RESUMEN
Melanoma clinical outcomes emerge from incompletely understood genetic mechanisms operating within the tumor and its microenvironment. Here, we used single-cell RNA-based spatial molecular imaging (RNA-SMI) in patient-derived archival tumors to reveal clinically relevant markers of malignancy progression and prognosis. We examined spatial gene expression of 203,472 cells inside benign and malignant melanocytic neoplasms, including melanocytic nevi and primary invasive and metastatic melanomas. Algorithmic cell clustering paired with intratumoral comparative two-dimensional analyses visualized synergistic, spatial gene signatures linking cellular proliferation, metabolism, and malignancy, validated by protein expression. Metastatic niches included up-regulation of CDK2 and FABP5, which independently predicted poor clinical outcome in 473 patients with melanoma via Cox regression analysis. More generally, our work demonstrates a framework for applying single-cell RNA-SMI technology toward identifying gene regulatory landscapes pertinent to cancer progression and patient survival.
Asunto(s)
Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Melanoma , Análisis de la Célula Individual , Humanos , Melanoma/patología , Melanoma/genética , Melanoma/metabolismo , Melanoma/mortalidad , Pronóstico , Análisis de la Célula Individual/métodos , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Microambiente Tumoral , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Masculino , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/mortalidad , Perfilación de la Expresión GénicaRESUMEN
High-grade serous ovarian carcinoma (HGSOC) is genetically unstable and characterised by the presence of subclones with distinct genotypes. Intratumoural heterogeneity is linked to recurrence, chemotherapy resistance, and poor prognosis. Here, we use spatial transcriptomics to identify HGSOC subclones and study their association with infiltrating cell populations. Visium spatial transcriptomics reveals multiple tumour subclones with different copy number alterations present within individual tumour sections. These subclones differentially express various ligands and receptors and are predicted to differentially associate with different stromal and immune cell populations. In one sample, CosMx single molecule imaging reveals subclones differentially associating with immune cell populations, fibroblasts, and endothelial cells. Cell-to-cell communication analysis identifies subclone-specific signalling to stromal and immune cells and multiple subclone-specific autocrine loops. Our study highlights the high degree of subclonal heterogeneity in HGSOC and suggests that subclone-specific ligand and receptor expression patterns likely modulate how HGSOC cells interact with their local microenvironment.
Asunto(s)
Neoplasias Ováricas , Microambiente Tumoral , Humanos , Femenino , Microambiente Tumoral/genética , Células Endoteliales/metabolismo , Neoplasias Ováricas/patología , Perfilación de la Expresión Génica , Variaciones en el Número de Copia de ADNRESUMEN
The utilization of single-cell resolved spatial transcriptomics to delineate immune responses during SARS-CoV-2 infection was able to identify M1 macrophages to have elevated expression of IFI27 in areas of infection.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , Perfilación de la Expresión Génica , Macrófagos , Proteínas de la MembranaRESUMEN
Ulcerative colitis and Crohn's disease are chronic inflammatory intestinal diseases with perplexing heterogeneity in disease manifestation and response to treatment. While the molecular basis for this heterogeneity remains uncharacterized, single-cell technologies allow us to explore the transcriptional states within tissues at an unprecedented resolution which could further understanding of these complex diseases. Here, we apply single-cell RNA-sequencing to human inflamed intestine and show that the largest differences among patients are present within the myeloid compartment including macrophages and neutrophils. Using spatial transcriptomics in human tissue at single-cell resolution (CosMx Spatial Molecular Imaging) we spatially localize each of the macrophage and neutrophil subsets identified by single-cell RNA-sequencing and unravel further macrophage diversity based on their tissue localization. Finally, single-cell RNA-sequencing combined with single-cell spatial analysis reveals a strong communication network involving macrophages and inflammatory fibroblasts. Our data sheds light on the cellular complexity of these diseases and points towards the myeloid and stromal compartments as important cellular subsets for understanding patient-to-patient heterogeneity.
Asunto(s)
Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Neutrófilos , Enfermedades Inflamatorias del Intestino/genética , Enfermedad de Crohn/genética , Macrófagos , ARNRESUMEN
PURPOSE: Cancer drug development is currently limited by a paradigm of preclinical evaluation that does not adequately recapitulate the complexity of the intact human tumor microenvironment (TME). To overcome this, we combined trackable intratumor microdosing (CIVO) with spatial biology readouts to directly assess drug effects in patient tumors in situ. EXPERIMENTAL DESIGN: In a first-of-its-kind phase 0 clinical trial, we explored the effects of an investigational stage SUMOylation-activating enzyme (SAE) inhibitor, subasumstat (TAK-981) in 12 patients with head and neck carcinoma (HNC). Patients scheduled for tumor resection received percutaneous intratumor injections of subasumstat and vehicle control 1 to 4 days before surgery, resulting in spatially localized and graded regions of drug exposure (â¼1,000-2,000 µm in diameter). Drug-exposed (n = 214) and unexposed regions (n = 140) were compared by GeoMx Digital Spatial Profiler, with evaluation at single-cell resolution in a subset of these by CosMx Spatial Molecular Imager. RESULTS: Localized regions of subasumstat exposure revealed SUMO pathway inhibition, elevation of type I IFN response, and inhibition of cell cycle across all tumor samples. Single-cell analysis by CosMx demonstrated cell-cycle inhibition specific to the tumor epithelium, and IFN pathway induction commensurate with a TME shift from immune-suppressive to immune-permissive. CONCLUSIONS: Pairing CIVO with spatial profiling enabled detailed investigation of response to subasumstat across a diverse sampling of native and intact TME. We demonstrate that drug mechanism of action can be directly evaluated in a spatially precise manner in the most translationally relevant setting: an in situ human tumor.
Asunto(s)
Antineoplásicos , Neoplasias de Cabeza y Cuello , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Microambiente TumoralRESUMEN
Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .
Asunto(s)
Proteínas , ARN , Humanos , Adhesión en Parafina , ARN/genética , Imagen Molecular , FormaldehídoRESUMEN
Ancestrally marine threespine stickleback fish (Gasterosteus aculeatus) have undergone an adaptive radiation into freshwater environments throughout the Northern Hemisphere, creating an excellent model system for studying molecular adaptation and speciation. Ecological and behavioral factors have been suggested to underlie stickleback reproductive isolation and incipient speciation, but reproductive proteins mediating gamete recognition during fertilization have so far remained unexplored. To begin to investigate the contribution of reproductive proteins to stickleback reproductive isolation, we have characterized the stickleback egg coat proteome. We find that stickleback egg coats are comprised of homologs to the zona pellucida (ZP) proteins ZP1 and ZP3, as in other teleost fish. Our molecular evolutionary analyses indicate that across teleosts, ZP3 but not ZP1 has experienced positive Darwinian selection. Mammalian ZP3 is also rapidly evolving, and surprisingly some residues under selection in stickleback and mammalian ZP3 directly align. Despite broad homology, however, we find differences between mammalian and stickleback ZP proteins with respect to glycosylation, disulfide bonding, and sites of synthesis. Taken together, the changes we observe in stickleback ZP protein architecture suggest that the egg coats of stickleback fish, and perhaps fish more generally, have evolved to fulfill a more protective functional role than their mammalian counterparts.
Asunto(s)
Proteínas del Huevo/fisiología , Oocitos/fisiología , Smegmamorpha/metabolismo , Animales , Citoprotección/fisiología , Proteínas del Huevo/metabolismo , Femenino , Oocitos/citología , Oocitos/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Proteómica , Zona Pelúcida/metabolismo , Zona Pelúcida/fisiología , Glicoproteínas de la Zona Pelúcida/análisis , Glicoproteínas de la Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/fisiologíaRESUMEN
All animal oocytes are surrounded by a glycoproteinaceous egg coat, a specialized extracellular matrix that serves both structural and species-specific roles during fertilization. Egg coat glycoproteins polymerize into the extracellular matrix of the egg coat using a conserved protein-protein interaction module-the zona pellucida (ZP) domain-common to both vertebrates and invertebrates, suggesting that the basic structural features of egg coats have been conserved across hundreds of millions of years of evolution. Egg coat proteins, as with other proteins involved in reproduction, are frequently found to be rapidly evolving. Given that gamete compatibility must be maintained for the fitness of sexually reproducing organisms, this finding is somewhat paradoxical and suggests a role for adaptive diversification in reproductive protein evolution. Here we review the structure and function of metazoan egg coat proteins, with an emphasis on the potential role their evolution has played in the creation and maintenance of species boundaries.
Asunto(s)
Evolución Biológica , Proteínas del Huevo/química , Proteínas del Huevo/metabolismo , Glicoproteínas de la Zona Pelúcida/química , Glicoproteínas de la Zona Pelúcida/metabolismo , Animales , Proteínas del Huevo/fisiología , Femenino , Humanos , Invertebrados/química , Invertebrados/embriología , Invertebrados/metabolismo , Dominios Proteicos , Multimerización de Proteína/fisiología , Vertebrados/embriología , Vertebrados/metabolismo , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/fisiologíaRESUMEN
The extent to which convergent adaptation to similar ecological niches occurs by a predictable genetic basis remains a fundamental question in biology. Threespine stickleback fish have undergone an adaptive radiation in which ancestral oceanic populations repeatedly colonized and adapted to freshwater habitats. In multiple lakes in British Columbia, two different freshwater ecotypes have evolved: a deep-bodied benthic form adapted to forage near the lake substrate, and a narrow-bodied limnetic form adapted to forage in open water. Here, we use genome-wide linkage mapping in marine × benthic F2 genetic crosses to test the extent of shared genomic regions underlying benthic adaptation in three benthic populations. We identify at least 100 Quantitative Trait Loci (QTL) harboring genes influencing skeletal morphology. The majority of QTL (57%) are unique to one cross. However, four genomic regions affecting eight craniofacial and armor phenotypes are found in all three benthic populations. We find that QTL are clustered in the genome and overlapping QTL regions are enriched for genomic signatures of natural selection. These findings suggest that benthic adaptation has occurred via both parallel and nonparallel genetic changes.
Asunto(s)
Adaptación Fisiológica/genética , Mapeo Cromosómico , Sitios de Carácter Cuantitativo , Smegmamorpha/genética , Animales , Evolución Biológica , Colombia Británica , Cruzamientos Genéticos , Ligamiento Genético , Lagos , Fenotipo , Selección GenéticaRESUMEN
Marine populations of the threespine stickleback (Gasterosteus aculeatus) have repeatedly colonized and rapidly adapted to freshwater habitats, providing a powerful system to map the genetic architecture of evolved traits. Here, we developed and applied a binned genotyping-by-sequencing (GBS) method to build dense genome-wide linkage maps of sticklebacks using two large marine by freshwater F2 crosses of more than 350 fish each. The resulting linkage maps significantly improve the genome assembly by anchoring 78 new scaffolds to chromosomes, reorienting 40 scaffolds, and rearranging scaffolds in 4 locations. In the revised genome assembly, 94.6% of the assembly was anchored to a chromosome. To assess linkage map quality, we mapped quantitative trait loci (QTL) controlling lateral plate number, which mapped as expected to a 200-kb genomic region containing Ectodysplasin, as well as a chromosome 7 QTL overlapping a previously identified modifier QTL. Finally, we mapped eight QTL controlling convergently evolved reductions in gill raker length in the two crosses, which revealed that this classic adaptive trait has a surprisingly modular and nonparallel genetic basis.