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1.
PLoS One ; 15(8): e0236839, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780746

RESUMO

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 µM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteína Adaptadora GRB2/antagonistas & inibidores , Quinoxalinas/farmacologia , Animais , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/metabolismo , Humanos , Células K562 , Rim/citologia , Cinética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ligação Proteica , Quinoxalinas/química , Quinoxalinas/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Ressonância de Plasmônio de Superfície , Peixe-Zebra , Domínios de Homologia de src
2.
PLoS One ; 15(8): e0238466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857809

RESUMO

PURPOSE: Phyllodes tumors (PTs) are biphasic tumors accounting for 0.3-1.5% of all breast tumors. Epithelial membrane proteins (EMPs) have been reported in various malignant tumors but their expression in PTs is unclear. In this study, we aimed to evaluate the expression of EMP1, EMP2, and EMP3 in breast phyllodes tumors (PTs), and to investigate their clinical implications. METHODS: In total, 185 PTs were used for constructing a tissue microarray. Immunohistochemical staining for EMP1, EMP2, and EMP3 was performed, and the results were analyzed along with the clinicopathologic parameters. RESULTS: In total, 185 PTs were included in this study, and comprised 138 benign, 32 borderline, and 15 malignant PTs. In malignant PTs, the epithelial component showed decreased expression of EMP1 (P = 0.027), EMP2 (P = 0.004), and EMP3 (P = 0.032), compared to the benign and borderline PTs. Conversely, stromal component of borderline and malignant PTs showed higher expression of EMP1 (P = 0.027), EMP2 (P = 0.004), and EMP3 (P = 0.032) compared to benign PTs. Expression of EMP1 and EMP3 correlated positively with stromal cellularity and cellular atypia (P < 0.001). In the univariate analysis, stromal EMP3 was associated with shorter disease-free survival (P < 0.001), and shorter overall survival (P = 0.034). CONCLUSION: The expression of EMP1, EMP2, and EMP3 is decreased in the epithelial component and is increased in the stromal component of PT with higher histologic grade. Thus, stromal EMP3 expression may serve as an independent prognostic factor in PT.


Assuntos
Neoplasias da Mama/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Tumor Filoide/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Gradação de Tumores , Tumor Filoide/patologia , Prognóstico , Células Estromais/metabolismo , Células Estromais/patologia , Análise de Sobrevida , Análise Serial de Tecidos
3.
Gene ; 761: 145028, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32763490

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies and inflicts high mortality worldwide. The effect of tumor microenvironment components on HCC oncogenesis remains unknown. In particular, the nonleukocyte portion of the stromal fraction (SF) is poorly understood. METHODS: We comprehensively evaluated the proportional cell counts and gene expression data from The Cancer Genome Atlas (TCGA) to examine the contributions of cell components to the tumor microenvironment. Single-cell sequencing data from the Gene Expression Omnibus (GEO) were also analyzed to verify the association between the nonleukocyte SF and genes. We classified HCC using a hierarchical clustering method based on diversity of nonleukocyte SF-related gene expression among different components, and we used an appropriate GEO dataset to verify the clusters with a support vector machine (SVM) model. The prognosis of subtypes and their relationship with tumor microenvironmental cell proportions, clinicopathogenesis factors, and other indicators were evaluated. RESULTS: Based on linear regression, 711 genes related to nonleukocyte SF were selected from the TCGA dataset. We classified HCC into three subtypes using genes related to the nonleukocyte SF. Additionally, the GEO single-cell sequencing data confirmed the relationship between genes and the nonleukocyte SF. The tumor microenvironment of Type 2 contained the most significant mutually reinforcing interaction between the nonleukocyte SF and tumor cells. Meanwhile, Type 2 patients had the poorest prognosis and the most severe tumor-node-metastasis (TNM) stages, histological grades, etc. The analysis based on the GEO dataset verified the classification results with an SVM model. Type 2 was associated with worse clinicopathological characteristics, including tumor grading and staging, than the other types. In addition, the pathway analysis revealed that signals related to the SF and cell proliferation were significantly enhanced in Type 2 compared to the other group, which consisted of Types 1 and 3. CONCLUSION: The nonleukocyte SF in the tumor microenvironment contributed greatly to HCC oncogenesis. We can use these HCC classification criteria to stratify patients into subtypes for personalized treatment.


Assuntos
Carcinoma Hepatocelular/genética , Células Estromais/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genômica , Humanos , Neoplasias Hepáticas/genética , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Microambiente Tumoral/genética
4.
PLoS One ; 15(7): e0234086, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658928

RESUMO

Based on microRNA (miR) microarray analysis, we previously found that miR22-5p expression is decreased in the mid-luteal endometrium of women with minimal/mild endometriosis. Bioinformatics analysis predicted that miR22-5p targets ten-eleven translocation (TET2) 3'-untranslated region. This study aimed to determine the regulation and roles of miR22-5p in the pathogenesis of minimal/mild endometriosis-associated infertility. MiR22-5p and TET2 expression in the mid-luteal endometrium from women with or without minimal/mild endometriosis was analyzed. After transfection with miR22-5p mimics or inhibitor, TET2 expression was analyzed by quantitative reverse transcription (RT-q) PCR, western blotting and immunohistochemistry. 5-Hydroxymethylcytosine was determined by immunofluorescence and dot blotting. Expression and promoter methylation of estrogen receptor 2 (ESR2) was measured by RT-qPCR and western blotting, and by bisulfite sequencing, respectively. We first established that miR22-5p expression decreased and TET2 expression increased in minimal/mild endometriosis during implantation window. TET2 was found to be a direct target of miR22-5p. MiR22-5p regulated the expression of ESR2, but did not directly affect methylation of its promoter region (-197/+359). Our results suggest that an imbalance in miR22-5p expression in the mid-luteal endometrium may be involved in minimal/mild endometriosis-associated infertility.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Implantação do Embrião , Endometriose/complicações , Endométrio/metabolismo , Receptor beta de Estrogênio/biossíntese , Infertilidade Feminina/etiologia , Fase Luteal/fisiologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas/biossíntese , Regiões 3' não Traduzidas/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/análise , Adulto , Células Cultivadas , Metilação de DNA , Proteínas de Ligação a DNA/genética , Receptor beta de Estrogênio/genética , Feminino , Regulação da Expressão Gênica , Genes Reporter , Humanos , Infertilidade Feminina/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , Regiões Promotoras Genéticas/genética , Transporte Proteico/genética , Proteínas Proto-Oncogênicas/genética , Células Estromais/metabolismo , Adulto Jovem
5.
Nat Commun ; 11(1): 3677, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699279

RESUMO

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.


Assuntos
Linfócitos B/imunologia , Microambiente Celular/imunologia , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Linhagem Celular , Quimiocina CXCL13/imunologia , Simulação por Computador , Células Dendríticas Foliculares/citologia , Células Dendríticas Foliculares/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Modelos Biológicos , Tonsila Palatina/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo
6.
Prostate ; 80(13): 1087-1096, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32609927

RESUMO

BACKGROUND: Prostate cancer is the second most common cancer worldwide. Tumor microenvironment is composed of activated fibroblasts, the so called carcinoma-associated fibroblasts (CAFs). They express high levels of α-smooth muscle actin (α-SMA) and type I collagen (COL1), and support proliferation and migration of tumor epithelial cells. Extracorporeal shock waves (ESWs), acoustic waves, are effective in the treatment of hypertrophic scars, due to their ability to modulate fibrosis. Based on this rationale, the study evaluated the effects of ESWs on CAF activation and the influence of ESW-treated CAFs on the growth and migration of epithelial prostatic carcinoma cells. METHODS: Primary cultures of CAFs (n = 10) were prepared from tumors of patients undergoing surgery for high-risk prostate carcinoma. CAFs were treated with ESWs (energy levels: 0.32 mJ/mm2 , 1000 pulses; 0.59 mJ/mm2 , 250 pulses). After treatment, the messenger RNA and protein levels of the stromal activation markers α-SMA and COL1 were determined. Subsequently, two different stabilized cell lines (PC3 and DU145) of androgen-resistant prostate cancer were treated with the conditioned media produced by ESW-treated CAFs. At different times, viability and migration of PC3 and DU145 cells were evaluated. Viability was also assessed by coculture system using CAFs and PC3 or DU145 cells. RESULTS: ESWs reduced gene expression and protein level of α-SMA and COL1 in CAFs. The treatment of PC3 and DU145 with conditioned media of ESW-treated CAFs determined a reduction of their growth and invasive potential. Coculture systems between ESW-treated CAFs and PC3 or DU145 cells confirmed the epithelial cell number reduction. CONCLUSIONS: This in vitro study demonstrates for the first time that ESWs are able to modulate the activation of prostate CAFs in favor of a less "reactive" stroma, with consequent slowing of the growth and migration of prostate cancer epithelial cells. However, only further studies to be performed in vivo will confirm the possibility of using this new therapy in patients with prostate cancer.


Assuntos
Tratamento por Ondas de Choque Extracorpóreas/métodos , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/terapia , Células Estromais/patologia , Actinas/genética , Actinas/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Progressão da Doença , Humanos , Masculino , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/metabolismo
7.
Adv Exp Med Biol ; 1263: 1-11, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32588319

RESUMO

From a general perspective, in the context of solid tumors, we can distinguish metabolic alterations of cancer cells from those of the stroma. These two components interact with each other and with the extracellular matrix (ECM), and these interactions can take the form of either metabolic competition or metabolic symbiosis. The aim of this chapter is to overview the canonical metabolic alterations of tumor and stroma cells and to present specific examples of metabolic competition and symbiosis. We will also discuss the complexity and plasticity of metabolism, which pose indeed a serious threat to our ability to target selective metabolic features of tumor microenvironment with drugs. Finally, we will highlight some limitations of state-of-the-art techniques used to study tumor metabolism and propose some innovative solutions to investigate the clinical relevance of metabolic alterations for patient management and treatment.


Assuntos
Neoplasias/metabolismo , Microambiente Tumoral , Matriz Extracelular/metabolismo , Humanos , Neoplasias/patologia , Células Estromais/metabolismo
8.
Adv Exp Med Biol ; 1263: 175-202, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32588328

RESUMO

The tumor microenvironment (TME) is an evolutionally low-level and embryonically featured tissue comprising heterogenic populations of malignant and stromal cells as well as noncellular components. Under radiotherapy (RT), the major modality for the treatment of malignant diseases [1], TME shows an adaptive response in multiple aspects that affect the efficacy of RT. With the potential clinical benefits, interests in RT combined with immunotherapy (IT) are intensified with a large scale of clinical trials underway for an array of cancer types. A better understanding of the multiple molecular aspects, especially the cross talks of RT-mediated energy reprogramming and immunoregulation in the irradiated TME (ITME), will be necessary for further enhancing the benefit of RT-IT modality. Coming studies should further reveal more mechanistic insights of radiation-induced instant or permanent consequence in tumor and stromal cells. Results from these studies will help to identify critical molecular pathways including cancer stem cell repopulation, metabolic rewiring, and specific communication between radioresistant cancer cells and the infiltrated immune active lymphocytes. In this chapter, we will focus on the following aspects: radiation-repopulated cancer stem cells (CSCs), hypoxia and re-oxygenation, reprogramming metabolism, and radiation-induced immune regulation, in which we summarize the current literature to illustrate an integrated image of the ITME. We hope that the contents in this chapter will be informative for physicians and translational researchers in cancer radiotherapy or immunotherapy.


Assuntos
Neoplasias/radioterapia , Microambiente Tumoral , Humanos , Imunoterapia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Estromais/metabolismo
9.
Nat Commun ; 11(1): 2768, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488016

RESUMO

Fibrotic disorders are some of the most devastating and poorly treated conditions in developed nations, yet effective therapeutics are not identified for many of them. A major barrier for the identification of targets and successful clinical translation is a limited understanding of the human fibrotic microenvironment. Here, we construct a stromal cell atlas of human fibrosis at single cell resolution from patients with Dupuytren's disease, a localized fibrotic condition of the hand. A molecular taxonomy of the fibrotic milieu characterises functionally distinct stromal cell types and states, including a subset of immune regulatory ICAM1+ fibroblasts. In developing fibrosis, myofibroblasts exist along an activation continuum of phenotypically distinct populations. We also show that the tetraspanin CD82 regulates cell cycle progression and can be used as a cell surface marker of myofibroblasts. These findings have important implications for targeting core pathogenic drivers of human fibrosis.


Assuntos
Contratura de Dupuytren/imunologia , Contratura de Dupuytren/metabolismo , Fibrose/imunologia , Fibrose/metabolismo , Células Estromais/metabolismo , Actinas/metabolismo , Biomarcadores/metabolismo , Quimiocinas CXC/metabolismo , Contratura de Dupuytren/patologia , Fibrose/patologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Medicina Molecular , Miofibroblastos/metabolismo , Tetraspaninas/metabolismo , Microambiente Tumoral/fisiologia
10.
Proc Natl Acad Sci U S A ; 117(27): 15874-15883, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571916

RESUMO

After acute kidney injury (AKI), patients either recover or alternatively develop fibrosis and chronic kidney disease. Interactions between injured epithelia, stroma, and inflammatory cells determine whether kidneys repair or undergo fibrosis, but the molecular events that drive these processes are poorly understood. Here, we use single nucleus RNA sequencing of a mouse model of AKI to characterize cell states during repair from acute injury. We identify a distinct proinflammatory and profibrotic proximal tubule cell state that fails to repair. Deconvolution of bulk RNA-seq datasets indicates that this failed-repair proximal tubule cell (FR-PTC) state can be detected in other models of kidney injury, increasing during aging in rat kidney and over time in human kidney allografts. We also describe dynamic intercellular communication networks and discern transcriptional pathways driving successful vs. failed repair. Our study provides a detailed description of cellular responses after injury and suggests that the FR-PTC state may represent a therapeutic target to improve repair.


Assuntos
Lesão Renal Aguda/metabolismo , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Transcriptoma , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Aloenxertos , Animais , Modelos Animais de Doenças , Fibrose , Redes Reguladoras de Genes , Humanos , Rim/lesões , Túbulos Renais Proximais/lesões , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Análise de Sequência de RNA , Células Estromais/metabolismo , Células Estromais/patologia
11.
Nat Cell Biol ; 22(7): 758-766, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483388

RESUMO

Cancer-associated fibroblasts (CAFs) perform diverse roles and can modulate therapy responses1. The inflammatory environment within tumours also influences responses to many therapies, including the efficacy of oncolytic viruses2; however, the role of CAFs in this context remains unclear. Furthermore, little is known about the cell signalling triggered by heterotypic cancer cell-fibroblast contacts and about what activates fibroblasts to express inflammatory mediators1,3. Here, we show that direct contact between cancer cells and CAFs triggers the expression of a wide range of inflammatory modulators by fibroblasts. This is initiated following transcytosis of cytoplasm from cancer cells into fibroblasts, leading to the activation of STING and IRF3-mediated expression of interferon-ß1 and other cytokines. Interferon-ß1 then drives interferon-stimulated transcriptional programs in both cancer cells and stromal fibroblasts and ultimately undermines the efficacy of oncolytic viruses, both in vitro and in vivo. Further, targeting IRF3 solely in stromal fibroblasts restores oncolytic herpes simplex virus function.


Assuntos
Fibroblastos Associados a Câncer/imunologia , Instabilidade Genômica , Fator Regulador 3 de Interferon/metabolismo , Proteínas de Membrana/metabolismo , Terapia Viral Oncolítica , Neoplasias Cutâneas/imunologia , Células Estromais/imunologia , Adulto , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Células Cultivadas , Citocinas , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Vírus Oncolíticos/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Células Estromais/metabolismo , Células Estromais/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
PLoS One ; 15(5): e0230354, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413029

RESUMO

Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in αSMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1's role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.


Assuntos
Neoplasias Ósseas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Neoplasias da Próstata/metabolismo , Sulfotransferases/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte/química , Via de Sinalização Wnt , Neoplasias Ósseas/secundário , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Hidrogéis/química , Macrófagos/metabolismo , Masculino , Neoplasias da Próstata/patologia , Células Estromais/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(21): 11387-11398, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385149

RESUMO

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.


Assuntos
Colágeno/ultraestrutura , Miofibroblastos/citologia , Células Estromais/citologia , Tecido Adiposo/citologia , Fenômenos Biomecânicos , Diferenciação Celular , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/ultraestrutura , Fibronectinas/metabolismo , Humanos , Mecanotransdução Celular , Miofibroblastos/metabolismo , Miofibroblastos/ultraestrutura , Células Estromais/metabolismo , Células Estromais/ultraestrutura
14.
Nat Commun ; 11(1): 2285, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385277

RESUMO

Advanced metastatic cancer poses utmost clinical challenges and may present molecular and cellular features distinct from an early-stage cancer. Herein, we present single-cell transcriptome profiling of metastatic lung adenocarcinoma, the most prevalent histological lung cancer type diagnosed at stage IV in over 40% of all cases. From 208,506 cells populating the normal tissues or early to metastatic stage cancer in 44 patients, we identify a cancer cell subtype deviating from the normal differentiation trajectory and dominating the metastatic stage. In all stages, the stromal and immune cell dynamics reveal ontological and functional changes that create a pro-tumoral and immunosuppressive microenvironment. Normal resident myeloid cell populations are gradually replaced with monocyte-derived macrophages and dendritic cells, along with T-cell exhaustion. This extensive single-cell analysis enhances our understanding of molecular and cellular dynamics in metastatic lung cancer and reveals potential diagnostic and therapeutic targets in cancer-microenvironment interactions.


Assuntos
Adenocarcinoma de Pulmão/genética , Reprogramação Celular/genética , Neoplasias Pulmonares/genética , Análise de Sequência de RNA , Análise de Célula Única , Imunidade Adaptativa , Adenocarcinoma de Pulmão/irrigação sanguínea , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Linhagem da Célula , Progressão da Doença , Células Endoteliais/patologia , Humanos , Ligantes , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Células Mieloides/patologia , Miofibroblastos/patologia , Metástase Neoplásica , Estadiamento de Neoplasias , Neovascularização Patológica/patologia , Fenótipo , Receptores de Superfície Celular/metabolismo , Células Estromais/metabolismo , Análise de Sobrevida
15.
Prostate ; 80(10): 731-741, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32356572

RESUMO

BACKGROUND: Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. METHODS: Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1ß and TGF-ß1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN. RESULTS: OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-ß1 stimulated OPN secretion by NHPrE-1 cells and both IL-1ß and TGF-ß1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines. CONCLUSIONS: OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.


Assuntos
Osteopontina/biossíntese , Hiperplasia Prostática/metabolismo , Quimiocinas CXC/biossíntese , Quimiocinas CXC/genética , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Humanos , Imuno-Histoquímica , Interleucina-6/biossíntese , Interleucina-6/genética , Masculino , Osteopontina/genética , Hiperplasia Prostática/genética , Hiperplasia Prostática/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Estromais/metabolismo , Células Estromais/patologia
16.
Nat Commun ; 11(1): 2054, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345968

RESUMO

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.


Assuntos
Células Dendríticas/citologia , Nicho de Células-Tronco , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CXCL12/farmacologia , Análise por Conglomerados , Colágeno/farmacologia , Células Dendríticas/efeitos dos fármacos , Combinação de Medicamentos , Humanos , Laminina/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteoglicanas/farmacologia , Nicho de Células-Tronco/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo
17.
Nat Commun ; 11(1): 1936, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321913

RESUMO

The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis.


Assuntos
Enterócitos/metabolismo , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Enterócitos/citologia , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas-G/genética , Células Estromais/metabolismo , Proteína Wnt-5a/metabolismo
18.
Cancer Immunol Immunother ; 69(8): 1549-1564, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32303794

RESUMO

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) and their subsets contribute to breast cancer prognosis. We investigated the prognostic impact of CD3+, CD8+ and FOXP3+ TILs in patients with early intermediate/high-risk breast cancer treated with adjuvant anthracycline-based chemotherapy within two randomized trials conducted by our Group. METHODS: We examined 1011 patients (median follow-up 130.9 months) and their tumors for total, stromal (s) and intratumoral (i) CD3, CD8 and FOXP3 lymphocyte density (counts/mm2) on tissue-microarray cores by immunohistochemistry. Morphological sTIL density on whole H&E-stained sections was also evaluated. RESULTS: The majority of TILs were CD3+. Total CD3 and CD8, sCD3 and sCD8, iCD3 and iCD8, sFOXP3 and iFOXP3 were strongly correlated (Spearman's rho values > 0.6). High individual lymphocytic subsets and sTIL density were strongly associated with high tumor grade, higher proliferation and HER2-positive and triple-negative tumors (all p values < 0.001). Higher sTIL density (10% increments), high density of almost each individual marker and all-high profiles conferred favorable prognosis. However, when adjusted for sTIL density, stromal and intratumoral lymphocytic subsets lost their prognostic significance, while higher sTIL density conferred up to 15% lower risk for relapse. Independently of sTIL density, higher total CD3+ and CD8+ TILs conferred 35% and 28% lower risk for relapse, respectively. CONCLUSIONS: Stromal and intratumoral CD3+, CD8+ and FOXP3+ TIL density do not seem to add prognostic information over the morphologically assessed sTIL density, which is worth introducing in routine histology reports.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Complexo CD3/metabolismo , Antígenos CD8/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Células Estromais/patologia , Adulto , Idoso , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Quimioterapia Adjuvante , Feminino , Seguimentos , Humanos , Subpopulações de Linfócitos , Pessoa de Meia-Idade , Prognóstico , Radioterapia Adjuvante , Células Estromais/imunologia , Células Estromais/metabolismo , Adulto Jovem
19.
Cancer Immunol Immunother ; 69(8): 1577-1588, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32306077

RESUMO

HLA-DR, an MHC class II molecule that mediates antigen presentation, is a favourable prognostic indicator in colorectal cancer (CRC). However, the dynamics and location of HLA-DR expression during CRC development are unclear. We aimed to define HLA-DR expression by immunohistochemistry in colorectal epithelium and stromal tissue at different stages of cancer development, assessing non-neoplastic colorectal adenocarcinoma-adjacent tissue, adenomas and carcinoma tissues, and to associate HLA-DR levels with clinical outcomes. Patients with higher than median HLA-DR expression survived at least twice as long as patients with lower expression. This association was significant for HLA-DR staining in the colorectal carcinoma epithelium (n = 152, p = 0.011, HR 1.9, 95% CI 1.15-3.15) and adjacent non-neoplastic epithelium (n = 152, p < 0.001, HR 2.7, 95% CI 1.59-4.66), but not stroma. In stage II cases, however, the prognostic value of HLA-DR expression was significant only in adjacent non-neoplastic tissues, for both epithelium (n = 63, p = 0.015, HR 3.6, 95% CI 1.279-10.25) and stroma (n = 63, p = 0.018, HR 5.07, 95% CI 1.32-19.49). HLA-DR was lower in carcinoma tissue compared to matched adenomas (n = 35), in epithelium (p < 0.01) and stroma (p < 0.001). HLA-DR was further reduced in late-stage carcinoma (n = 101) compared to early stage (n = 105), in epithelium (p < 0.001) and stroma (p < 0.01). HLA-DR expression was lower (p < 0.05) in the adjacent non-neoplastic epithelium of patients with cancer recurrence. We demonstrate a progressive loss of HLA-DR in epithelial and stromal tissue compartments during CRC development and show prognostic ability in carcinoma-adjacent non-neoplastic tissues, highlighting the importance of this molecule in the anti-cancer immune response. These findings may have wider implications for immunotherapeutic interventions.


Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Antígenos HLA-DR/metabolismo , Recidiva Local de Neoplasia/patologia , Células Estromais/metabolismo , Adenocarcinoma/metabolismo , Adenoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
20.
J Vis Exp ; (157)2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32281982

RESUMO

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.


Assuntos
Leucócitos/patologia , Microambiente Tumoral/imunologia , Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Automação , Temperatura Alta , Humanos , Processamento de Imagem Assistida por Computador , Inclusão em Parafina , Coloração e Rotulagem , Células Estromais/metabolismo , Fixação de Tecidos
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