Configurational entropy is an intrinsic driver of tissue structural heterogeneity.

Tissues comprise ordered arrangements of cells that can be surprisingly disordered in their details. How the properties of single cells and their microenvironment contribute to the balance between order and disorder at the tissue-scale remains poorly understood. Here, we address this question using the self-organization of human mammary organoids as a model. We find that organoids behave like a dynamic structural ensemble at the steady state. We apply a maximum entropy formalism to derive the ensemble distribution from three measurable parameters - the degeneracy of structural states, interfacial energy, and tissue activity (the energy associated with positional fluctuations). We link these parameters with the molecular and microenvironmental factors that control them to precisely engineer the ensemble across multiple conditions. Our analysis reveals that the entropy associated with structural degeneracy sets a theoretical limit to tissue order and provides new insight for tissue engineering, development, and our understanding of disease progression.

A Mammary Organoid Model to Study Branching Morphogenesis.

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.

In vivo genome-editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis.

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53+/- heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.

Current Status of Breast Organoid Models.

Breast cancer (BC) is the most frequently diagnosed malignancy among women globally. Although mouse models have been critical in advancing the knowledge of BC tumorigenesis and progression, human breast models comprising the breast tissue microenvironment are needed to help elucidate the underlying mechanisms of BC risk factors. As such, it is essential to identify an ex vivo human breast tissue mimetic model that can accurately pinpoint the effects of these factors in BC development. While two-dimensional models have been invaluable, they are not suitable for studying patient-specific tumor biology and drug response. Recent developments in three-dimensional (3D) models have led to the prominence of organized structures grown in a 3D environment called "organoids." Breast organoids can accurately recapitulate the in vivo breast microenvironment and have been used to examine factors that affect signaling transduction, gene expression, and tissue remodeling. In this review, the applications, components, and protocols for development of breast organoids are discussed. We summarize studies that describe the utility of breast organoids, including in the study of normal mammary gland development and tumorigenesis. Finally, we provide an overview of protocols for development of breast organoids, and the advantages and disadvantages of different techniques in studies are described. The included studies have shown that breast organoids will continue to serve as a crucial platform for understanding of progression of BC tumors and the testing of novel therapeutics.

Characterization of Organoid Cultures to Study the Effects of Pregnancy Hormones on the Epigenome and Transcriptional Output of Mammary Epithelial Cells.

The use of mouse derived mammary organoids can provide a unique strategy to study mammary gland development across a normal life cycle, as well as offering insights into how malignancies form and progress. Substantial cellular and epigenomic changes are triggered in response to pregnancy hormones, a reaction that engages molecular and cellular changes that transform the mammary epithelial cells into "milk producing machines". Such epigenomic alterations remain stable in post-involution mammary epithelial cells and control the reactivation of gene transcription in response to re-exposure to pregnancy hormones. Thus, a system that tightly controls exposure to pregnancy hormones, epigenomic alterations, and activation of transcription will allow for a better understanding of such molecular switches. Here, we describe the characterization of ex vivo cultures to mimic the response of mammary organoid cultures to pregnancy hormones and to understand gene regulation and epigenomic reprogramming on consecutive hormone exposure. Our findings suggest that this system yields similar epigenetic modifications to those reported in vivo, thus representing a suitable model to closely track epigenomic rearrangement and define unknown players of pregnancy-induced development.

Optimal, Large-Scale Propagation of Mouse Mammary Tumor Organoids.

Tumor organoids mimic the architecture and heterogeneity of in vivo tumors and enable studies of collective interactions between tumor cells as well as with their surrounding microenvironment. Although tumor organoids hold significant promise as cancer models, they are also more costly and labor-intensive to cultivate than traditional 2D cell culture. We sought to identify critical factors regulating organoid growth ex vivo, and to use these observations to develop a more efficient organoid expansion method. Using time-lapse imaging of mouse mammary tumor organoids in 3D culture, we observed that outgrowth potential varies non-linearly with initial organoid size. Maximal outgrowth occurred in organoids with a starting size between ~10 to 1000 cells. Based on these observations, we developed a suspension culture method that maintains organoids in the ideal size range, enabling expansion from 1 million to over 100 million cells in less than 2 weeks and less than 3 hours of hands-on time. Our method facilitates the rapid, cost-effective expansion of organoids for CRISPR based studies and other assays requiring a large amount of organoid starting material.

Organoid models for mammary gland dynamics and breast cancer.

The mammary gland is a highly dynamic tissue that undergoes repeated cycles of growth and involution during pregnancy and menstruation. It is also the site from which breast cancers emerge. Organoids provide an in vitro model that preserves several of the cellular, structural, and microenvironmental features that dictate mammary gland function in vivo and have greatly advanced our understanding of glandular biology. Their tractability for genetic manipulation, live imaging, and high throughput screening have facilitated investigation into the mechanisms of glandular morphogenesis, structural maintenance, tumor progression, and invasion. Opportunities remain to enhance cellular and structural complexity of mammary organoid models, including incorporating additional cell types and hormone signaling.

Derivation of a robust mouse mammary organoid system for studying tissue dynamics.

Advances in stem cell research have enabled the generation of 'mini organs' or organoids that recapitulate phenotypic traits of the original biological specimen. Although organoids have been demonstrated for multiple organ systems, there are more limited options for studying mouse mammary gland formation in vitro Here, we have built upon previously described culture assays to define culture conditions that enable the efficient generation of clonal organoid structures from single sorted basal mammary epithelial cells (MECs). Analysis of Confetti-reporter mice revealed the formation of uni-colored structures and thus the clonal nature of these organoids. High-resolution 3D imaging demonstrated that basal cell-derived complex organoids comprised an inner compartment of polarized luminal cells with milk-producing capacity and an outer network of elongated myoepithelial cells. Conversely, structures generated from luminal MECs rarely contained basal/myoepithelial cells. Moreover, flow cytometry and 3D microscopy of organoids generated from lineage-specific reporter mice established the bipotent capacity of basal cells and the restricted potential of luminal cells. In summary, we describe optimized in vitro conditions for the efficient generation of mouse mammary organoids that recapitulate features of mammary tissue architecture and function, and can be applied to understand tissue dynamics and cell-fate decisions.