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1.
Development ; 143(3): 427-36, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26721501

RESUMO

In the intestine, finger-like villi provide abundant surface area for nutrient absorption. During murine villus development, epithelial Hedgehog (Hh) signals promote aggregation of subepithelial mesenchymal clusters that drive villus emergence. Clusters arise first dorsally and proximally and spread over the entire intestine within 24 h, but the mechanism driving this pattern in the murine intestine is unknown. In chick, the driver of cluster pattern is tensile force from developing smooth muscle, which generates deep longitudinal epithelial folds that locally concentrate the Hh signal, promoting localized expression of cluster genes. By contrast, we show that in mouse, muscle-induced epithelial folding does not occur and artificial deformation of the epithelium does not determine the pattern of clusters or villi. In intestinal explants, modulation of Bmp signaling alters the spatial distribution of clusters and changes the pattern of emerging villi. Increasing Bmp signaling abolishes cluster formation, whereas inhibiting Bmp signaling leads to merged clusters. These dynamic changes in cluster pattern are faithfully simulated by a mathematical model of a Turing field in which an inhibitor of Bmp signaling acts as the Turing activator. In vivo, genetic interruption of Bmp signal reception in either epithelium or mesenchyme reveals that Bmp signaling in Hh-responsive mesenchymal cells controls cluster pattern. Thus, unlike in chick, the murine villus patterning system is independent of muscle-induced epithelial deformation. Rather, a complex cocktail of Bmps and Bmp signal modulators secreted from mesenchymal clusters determines the pattern of villi in a manner that mimics the spread of a self-organizing Turing field.


Assuntos
Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Intestinos/embriologia , Microvilosidades/metabolismo , Transdução de Sinais , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Epitélio/embriologia , Proteínas Hedgehog/metabolismo , Hibridização In Situ , Ligantes , Mesoderma/embriologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Músculo Liso/embriologia , Tamanho do Órgão , Resistência à Tração
2.
Proc Natl Acad Sci U S A ; 109(39): 15817-22, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-23019366

RESUMO

In the adult intestine, an organized array of finger-like projections, called villi, provide an enormous epithelial surface area for absorptive function. Villi first emerge at embryonic day (E) 14.5 from a previously flat luminal surface. Here, we analyze the cell biology of villus formation and examine the role of paracrine epithelial Hedgehog (Hh) signals in this process. We find that, before villus emergence, tight clusters of Hh-responsive mesenchymal cells form just beneath the epithelium. Cluster formation is dynamic; clusters first form dorsally and anteriorly and spread circumferentially and posteriorly. Statistical analysis of cluster distribution reveals a patterned array; with time, new clusters form in spaces between existing clusters, promoting approximately four rounds of villus emergence by E18.5. Cells within mesenchymal clusters express Patched1 and Gli1, as well as Pdgfrα, a receptor previously shown to participate in villus development. BrdU-labeling experiments show that clusters form by migration and aggregation of Hh-responsive cells. Inhibition of Hh signaling prevents cluster formation and villus development, but does not prevent emergence of villi in areas where clusters have already formed. Conversely, increasing Hh signaling increases the size of villus clusters and results in exceptionally wide villi. We conclude that Hh signals dictate the initial aspects of the formation of each villus by controlling mesenchymal cluster aggregation and regulating cluster size.


Assuntos
Proteínas Hedgehog/metabolismo , Mucosa Intestinal/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas Hedgehog/genética , Humanos , Mucosa Intestinal/citologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Receptores Patched , Receptor Patched-1 , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteína GLI1 em Dedos de Zinco
3.
Ann Otol Rhinol Laryngol ; 123(9): 662-72, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24816422

RESUMO

OBJECTIVE: A new head and neck cancer cell line was developed from a highly aggressive HNSCC of the oral cavity diagnosed in a 26-year-old pregnant woman. METHODS: Cells from the primary tumor were passaged in culture and genotyped as a unique cell line. The resultant cell line was assessed for its ability to replicate the primary tumor. RESULTS: The primary tumor and cell line contained 19.03% and 19.62% CD44(high) cells, respectively. CD44(high) cancer stem cells from UM-SCC-103 formed tumors after flank injections in mice that reconstituted the heterogeneity of the primary tumor. CD44 staining and histology in the primary tumor and tumors grown in vivo from the cell line were similar. CD44(high) cells from the primary tumor resulted in lung colony formation in 2 out of 2 tail vein injections in mice, whereas CD44(low) cells did not. Similarly, CD44(high) cells from UM-SCC-103 formed lung tumors in 2 out of 4 mice, whereas CD44(low) cells did not. CONCLUSION: The similarity in marker expression and tumorigenic behavior between the primary tumor and the resulting cell line strongly suggests that the cell line resembles the primary tumor that it was derived from and provides an important new research tool for the study of head and neck carcinomas in young patients.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Neoplasias da Língua/genética , Adulto , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral/metabolismo , Feminino , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Gravidez , Neoplasias da Língua/metabolismo , Neoplasias da Língua/patologia
4.
bioRxiv ; 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36778265

RESUMO

Background & Aims: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or conditions like Inflammatory Bowel Disease (IBD) where immune cell infiltration produces 'inflammatory hypoxia', a chronic condition that starves the mucosa of oxygen. Epithelial regeneration after ischemia and IBD suggests intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of acute and chronic hypoxia on human ISC (hISC) properties have not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs isolated from healthy human tissues. We then test the hypothesis that some inflammation-associated interleukins protect hISCs during prolonged hypoxia. Methods: hISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72hrs. Viability, HIF1α response, transcriptomics, cell cycle dynamics, and hISC response to cytokines were evaluated. Results: The novel MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs remain viable until 72hrs and exhibit peak HIF1α at 24hrs. hISCs lose stem cell activity at 24hrs that recovers at 48hrs of hypoxia. Hypoxia increases the proportion of hISCs in G1 and regulates hISC capacity to respond to multiple inflammatory signals. Hypoxia induces hISCs to upregulate many interleukin receptors and hISCs demonstrate hypoxia-dependent cell cycle regulation and increased organoid forming efficiency when treated with specific interleukins. Conclusions: Hypoxia primes hISCs to respond differently to interleukins than hISCs in normoxia through a transcriptional response. hISCs slow cell cycle progression and increase hISC activity when treated with hypoxia and specific interleukins. These findings have important implications for epithelial regeneration in the gut during inflammatory events.

5.
Cell Mol Gastroenterol Hepatol ; 16(5): 823-846, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37562653

RESUMO

BACKGROUND AND AIMS: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). METHODS: hISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses. RESULTS: The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1ß, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. CONCLUSIONS: Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.


Assuntos
Inflamação , Interleucinas , Humanos , Interleucinas/metabolismo , Inflamação/metabolismo , Células-Tronco/metabolismo , Hipóxia , Oxigênio/metabolismo
6.
Cell Mol Gastroenterol Hepatol ; 14(2): 409-434, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35489715

RESUMO

BACKGROUND & AIMS: Fatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin. METHODS: Single-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)-labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography. RESULTS: Single-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes. CONCLUSIONS: Primary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.


Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Células CACO-2 , Diabetes Mellitus Tipo 2/metabolismo , Enterócitos/metabolismo , Ácidos Graxos/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Metformina/farmacologia , RNA
7.
Dev Biol ; 318(1): 38-51, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18439994

RESUMO

As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.


Assuntos
Caenorhabditis elegans/fisiologia , Temperatura Alta , Hipóxia , Oócitos/fisiologia , Ovulação/fisiologia , Estresse Oxidativo , Ribonucleoproteínas/metabolismo , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Grânulos Citoplasmáticos/metabolismo , Feminino , Fertilização/fisiologia , Masculino , Oócitos/citologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/genética , Espermatozoides/citologia , Espermatozoides/metabolismo
8.
Head Neck ; 35(11): 1542-50, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23322448

RESUMO

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) have devastating morbidity rates with mortality mainly because of metastasis. METHODS: Multiplex enzyme-linked immunosorbent assay (ELISA) to assay a variety of cytokine levels secreted by a panel of stage-specific and anatomic site-specific primary, and recurrent and metastatic University of Michigan-HNSCC cell lines over a 72-hour time course. RESULTS: Conditioned medium from metastatic or recurrent HNSCC showed significantly higher amounts of interleukin (IL)-6, IL-6 receptor, tumor growth factor-beta (TGF-ß) and vascular endothelial growth factor (VEGF) than nonmetastatic cells or normal oral keratinocytes. Tumor necrosis factor (TNF) was only secreted by stage IV, metastatic, or recurrence-derived cell lines. CONCLUSION: The cytokine profile of cultured HNSCC cells suggests that high levels of IL-6 and IL-6R, TGF-ß, and VEGF are significantly related with their metastatogenic potential and provide rationale for determining if serum testing for a combination of these 4 soluble factors could be of predictive value for the HNSCC tumor progression and clinical outcome.


Assuntos
Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/secundário , Citocinas/sangue , Neoplasias de Cabeça e Pescoço/sangue , Ensaio de Imunoadsorção Enzimática , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/secundário , Humanos , Técnicas In Vitro , Interleucina-6/sangue , Valor Preditivo dos Testes , Prognóstico , Valores de Referência , Carcinoma de Células Escamosas de Cabeça e Pescoço , Estatísticas não Paramétricas , Fator de Crescimento Transformador beta1/sangue , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismo , Fator A de Crescimento do Endotélio Vascular/sangue
9.
Otolaryngol Head Neck Surg ; 149(1): 97-104, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23558285

RESUMO

OBJECTIVE: To evaluate in vitro the potential links between sialyl Lewis X (sLeX) and cancer stem cells (CSC) in head and neck squamous cell carcinoma (HNSCC). HNSCC is an aggressive malignancy with high mortality mainly due to metastasis. CSC have emerged as important players in HNSCC metastasis. sLeX is a tetrasaccharide carbohydrate known to play a key role in metastatic dissemination by promoting binding of the tumor cells to the endothelium. STUDY DESIGN: Experimental, in vitro. SETTING: Laboratory of Head and Neck Cancer Metastasis, University of Michigan. SUBJECTS AND METHODS: A panel of stage- and anatomic-site specific primary and metastatic HNSCC cell lines was assessed by flow cytometry to quantify sLeX relative expression levels. Serum-free conditioned media from the same HNSCC lines was collected over a time course of 72 hours and assessed by Western blot for secreted sLeX expression. Representative HNSCC cell lines were cultured as floating orospheres (condition that enhance CSC growth) or under normal adherent conditions and characterized by flow cytometry for CSC markers (CD44, aldehyde dehydrogenase [ALDH]) comparatively with sLeX expression. RESULTS: sLeX is predominantly expressed in carcinomas originating from the oral cavity. Secreted sLeX is also found to be high in oral carcinomas and increased over the analyzed time course. Floating orospheres were strongly positive for CD44 and ALDH, confirming CSC enrichment of the orospheres. Tumor cells grown as orospheres are 95% to 100% positive for sLeX compared to 10% to 40% of adherent counterpart. CONCLUSION: These studies provide the first evidence of sLeX relationship with CSC in HNSCC.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Células-Tronco Neoplásicas/metabolismo , Oligossacarídeos/metabolismo , Aldeído Desidrogenase/fisiologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Receptores de Hialuronatos/fisiologia , Células-Tronco Neoplásicas/patologia , Antígeno Sialil Lewis X
10.
Head Neck ; 35(3): 335-42, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22422571

RESUMO

BACKGROUND: The aim of this study was to investigate oral cancer in pregnant women, a rare but therapeutically challenging patient subset. METHODS: After institutional review board approval, an EMERSE search was used to identify all women treated at the University of Michigan from 1998 to 2010 with head and neck squamous cell carcinoma (HNSCC) during pregnancy. This identified 4 patients with tongue cancer. Biomarkers and human papillomavirus (HPV) were assessed by immunohistochemistry and multiplex PCR/mass spectrometry, respectively. RESULTS: Two patients responded well to therapy and are alive more than 10 years after diagnosis; 2 patients died of disease. All tumors overexpressed EGFR and Bcl-xL, 3 of 4 overexpressed c-Met, both tumors that progressed overexpressed p53. All tumors were negative for HPV, p16, estrogen receptor, progesterone receptor, and HER-2. CONCLUSIONS: Biomarkers of aggressive tumors (high EGFR, c-Met; high Bcl-xL-low p53) did not correlate with outcome. Additional studies are needed to determine whether perineural invasion, delay in diagnosis, and p53 overexpression are factors in poor survival.


Assuntos
Carcinoma de Células Escamosas/diagnóstico , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias Bucais/diagnóstico , Complicações Neoplásicas na Gravidez/diagnóstico , Adulto , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/virologia , Feminino , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Neoplasias Bucais/mortalidade , Neoplasias Bucais/virologia , Reação em Cadeia da Polimerase Multiplex , Papillomaviridae/isolamento & purificação , Gravidez , Complicações Neoplásicas na Gravidez/mortalidade , Complicações Neoplásicas na Gravidez/virologia , Fatores de Risco , Carcinoma de Células Escamosas de Cabeça e Pescoço
11.
Head Neck ; 34(10): 1480-91, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22162267

RESUMO

BACKGROUND: Few human papillomavirus (HPV)(+) head and neck squamous cell carcinoma (HNSCC) cell lines exist. We established University of Michigan-squamous cell carcinoma-104 (UM-SCC-104), a new HPV(+) HNSCC cell line from a recurrent oral cavity tumor, and characterized it for the presence of cancer stem cells (CSCs). METHODS: Tumor cells were tested for biomarker expression by immunohistology, and the presence of HPV was assessed by several methods. RESULTS: UM-SCC-104 has a unique genotype, contains HPV-16, and expresses E6/E7. Inoculation of aldehyde dehydrogenase (ALDH)(+) and ALDH(-) cells in an immunocompromised mouse resulted in tumor growth from the ALDH(+) cells after 6 weeks that recapitulated the histology of the primary, whereas ALDH(-) cells did not produce tumors. CONCLUSION: UM-SCC-104, a new HPV-16, CSC-containing HNSCC cell line will aid in studying recurrent HPV(+) tumors. The aggressive nature of this tumor is consistent with high uniform expression of epidermal growth factor receptor (EGFR) and a functionally significant proportion of ALDH(+) CSCs.


Assuntos
Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Papillomavirus Humano 16/genética , Células-Tronco Neoplásicas/patologia , Infecções por Papillomavirus/genética , Animais , Biópsia por Agulha , Carcinoma de Células Escamosas/patologia , Genes p53/genética , Genótipo , Neoplasias de Cabeça e Pescoço/patologia , Papillomavirus Humano 16/isolamento & purificação , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Células-Tronco Neoplásicas/metabolismo , Infecções por Papillomavirus/patologia , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Carcinoma de Células Escamosas de Cabeça e Pescoço , Transplante Heterólogo
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