RESUMEN
BACKGROUND: Low success rates in oncology drug development are prompting re-evaluation of preclinical models, including orthotopic tumor engraftment. In breast cancer models, tumor cells are typically injected into mouse mammary fat pads (MFP). However, this approach bypasses the epithelial microenvironment, potentially altering tumor properties in ways that affect translational application. MATERIALS AND METHODS: Tumors were generated by mammary intraductal (MIND) engraftment of 4T1 carcinoma cells. Growth, histopathology, and molecular features were quantified. RESULTS: Despite growth similar to that of 4T1 MFP tumors, 4T1 MIND tumors exhibit distinct histopathology and increased metastasis. Furthermore, >6,000 transcripts were found to be uniquely up-regulated in 4T1 MIND tumor cells, including genes that drive several cancer hallmarks, in addition to two known therapeutic targets that were not up-regulated in 4T1 MFP tumor cells. CONCLUSION: Engraftment into the epithelial microenvironment generates tumors that more closely recapitulate the complexity of malignancy, suggesting that intraductal adaptation of orthotopic mammary models may be an important step towards improving outcomes in preclinical drug screening and development.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias Mamarias Animales/genética , Proteínas de Neoplasias/genética , Neovascularización Patológica/genética , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/patología , Ratones , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Microambiente Tumoral/genéticaRESUMEN
Left-right (L-R) differences in mammographic parenchymal patterns are an early predictor of breast cancer risk; however, the basis for this asymmetry is unknown. Here, we use retinoid X receptor alpha heterozygous null (RXRα+/-) mice to propose a developmental origin: perturbation of coordinated anterior-posterior (A-P) and L-R axial body patterning. We hypothesized that by analogy to somitogenesis-in which retinoic acid (RA) attenuation causes anterior somite pairs to develop L-R asynchronously-that RA pathway perturbation would likewise result in asymmetric mammary development. To test this, mammary glands of RXRα+/- mice were quantitatively assessed to compare left- versus right-side ductal epithelial networks. Unlike wild-type controls, half of the RXRα+/- thoracic mammary gland (TMG) pairs exhibited significant L-R asymmetry, with left-side reduction in network size. In RXRα+/- TMGs in which symmetry was maintained, networks had bilaterally increased size, with left networks showing greater variability in area and pattern. Reminiscent of posterior somites, whose bilateral symmetry is refractory to RA attenuation, inguinal mammary glands (IMGs) also had bilaterally increased network size, but no loss of symmetry. Together, these results demonstrate that mammary glands exhibit differential A-P sensitivity to RXRα heterozygosity, with ductal network symmetry markedly compromised in anterior but not posterior glands. As TMGs more closely model human breast development than IMGs, these findings raise the possibility that for some women, breast cancer risk may initiate with subtle axial patterning defects that result in L-R asymmetric growth and pattern of the mammary ductal epithelium.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.
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Tipificación del Cuerpo/genética , Glándulas Mamarias Animales/embriología , Organogénesis , Receptor alfa X Retinoide/genética , Animales , Femenino , Ratones , Receptor alfa X Retinoide/metabolismoRESUMEN
Left-right asymmetry is a phenomenon that has a broad appeal-to anatomists, developmental biologists and evolutionary biologists-because it is a morphological feature of organisms that spans scales of size and levels of organization, from unicellular protists, to vertebrate organs, to social behaviour. Here, we highlight a number of important aspects of asymmetry that encompass several areas of biology-cell-level, physiological, genetic, anatomical and evolutionary components-and that are based on research conducted in diverse model systems, ranging from single cells to invertebrates to human developmental disorders. Together, the contributions in this issue reveal a heretofore-unsuspected variety in asymmetry mechanisms, including ancient chirality elements that could underlie a much more universal basis to asymmetry development, and provide much fodder for thought with far reaching implications in biomedical, developmental, evolutionary and synthetic biology. The new emerging theme of binary cell-fate choice, promoted by asymmetric cell division of a deterministic cell, has focused on investigating asymmetry mechanisms functioning at the single cell level. These include cytoskeleton and DNA chain asymmetry-mechanisms that are amplified and coordinated with those employed for the determination of the anterior-posterior and dorsal-ventral axes of the embryo.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.
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Tipificación del Cuerpo , Desarrollo Embrionario , Eucariontes/crecimiento & desarrollo , Animales , Plantas/embriologíaRESUMEN
Fractal dimension has emerged as a clinically useful tool in the diagnosis and management of breast cancer. The aim of the present study was to determine if fractal dimension can be applied for the analysis of a pre-clinical breast cancer mouse model, MMTV-cNeu. Using fractal dimension in conjunction with conventional morphometric measurements, the ductal epithelial networks of pubertal-stage MMTV-cNeu mice were quantitatively compared with those of wild-type mice. Significant alterations in ductal epithelial network growth and organization were detected during early neoplasia in MMTV-cNeu mice. Moreover, the left-side networks were significantly more affected relative to their wild-type counterparts than were the right-side networks, a finding that is consistent with elevated left-side tumor incidence reported for breast cancer patients. Taken together these results demonstrate that combined fractal dimension and morphometric analysis is an objective and sensitive approach to quantitatively identify ductal epithelial aberrancies that precede overt mammary carcinoma formation.
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Epitelio/patología , Genes erbB-2/genética , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/patología , Virus del Tumor Mamario del Ratón/genética , Lesiones Precancerosas/patología , Animales , Femenino , Procesamiento de Imagen Asistido por Computador , RatonesRESUMEN
Most mammals develop their mammary glands in pairs of which the two counterparts are symmetrically displaced away from the ventral midline. Based on this symmetry and the same functional outcome as a milk-producing organ, the mammary glands are easily presumed to be mere copies of one another. Based on our analysis of published data with inclusion of new results related to mammary development and pathology in mice, we argue that this presumption is incorrect: Between and within pairs, mammary glands differ from one another, and tumor incidence and biology depend on the position along the anterior-posterior and the left-right axis as well. This insight has implications for experimental designs with mouse models and for data extrapolation between mammary glands within and between species. We suggest that improved documentation of location-specific mammary gland features will lead to more insights into the molecular mechanisms of mammary gland development and cancer biology in both mice and humans.
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Neoplasias de la Mama/patología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Humanas/crecimiento & desarrollo , Neoplasias Mamarias Experimentales/patología , Animales , Femenino , HumanosRESUMEN
Lithium is a commonly used drug for the treatment of bipolar disorder. At high doses, lithium becomes teratogenic, which is a property that has allowed this agent to serve as a useful tool for dissecting molecular pathways that regulate embryogenesis. This study was designed to examine the impact of lithium on heart formation in the developing frog for insights into the molecular regulation of cardiac specification. Embryos were exposed to lithium at the beginning of gastrulation, which produced severe malformations of the anterior end of the embryo. Although previous reports characterized this deformity as a posteriorized phenotype, histological analysis revealed that the defects were more comprehensive, with disfigurement and disorganization of all interior tissues along the anterior-posterior axis. Emerging tissues were poorly segregated and cavity formation was decreased within the embryo. Lithium exposure also completely ablated formation of the heart and prevented myocardial cell differentiation. Despite the complete absence of cardiac tissue in lithium treated embryos, exposure to lithium did not prevent myocardial differentiation of precardiac dorsal marginal zone explants. Moreover, precardiac tissue freed from the embryo subsequent to lithium treatment at gastrulation gave rise to cardiac tissue, as demonstrated by upregulation of cardiac gene expression, display of sarcomeric proteins, and formation of a contractile phenotype. Together these data indicate that lithium's effect on the developing heart was not due to direct regulation of cardiac differentiation, but an indirect consequence of disrupted tissue organization within the embryo.
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Embrión no Mamífero/efectos de los fármacos , Corazón/embriología , Litio/farmacología , Animales , Embrión no Mamífero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xenopus laevisRESUMEN
WNT signaling has been shown to influence the development of the heart. Although recent data suggested that canonical WNTs promote the emergence and expansion of cardiac progenitors in the pregastrula embryo, it has long been accepted that once gastrulation begins, canonical WNT signaling needs to be suppressed for cardiac development to proceed. Yet, this latter supposition appears to be odds with the expression of multiple canonical WNTs in the developing heart. The present study examining the effect of ectopic canonical WNT signaling on cardiogenesis in the developing frog was designed to test the hypothesis that heart formation is dependent on the inhibition of canonical WNT activity at the onset of gastrulation. Here we report that cardiac differentiation of explanted precardiac tissue from the dorsal marginal zone was not suppressed by exposure to WNT1 protein, although expression of Tbx5, Tbx20, and Nkx2.5 was selectively reduced. Pharmacological activation of WNT signaling in intact embryos using the GSK3 inhibitor SB415286 did not prevent the formation of an anatomically normal and functionally sound heart, with the only defect observed being lower levels of the cardiac transcription factor Nkx2.5. In both the explant and whole embryo studies, expression of muscle genes and proteins was unaffected by ectopic canonical WNT signaling. In contrast, canonical Wnt signaling upregulated expression of the cardiac stem cell marker c-kit and pluripotency genes Oct25 and Oct60. However, this regulatory stimulation of stem cells did not come at the expense of blocking cardiac progenitors from differentiating.
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Diferenciación Celular , Corazón/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Miocardio/citología , Transducción de Señal , Células Madre/fisiología , Vía de Señalización Wnt , Xenopus laevis/crecimiento & desarrollo , Aminofenoles/farmacología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Blástula/citología , Blástula/metabolismo , Femenino , Gastrulación , Expresión Génica , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Larva/genética , Larva/metabolismo , Maleimidas/farmacología , Miocardio/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Sarcómeros/metabolismo , Células Madre/metabolismo , Técnicas de Cultivo de Tejidos , Proteína Wnt1/farmacología , Proteína Wnt1/fisiología , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
The significant morbidity and mortality associated with laterality disease almost always are attributed to complex congenital heart defects (CHDs), reflecting the extreme susceptibility of the developing heart to disturbances in the left-right (LR) body plan. To determine how LR positional information becomes ;translated' into anatomical asymmetry, left versus right side cardiomyocyte cell lineages were traced in normal and laterality defective embryos of the frog, Xenopus laevis. In normal embryos, myocytes in some regions of the heart were derived consistently from a unilateral lineage, whereas other regions were derived consistently from both left and right side lineages. However, in heterotaxic embryos experimentally induced by ectopic activation or attenuation of ALK4 signaling, hearts contained variable LR cell composition, not only compared with controls but also compared with hearts from other heterotaxic embryos. In most cases, LR cell lineage defects were associated with abnormal cardiac morphology and were preceded by abnormal Pitx2c expression in the lateral plate mesoderm. In situs inversus embryos there was a mirror image reversal in Pitx2c expression and LR lineage composition. Surprisingly, most of the embryos that failed to develop heterotaxy or situs inversus in response to misregulated ALK4 signaling nevertheless had altered Pitx2c expression, abnormal cardiomyocyte LR lineage composition and abnormal heart structure, demonstrating that cardiac laterality defects can occur even in instances of otherwise normal body situs. These results indicate that: (1) different regions of the heart contain distinct LR myocyte compositions; (2) LR cardiomyocyte lineages and Pitx2c expression are altered in laterality defective embryos; and (3) abnormal LR cardiac lineage composition frequently is associated with cardiac malformations. We propose that proper LR cell composition is necessary for normal morphogenesis, and that misallocated LR cell lineages may be causatively linked with CHDs that are present in heterotaxic individuals, as well as some 'isolated' CHDs that are found in individuals lacking overt features of laterality disease.
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Linaje de la Célula , Corazón Fetal/embriología , Lateralidad Funcional , Cardiopatías Congénitas/embriología , Situs Inversus/embriología , Xenopus/embriología , Receptores de Activinas/genética , Receptores de Activinas/metabolismo , Receptores de Activinas Tipo I , Animales , Tipificación del Cuerpo/genética , Embrión no Mamífero , Corazón Fetal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Mesodermo/citología , Mesodermo/metabolismo , Microscopía Confocal , Situs Inversus/genética , Situs Inversus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
Cellular and molecular left-right differences that are present in the mesodermal heart fields suggest that the heart is lateralized from its inception. Left-right asymmetry persists as the heart fields coalesce to form the primary heart tube, and overt, morphological asymmetry first becomes evident when the heart tube undergoes looping morphogenesis. Thereafter, chamber formation, differentiation of the inflow and outflow tracts, and position of the heart relative to the midline are additional features of heart development that exhibit left-right differences. Observations made in human clinical studies and in animal models of laterality disease suggest that all of these features of cardiac development are influenced by the embryonic left-right body axis. When errors in left-right axis determination happen, they almost always are associated with complex congenital heart malformations. The purpose of this review is to highlight what is presently known about cardiac development and upstream processes of left-right axis determination, and to consider how perturbation of the left-right body plan might ultimately result in particular types of congenital heart defects.
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Tipificación del Cuerpo/fisiología , Cardiopatías Congénitas/embriología , Corazón/embriología , Animales , Cardiopatías Congénitas/fisiopatología , HumanosRESUMEN
The majority of complex congenital heart defects occur in individuals who are afflicted by laterality disease. We hypothesize that the prevalence of valvuloseptal defects in this population is due to defective left-right patterning of the embryonic atrioventricular (AV) canal cushions, which are the progenitor tissue for valve and septal structures in the mature heart. Using embryos of the frog Xenopus laevis, this hypothesis was tested by performing left-right lineage analysis of myocytes and cushion mesenchyme cells of the superior and inferior cushion regions of the AV canal. Lineage analyses were conducted in both wild-type and laterality mutant embryos experimentally induced by misexpression of ALK4, a type I TGF-beta receptor previously shown to modulate left-right axis determination in Xenopus. We find that abnormalities in overall amount and left-right cell lineage composition are present in a majority of ALK4-induced laterality mutant embryos and that much variation in the nature of these abnormalities exists in embryos that exhibit the same overall body situs. We propose that these two parameters of cushion tissue formation-amount and left-right lineage origin-are important for normal processes of valvuloseptal morphogenesis and that defective allocation of cells in the AV canal might be causatively linked to the high incidence of valvuloseptal defects associated with laterality disease.
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Defectos de la Almohadilla Endocárdica/embriología , Corazón Fetal/embriología , Mesodermo/citología , Miocitos Cardíacos/citología , Situs Inversus/embriología , Receptores de Activinas/genética , Receptores de Activinas/metabolismo , Receptores de Activinas Tipo I , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular , Linaje de la Célula , Defectos de la Almohadilla Endocárdica/genética , Defectos de la Almohadilla Endocárdica/metabolismo , Corazón Fetal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Miocitos Cardíacos/metabolismo , ARN Mensajero/análisis , Situs Inversus/genética , Situs Inversus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Domoic acid (DA) is a rigid analog of the excitatory amino acid glutamate. It is produced by the diatom genus Pseudo-nitzschia and is a potent neurotoxin in both adult and developing animals. We have used zebrafish (Danio rerio) as a model to investigate and characterize the developmental toxicity of DA. Domoic acid was administered by microinjection to fertilized eggs at the 128- to 512-cell stages in concentrations ranging from 0.12 to 17 mg/kg (DA/egg weight). DA reduced hatching success by 40% at 0.4 mg/kg and by more than 50% at doses of 1.2 mg/kg and higher. Fifty percent of embryos treated with 1.2 mg/kg DA showed marked tonic-clonic type convulsions at 2 days post fertilization. Four days post fertilization (dpf), all embryos treated with 4.0 mg/kg DA and higher showed a complete absence of touch response reflexes. Commencing 5 dpf, rapid and constant pectoral fin movements were observed, a response which may be related to the hallmark effect in rodents of stereotypic scratching. These data indicate that zebrafish show symptoms of developmental DA toxicity as well as a similar sensitivity comparable to the effects of DA characterized in laboratory rodents.
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Ácido Kaínico/análogos & derivados , Neurotoxinas/toxicidad , Pez Cebra/crecimiento & desarrollo , Anomalías Inducidas por Medicamentos/patología , Animales , Conducta Animal/efectos de los fármacos , Sistema Cardiovascular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Embrión no Mamífero/efectos de los fármacos , Femenino , Ácido Kaínico/administración & dosificación , Ácido Kaínico/toxicidad , Masculino , Microinyecciones , Reflejo/efectos de los fármacos , NataciónRESUMEN
The type I transforming growth factor-beta (TGFbeta) receptor, activin-like kinase-4 (ALK4), is an important regulator of vertebrate development, with roles in mesoderm induction, primitive streak formation, gastrulation, dorsoanterior patterning, and left-right axis determination. To complement previous ALK4 functional studies, we have analyzed ALK4 expression in embryos of the frog, Xenopus laevis. Results obtained with reverse transcriptase-polymerase chain reaction indicate that ALK4 is present in both the animal and vegetal poles of blastula stage embryos and that expression levels are relatively constant amongst embryos examined at blastula, gastrula, neurula, and early tail bud stages. However, the tissue distribution of ALK4 mRNA, as assessed by whole-mount in situ hybridization, was found to change over this range of developmental stages. In the blastula stage embryo, ALK4 is detected in cells of the animal pole and the marginal zone. During gastrulation, ALK4 is detected in the outer ectoderm, involuting mesoderm, blastocoele roof, dorsal lip, and to a lesser extent, in the endoderm. At the onset of neurulation, ALK4 expression is prominent in the dorsoanterior region of the developing head, the paraxial mesoderm, and midline structures, including the prechordal plate and neural folds. Expression in older neurula stage embryos resolves to the developing brain, somites, notochord, and neural crest; thereafter, additional sites of ALK4 expression in tail bud stage embryos include the spinal cord, otic placode, developing eye, lateral plate mesoderm, branchial arches, and the bilateral heart fields. Together, these results not only reflect the multiple developmental roles that have been proposed for this TGFbeta receptor but also define spatiotemporal windows in which ALK4 may function to modulate fundamental embryological events.
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Receptores de Activinas/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Xenopus/fisiología , Receptores de Activinas/biosíntesis , Receptores de Activinas Tipo I , Animales , Blástula/metabolismo , Femenino , Gástrula/metabolismo , Hibridación in Situ , Mesodermo/metabolismo , Cresta Neural/metabolismo , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cola (estructura animal)/embriología , Factores de Tiempo , Proteínas de Xenopus/biosíntesis , Xenopus laevisRESUMEN
In Xenopus, several TGF betas, including nodal-related 1 (Xnr1), derriere, and chimeric forms of Vg1, elicit cardiac and visceral organ left-right (LR) defects when ectopically targeted to right mesendoderm cell lineages, suggesting that LR axis determination may require activity of one or more TGF betas. However, it is not known which, if any, of these ligands is required for LR axis determination, nor is it known which type I TGF beta receptor(s) are involved in mediating left-side TGF beta signaling. We report here that similar to effects of ectopic TGF betas, right-side expression of constitutively active activin-like kinase (ALK) 4 results in LR organ reversals as well as altered Pitx2 expression in the lateral plate mesoderm. Moreover, left-side expression of a kinase-deficient, dominant-negative ALK4 (DN-ALK4) or an ALK4 antisense morpholino also results in abnormal embryonic body situs, demonstrating a left-side requirement for ALK4 signaling. To determine which TGF beta(s) utilize the ALK4 pathway to mediate LR development, biochemical and functional assays were performed using an Activin-Vg1 chimera (AVg), Xnr1, and derriere. Whereas ALK4 can co-immunoprecipitate all of these TGF betas, including endogenous Vg1 protein from embryo homogenates, functional assays demonstrate that not all of these ligands require an intact ALK4 signaling pathway to modulate LR asymmetry. When AVg and DN-ALK4 are co-expressed, LR defects otherwise induced by AVg alone are attenuated by DN-ALK4; however, when functional assays are performed with Xnr1 or derriere, LR defects otherwise elicited by these ligands alone still occur in the presence of DN-ALK4. Intriguingly, when any of these TGF betas is expressed at a higher concentration to elicit primary axis defects, DN-ALK4 blocks gastrulation and dorsoanterior/ventroposterior defects that otherwise occur following ligand-only expression. Together, these results suggest not only that ALK4 interacts with multiple TGF betas to generate embryonic pattern, but also that ALK4 ligands differentially utilize the ALK4 pathway to regulate distinct aspects of axial pattern, with Vg1 as a modulator of ALK4 function in LR axis determination and Vg1, Xnr1, and derriere as modulators of ALK4 function in mesoderm induction during primary axis formation.