Exposure to pathogens throughout a lifetime influences immunity and organ function. Here, we explore how the systemic host-response to bacterial urinary tract infection (UTI) induces tissue-specific alterations to the mammary gland. Utilizing a combination of histological tissue analysis, single cell transcriptomics, and flow cytometry, we identify that mammary tissue from UTI-bearing mice displays collagen deposition, enlarged ductal structures, ductal hyperplasia with atypical epithelial transcriptomes and altered immune composition. Bacterial cells are absent in the mammary tissue and blood of UTI-bearing mice, therefore, alterations to the distal mammary tissue are mediated by the systemic host response to local infection. Furthermore, broad spectrum antibiotic treatment resolves the infection and restores mammary cellular and tissue homeostasis. Systemically, unresolved UTI correlates with increased plasma levels of the metalloproteinase inhibitor, TIMP1, which controls extracellular matrix remodeling and neutrophil function. Treatment of nulliparous and post-lactation UTI-bearing female mice with a TIMP1 neutralizing antibody, restores mammary tissue normal homeostasis, thus providing evidence for a link between the systemic host response during UTI and mammary gland alterations. Urinary tract infections (UTIs) can elicit systemic host-responses. Here the authors report that, in a mouse model, unresolved UTI is associated with alterations of the mammary tissue, including collagen deposition and hyperplasia.

During female adolescence and pregnancy, rising levels of hormones result in a cyclic source of signals that control the development of mammary tissue. While such alterations are well understood from a whole-gland perspective, the alterations that such hormones bring to organoid cultures derived from mammary glands have yet to be fully mapped. This is of special importance given that organoids are considered suitable systems to understand cross species breast development. Here we utilized single-cell transcriptional profiling to delineate responses of murine and human normal breast organoid systems to female hormones across evolutionary distinct species. Collectively, our study represents a molecular atlas of epithelial dynamics in response to estrogen and pregnancy hormones.

Abstract Chromatin remodeling controls transcriptional programs that drive mammary epithelial lineage commitment and cellular differentiation. This process is also important for the regulation of highly dynamic cellular states during mammary tumorigenesis, and perturbing the chromatin landscape of cancer cells can halt cellular and molecular mechanisms of mammary cancer development and progression. However, this paradigm assumes that all cancer cells that develop in the mammary tissue bear the same chromatin dependencies, and rely on similar programs throughout cancer progression. Here, we show that molecular dependencies regulated by the epigenetic factor BPTF control tumor development and progression across distinct mammary cancer cell states. With the utilization of classical models of mammary tumorigenesis, single cell transcriptional profiling, and organoid cultures, we demonstrate that BPTF loss results in the development of mammary tumors with a longer latency of ER•+ expression and tamoxifen sensitivity and the inhibition of lung metastasis development. While gain of ER• expression was associated with gain of luminal markers in Krt5+ BPTF KO mammary epithelial cells (MECs), metastasis-like signatures were significantly perturbed in cells with defined luminal fate. In addition, we detected a partial rescue of metastatic features via the activation of transcriptional programs controlled by TGF-•, thus indicating that BPTF sits atop a canonical metastatic pathway. Collectively, our studies illustrate how BPTF-specific chromatin remodeling inhibition induces phenotypes that differentially affect tumor development and progression, while allowing for the development of a murine model of ER•+ mammary tumorigenesis. Citation Format: Chen Chen, Michael F. Ciccone, Marygrace c. Trousdell, Deeptiman Chatterjee, Yixin Zhao, Steven M. Lewis, Dhivya Anandan, John E. Wilkinson, William C. K. Pomerantz, Adam Siepel, Camila O. dos Santos. Loss of BPTF restores estrogen-regulated programs and suppress metastatic development of mammary tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A079.

In proliferating neoplasms, microenvironment-derived selective pressures promote tumor heterogeneity by imparting diverse capacities for growth, differentiation, and invasion. However, what makes a tumor cell respond to signaling cues differently from a normal cell is not well understood. In the Drosophila ovarian follicle cells, apicobasal-polarity loss induces heterogeneous epithelial multilayering. When exacerbated by oncogenic-Notch expression, this multilayer displays an increased consistency in the occurrence of morphologically distinguishable cells adjacent to the polar follicle cells. Polar cells release the Jak/STAT ligand Unpaired (Upd), in response to which neighboring polarity-deficient cells exhibit a precursor-like transcriptomic state. Among the several regulons active in these cells, we could detect and further validate the expression of Snail family transcription factor Escargot (Esg). We also ascertain a similar relationship between Upd and Esg in normally developing ovaries, where establishment of polarity determines early follicular differentiation. Overall, our results indicate that epithelial-cell polarity acts as a gatekeeper against microenvironmental selective pressures that drive heterogeneity.

Developments in single-cell technology have considerably changed the way we study biology. Significant efforts have been made over the last few years to build comprehensive cell-type-specific transcriptomic atlases for a wide range of tissues in several model organisms in order to discover cell-type-specific markers and drivers of gene expression. One such tissue is the ovary of the fruit-fly Drosophila melanogaster, which is a popular model system with wide-ranging applications in the study of both development and disease. Three independent studies have recently produced comprehensive maps of cell-type-specific gene expression that describe both spatiotemporal regulation of the process of oogenesis and unique transcriptomic profiles of different cell types that constitute the ovary. In this chapter, we outlined the wet-lab protocol that was followed in our recent study for sample preparation and reanalyze the resultant dataset to discuss the benchmarks in data analysis, which are fundamental to comprehensive curation of the single-cell dataset representing the fly ovary.

Apicobasal cell polarity loss is a founding event in epithelial–mesenchymal transition and epithelial tumorigenesis, yet how pathological polarity loss links to plasticity remains largely unknown. To understand the mechanisms and mediators regulating plasticity upon polarity loss, we performed single-cell RNA sequencing of Drosophila ovaries, where inducing polarity-gene l(2)gl-knockdown (Lgl-KD) causes invasive multilayering of the follicular epithelia. Analyzing the integrated Lgl-KD and wildtype transcriptomes, we discovered the cells specific to the various discernible phenotypes and characterized the underlying gene expression. A genetic requirement of Keap1-Nrf2 signaling in promoting multilayer formation of Lgl-KD cells was further identified. Ectopic expression of Keap1 increased the volume of delaminated follicle cells that showed enhanced invasive behavior with significant changes to the cytoskeleton. Overall, our findings describe the comprehensive transcriptome of cells within the follicle cell tumor model at the single-cell resolution and identify a previously unappreciated link between Keap1-Nrf2 signaling and cell plasticity at early tumorigenesis.

ABSTRACT Apicobasal cell-polarity loss is a founding event in Epithelial-Mesenchymal Transition (EMT) and epithelial tumorigenesis, yet how pathological polarity loss induces plasticity changes remains largely unknown. To understand the mechanisms and mediators regulating plasticity upon polarity loss, we performed single-cell (sc) RNA sequencing of Drosophila ovaries, where inducing polarity-gene l(2)gl knockdown (Lgl-KD) causes invasive delamination of the follicular epithelia. Integrating Lgl-KD with the corresponding wild-type sc-transcriptome, we discovered clusters specific to various discernible phenotype-specific cell types and further characterized the regulons active in those cells. A genetic requirement of Keap1-Nrf2 signaling in promoting multilayer formation of Lgl-KD cells was further identified. Elevated expression of Keap1 increased the volume of delaminated follicle cells that undergo enhanced collective invasion via cytoskeletal remodeling. Overall, our findings describe the comprehensive transcriptome of the follicle-cell tumor model at the single-cell resolution and identify a previously unappreciated link between stress signaling and cell plasticity in early tumorigenesis.

Ploidy variation is a cancer hallmark and is frequently associated with poor prognosis in high-grade cancers. Using a Drosophila solid-tumor model where oncogenic Notch drives tumorigenesis in a transition-zone microenvironment in the salivary gland imaginal ring, we find that the tumor-initiating cells normally undergo endoreplication to become polyploid. Upregulation of Notch signaling, however, induces these polyploid transition-zone cells to re-enter mitosis and undergo tumorigenesis. Growth and progression of the transition-zone tumor are fueled by a combination of polyploid mitosis, endoreplication, and depolyploidization. Both polyploid mitosis and depolyploidization are error prone, resulting in chromosomal copy-number variation and polyaneuploidy. Comparative RNA-seq and epistasis analysis reveal that the DNA-damage response genes, also active during meiosis, are upregulated in these tumors and are required for the ploidy-reduction division. Together, these findings suggest that polyploidy and associated cell-cycle variants are critical for increased tumor-cell heterogeneity and genome instability during cancer progression.

Notch is a conserved developmental signaling pathway that is dysregulated in many cancer types, most often through constitutive activation. Tumor cells with nuclear accumulation of the active Notch receptor, NICD, generally exhibit enhanced survival while patients experience poorer outcomes. To understand the impact of NICD accumulation during tumorigenesis, we developed a tumor model using the Drosophila ovarian follicular epithelium. Using this system we demonstrated that NICD accumulation contributed to larger tumor growth, reduced apoptosis, increased nuclear size, and fewer incidents of DNA damage without altering ploidy. Using bulk RNA sequencing we identified key genes involved in both a pre- and post- tumor response to NICD accumulation. Among these are genes involved in regulating double-strand break repair, chromosome organization, metabolism, like raptor, which we experimentally validated contributes to early Notch-induced tumor growth. Finally, using single-cell RNA sequencing we identified follicle cell-specific targets in NICD-overexpressing cells which contribute to DNA repair and negative regulation of apoptosis. This valuable tumor model for nuclear NICD accumulation in adult Drosophila follicle cells has allowed us to better understand the specific contribution of nuclear NICD accumulation to cell survival in tumorigenesis and tumor progression.

Table of marker genes used in this study to identify cell types with selected references. (PDF)

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, we identified the transcriptional trajectory of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. We further identify expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to our identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types.

SYSTEMS TECHNOLOGY, the IEEE Control Systems Society publishes high-quality papers on technological advances in the design, realization, and operation of control systems.Submissions should emphasize novel contributions to the solution of control engineering problems arising in specific application areas.The official scope can be found at the following link: http://ieeexplore.ieee.org/xpl/aboutJournal.jsp?punumber=87#AimsScope.This TRANSACTIONS is published bimonthly.Three types of contributions are regularly considered:1) Papers-Presentation of significant research, development, or application of control systems technology.2) Brief Papers-Concise descriptions of a contribution to a specific aspect of design, realization, or operation of control systems technology.3) Letters-Significant remarks of interest to control systems engineers, and comments on previously published papers.In addition, special papers (tutorials, surveys, and perspectives on the current trends in control systems technology) are solicited.

Table of marker genes previously identified in different cell types with the associated references. Cells expressing 2 or more markers from different cell types were considered doublets and removed from the cluster (marked by X) to retain only high-quality cells. (PDF)

AbstractOogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modelled extensively using theDrosophilaovary. While different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic dataset for the adultDrosophilaovary and connected tissues. This dataset captures the entire transcriptional trajectory of the developing follicle cell population over time. Our findings provide detailed insight into processes such as cell-cycle switching, migration, symmetry breaking, nurse cell engulfment, egg-shell formation, and signaling during corpus luteum formation, marking a newly identified oogenesis-to-ovulation transition. Altogether, these findings provide a broad perspective on oogenesis at a single-cell resolution while revealing new genetic markers and fate-specific transcriptional signatures to facilitate future studies.

Cancer is a cumulative manifestation of several complicated disease states that affect multiple organs. Over the last few decades, the fruit fly Drosophila melanogaster, has become a successful model for studying human cancers. The genetic simplicity and vast arsenal of genetic tools available in Drosophila provides a unique opportunity to address questions regarding cancer initiation and progression that would be extremely challenging in other model systems. In this chapter we provide a historical overview of Drosophila as a model organism for cancer research, summarize the multitude of genetic tools available, offer a brief comparison between different model organisms and cell culture platforms used in cancer studies and briefly discuss some of the latest models and concepts in recent Drosophila cancer research.

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modelled extensively using the ovary. While different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic dataset for the adult ovary and connected tissues. This dataset captures the entire transcriptional trajectory of the developing follicle cell population over time. Our findings provide detailed insight into processes such as cell-cycle switching, migration, symmetry breaking, nurse cell engulfment, egg-shell formation, and signaling during corpus luteum formation, marking a newly identified oogenesis-to-ovulation transition. Altogether, these findings provide a broad perspective on oogenesis at a single-cell resolution while revealing new genetic markers and fate-specific transcriptional signatures to facilitate future studies.