Mammary Organoids Research Articles

Primary Mammary Organoid Model of Lactation and Involution.

Mammary gland development occurs mainly after birth and is composed of three successive stages: puberty, pregnancy and lactation, and involution. These developmental stages are associated with major tissue remodeling, including extensive changes in mammary epithelium, as well as surrounding stroma. Three-dimensional (3D) mammary organoid culture has become an important tool in mammary gland biology and enabled invaluable discoveries on pubertal mammary branching morphogenesis and breast cancer. However, a suitable 3D organoid model recapitulating key aspects of lactation and involution has been missing. Here, we describe a robust and straightforward mouse mammary organoid system modeling lactation and involution-like process, which can be applied to study mechanisms of physiological mammary gland lactation and involution as well as pregnancy-associated breast cancer.

Generation of Mosaic Mammary Organoids by Differential Trypsinization.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Abstract P3-01-01: A dual role of natural killer cells in breast cancer metastasis

Abstract BACKGROUND: Metastatic disease drives breast cancer mortality. We recently discovered that leading cells at the invasive edge of mammary tumor organoids retain a conserved basal epithelial program defined by their expression of keratin-14 (K14), establishing K14 as a good marker of invasive breast cancer cells. K14-positive invasive cells also exhibit characteristics that make them targets of immunosurveillance by natural killer (NK) cells. While NK cells are key immune mediators in the control of metastasis, our understanding of the specific mechanisms behind this regulation and its eventual evasion by metastatic cells remains incomplete. METHODS: We have developed a novel preclinical 3D co-culture assay to discover mechanisms behind interactions between K14+ invasive breast cancer cells and NK cells. Combined with in vivo assays of metastasis, we are able to determine how NK cells limit the early stages of metastasis and also how tumor cells can influence key NK cell properties. RESULTS: In ex vivo co-culture assays of NK cells isolated from healthy mouse donors and mammary tumor organoids from MMTV-PyMT and C31T mouse models of breast cancer, we demonstrate that NK cells limit the early stages of metastasis, namely invasion and colony formation. Antibodies to invasive K14+ cells were able to enhance the ability of NK cells to limit colony formation, suggesting antibody-dependent cell mediated cytotoxicity. Surprisingly, when isolated from tumor bearing mice, NK cells did not limit invasion and instead promoted colony formation. The in vivo adoptive transfer of NK cells from healthy donors prevents the progression of early lung metastatic seeds to macrometastases, while the adoptive transfer of cells isolated from tumor bearing donors promotes macrometastatic development. This growth promoting phenotype can be neutralized with antibodies targeting inhibitory cell surface receptors. CONCLUSIONS: Our ex vivo and in vivo data demonstrate that healthy donor NK cells can limit metastasis through the directed cytotoxicity against pioneering K14+ invasive cells. However, prolonged exposure to tumors reprogram NK cells from tumor killing to tumor promoting, specifically in promoting the outgrowth of macrometastases. Further, we can neutralize this effect using NK cell specific inhibitory antibodies. This is the first time inhibitory signaling on NK cells have been linked with a growth promoting phenotype. These data can provide insight into when the use of NK cell directed therapies can be used to treat or prevent clinically relevant metastatic disease. Citation Format: Isaac S. Chan, Hildur Knútsdoóttir, Gayathri Ramakrishnan, Veena Padmanaban, Joel S. Bader, Elizabeth M. Jaffee, Andrew J. Ewald. A dual role of natural killer cells in breast cancer metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-01.

Abstract P3-02-07: Fatty acid metabolism is associated with chromatin remodeling in mammary epithelial cells

Abstract Introduction: Altered metabolism is a hallmark of cancer. We have reported that a set of lipid metabolism (LiMe) genes is over-expressed in the contralateral unaffected breasts (CUBs) of women with unilateral estrogen receptor negative (ER-) breast cancer, who are at high risk for future ER- tumors. We hypothesized that a potential mechanism connecting lipid metabolism to risk of ER- disease may involve epigenetic regulation of gene expression in breast cells by lipids, as shown by McDonnel et. al., Cell reports 2016 in hepatocytes. Methods: Mass spectrometry was performed to profile the composition of lipid species in the sera of patients with ER+ versus ER- disease. MCF-10A cells and mammary organoids from reduction mammoplasty patients were treated with Octanoic (OA) and Linoleic acid (LA) followed by western blot analysis of acetylated histones and sequencing of RNA with qPCR confirmation of significant findings. Partial wave spectroscopic (PWS) microscopy was employed to study change in chromatin. Epithelial RNA from microdissected benign breast samples from CUBs of 84 patients (28 ER+, 28 ER- and 28 healthy controls) were subjected to qPCR. Results: Sera from ER- patients showed higher levels of two triacylglycerides: TAG46:4-FA18:2 (1.25-fold, p=0.05) and TAG 47:2-FA18:2 (1.35-fold, p=0.07), both of which contain LA. MCF10A cells and human mammary organoids showed induction of H3 acetylation at lysine 9 & 14 after treatment with LA and OA treatment at physiological levels. This was accompanied by evidence of chromatin remodeling and 2-fold increase in DNA accessibility on PWS. RNA-Seq of OA treated MCF-10A cells revealed significant upregulation of LiMe genes together with pathways associated with breast malignancy: Notch, Wnt, EMT and adenylate cyclase. The adenylate cyclase pathway (known to be specifically associated with ER- breast cancer risk), was enriched 3.7-fold (p=0.007). In RNA from CUBs of 84 patients, we found overexpression of Notch pathway intermediates that responded to lipid exposure in MCF10A in the ER- group, specifically Notch1 (1.8-fold, p=0.02) and Hey1 (2.9-fold, p=0.05). Conclusion: Specific classes of dietary lipids are the source of acetyl-CoA which acetylates histones and remodels chromatin to modulate gene transcription in ER- MCF10A cells. Thus revved-up fatty acid oxidation may play a role in ER- breast oncogenesis via Notch signaling. Our novel findings point to potential interception strategies that target connections between lipid metabolism and histone modifications. Citation Format: Shivangi Yadav, MiRan Choi, David Van Derway, Greta Braur, Vadim Backman, Seema Ahsan Khan, Susan Clare. Fatty acid metabolism is associated with chromatin remodeling in mammary epithelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-07.

An organoid-based carcinogenesis model induced by in vitro chemical treatment.

Animal carcinogenesis models induced by environmental chemicals have been widely used for basic and applied cancer research. However, establishment of in vitro or ex vivo models is essential for molecular mechanistic elucidation of early events in carcinogenesis, leading to clarification of the total mode of action. In the present study, to establish an organoid-based chemical carcinogenesis model, mouse organoids were treated in vitro with 4 genotoxic chemicals, e.g. ethyl methanesulfonate (EMS), acrylamide (AA), diethylnitrosamine (DEN) and 7,12-dimethylbenz[a]anthracene (DMBA) to examine their tumorigenicity after injection to nude mice. The four chemicals were reported to induce lung, liver or mammary carcinomas in mouse models. DMBA-treated mammary tissue-derived organoids with Trp53 heterozygous knockout exhibited tumorigenicity, but not those with wild-type Trp53, reflecting previous reports of corresponding animal models. Treatment of lung organoids with or without Trp53 knockout with EMS or AA resulted in carcinogenic histopathological characteristics, and the activation of oncogenic kinases was demonstrated in the nodules from the nude mouse subcutis. DEN-treated liver (biliary tract) organoids also had an increased number of similar changes. In conclusion, an ex vivo model for chemical carcinogenesis was established using normal mouse tissue-derived organoids. This model will be applied to detect early molecular events, leading to clarification of the mode of action of chemical carcinogenesis.

Twist1-Induced Epithelial Dissemination Requires Prkd1 Signaling.

Dissemination is an essential early step in metastasis but its molecular basis remains incompletely understood. To define the essential targetable effectors of this process, we developed a 3D mammary epithelial culture model, in which dissemination is induced by overexpression of the transcription factor Twist1. Transcriptomic analysis and ChIP-PCR together demonstrated that protein kinase D1 (Prkd1) is a direct transcriptional target of Twist1 and is not expressed in the normal mammary epithelium. Pharmacologic and genetic inhibition of Prkd1 in the Twist1-induced dissemination model demonstrated that Prkd1 was required for cells to initiate extracellular matrix (ECM)-directed protrusions, release from the epithelium, and migrate through the ECM. Antibody-based protein profiling revealed that Prkd1 induced broad phosphorylation changes, including an inactivating phosphorylation of β-catenin and two microtubule depolymerizing phosphorylations of Tau, potentially explaining the release of cell-cell contacts and persistent activation of Prkd1. In patients with breast cancer, TWIST1 and PRKD1 expression correlated with metastatic recurrence, particularly in basal breast cancer. Prkd1 knockdown was sufficient to block dissemination of both murine and human mammary tumor organoids. Finally, Prkd1 knockdown in vivo blocked primary tumor invasion and distant metastasis in a mouse model of basal breast cancer. Collectively, these data identify Prkd1 as a novel and targetable signaling node downstream of Twist1 that is required for epithelial invasion and dissemination. SIGNIFICANCE: Twist1 is a known regulator of metastatic cell behaviors but not directly targetable. This study provides a molecular explanation for how Twist1-induced dissemination works and demonstrates that it can be targeted. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/2/204/F1.large.jpg.

Characterizing optical coherence tomography speckle fluctuation spectra of mammary organoids during suppression of intracellular motility.

An understanding of how the mammary gland responds to toxicant and drug exposures can shed light on mechanisms of breast cancer initiation/progression and therapeutic effectiveness, respectively. In this study, we employed noninvasive, label-free and high-throughput optical coherence tomography speckle fluctuation spectroscopy (OCT-SFS) to track exposure-response relationships in three-dimensional (3D) mammary epithelial organoid models. OCT-SFS is sensitive to relatively high speed (~0.16-8 µm/min) motions of subcellular light scattering components occurring over short (~2-114 s) time scales, termed "intracellular motility." In this study, OCT speckle fluctuation spectra are quantified by two metrics: the intracellular motility amplitude, M, and frequency-dependent motility roll-off, α. OCT-SFS was performed on human mammary organoid models comprised of pre-malignant MCF10DCIS.com cells or MCF7 adenocarcinoma cells over 6 days of exposure to either a microtubule inhibitor (Paclitaxel, Taxol) or a myosin II inhibitor (Blebbistatin). Raw values of α and M were normalized to a dynamic range corresponding to fixed (0%) and live/homeostatic (100%) organoids for each cell line. In this work, we observed a significant decrease in both M and α of MCF10DCIS.com organoids after 24 hours of exposure to Taxol (P<0.001), and a significant decrease only in α for MCF7 organoids after 48 hours of exposure (P<0.0001). We also observed a significant decrease in both M and α of MCF7 organoids at the longest exposure time of 6 days to Blebbistatin (P<0.0001), and a significant decrease only in M for MCF10DCIS.com organoids after 24 hours of exposure (P<0.01). OCT-SFS revealed cell line-specific response patterns, in terms of intracellular motility, to different motility suppression mechanisms. This provides a foundation for future OCT-SFS studies of longitudinal responses of the mammary gland in toxicology and drug research.

Characterizing optical coherence tomography speckle fluctuation spectra of mammary organoids during suppression of intracellular motility

Characterizing optical coherence tomography speckle fluctuation spectra of mammary organoids during suppression of intracellular motility

A new pipeline for pathophysiological analysis of the mammary gland based on organoid transplantation and organ clearing.

ABSTRACTRecent developments in techniques for tissue clearing and size reduction have enabled optical imaging of whole organs and the study of rare tumorigenic events in vivo. The adult mammary gland provides a unique model for investigating physiological or pathological processes such as morphogenesis or epithelial cell dissemination. Here, we establish a new pipeline to study rare cellular events occurring in the mammary gland, by combining orthotopic transplantation of mammary organoids with the uDISCO organ size reduction and clearing method. This strategy allows us to analyze the behavior of individually labeled cells in regenerated mammary gland. As a proof of concept, we analyzed the localization of rare epithelial cells overexpressing atypical protein kinase C iota (also known as PRKCI, referred to here as aPKCι) with an N-terminal eGFP fusion (GFP-aPKCι+) in the normal mammary gland. Using this analytical pipeline, we were able to visualize epithelial aPKCι+ cells escaping from the normal mammary epithelium and disseminating into the surrounding stroma. This technical resource should benefit mammary development and tumor progression studies.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Microscale pressure measurements based on an immiscible fluid/fluid interface

A method of microscale pressure measurement based on immiscible fluid/fluid interface is proposed. This method utilizes observed curvature changes in a fluid/fluid interface, and can accurately report hydraulic pressure in fluids at length scales of 10 microns. The method is especially suited for measuring fluid pressure in micro-scale biological samples. Using this method, we probe fluid pressure build up in epithelial domes, murine mammary gland organoids embedded in hydrogel, and lumen pressure in the developing mouse embryo. Results reveal that the pressure developed across epithelial barriers is on the order of 100~300 Pa, and is modulated by ion channel activity.

BRCA1 mutation influences progesterone response in human benign mammary organoids

BackgroundWomen, who carry a germline BRCA1 gene mutation, have a markedly increased risk of developing breast cancer during their lifetime. While BRCA1 carriers frequently develop triple-negative, basal-like, aggressive breast tumors, hormone signaling is important in the genesis of BRCA1 mutant breast cancers. We investigated the hormone response in BRCA1-mutated benign breast tissue using an in vitro organoid system.MethodsScaffold-free, multicellular human breast organoids generated from benign breast tissues from non-carrier or BRCA1 mutation carriers were treated in vitro with a stepwise menstrual cycle hormone regimen of estradiol (E2) and progesterone (P4) over the course of 28 days.ResultsBreast organoids exhibited characteristics of the native breast tissue, including expression of hormone receptors, collagen production, and markers of luminal and basal epithelium, and stromal fibroblasts. RNA sequencing analysis revealed distinct gene expression in response to hormone treatment in the non-carrier and BRCA1-mutated organoids. The selective progesterone receptor modulator, telapristone acetate (TPA), was used to identify specifically PR regulated genes. Specifically, extracellular matrix organization genes were regulated by E2+P4+TPA in the BRCA1-mutated organoids but not in the non-carrier organoids. In contrast, in the non-carrier organoids, known PR target genes such as the cell cycle genes were inhibited by TPA.ConclusionsThese data show that BRCA1 mutation influences hormone response and in particular PR activity which differs from that of non-carrier organoids. Our organoid model system revealed important insights into the role of PR in BRCA1-mutated benign breast cells and the critical paracrine actions that modify hormone receptor (HR)-negative cells. Further analysis of the molecular mechanism of BRCA1 and PR crosstalk is warranted using this model system.

Cell swelling, softening and invasion in a three-dimensional breast cancer model.

Sculpting of structure and function of three-dimensional multicellular tissues depend critically on the spatial and temporal coordination of cellular physical properties, yet the organizational principles that govern these events, and their disruption in disease, remain poorly understood. Using a multicellular mammary cancer organoid model, here we map in three dimensions the spatial and temporal evolution of positions, motions, and physical characteristics of individual cells. Compared with cells in the organoid core, cells at the organoid periphery and the invasive front are found to be systematically softer, larger and more dynamic. These mechanical changes are shown to arise from supracellular fluid flow through gap junctions, suppression of which delays transition to an invasive phenotype. Together, these findings highlight the role of spatiotemporal coordination of cellular physical properties in tissue organization and disease progression.

Abstract 51: Propagation of human colon, mammary, and lung cancer organoids in growth medium utilizing tissue-specific reagent kits and ready-to-use Wnt-3a and R-Spondin1 conditioned media

Abstract Three-dimensional “organoid” growth of tumors may represent a more physiologically relevant in vitro model system than traditional two-dimensional monolayer cultures of cancer cell lines. With the increased availability of cryopreserved human cancer organoids generated by academic laboratories, large-scale biobanking initiatives, and commercial sources, there is an unmet need for simplified, standardized, and cost-effective methods for preparation of the complex growth media required by these models. Human organoid culture media contains a variety of recombinant proteins, small molecules, and other growth factors that are costly to purchase in small-scale, time consuming to reconstitute and aliquot, and have varying stability and shelf life once prepared. Organoid culture media often also utilizes undefined conditioned media (CM) from one or more engineered cell lines that must be cultured separately, requiring additional time and resources to maintain. These lines secrete critical growth factors and the CM generated must be carefully prepared, collected, and stored. CM is subject to variability in activity levels due to batch-to-batch and protocol-to-protocol differences that can affect subsequent organoid culture performance. To address these challenges and to facilitate the wide-spread adoption of human organoid culture we are developing reagent kits containing essential growth medium components in an individually lyophilized format for long-term storage and easy single-use preparation of organoid growth media for a variety of tissue types including human colon, mammary, and lung. We compared growth medium formulated with our components with small-scale “homebrew” preparations and found equivalent or better culture performance (as calculated by doubling times) and similar morphology. Both approaches were able to generate media capable of supporting growth for at least three passages (~30 days) in 15 different organoid models. Additionally, we compared protocols and cell lines for CM generation to support organoid culture and found that an optimized, scaled-up production method could generate Wnt-3a and R-spondin1 CM with higher activity levels (up to 52%) and reduced lot-lot variability in comparison with commonly used small-scale protocols. We found that the optimized protocols produced CM that was stable for at least 2 months at 4°C and at least 6 months at -20°C, and was resilient against freezing and thawing. Previously frozen CM was also able to support organoid growth in culture. These results show that our tissue-specific kits and ready-to-use CM can provide a simplified, cost effective method to support the long-term propagation of human cancer organoids from multiple tissues and cancer types. Citation Format: James Clinton, Allison Ruchinskas, Himanshi Desai, Nanda Mahashetty, Dezhong Yin. Propagation of human colon, mammary, and lung cancer organoids in growth medium utilizing tissue-specific reagent kits and ready-to-use Wnt-3a and R-Spondin1 conditioned media [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 51.

Abstract 2649: A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells

Abstract Hypoxia occurs in 90% of solid tumors due to inefficient vasculature and has been demonstrated to promote a more aggressive phenotype, correlating with worse patient prognosis. Current methods to measure hypoxia in human tumors only identify cells that are hypoxic at the time the tissue is collected and do not delineate cells that were transiently exposed to hypoxia but survived and adapted. To overcome this challenge, we generated a Lox-Cre dual-vector oxygen-dependent reporter system. Under hypoxia, Cre expression leads to the cleavage of a DsRed reporter and permanent expression of a GFP reporter. We stably transduced breast cancer cell lines and demonstrated an irreversible reporter-switch that occurs when cells are exposed to 0.5% O2, the typical O2 level in human breast tumors, but does not occur under physiological conditions (8% O2). To explore the utility of our system, we embedded spheroids derived from MDA-MB-231 reporter cells in collagen and cultured them under 20% O2 conditions. A defined hypoxic core (GFP+) was visible after 15 days in culture and it correlated with hypoxic O2 measurements. We then generated orthotopic breast tumors by transplanting MDA-MB-231 reporter cells into the mammary fat pad of mice. Hypoxic cells (GFP+) were localized in the peri-necrotic region of the tumor and co-localized with hypoxyprobe and HIF1α labeling. RNA sequencing of GFP+ compared to DsRed+ tumor cells demonstrated that GFP+ cells were enriched for the hallmark signature of hypoxia. Additionally, we developed a transgenic mouse expressing the hypoxia-driven construct that was bred to a tdTomato-floxed GFP mouse. A female double fluorescent/hypoxia-reporter mouse was then bred to a male FVB/N-Tg(MMTV-PyMT) mouse in order to generate triple-transgenic mice that form breast tumors that spontaneously metastasize. We systematically studied hypoxia in spontaneous breast tumors from the development of ductal hyperplasia to late stage invasive carcinoma. Tumor hypoxia was first detected in early stage ductal carcinoma in situ (DCIS) lesions. Similarly to the spheroid model, mammary organoids derived from triple-transgenic mice tumors cultured under 20% O2 conditions also exhibited a defined hypoxic core after 15 days in culture. Both orthotopic and transgenic mouse models revealed an increased potency of post-hypoxic cells at arriving to distant sites in early stage metastasis. We utilized RNA sequencing to identify potential mechanisms of enhanced metastatic capacity. Ultimately, our system allowed the fate-map of hypoxic cells during tumor progression to metastasis. We show for the first time that 3D in vitro organoids derived from PyMT tumors develop regions of hypoxia under normal tissue culture conditions. In vivo, we show that hypoxic cells are more potent at arriving at distant sites and that they retain a different gene expression that potentially confers a metastatic advantage. Citation Format: Inês Godet, Yu Jung Shin, Julia A. Ju, Soumitra Bhoyar, I Chae Ye, Guannan Wang, Saraswati Sukumar, Daniele M. Gilkes. A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2649.

DC-SCRIPT deficiency delays mouse mammary gland development and branching morphogenesis

Mammary glands are unique organs in which major adaptive changes occur in morphogenesis and development after birth. Breast cancer is the most common cancer and a major cause of mortality in females worldwide. We have previously identified the loss of expression of the transcription regulator DC-SCRIPT (Zfp366) as a prominent prognostic event in estrogen receptor positive breast cancer patients. DC-SCRIPT affects multiple transcriptional events in breast cancer cells, including estrogen and progesterone receptor-mediated transcription, and promotes CDKN2B-related cell cycle arrest. As loss of DC-SCRIPT expression appears an early event in breast cancer development, we here investigated the role of DC-SCRIPT in mammary gland development using wild-type and DC-SCRIPT knockout mice. Mice lacking DC-SCRIPT exhibited severe breeding problems and showed significant growth delay relative to littermate wild-type mice. Subsequent analysis revealed that DC-SCRIPT was expressed in mouse mammary epithelium and that DC-SCRIPT deficiency delayed mammary gland morphogenesis in vivo. Finally, analysis of 3D mammary gland organoid cultures confirmed that loss of DC-SCRIPT dramatically delayed mammary organoid branching in vitro. The study shows for the first time that DC-SCRIPT deficiency delays mammary gland morphogenesis in vivo and in vitro. These data define DC-SCRIPT as a novel modulator of mammary gland development.

3D bioprinted mammary organoids and tumoroids in human mammary derived ECM hydrogels

The extracellular matrix (ECM) of tissues is an important mediator of cell function. Moreover, understanding cellular dynamics within their specific tissue context is also important for developmental biology, cancer research, and regenerative medicine. However, robust in vitro models that incorporate tissue-specific microenvironments are lacking. Here we describe a novel mammary-specific culture protocol that combines a self-gelling hydrogel comprised solely of ECM from decellularized rat or human breast tissue with the use of our previously described 3D bioprinting platform. We initially demonstrate that undigested and decellularized mammary tissue can support mammary epithelial and tumor cell growth. We then describe a methodology for generating mammary ECM extracts that can spontaneously gel to form hydrogels. These ECM hydrogels retain unique structural and signaling profiles that elicit differential responses when normal mammary and breast cancer cells are cultured within them. Using our bioprinter, we establish that we can generate large organoids/tumoroids in the all mammary-derived hydrogel. These findings demonstrate that our system allows for growth of organoids/tumoroids in a tissue-specific matrix with unique properties, thus providing a suitable platform for ECM and epithelial/cancer cell studies. Statement of SignificanceFactors within extracellular matrices (ECMs) are specific to their tissue of origin. It has been shown that tissue specific factors within the mammary gland’s ECM have pronounced effects on cellular differentiation and cancer behavior. Understanding the role of the ECM in controlling cell fate has major implications for developmental biology, tissue engineering, and cancer therapy. However, in vitro models to study cellular interactions with tissue specific ECM are lacking. Here we describe the generation of 3D hydrogels consisting solely of human or mouse mammary ECM. We demonstrate that these novel 3D culture substrates can sustain large 3D bioprinted organoid and tumoroid formation. This is the first demonstration of an all mammary ECM culture system capable of sustaining large structural growths.

A 3D bioprinter platform for mechanistic analysis of tumoroids and chimeric mammary organoids

The normal mammary microenvironment can suppress tumorigenesis and redirect cancer cells to adopt a normal mammary epithelial cell fate in vivo. Understanding of this phenomenon offers great promise for novel treatment and detection strategies in cancer, but current model systems make mechanistic insights into the process difficult. We have recently described a low-cost bioprinting platform designed to be accessible for basic cell biology laboratories. Here we report the use of this system for the study of tumorigenesis and microenvironmental redirection of breast cancer cells. We show our bioprinter significantly increases tumoroid formation in 3D collagen gels and allows for precise generation of tumoroid arrays. We also demonstrate that we can mimic published in vivo findings by co-printing cancer cells along with normal mammary epithelial cells to generate chimeric organoids. These chimeric organoids contain cancer cells that take part in normal luminal formation. Furthermore, we show for the first time that cancer cells within chimeric structures have a significant increase in 5-hydroxymethylcytosine levels as compared to bioprinted tumoroids. These results demonstrate the capacity of our 3D bioprinting platform to study tumorigenesis and microenvironmental control of breast cancer and highlight a novel mechanistic insight into the process of microenvironmental control of cancer.

High-resolution 3D imaging of fixed and cleared organoids.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.