RESUMEN
Medulloblastomas with extensive nodularity are cerebellar tumors characterized by two distinct compartments and variable disease progression. The mechanisms governing the balance between proliferation and differentiation in MBEN remain poorly understood. Here, we employ a multi-modal single cell transcriptome analysis to dissect this process. In the internodular compartment, we identify proliferating cerebellar granular neuronal precursor-like malignant cells, along with stromal, vascular, and immune cells. In contrast, the nodular compartment comprises postmitotic, neuronally differentiated malignant cells. Both compartments are connected through an intermediate cell stage resembling actively migrating CGNPs. Notably, we also discover astrocytic-like malignant cells, found in proximity to migrating and differentiated cells at the transition zone between the two compartments. Our study sheds light on the spatial tissue organization and its link to the developmental trajectory, resulting in a more benign tumor phenotype. This integrative approach holds promise to explore intercompartmental interactions in other cancers with varying histology.
Asunto(s)
Neoplasias Cerebelosas , Meduloblastoma , Humanos , Meduloblastoma/genética , Diferenciación Celular , Neoplasias Cerebelosas/genética , Progresión de la Enfermedad , Técnicas HistológicasRESUMEN
In multiple myeloma spatial differences in the subclonal architecture, molecular signatures and composition of the microenvironment remain poorly characterized. To address this shortcoming, we perform multi-region sequencing on paired random bone marrow and focal lesion samples from 17 newly diagnosed patients. Using single-cell RNA- and ATAC-seq we find a median of 6 tumor subclones per patient and unique subclones in focal lesions. Genetically identical subclones display different levels of spatial transcriptional plasticity, including nearly identical profiles and pronounced heterogeneity at different sites, which can include differential expression of immunotherapy targets, such as CD20 and CD38. Macrophages are significantly depleted in the microenvironment of focal lesions. We observe proportional changes in the T-cell repertoire but no site-specific expansion of T-cell clones in intramedullary lesions. In conclusion, our results demonstrate the relevance of considering spatial heterogeneity in multiple myeloma with potential implications for models of cell-cell interactions and disease progression.
Asunto(s)
Mieloma Múltiple , Humanos , Mieloma Múltiple/genética , Comunicación Celular , Secuenciación de Inmunoprecipitación de Cromatina , Células Clonales , Progresión de la Enfermedad , Microambiente Tumoral/genéticaRESUMEN
Intratumor heterogeneity as a clinical challenge becomes most evident after several treatment lines, when multidrug-resistant subclones accumulate. To address this challenge, the characterization of resistance mechanisms at the subclonal level is key to identify common vulnerabilities. In this study, we integrate whole-genome sequencing, single-cell (sc) transcriptomics (scRNA sequencing), and chromatin accessibility (scATAC sequencing) together with mitochondrial DNA mutations to define subclonal architecture and evolution for longitudinal samples from 15 patients with relapsed or refractory multiple myeloma. We assess transcriptomic and epigenomic changes to resolve the multifactorial nature of therapy resistance and relate it to the parallel occurrence of different mechanisms: (1) preexisting epigenetic profiles of subclones associated with survival advantages, (2) converging phenotypic adaptation of genetically distinct subclones, and (3) subclone-specific interactions of myeloma and bone marrow microenvironment cells. Our study showcases how an integrative multiomics analysis can be applied to track and characterize distinct multidrug-resistant subclones over time for the identification of molecular targets against them.
Asunto(s)
Mieloma Múltiple , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Multiómica , Mutación , Transcriptoma , Microambiente Tumoral/genéticaRESUMEN
Alternative lengthening of telomeres (ALT) occurs in â¼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.
Asunto(s)
Telómero/metabolismo , Línea Celular , ADN de Cadena Simple/genética , Humanos , Hibridación Fluorescente in Situ , Neoplasias/genética , Telomerasa/genética , Telómero/genética , Homeostasis del TelómeroRESUMEN
Colorectal cancer is among the leading causes of cancer-associated deaths worldwide. Treatment failure and tumor recurrence due to survival of therapy-resistant cancer stem/initiating cells represent major clinical issues to overcome. In this study, we identified lysine methyltransferase 9 (KMT9), an obligate heterodimer composed of KMT9α and KMT9ß that monomethylates histone H4 at lysine 12 (H4K12me1), as an important regulator in colorectal tumorigenesis. KMT9α and KMT9ß were overexpressed in colorectal cancer and colocalized with H4K12me1 at promoters of target genes involved in the regulation of proliferation. Ablation of KMT9α drastically reduced colorectal tumorigenesis in mice and prevented the growth of murine as well as human patient-derived tumor organoids. Moreover, loss of KMT9α impaired the maintenance and function of colorectal cancer stem/initiating cells and induced apoptosis specifically in this cellular compartment. Together, these data suggest that KMT9 is an important regulator of colorectal carcinogenesis, identifying KMT9 as a promising therapeutic target for the treatment of colorectal cancer. SIGNIFICANCE: The H4K12 methyltransferase KMT9 regulates tumor cell proliferation and stemness in colorectal cancer, indicating that targeting KMT9 could be a useful approach for preventing and treating this disease.
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Carcinogénesis/genética , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/genética , Estudios de Casos y Controles , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Organoides/metabolismo , Multimerización de Proteína , ARN Mensajero/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/químicaRESUMEN
Virtually all patients with multiple myeloma become unresponsive to treatment over time. Relapsed/refractory multiple myeloma (RRMM) is accompanied by the clonal evolution of myeloma cells with heterogeneous genomic aberrations and profound changes of the bone marrow microenvironment (BME). However, the molecular mechanisms that drive drug resistance remain elusive. Here, we analyze the heterogeneous tumor cell population and its complex interaction network with the BME of 20 RRMM patients by single cell RNA-sequencing before/after treatment. Subclones with chromosome 1q-gain express a specific transcriptomic signature and frequently expand during treatment. Furthermore, RRMM cells shape an immune suppressive BME by upregulation of inflammatory cytokines and close interaction with the myeloid compartment. It is characterized by the accumulation of PD1+ γδ T-cells and tumor-associated macrophages as well as the depletion of hematopoietic progenitors. Thus, our study resolves transcriptional features of subclones in RRMM and mechanisms of microenvironmental reprogramming with implications for clinical decision-making.
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Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple/genética , Transcriptoma , Microambiente Tumoral/genética , Antineoplásicos/uso terapéutico , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Médula Ósea/patología , Citocinas/genética , Citocinas/inmunología , Resistencia a Antineoplásicos/inmunología , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Humanos , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/patología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Recurrencia , Análisis de Secuencia de ARN , Transducción de Señal , Análisis de la Célula Individual , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC.
RESUMEN
BACKGROUND: Glioblastoma (GBM) consists of devastating neoplasms with high invasive capacity, which have been difficult to study in vitro in a human-derived model system. Therapeutic progress is also limited by cellular heterogeneity within and between tumors, among other factors such as therapy resistance. To address these challenges, we present an experimental model using human cerebral organoids as a scaffold for patient-derived GBM cell invasion. METHODS: This study combined tissue clearing and confocal microscopy with single-cell RNA sequencing of GBM cells before and after co-culture with organoid cells. RESULTS: We show that tumor cells within organoids extend a network of long microtubes, recapitulating the in vivo behavior of GBM. Transcriptional changes implicated in the invasion process are coherent across patient samples, indicating that GBM cells reactively upregulate genes required for their dispersion. Potential interactions between GBM and organoid cells identified by an in silico receptor-ligand pairing screen suggest functional therapeutic targets. CONCLUSIONS: Taken together, our model has proven useful for studying GBM invasion and transcriptional heterogeneity in vitro, with applications for both pharmacological screens and patient-specific treatment selection on a time scale amenable to clinical practice.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Organoides , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/patología , Humanos , Invasividad Neoplásica , Organoides/patología , Transcriptoma , Células Tumorales CultivadasRESUMEN
Patient-derived 3D cell culture systems are currently advancing cancer research since they potentiate the molecular analysis of tissue-like properties and drug response under well-defined conditions. However, our understanding of the relationship between the heterogeneity of morphological phenotypes and the underlying transcriptome is still limited. To address this issue, we here introduce "pheno-seq" to directly link visual features of 3D cell culture systems with profiling their transcriptome. As prototypic applications breast and colorectal cancer (CRC) spheroids were analyzed by pheno-seq. We identified characteristic gene expression signatures of epithelial-to-mesenchymal transition that are associated with invasive growth behavior of clonal breast cancer spheroids. Furthermore, we linked long-term proliferative capacity in a patient-derived model of CRC to a lowly abundant PROX1-positive cancer stem cell subtype. We anticipate that the ability to integrate transcriptome analysis and morphological patho-phenotypes of cancer cells will provide novel insight on the molecular origins of intratumor heterogeneity.
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Técnicas de Cultivo de Célula/métodos , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Mama/patología , Línea Celular Tumoral , Linaje de la Célula/genética , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Genes Relacionados con las Neoplasias , Humanos , Células Madre Neoplásicas/patología , Fenotipo , Análisis de la Célula IndividualRESUMEN
Streptococcus pneumoniae is a major human pathogen that successfully adapts to the host environment via an efficient uptake system for free DNA liberated from other organisms in the upper respiratory tract, facilitating immune evasion and drug resistance. Although the initial signaling events leading to pneumococcal competence for DNA transformation and the fate of DNA when it has been taken up have been extensively studied, the actual mechanism by which DNA in the environment may traverse the thick capsular and cell wall layers remains unknown. Here we visualize that induction of competence results in the formation of a native morphologically distinct pilus structure on the bacterial surface. This plaited pilus is encoded by the competence (com)G locus, and, after assembly, it is rapidly released into the surrounding medium. Heterologous pneumococcal pilus expression in Escherichia coli was obtained by replacing the pulE-K putative pilin genes of the Klebsiella oxytoca type II secretion system with the complete comG locus. In the pneumococcus, the coordinated secretion of pili from the cells correlates to DNA transformation. A model for DNA transformation is proposed whereby pilus assembly "drills" a channel across the thick cell wall that becomes transiently open by secretion of the pilus, providing the entry port for exogenous DNA to gain access to DNA receptors associated with the cytoplasmic membrane.
Asunto(s)
Sistemas de Secreción Bacterianos/fisiología , Competencia de la Transformación por ADN/genética , ADN/metabolismo , Fimbrias Bacterianas/metabolismo , Streptococcus pneumoniae/metabolismo , Transformación Bacteriana/fisiología , Electroforesis en Gel de Poliacrilamida , Fimbrias Bacterianas/ultraestructura , Microscopía Electrónica de Transmisión , Transformación Bacteriana/genética , Ácido TricloroacéticoRESUMEN
Phase variation of hypermutable simple sequence repeats (SSRs) is a widespread and stochastic mechanism to generate phenotypic variation within a population and thereby contributes to host adaptation of bacterial pathogens. Although several examples of SSRs that affect transcription or coding potential have been reported, we now show that a SSR also impacts small RNA-mediated posttranscriptional regulation. Based on in vitro and in vivo analyses, we demonstrate that a variable homopolymeric G-repeat in the leader of the TlpB chemotaxis receptor mRNA of the human pathogen Helicobacter pylori is directly targeted by a small RNA (sRNA), RepG (Regulator of polymeric G-repeats). Whereas RepG sRNA is highly conserved, the tlpB G-repeat length varies among diverse H. pylori strains, resulting in strain-specific RepG-mediated tlpB regulation. Based on modification of the G-repeat length within one strain, we demonstrate that the G-repeat length determines posttranscriptional regulation and can mediate both repression and activation of tlpB through RepG. In vitro translation assays show that this regulation occurs at the translational level and that RepG influences tlpB translation dependent on the G-repeat length. In contrast to the digital ON-OFF switches through frame-shift mutations within coding sequences, such modulation of posttranscriptional regulation allows for a gradual control of gene expression. This connection to sRNA-mediated posttranscriptional regulation might also apply to other genes with SSRs, which could be targeting sites of cis- or trans-encoded sRNAs, and thereby could facilitate host adaptation through sRNA-mediated fine-tuning of virulence gene expression.