RESUMEN
The morphogenesis of branched organs remains a subject of abiding interest. Although much is known about the underlying signaling pathways, it remains unclear how macroscopic features of branched organs, including their size, network topology, and spatial patterning, are encoded. Here, we show that, in mouse mammary gland, kidney, and human prostate, these features can be explained quantitatively within a single unifying framework of branching and annihilating random walks. Based on quantitative analyses of large-scale organ reconstructions and proliferation kinetics measurements, we propose that morphogenesis follows from the proliferative activity of equipotent tips that stochastically branch and randomly explore their environment but compete neutrally for space, becoming proliferatively inactive when in proximity with neighboring ducts. These results show that complex branched epithelial structures develop as a self-organized process, reliant upon a strikingly simple but generic rule, without recourse to a rigid and deterministic sequence of genetically programmed events.
Asunto(s)
Riñón/crecimiento & desarrollo , Glándulas Mamarias Humanas/crecimiento & desarrollo , Modelos Biológicos , Morfogénesis , Próstata/crecimiento & desarrollo , Animales , Femenino , Humanos , Riñón/embriología , Masculino , Glándulas Mamarias Humanas/embriología , Ratones , Próstata/embriologíaRESUMEN
BACKGROUND: Neoadjuvant chemotherapy (NAC) is the standard of care for patients with early-stage triple negative breast cancers (TNBC). However, more than half of TNBC patients do not achieve a pathological complete response (pCR) after NAC, and residual cancer burden (RCB) is associated with dismal long-term prognosis. Understanding the mechanisms underlying differential treatment outcomes is therefore critical to limit RCB and improve NAC efficiency. METHODS: Human TNBC cell lines and patient-derived organoids were used in combination with real-time metabolic assays to evaluate the effect of NAC (paclitaxel and epirubicin) on tumor cell metabolism, in particular glycolysis. Diagnostic biopsies (pre-NAC) from patients with early TNBC were analyzed by bulk RNA-sequencing to evaluate the predictive value of a glycolysis-related gene signature. RESULTS: Paclitaxel induced a consistent metabolic switch to glycolysis, correlated with a reduced mitochondrial oxidative metabolism, in TNBC cells. In pre-NAC diagnostic biopsies from TNBC patients, glycolysis was found to be upregulated in non-responders. Furthermore, glycolysis inhibition greatly improved response to NAC in TNBC organoid models. CONCLUSIONS: Our study pinpoints a metabolic adaptation to glycolysis as a mechanism driving resistance to NAC in TNBC. Our data pave the way for the use of glycolysis-related genes as predictive biomarkers for NAC response, as well as the development of inhibitors to overcome this glycolysis-driven resistance to NAC in human TNBC patients.
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Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Terapia Neoadyuvante , Pronóstico , Resultado del Tratamiento , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
The functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a tradeoff between spatial resolution and cell profiling depth. Here, we develop a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran) cage coupled to a set of nanobodies allows high-resolution photo-uncaging of different cell types in areas of interest. Single-cell RNA-sequencing of spatially defined CD8+ T cells is used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.
Asunto(s)
Benzofuranos/química , Linfocitos T CD8-positivos/citología , Nitrofenoles/química , Análisis de la Célula Individual , HumanosRESUMEN
During puberty, the mouse mammary gland develops into a highly branched epithelial network. Owing to the absence of exclusive stem cell markers, the location, multiplicity, dynamics and fate of mammary stem cells (MaSCs), which drive branching morphogenesis, are unknown. Here we show that morphogenesis is driven by proliferative terminal end buds that terminate or bifurcate with near equal probability, in a stochastic and time-invariant manner, leading to a heterogeneous epithelial network. We show that the majority of terminal end bud cells function as highly proliferative, lineage-committed MaSCs that are heterogeneous in their expression profile and short-term contribution to ductal extension. Yet, through cell rearrangements during terminal end bud bifurcation, each MaSC is able to contribute actively to long-term growth. Our study shows that the behaviour of MaSCs is not directly linked to a single expression profile. Instead, morphogenesis relies upon lineage-restricted heterogeneous MaSC populations that function as single equipotent pools in the long term.
Asunto(s)
Linaje de la Célula , Glándulas Mamarias Animales/citología , Morfogénesis , Células Madre/citología , Células Madre/metabolismo , Animales , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Ratones , Modelos Moleculares , Maduración Sexual , Análisis de la Célula Individual , Procesos EstocásticosRESUMEN
Understanding to what extent stem cell potential is a cell-intrinsic property or an emergent behavior coming from global tissue dynamics and geometry is a key outstanding question of systems and stem cell biology. Here, we propose a theory of stem cell dynamics as a stochastic competition for access to a spatially localized niche, giving rise to a stochastic conveyor-belt model. Cell divisions produce a steady cellular stream which advects cells away from the niche, while random rearrangements enable cells away from the niche to be favorably repositioned. Importantly, even when assuming that all cells in a tissue are molecularly equivalent, we predict a common ("universal") functional dependence of the long-term clonal survival probability on distance from the niche, as well as the emergence of a well-defined number of functional stem cells, dependent only on the rate of random movements vs. mitosis-driven advection. We test the predictions of this theory on datasets of pubertal mammary gland tips and embryonic kidney tips, as well as homeostatic intestinal crypts. Importantly, we find good agreement for the predicted functional dependency of the competition as a function of position, and thus functional stem cell number in each organ. This argues for a key role of positional fluctuations in dictating stem cell number and dynamics, and we discuss the applicability of this theory to other settings.
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Linaje de la Célula , Autorrenovación de las Células , Nicho de Células Madre , Animales , Supervivencia Celular , Femenino , Homeostasis , Intestinos/citología , Intestinos/crecimiento & desarrollo , Riñón/citología , Riñón/crecimiento & desarrollo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Modelos Teóricos , Relación Señal-Ruido , Células Madre/citología , Células Madre/fisiologíaRESUMEN
The architecture of the mouse mammary gland is highly dynamic and constantly remodeled during pubertal development and estrous cycle-driven sprouting and regression of alveolar side branches. During each of these developmental stages, turnover is driven by distinct subsets of mammary epithelial cells. Extensive previous research has shed light on the unique morphological and cell biological characteristics of each stage. However, technological shortcomings failed to capture the dynamics and single-cell contributions to mammary remodeling. Here, we developed in vivo imaging strategies to follow the same mammary ducts over time and quantify the dynamics of mammary gland growth and remodeling from single-cell level to organ scale. Using a combination of intravital microscopy and genetic reporter systems we show how proliferative heterogeneity drives ductal morphogenesis during different developmental stages. To visualize pubertal growth at the cellular level, we performed long-term time-lapse imaging of extending terminal end buds through a mammary imaging window. We show that single-cells within the terminal end buds are extremely motile and continuously exchange position whilst the duct is elongating. To visualize short-term remodeling in the adult mammary gland at the single cell level, we performed multi-day intravital imaging in photoconvertible Kikume Green-Red mice and fluorescent ubiquitination-based cell cycle indicator mice. We demonstrate that the contribution of single-cells to estrous-driven remodeling is highly variable between cells in the same micro-environment. To assess the effects of this dynamic proliferative contribution on the long-term stability of tissue architecture, we developed a repeated skin flap method to assess mammary gland morphology by intravital microscopy over extended time spans for up to six months. Interestingly, in contrast to the short-term dynamic remodeling, the long-term morphology of the mammary gland remains remarkably stable. Together, our tool box of imaging strategies allows to identify and map transient and continuing dynamics of single cells to the architecture of the mammary gland.
Asunto(s)
Células Epiteliales/fisiología , Microscopía Intravital/métodos , Glándulas Mamarias Animales/fisiología , Animales , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Células Epiteliales/citología , Ciclo Estral/fisiología , Femenino , Homeostasis/fisiología , Glándulas Mamarias Animales/citología , Ratones , Pubertad/fisiologíaRESUMEN
The E2F family of transcription factors plays an important role in controlling cell-cycle progression. While this is their best-known function, we report here novel functions for the newest members of the E2F family, E2F7 and E2F8 (E2F7/8). We show that simultaneous deletion of E2F7/8 in zebrafish and mice leads to severe vascular defects during embryonic development. Using a panel of transgenic zebrafish with fluorescent-labelled blood vessels, we demonstrate that E2F7/8 are essential for proper formation of blood vessels. Despite their classification as transcriptional repressors, we provide evidence for a molecular mechanism through which E2F7/8 activate the transcription of the vascular endothelial growth factor A (VEGFA), a key factor in guiding angiogenesis. We show that E2F7/8 directly bind and stimulate the VEGFA promoter independent of canonical E2F binding elements. Instead, E2F7/8 form a transcriptional complex with the hypoxia inducible factor 1 (HIF1) to stimulate VEGFA promoter activity. These results uncover an unexpected link between E2F7/8 and the HIF1-VEGFA pathway providing a molecular mechanism by which E2F7/8 control angiogenesis.
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Factores de Transcripción E2F/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Fisiológica/genética , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Factores de Transcripción E2F/genética , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Eliminación de Gen , Humanos , Ratones , Regiones Promotoras Genéticas , Pez CebraRESUMEN
Over the past 50 years, researchers from the mammary gland field have launched a collection of distinctive 3D cell culture systems to study multiple aspects of mammary gland physiology and disease. As our knowledge about the mammary gland evolves, more sophisticated 3D cell culture systems are required to answer more and more complex questions. Nowadays, morphologically complex mammary organoids can be generated in distinct 3D settings, along with reproduction of multiple aspects of the gland microenvironment. Yet, each 3D culture protocol comes with its advantages and limitations, where some culture systems are best suited to study stemness potential, whereas others are tailored towards the study of mammary gland morphogenesis. Therefore, prior to starting a 3D mammary culture experiment, it is important to consider and select the ideal culture model to address the biological question of interest. The number and technical requirements of novel 3D cell culture methods vastly increased over the past decades, making it currently challenging and time consuming to identify the best experimental testing. In this chapter, we provide a summary of the most promising murine and human 3D organoid models that are currently used in mammary gland biology research. For each model, we will provide a brief description of the protocol and an overview of the expected morphological outcome, the advantages of the model, and the potential pitfalls, to guide the reader to the best model of choice for specific applications.
Asunto(s)
Glándulas Mamarias Animales , Glándulas Mamarias Humanas , Humanos , Ratones , Animales , Mama , Organoides , Técnicas de Cultivo de Célula/métodos , Árboles de DecisiónRESUMEN
Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.
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Autofagia , Inhibidores de Puntos de Control Inmunológico , Ganglios Linfáticos , Esfingosina/análogos & derivados , Linfocitos T , Autofagia/efectos de los fármacos , Animales , Ganglios Linfáticos/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones Endogámicos C57BL , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Células Endoteliales/metabolismo , Esfingosina/farmacología , Esfingosina/metabolismo , Humanos , Lisofosfolípidos/metabolismo , Inmunoterapia/métodos , Movimiento CelularRESUMEN
Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.
RESUMEN
The mammary gland consists of a bilayered epithelial structure with an extensively branched morphology. The majority of this epithelial tree is laid down during puberty, during which actively proliferating terminal end buds repeatedly elongate and bifurcate to form the basic structure of the ductal tree. Mammary ducts consist of a basal and luminal cell layer with a multitude of identified sub-lineages within both layers. The understanding of how these different cell lineages are cooperatively driving branching morphogenesis is a problem of crossing multiple scales, as this requires information on the macroscopic branched structure of the gland, as well as data on single-cell dynamics driving the morphogenic program. Here we describe a method to combine genetic lineage tracing with whole-gland branching analysis. Quantitative data on the global organ structure can be used to derive a model for mammary gland branching morphogenesis and provide a backbone on which the dynamics of individual cell lineages can be simulated and compared to lineage-tracing approaches. Eventually, these quantitative models and experiments allow to understand the couplings between the macroscopic shape of the mammary gland and the underlying single-cell dynamics driving branching morphogenesis.
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Células Epiteliales , Glándulas Mamarias Animales , Animales , Morfogénesis/genética , Linaje de la CélulaRESUMEN
Breast cancer is a pathological condition characterized by high morphological and molecular heterogeneity. Not only the breast cancer cells, but also their tumor micro-environment consists of a multitude of cell types and states, which continuously evolve throughout progression of the disease. To understand breast cancer evolution within this complex environment, in situ analysis of breast cancer and their co-evolving cells and structures in space and time are essential. In this review, recent technical advances in three-dimensional (3D) and intravital imaging of breast cancer are discussed. Moreover, we highlight the resulting new knowledge on breast cancer biology obtained through these innovative imaging technologies. Finally, we discuss how multidimensional imaging technologies can be integrated with molecular profiling to understand the full complexity of breast cancer and the tumor micro-environment during tumor progression and treatment response.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Diagnóstico por Imagen , Microambiente TumoralRESUMEN
Mouse intraductal modeling enables efficient in vivo propagation of pre-invasive breast cancer lesions and provides a suitable micro-environment for creating patient-derived tumor xenograft models of estrogen-receptor-positive breast cancer. Here, we present a protocol for mouse intraductal modeling of primary ductal carcinoma in situ (DCIS). We describe steps for processing primary DCIS tissues and performing intraductal injections. We then detail procedures for processing intraductal lesions for 3D whole-mount imaging or serial transplantation using magnetic bead sorting. For complete details on the use and execution of this protocol, please refer to Hutten et al. (2023).1.
Asunto(s)
Neoplasias de la Mama , Carcinoma Intraductal no Infiltrante , Humanos , Ratones , Animales , Femenino , Carcinoma Intraductal no Infiltrante/patología , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Microambiente TumoralRESUMEN
Oriented cell divisions are critical for the formation and maintenance of structured epithelia. Proper mitotic spindle orientation relies on polarised anchoring of force generators to the cell cortex by the evolutionarily conserved protein complex formed by the Gαi subunit of heterotrimeric G proteins, the Leucine-Glycine-Asparagine repeat protein (LGN) and the nuclear mitotic apparatus protein. However, the polarity cues that control cortical patterning of this ternary complex remain largely unknown in mammalian epithelia. Here we identify the membrane-associated protein Annexin A1 (ANXA1) as an interactor of LGN in mammary epithelial cells. Annexin A1 acts independently of Gαi to instruct the accumulation of LGN and nuclear mitotic apparatus protein at the lateral cortex to ensure cortical anchoring of Dynein-Dynactin and astral microtubules and thereby planar alignment of the mitotic spindle. Loss of Annexin A1 randomises mitotic spindle orientation, which in turn disrupts epithelial architecture and luminogenesis in three-dimensional cultures of primary mammary epithelial cells. Our findings establish Annexin A1 as an upstream cortical cue that regulates LGN to direct planar cell divisions during mammalian epithelial morphogenesis.
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Anexina A1 , Polaridad Celular , Células Epiteliales , Huso Acromático , Animales , Humanos , Ratones , Anexina A1/metabolismo , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , División Celular/fisiología , Polaridad Celular/genética , Polaridad Celular/fisiología , Células Epiteliales/metabolismo , Mamíferos/metabolismo , Morfogénesis , Huso Acromático/genética , Huso Acromático/metabolismoRESUMEN
Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination.
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Neoplasias de la Mama , Células Endoteliales , Humanos , Femenino , Células Endoteliales/metabolismo , Ligandos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Apoptosis/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice, we show that CAFs in both invasive lobular breast cancer and triple-negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo and in vitro studies reveal the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. Functional co-culture experiments show that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data suggest that CD26+ and CD26- NFs transform into distinct CAF subpopulations in mouse models of breast cancer.
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Neoplasias de la Mama , Fibroblastos Asociados al Cáncer , Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Femenino , Dipeptidil Peptidasa 4/genética , Fibroblastos , Fibroblastos Asociados al Cáncer/patología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Miofibroblastos/patología , Microambiente Tumoral , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular TumoralRESUMEN
Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer (IBC). Due to a lack of biomarkers able to distinguish high- from low-risk cases, DCIS is treated similar to early IBC even though the minority of untreated cases eventually become invasive. Here, we characterized 115 patient-derived mouse-intraductal (MIND) DCIS models reflecting the full spectrum of DCIS observed in patients. Utilizing the possibility to follow the natural progression of DCIS combined with omics and imaging data, we reveal multiple prognostic factors for high-risk DCIS including high grade, HER2 amplification, expansive 3D growth, and high burden of copy number aberrations. In addition, sequential transplantation of xenografts showed minimal phenotypic and genotypic changes over time, indicating that invasive behavior is an intrinsic phenotype of DCIS and supporting a multiclonal evolution model. Moreover, this study provides a collection of 19 distributable DCIS-MIND models spanning all molecular subtypes.
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Neoplasias de la Mama , Carcinoma Intraductal no Infiltrante , Humanos , Animales , Ratones , Femenino , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Bancos de Muestras Biológicas , Xenoinjertos , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Factores de Riesgo , Progresión de la EnfermedadRESUMEN
Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.
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Técnicas Biosensibles , Proteínas Proto-Oncogénicas c-akt , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Técnicas Biosensibles/métodosRESUMEN
Branching morphogenesis is the process that gives rise to branched structures in several organs, such as the lung, the kidney, and the mammary gland. Although morphologically well described, the exact mechanisms driving branch elongation and bifurcation are still poorly understood. Signaling cues from the stroma and extracellular matrix have an important role in driving branching morphogenesis. Organoid models derived from primary mammary epithelial cells have emerged as a powerful tool to gain insight into branching morphogenesis of the mammary gland. However, current available mammary organoid culture protocols result in morphologically simple structures which do not resemble the complex branched structure of the in vivo mammary gland. Supplementation of growth factors to mammary organoids cultured in basement membrane extract or collagen I were shown to induce bud formation and elongation but are not sufficient to drive true branching events. Here, we present an improved culture approach based on 3D primary mammary epithelial cell culture to develop branched organoids with a complex morphology. By alternating the addition of fibroblast growth factor 2 and epidermal growth factor to mammary organoids cultured in a basement membrane extract matrix enriched with collagen type I fibers, we obtain complex mammary organoid structures with primary, secondary, and tertiary branches over a period of 15-20 days. Mammary organoid structures grow >1 mm in size and show an elongated and branched shape which resembles in vivo mammary gland morphology. This novel branched mammary organoid model offers many possibilities to study the mechanisms of branching in the developing mammary gland.
RESUMEN
The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.