME).We dissected these influences in isocitrate dehydrogenase (IDH)-mutant gliomas by combining 14,226 single-cell RNA sequencing (RNA-seq) profiles from 16 patient samples with bulk RNA-seq profiles from 165 patient samples. Differences in bulk profiles between IDH-mutant astrocytoma and oligodendroglioma can be primarily explained by distinct TME and signature genetic events, whereas both tumor types share similar developmental hierarchies and lineages of glial differentiation. As tumor grade increases, we find enhanced proliferation of malignant cells, larger pools of undifferentiated glioma cells, and an increase in macrophage over microglia expression programs in TME. Our work provides a unifying model for IDH-mutant gliomas and a general framework for dissecting the differences among human tumor subclasses.National Cancer Institute (U.S.) (Grant P30-CA14051

Uncovering tumor heterogeneity, cell plasticity and characterization of the tumor microenvironment by single cell analysis.

The study of cancer genomics uses our constant technological advances in sequencing the human genome. To-date, most of genetic and transcriptional sequencing analyses have been performed in bulk tumor tissue, representing the grounds of our current understanding of cancer biology. However, tumors represent a complex ecosystem, with heterogeneous subpopulations of tumor cells but also different types of stromal and immune cells coexisting together, and this heterogeneity is increasingly recognized as a key determining factor in cancer therapeutic failure. Thus, we propose to leverage single-cell transcriptomic technologies, that our lab has pioneered in human tumors, to reconstruct the cellular, transcriptional and genetic architecture of human tumors, at unprecedented resolution. Firstly, we focused on glioblastoma, a lethal form of brain cancer, known for important tumor heterogeneity. By sequencing ~24K single cells from 28 patients, integrating our analysis with the TCGA dataset following up with functional experiments, we showed that glioblastoma's heterogeneity is mainly represented by four different cellular states. The frequency of each of those four states varies across patient and is dictated by specific genetic alterations. Using patient-derived xenografts, we showed that different subpopulations generate tumors of similar composition, suggesting cell plasticity between the different states. Then, we focused on synovial sarcoma (SyS), an aggressive neoplasm driven by a chromosomal translocation, known to act as an aberrant transcriptional regulator. By sequencing ~16K single cells from 12 tumors, we identified a common transcriptional program shared across all tumors capturing intra-tumor heterogeneity, with an overall expression associated with prognosis and risk of metastasis. We showed that this core oncogenic program was directly regulated by the translocation, inducing all the hallmarks of cancer. We identified some mechanisms of interaction between malignant and immune cells, with the core oncogenic program expressed by malignant cells inhibiting immune infiltration, while T-cells and macrophages from the tumor microenvironment canin turn repress this program. Finally, we identified pharmaceutical agents, such as HDAC and CDK4/6 inhibitors, that couId be good candidates in SyS therapy by inhibiting specific mediators of SyS oncogenic pathways. -- Les importantes avancées technologiques dans le séquençage du génome humain ont permis de construire les bases de nos connaissances actuelles en oncologie. Cependant, les tumeurs représentent un écosystème complexe comprenant des cellules tumorales, stromales et immunitaires hétérogènes, et la majorité du séquençage se fait à partir d'échantillons tumoraux, masquant cette hétérogénéité. C'est pourquoi, nous proposons d'utiliser des nouvelles techniques de séquençage de cellules individuelles que notre laboratoire a développé dans les tumeurs humaines, afin de reconstruire l'architecture cellulaire, transcriptomique et génétique des tumeurs humaines. Tout d'abord, nous nous sommes concentrés sur le glioblastome, une tumeur cérébrale agressive et très hétérogène. En séquençant ~24'000 cellules isolées de 28 patients, en intégrant nos analyses avec les données du TCGA et grâce à des expériences fonctionnelles, nous avons montré que l'hétérogénéité du glioblastome est représentée par quatre sous­ populations de cellules. Leur proportion est influencée par des facteurs génétiques et environnementaux et l'injection de différentes sous-populations utilisant des xénogreffes dans des souris a montré que chaque sous-population avait la capacité de reproduire les sous­ populations présentes dans la tumeur d'origine, suggérant une importante plasticité cellulaire. Ensuite, nous nous sommes intéressé au sarcome synovial (SyS), une tumeur induite par une translocation qui dérégule la transcription. En séquençant ~16'000 cellules isolées de 12 tumeurs, nous avons identifié un programme de transcription qui identifie une sous-population de cellules dans toutes les tumeurs analysées, et l'expression globale de ce programme est associée avec le pronostic des patients. Nous avons montré que son expression est directement induite par la translocation, induisant les caractéristiques principales des cellules cancéreuses. Nous avons identifié des mécanismes d'interaction entre les cellules cancéreuses et immunitaires, avec un effet anti-immunitaire de ce programme, et la capacité des cellules immunitaires d'inhiber ce programme. Finalement, nous avons identifié des thérapies, telle que les inhibiteurs de HDAC et CDK4/6, qui pourraient être efficaces grâce à leur effet sur les intermédiaires des voies de signalisation identifiées dans le SyS

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish