RESUMEN
We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.
Asunto(s)
Biomarcadores/metabolismo , Técnicas de Cultivo de Célula/métodos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/fisiología , Morfogénesis/fisiología , Organoides/fisiología , Regeneración/fisiología , Separación Celular/métodos , Colágeno , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Técnicas de Dilución del Indicador , Neprilisina/metabolismoRESUMEN
Basic helix-loop-helix transcription factor Twist1 is a master regulator of Epithelial-Mesenchymal Transition (EMT), a cellular program implicated in different stages of development as well as metastatic dissemination of carcinomas. Here, we show that Twist1 requires TGF-beta type-I receptor (TGFBR1)-activation to bind an enhancer region of downstream effector ZEB1, thereby inducing ZEB1 transcription and EMT. When TGFBR1-phosphorylation is inhibited, Twist1 generates a distinct cell state characterized by collective invasion, simultaneous proliferation and expression of endothelial markers. By contrast, TGFBR1-activation directs Twist1 to induce stable mesenchymal transdifferentiation through EMT, thereby generating cells that display single-cell invasion, but lose their proliferative capacity. In conclusion, preventing Twist1-induced EMT by inhibiting TGFß-signaling does not generally block acquisition of invasion, but switches mode from single-cell/non-proliferative to collective/proliferative. Together, these data reveal that transient Twist1-activation induces distinct cell states depending on signaling context and caution against the use of TGFß-inhibitors as a therapeutic strategy to target invasiveness.
Asunto(s)
Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteína 1 Relacionada con Twist/genética , Células A549 , Células Cultivadas , Células Epiteliales/metabolismo , Células HEK293 , Humanos , Immunoblotting , Glándulas Mamarias Humanas/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
Master regulators of the epithelial-mesenchymal transition such as Twist1 and Snail1 have been implicated in invasiveness and the generation of cancer stem cells, but their persistent activity inhibits stem-cell-like properties and the outgrowth of disseminated cancer cells into macroscopic metastases. Here, we show that Twist1 activation primes a subset of mammary epithelial cells for stem-cell-like properties, which only emerge and stably persist following Twist1 deactivation. Consequently, when cells undergo a mesenchymal-epithelial transition (MET), they do not return to their original epithelial cell state, evidenced by acquisition of invasive growth behavior and a distinct gene expression profile. These data provide an explanation for how transient Twist1 activation may promote all steps of the metastatic cascade; i.e., invasion, dissemination, and metastatic outgrowth at distant sites.