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1.
Cell ; 184(11): 2988-3005.e16, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34019793

RESUMO

Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.


Assuntos
Carcinoma de Células Renais/metabolismo , Recidiva Local de Neoplasia/genética , Macrófagos Associados a Tumor/metabolismo , Adulto , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Estudos de Coortes , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Rim/metabolismo , Neoplasias Renais/patologia , Linfócitos do Interstício Tumoral/patologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Microambiente Tumoral , Macrófagos Associados a Tumor/fisiologia
2.
Entropy (Basel) ; 25(3)2023 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-36981431

RESUMO

Gene sets are being increasingly leveraged to make high-level biological inferences from transcriptomic data; however, existing gene set analysis methods rely on overly conservative, heuristic approaches for quantifying the statistical significance of gene set enrichment. We created Nonparametric analytical-Rank-based Enrichment Analysis (NaRnEA) to facilitate accurate and robust gene set analysis with an optimal null model derived using the information theoretic Principle of Maximum Entropy. By measuring the differential activity of ~2500 transcriptional regulatory proteins based on the differential expression of each protein's transcriptional targets between primary tumors and normal tissue samples in three cohorts from The Cancer Genome Atlas (TCGA), we demonstrate that NaRnEA critically improves in two widely used gene set analysis methods: Gene Set Enrichment Analysis (GSEA) and analytical-Rank-based Enrichment Analysis (aREA). We show that the NaRnEA-inferred differential protein activity is significantly correlated with differential protein abundance inferred from independent, phenotype-matched mass spectrometry data in the Clinical Proteomic Tumor Analysis Consortium (CPTAC), confirming the statistical and biological accuracy of our approach. Additionally, our analysis crucially demonstrates that the sample-shuffling empirical null models leveraged by GSEA and aREA for gene set analysis are overly conservative, a shortcoming that is avoided by the newly developed Maximum Entropy analytical null model employed by NaRnEA.

3.
Circulation ; 142(21): 2060-2075, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-32962412

RESUMO

BACKGROUND: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive. METHODS: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro. RESULTS: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability. CONCLUSIONS: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.


Assuntos
Aterosclerose/genética , Aterosclerose/patologia , Diferenciação Celular/fisiologia , Genômica/métodos , Miócitos de Músculo Liso/patologia , Fenótipo , Animais , Aterosclerose/terapia , Desdiferenciação Celular/fisiologia , Movimento Celular/fisiologia , Transdiferenciação Celular/fisiologia , Células Cultivadas , Feminino , Terapia Genética/tendências , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miócitos de Músculo Liso/fisiologia , Análise de Sequência de RNA/métodos
4.
Genet Med ; 21(8): 1772-1780, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30700791

RESUMO

PURPOSE: Develop an automated exome analysis workflow that can produce a very small number of candidate variants yet still detect different numbers of deleterious variants between probands and unaffected siblings. METHODS: Ninety-seven outbred nuclear families from the Undiagnosed Diseases Program/Network included single probands and the corresponding unaffected sibling(s). Single-nucleotide polymorphism (SNP) chip and exome analyses were performed on all, with proband and unaffected sibling considered independently as the target. The total burden of candidate genetic variants was summed for probands and siblings over all considered disease models. RESULTS: Exome analysis workflow include automated programs for ethnicity-matched genotype calling, salvage pathway for Mendelian inconsistency, compound heterozygous recessive detection, BAM file regional curation, population frequency filtering, pedigree-aware BAM file noise evaluation, and exon deletion filtration. This workflow relied heavily on BAM file analysis. A greater average pathogenic variant number was found compared with unaffected siblings. This was significant (p < 0.05) when using published recommended thresholds, and implies that causal variants are retained in many probands' lists. CONCLUSION: Using Mendelian and non-Mendelian models, this agnostic exome analysis shows a difference between a small group of probands and their unaffected siblings. This workflow produces candidate lists small enough to pursue with laboratory validation.


Assuntos
Variações do Número de Cópias de DNA/genética , Processamento Eletrônico de Dados , Doenças Genéticas Inatas/diagnóstico , Análise de Sequência de DNA , Exoma/genética , Éxons/genética , Feminino , Doenças Genéticas Inatas/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Deleção de Sequência/genética , Irmãos
5.
Cancer Discov ; 14(2): 348-361, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-37966260

RESUMO

The sparse vascularity of pancreatic ductal adenocarcinoma (PDAC) presents a mystery: What prevents this aggressive malignancy from undergoing neoangiogenesis to counteract hypoxia and better support growth? An incidental finding from prior work on paracrine communication between malignant PDAC cells and fibroblasts revealed that inhibition of the Hedgehog (HH) pathway partially relieved angiosuppression, increasing tumor vascularity through unknown mechanisms. Initial efforts to study this phenotype were hindered by difficulties replicating the complex interactions of multiple cell types in vitro. Here we identify a cascade of paracrine signals between multiple cell types that act sequentially to suppress angiogenesis in PDAC. Malignant epithelial cells promote HH signaling in fibroblasts, leading to inhibition of noncanonical WNT signaling in fibroblasts and epithelial cells, thereby limiting VEGFR2-dependent activation of endothelial hypersprouting. This cascade was elucidated using human and murine PDAC explant models, which effectively retain the complex cellular interactions of native tumor tissues. SIGNIFICANCE: We present a key mechanism of tumor angiosuppression, a process that sculpts the physiologic, cellular, and metabolic environment of PDAC. We further present a computational and experimental framework for the dissection of complex signaling cascades that propagate among multiple cell types in the tissue environment. This article is featured in Selected Articles from This Issue, p. 201.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Hedgehog/genética , Neoplasias Pancreáticas/patologia , Fator A de Crescimento do Endotélio Vascular
6.
bioRxiv ; 2024 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-38559080

RESUMO

Diffuse Midline Gliomas (DMGs) are universally fatal, primarily pediatric malignancies affecting the midline structures of the central nervous system. Despite decades of clinical trials, treatment remains limited to palliative radiation therapy. A major challenge is the coexistence of molecularly distinct malignant cell states with potentially orthogonal drug sensitivities. To address this challenge, we leveraged established network-based methodologies to elucidate Master Regulator (MR) proteins representing mechanistic, non-oncogene dependencies of seven coexisting subpopulations identified by single-cell analysis-whose enrichment in essential genes was validated by pooled CRISPR/Cas9 screens. Perturbational profiles of 372 clinically relevant drugs helped identify those able to invert the activity of subpopulation-specific MRs for follow-up in vivo validation. While individual drugs predicted to target individual subpopulations-including avapritinib, larotrectinib, and ruxolitinib-produced only modest tumor growth reduction in orthotopic models, systemic co-administration induced significant survival extension, making this approach a valuable contribution to the rational design of combination therapy.

7.
medRxiv ; 2024 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-39417133

RESUMO

IgA vasculitis (IgAV) is a pediatric disease with skin and systemic manifestations. Here, we conducted genome, transcriptome, and proteome-wide association studies in 2,170 IgAV cases and 5,928 controls, generated IgAV-specific maps of gene expression and splicing from blood of 255 pediatric cases, and reconstructed myeloid-specific regulatory networks to define disease master regulators modulated by the newly identified disease driver genes. We observed significant association at the HLA-DRB1 (OR=1.55, P=1.1×10-25) and fine-mapped specific amino-acid risk substitutions in DRß1. We discovered two novel non-HLA loci: FCAR (OR=1.51, P=1.0×10-20) encoding a myeloid IgA receptor FcαR, and INPP5D (OR=1.34, P=2.2×10-9) encoding a known inhibitor of FcαR signaling. The FCAR risk locus co-localized with a cis-eQTL increasing FCAR expression; the risk alleles disrupted a PRDM1 binding motif within a myeloid enhancer of FCAR. Another risk locus was associated with a higher genetically predicted levels of plasma IL6R. The IL6R risk haplotype carried a missense variant contributing to accelerated cleavage of IL6R into a soluble form. Using systems biology approaches, we prioritized IgAV master regulators co-modulated by FCAR, INPP5D and IL6R in myeloid cells. We additionally identified 21 shared loci in a cross-phenotype analysis of IgAV with IgA nephropathy, including novel loci PAID4, WLS, and ANKRD55.

8.
Nat Commun ; 14(1): 2947, 2023 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-37268690

RESUMO

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/ß-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.


Assuntos
Barreira Hematoencefálica , Receptores Frizzled , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Retina/metabolismo , Barreira Hematorretiniana/metabolismo , Via de Sinalização Wnt
9.
Cell Rep ; 33(10): 108474, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33296649

RESUMO

Bi-species, fusion-mediated, somatic cell reprogramming allows precise, organism-specific tracking of unknown lineage drivers. The fusion of Tcf7l1-/- murine embryonic stem cells with EBV-transformed human B cell lymphocytes, leads to the generation of bi-species heterokaryons. Human mRNA transcript profiling at multiple time points permits the tracking of the reprogramming of B cell nuclei to a multipotent state. Interrogation of a human B cell regulatory network with gene expression signatures identifies 8 candidate master regulator proteins. Of these 8 candidates, ectopic expression of BAZ2B, from the bromodomain family, efficiently reprograms hematopoietic committed progenitors into a multipotent state and significantly enhances their long-term clonogenicity, stemness, and engraftment in immunocompromised mice. Unbiased systems biology approaches let us identify the early driving events of human B cell reprogramming.


Assuntos
Reprogramação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Reprogramação Celular/fisiologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Feminino , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Multipotentes/metabolismo , Fatores de Transcrição/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/fisiologia
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