A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin ß5) as the essential integrin α/ß pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the integral pocket within the ß-propeller domain of ITGAV for integrin αVß5 dimerization. Combined with in silico compound docking, we developed a CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) pipeline for drug discovery and identified Cpd_AV2 as a lead inhibitor targeting the ß-propeller central pocket of ITGAV. Cpd_AV2 treatment led to rapid uncoupling of integrin αVß5 and cellular apoptosis, providing a unique class of therapeutic action that eliminates the integrin signaling via heterodimer dissociation. We also foresee the CRISPR-TICA approach to be an accessible method for future drug discovery studies.

ELF5: A Molecular Clock for Breast Aging and Cancer Susceptibility.

Breast cancer is predominantly an age-related disease, with aging serving as the most significant risk factor, compounded by germline mutations in high-risk genes like BRCA1/2. Aging induces architectural changes in breast tissue, particularly affecting luminal epithelial cells by diminishing lineage-specific molecular profiles and adopting myoepithelial-like characteristics. ELF5 is an important transcription factor for both normal breast and breast cancer development. This review focuses on the role of ELF5 in normal breast development, its altered expression throughout aging, and its implications in cancer. It discusses the lineage-specific expression of ELF5, its regulatory mechanisms, and its potential as a biomarker for breast-specific biological age and cancer risk.

Functional delineation of the luminal epithelial microenvironment in breast using cell-based screening in combinatorial microenvironments.

Microenvironment signals are potent determinants of cell fate and arbiters of tissue homeostasis, however understanding how different microenvironment factors coordinately regulate cellular phenotype has been experimentally challenging. Here we used a high-throughput microenvironment microarray comprised of 2640 unique pairwise signals to identify factors that support proliferation and maintenance of primary human mammary luminal epithelial cells. Multiple microenvironment factors that modulated luminal cell number were identified, including: HGF, NRG1, BMP2, CXCL1, TGFB1, FGF2, PDGFB, RANKL, WNT3A, SPP1, HA, VTN, and OMD. All of these factors were previously shown to modulate luminal cell numbers in painstaking mouse genetics experiments, or were shown to have a role in breast cancer, demonstrating the relevance and power of our high-dimensional approach to dissect key microenvironmental signals. RNA-sequencing of primary epithelial and stromal cell lineages identified the cell types that express these signals and the cognate receptors in vivo. Cell-based functional studies confirmed which effects from microenvironment factors were reproducible and robust to individual variation. Hepatocyte growth factor (HGF) was the factor most robust to individual variation and drove expansion of luminal cells via cKit+ progenitor cells, which expressed abundant MET receptor. Luminal cells from women who are genetically high risk for breast cancer had significantly more MET receptor and may explain the characteristic expansion of the luminal lineage in those women. In ensemble, our approach provides proof of principle that microenvironment signals that control specific cellular states can be dissected with high-dimensional cell-based approaches.

Comprehensive single cell aging atlas of mammary tissues reveals shared epigenomic and transcriptomic signatures of aging and cancer.

Aging is the greatest risk factor for breast cancer; however, how age-related cellular and molecular events impact cancer initiation is unknown. We investigate how aging rewires transcriptomic and epigenomic programs of mouse mammary glands at single cell resolution, yielding a comprehensive resource for aging and cancer biology. Aged epithelial cells exhibit epigenetic and transcriptional changes in metabolic, pro-inflammatory, or cancer-associated genes. Aged stromal cells downregulate fibroblast marker genes and upregulate markers of senescence and cancer-associated fibroblasts. Among immune cells, distinct T cell subsets (Gzmk+, memory CD4+, γδ) and M2-like macrophages expand with age. Spatial transcriptomics reveal co-localization of aged immune and epithelial cells in situ. Lastly, transcriptional signatures of aging mammary cells are found in human breast tumors, suggesting mechanistic links between aging and cancer. Together, these data uncover that epithelial, immune, and stromal cells shift in proportions and cell identity, potentially impacting cell plasticity, aged microenvironment, and neoplasia risk.

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.

Loss of epigenetic suppression of retrotransposons with oncogenic potential in aging mammary luminal epithelial cells.

A primary function of DNA methylation in mammalian genomes is to repress transposable elements (TEs). The widespread methylation loss that is commonly observed in cancer cells results in the loss of epigenetic repression of TEs. The aging process is similarly characterized by changes to the methylome. However, the impact of these epigenomic alterations on TE silencing and the functional consequences of this have remained unclear. To assess the epigenetic regulation of TEs in aging, we profiled DNA methylation in human mammary luminal epithelial cells (LEps)-a key cell lineage implicated in age-related breast cancers-from younger and older women. We report here that several TE subfamilies function as regulatory elements in normal LEps, and a subset of these display consistent methylation changes with age. Methylation changes at these TEs occurred at lineage-specific transcription factor binding sites, consistent with loss of lineage specificity. Whereas TEs mainly showed methylation loss, CpG islands (CGIs) that are targets of the Polycomb repressive complex 2 (PRC2) show a gain of methylation in aging cells. Many TEs with methylation loss in aging LEps have evidence of regulatory activity in breast cancer samples. We furthermore show that methylation changes at TEs impact the regulation of genes associated with luminal breast cancers. These results indicate that aging leads to DNA methylation changes at TEs that undermine the maintenance of lineage specificity, potentially increasing susceptibility to breast cancer.

Preneoplastic stromal cells promote BRCA1-mediated breast tumorigenesis.

Women with germline BRCA1 mutations (BRCA1+/mut) have increased risk for hereditary breast cancer. Cancer initiation in BRCA1+/mut is associated with premalignant changes in breast epithelium; however, the role of the epithelium-associated stromal niche during BRCA1-driven tumor initiation remains unclear. Here we show that the premalignant stromal niche promotes epithelial proliferation and mutant BRCA1-driven tumorigenesis in trans. Using single-cell RNA sequencing analysis of human preneoplastic BRCA1+/mut and noncarrier breast tissues, we show distinct changes in epithelial homeostasis including increased proliferation and expansion of basal-luminal intermediate progenitor cells. Additionally, BRCA1+/mut stromal cells show increased expression of pro-proliferative paracrine signals. In particular, we identify pre-cancer-associated fibroblasts (pre-CAFs) that produce protumorigenic factors including matrix metalloproteinase 3 (MMP3), which promotes BRCA1-driven tumorigenesis in vivo. Together, our findings demonstrate that precancerous stroma in BRCA1+/mut may elevate breast cancer risk through the promotion of epithelial proliferation and an accumulation of luminal progenitor cells with altered differentiation.

Sustained postconfluent culture of human mammary epithelial cells enriches for luminal and c-Kit+ subtypes.

BACKGROUND: A challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the composition of the culture with serial passaging. This drift in composition presents a challenge for studying luminal and progenitor cells, which are prospective cells of origin for most breast cancer subtypes. METHODS: We demonstrate the use of postconfluent culture on HMECs. Postconfluent culture entails culturing HMECs for 2-5 weeks without passaging but maintaining frequent feedings in low-stress M87A culture medium. In contrast, standard HMEC culture entails enzymatic subculturing every 3-5 days to maintain subconfluent density. RESULTS: When compared to standard HMEC culture, postconfluent culture yields increased proportions of luminal cells and c-Kit+ progenitor cells. Postconfluent cultures develop a distinct multilayered morphology with individual cells showing decreased physical deformability as compared to cells in standard culture. Gene expression analysis of postconfluent cells shows increased expression of lineage-specific markers and extracellular matrix components. CONCLUSIONS: Postconfluent culture is a novel, useful strategy for altering the lineage composition of HMECs, by increasing the proportional representation of luminal and progenitor cells. We speculate that postconfluent culture creates a microenvironment with cellular composition closer to the physiological state and eases the isolation of scarce cell subtypes. As such, postconfluent culture is a valuable tool for researchers using HMECs for breast cancer research.

Cellular and molecular mechanisms of breast cancer susceptibility.

There is a plethora of recognized risk factors for breast cancer (BC) with poorly understood or speculative biological mechanisms. The lack of prevention options highlights the importance of understanding the mechanistic basis of cancer susceptibility and finding new targets for breast cancer prevention. Until now, we have understood risk and cancer susceptibility primarily through the application of epidemiology and assessing outcomes in large human cohorts. Relative risks are assigned to various human behaviors and conditions, but in general the associations are weak and there is little understanding of mechanism. Aging is by far the greatest risk factor for BC, and there are specific forms of inherited genetic risk that are well-understood to cause BC. We propose that bringing focus to the biology underlying these forms of risk will illuminate biological mechanisms of BC susceptibility.

Protocol for computationally evaluating the loss of stoichiometry and coordinated expression of proteins.

Dysregulation of the transcriptional or translational machinery can alter the stoichiometry of multiprotein complexes and occurs in natural processes such as aging. Loss of stoichiometry has been shown to alter protein complex functions. We provide a protocol and associated code that use omics data to quantify these stoichiometric changes via statistical dispersion utilizing the interquartile range of expression values per grouping variable. This descriptive statistical approach enables the quantification of stoichiometry changes without additional data acquisition. For complete details on the use and execution of this protocol, please refer to Hinz et al. (2021).

High-Throughput Microenvironment Microarray (MEMA) High-Resolution Imaging.

The interaction between cells and their surrounding microenvironment has a crucial role in determining cell fate. In many pathological conditions, the microenvironment drives disease progression as well as therapeutic resistance. A number of challenges arise for researchers examining these cell-microenvironment interactions: (1) Tissue microenvironments are combinatorial and dynamic systems, and in pathological situations like cancer, microenvironments become infamously chaotic and highly heterogeneous. (2) Cells exhibit heterogeneous phenotypes, and even rare cell subpopulations can have a substantial role in tissue homeostasis and disease progression. This chapter discusses technical aspects relevant to dissecting cell-microenvironment interaction using the Microenvironment Microarray (MEMA) platform, which is a cell-based functional high-throughput screening of interactions between cells and combinatorial microenvironments at the single-cell level. MEMA provides insights into how cell phenotype and function is elicited by microenvironmental components. In this chapter, we describe automating a high-throughput and high-resolution imaging pipeline for single-cell-resolution analysis.

NCI's publication affiliation conundrum: Reframing innovation to incentivize an equitable path for advocate representation.

Advocacy engagement has been at the forefront of National Cancer Institute (NCI) efforts to advance scientific discoveries and transform medical interventions. Nonetheless, the journey for advocates has been uneven. Case in Point: NCI publication affiliation rules of engagement pose unique equity challenges while raising questions about structural representation in biomedical research. Abiding by the core rationale that publication affiliation should be tailored to employment status, the NCI has systematically denied research advocate volunteers the opportunity to specifically list NCI as an institutional affiliation on academic publications. Unpacking advocate NCI publication affiliation restrictions and its links with advocacy heritage preservation and convergent science goals poses unique diversity, equity, and inclusion challenges and opportunities. Improving the quality of structural representation in biomedical research requires new theories of action and flexible planning to advance, promote and build capacity for strategic advocacy inclusion and equity within publication affiliation initiatives. Here we highlight several opportunities for how leadership might formulate a radically different vision for NCI's approach. This perspective interrogates the best way forward for ensuring that biomedical employee and volunteer advocate workforce publication affiliation intersections are characterized by increased creativity and representation parity. Imbuing the scientist and clinical researcher archetype with social dimensions, we join NCI critical thinkers in urging employees, funded academics, and volunteer citizen scientists to collectively assume the role as paladins of science and integrity who view the triumphs of making a difference in science alongside the social responsibility of promoting transdisciplinary professionalism and the democratization of science.

Evaluating sources of technical variability in the mechano-node-pore sensing pipeline and their effect on the reproducibility of single-cell mechanical phenotyping.

Cellular mechanical properties can reveal physiologically relevant characteristics in many cell types, and several groups have developed microfluidics-based platforms to perform high-throughput single-cell mechanical testing. However, prior work has performed only limited characterization of these platforms' technical variability and reproducibility. Here, we evaluate the repeatability performance of mechano-node-pore sensing, a single-cell mechanical phenotyping platform developed by our research group. We measured the degree to which device-to-device variability and semi-manual data processing affected this platform's measurements of single-cell mechanical properties. We demonstrated high repeatability across the entire technology pipeline even for novice users. We then compared results from identical mechano-node-pore sensing experiments performed by researchers in two different laboratories with different analytical instruments, demonstrating that the mechanical testing results from these two locations are in agreement. Our findings quantify the expectation of technical variability in mechano-node-pore sensing even in minimally experienced hands. Most importantly, we find that the repeatability performance we measured is fully sufficient for interpreting biologically relevant single-cell mechanical measurements with high confidence.

Deep proteome profiling of human mammary epithelia at lineage and age resolution.

Age is the major risk factor in most carcinomas, yet little is known about how proteomes change with age in any human epithelium. We present comprehensive proteomes comprised of >9,000 total proteins and >15,000 phosphopeptides from normal primary human mammary epithelia at lineage resolution from ten women ranging in age from 19 to 68 years. Data were quality controlled and results were biologically validated with cell-based assays. Age-dependent protein signatures were identified using differential expression analyses and weighted protein co-expression network analyses. Upregulation of basal markers in luminal cells, including KRT14 and AXL, were a prominent consequence of aging. PEAK1 was identified as an age-dependent signaling kinase in luminal cells, which revealed a potential age-dependent vulnerability for targeted ablation. Correlation analyses between transcriptome and proteome revealed age-associated loss of proteostasis regulation. Age-dependent proteome changes in the breast epithelium identified heretofore unknown potential therapeutic targets for reducing breast cancer susceptibility.

Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression.

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age.

Breast-Specific Molecular Clocks Comprised of ELF5 Expression and Promoter Methylation Identify Individuals Susceptible to Cancer Initiation.

A robust breast cancer prevention strategy requires risk assessment biomarkers for early detection. We show that expression of ELF5, a transcription factor critical for normal mammary development, is downregulated in mammary luminal epithelia with age. DNA methylation of the ELF5 promoter is negatively correlated with expression in an age-dependent manner. Both ELF5 methylation and gene expression were used to build biological clocks to estimate chronological ages of mammary epithelia. ELF5 clock-based estimates of biological age in luminal epithelia from average-risk women were within three years of chronological age. Biological ages of breast epithelia from BRCA1 or BRCA2 mutation carriers, who were high risk for developing breast cancer, suggested they were accelerated by two decades relative to chronological age. The ELF5 DNA methylation clock had better performance at predicting biological age in luminal epithelial cells as compared with two other epigenetic clocks based on whole tissues. We propose that the changes in ELF5 expression or ELF5-proximal DNA methylation in luminal epithelia are emergent properties of at-risk breast tissue and constitute breast-specific biological clocks. PREVENTION RELEVANCE: ELF5 expression or DNA methylation level at the ELF5 promoter region can be used as breast-specific biological clocks to identify women at higher than average risk of breast cancer.

Volume-constrained microcontainers enable myoepithelial functional differentiation in highly parallel mammary organoid culture.

A long-standing constraint on organoid culture is the need to add exogenous substances to provide hydrogel matrix, which limits the study of fully human or fully native organoids. This paper introduces an approach to culture reconstituted mammary organoids without the impediment of exogenous matrix. We enclose organoids in nanoliter-scale, topologically enclosed, fluid compartments surrounded by agar. Organoids cultured in these "microcontainers" appear to secrete enough extracellular matrix to yield a self-sufficient microenvironment without exogenous supplements. In microcontainers, mammary organoids exhibit contractility and a high-level, physiological, myoepithelial (MEP) behavior that has not been previously reported in reconstituted organoids. The presence of contractility suggests that microcontainers elicit MEP functional differentiation, an important milestone. Microcontainers yield thousands of substantially identical and individually trackable organoids within a single culture vessel, enabling longitudinal studies and statistically powerful experiments, such as the evaluation of small effect sizes. Microcontainers open new doors for researchers who rely on organoid models.

Integration of mechanical and ECM microenvironment signals in the determination of cancer stem cell states.

PURPOSE OF REVIEW: Cancer stem cells (CSCs) are increasingly understood to play a central role in tumor progression. Growing evidence implicates tumor microenvironments as a source of signals that regulate or even impose CSC states on tumor cells. This review explores points of integration for microenvironment-derived signals that are thought to regulate CSCs in carcinomas. RECENT FINDINGS: CSC states are directly regulated by the mechanical properties and extra cellular matrix (ECM) composition of tumor microenvironments that promote CSC growth and survival, which may explain some modes of therapeutic resistance. CSCs sense mechanical forces and ECM composition through integrins and other cell surface receptors, which then activate a number of intracellular signaling pathways. The relevant signaling events are dynamic and context-dependent. SUMMARY: CSCs are thought to drive cancer metastases and therapeutic resistance. Cells that are in CSC states and more differentiated states appear to be reversible and conditional upon the components of the tumor microenvironment. Signals imposed by tumor microenvironment are of a combinatorial nature, ultimately representing the integration of multiple physical and chemical signals. Comprehensive understanding of the tumor microenvironment-imposed signaling that maintains cells in CSC states may guide future therapeutic interventions.

Evidence for accelerated aging in mammary epithelia of women carrying germline BRCA1 or BRCA2 mutations.

During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are susceptible to cancer initiation. In the present study, we show that histologically normal breast tissue from younger women who are susceptible to breast cancer, as a result of harboring a germline mutation in BRCA1, BRCA2 or PALB2 genes, exhibits hallmarks of accelerated aging. These include proportionately increased luminal epithelial cells that acquired myoepithelial markers, decreased proportions of myoepithelial cells and a basal differentiation bias or failure of differentiation of cKit+ progenitors. High-risk luminal and myoepithelial cells are transcriptionally enriched for genes of the opposite lineage, inflammatory- and cancer-related pathways. We have identified breast-aging hallmarks that reflect a convergent biology of cancer susceptibility, regardless of the specific underlying genetic or age-dependent risk or the associated breast cancer subtype.

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer.

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl + cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.