Interleukin-8: a tumor-agnostic biomarker integrating cancer biology and host response across solid tumors.

Endometrial stromal cells reportedly have a role in the initial invasion of endometrial tissue into the peritoneum. Hepatocyte growth factor (HGF), which is a ligand for the c-met protooncogene product (Met), stimulates proliferation and invasion of a large number of cells. In this study we investigated the role of the HGF/Met system in the pathogenesis of endometriosis. HGF concentrations in the peritoneal fluid of patients with endometriosis were significantly higher than in those without endometriosis and correlated positively with revised American Society of Reproductive Medicine scores. We showed that the peritoneum and endometriotic stromal cells may be major sources of HGF in peritoneal fluid. Endometrial and endometriotic stromal cells expressed the Met receptor, which was activated by endogenous and exogenous HGF. HGF enhanced stromal cell proliferation and invasion. We also demonstrated that the HGF-stimulated stromal cell invasion was due in part to the induction of urokinase-type plasminogen activator, a member of the extracellular proteolysis system. In conclusion, the HGF/Met system is involved in the pathogenesis of endometriosis by promoting stromal cell proliferation and invasion of shed endometria and endometrial lesions via autocrine and paracrine pathways.

Endometriosis is a debilitating disease affects up to 20% of reproductive age women characterized by the presence of functional endometrial glandular epithelium and stroma out side the uterine cavity. The pathophysiology of endometriosis remains as enigma in reproductive medicine. Accumulating evidence indicates that steroids, growth factors, and cytokines, and prostaglandins may promote establishment and maintenance of endometriosis. Treatment options include oral contraceptives, aromatase inhibitors, androgenic agents, and gonadotrophin releasing hormone analogues and nonsteroidal anti-inflammatory drugs and surgical removal. But the recurrence rate is up to 70% after cessation of therapy. Prostaglandin E2 (PGE2) promotes cell proliferation, migration, invasion, angiogenesis, anti-apoptosis, pain and immunomodulation. PGE2 mediated effects are primarily mediated through G-protein coupled membrane receptors designated EP that includes EP1, EP2, EP3 and EP4. EP2 and EP4 receptors are coupled to adenyl cyclase generating cAMP that in turn activates the protein kinase A signaling pathway. EP1 receptor is coupled to phospholipase C generating two second messengers, inositol triphosphate (IP3) involved in the liberation of intracellular calcium (Ca2+), and diacyl glycerol an activator of protein kinase C. There are several EP3 isoforms exhibiting a wide range of actions from inhibition of cAMP production to increases in Ca2+ and IP3. In this study, we used immortalized human endometriotic epithelial cells (11-Z, 12-Z, 49-Z, and 108-Z) and stromal cells (22-B) as in vitro models to unravel PGE2 signaling in pathophysiology of endometriosis in human. Normal endometrial epithelial and stromal cells were used as control. We investigated: (1) PGE2 production; (2) expression profiles of EP1, EP2, EP3, and EP4 receptors and (3) role of EP receptors in endometriotic cell proliferation, migration and invasion, and unraveled the underlined mechanisms. We used RT-PCR, western blot, zymography, matrigel migration and invasion assays, and ELISA. Endometriotic cells produced 8–10 fold more PGE2 than normal endometrial cells at basal condition. EP2 and EP4 receptor mRNAs and proteins were abundantly expressed whereas EP1 and EP3 receptor mRNAs and proteins are barely detectable in endometriotic epithelial and stromal cells. Inhibition of either EP2 or EP4 decreased (60%) the endometriotic epithelial and stromal cell proliferation, migration and invasion. Combined inhibition of EP2 and EP4 synergistically decreased (80–85%) endometriotic epithelial and stromal cell proliferation, migration and invasion. Inhibition of EP1 receptor did not have any effect. Inhibition of EP2 and/or EP4 decreased the expression and phosphorylation of Bcl2 protein. Inhibition of EP2 and/or EP4 decreased MMP2 and MMP9 activities. These results suggest that EP2 and/or EP4 mediated cell proliferation and invasion might be mediated respectively through Bcl2 and MMP2 and MMP9 pathways in endometriotic epithelial and stromal cells. Although endometriosis is not a malignancy its pathophysiology seems to be analogous to the mechanisms of cancer metastasis. In conclusion, these endometriotic epithelial and stromal cells can be used as ideal model to study the cellcell communications and interactions in the establishment and maintenance of endometriosis in human. Targeting PGE2 receptors and downstream signaling might be a novel therapy for endometriosis in human. (poster)

Estradiol and tamoxifen enhance invasion of endometrial stromal cells in a three-dimensional coculture model of adenomyosis

Factors that control recruitment of theca cells from ovarian stromal-interstitial cells are important for early follicle development in the ovary. During recruitment, theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. Recruitment of theca cells from ovarian stromal stem cells is proposed to involve cellular proliferation, as well as induction of theca cell-specific functional markers. Previously, the speculation was made that a granulosa cell-derived "theca cell organizer" is involved in theca cell recruitment. Granulosa cells have been shown to produce kit-ligand/stem cell factor (KL). KL is known to promote stem cell proliferation and differentiation in a number of tissues. Therefore, the hypothesis was tested in the current study that granulosa cell-derived KL may help recruit theca cells from undifferentiated stromal stem cells during early follicle development. The actions of KL were examined using adult bovine ovarian fragment organ culture and isolated ovarian stromal-interstitial cells. In organ culture KL significantly increased the number of theca cell layers around primary follicles. Experiments using purified stromal-interstitial cell cultures showed that KL stimulated ovarian stromal cell proliferation in a dose-dependent manner. Stromal cell differentiation into theca cells was analyzed by the induction of theca cell functional markers (i.e., androstenedione and progesterone production). Bovine ovarian stromal cells produced low levels of androstenedione (5-40 ng/microg DNA) and progesterone (5-30 ng/microg DNA) in vitro that were approximately 20-fold lower than theca cells under similar conditions. Treatment with KL did not affect ovarian stromal cell androstenedione or progesterone production. Interestingly, hormones such as estrogen and hCG did stimulate stromal cell steroid production. The results in this study suggest that granulosa cell-derived KL appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles. KL directly stimulated ovarian stromal cell proliferation but alone did not induce functional differentiation (i.e., high steroid production). Therefore, KL is proposed to promote early follicle development by inducing proliferation and organization of stromal stem cells around small follicles. Observations suggest that KL may act as a granulosa-derived "theca cell organizer" to promote stem cell recruitment of ovarian stromal cells in a manner similar to the way that KL promotes hematopoietic and lymphoid stem cells in bone marrow and the thymus.

Objective This study aimed to analyze the effect of low-molecular-weight heparin (LMWH) on the decidualization of stromal cells in early pregnancy and explore the effect of LMWH on pregnancy outcomes. Methods Recurrent spontaneous abortion (RSA) mouse model (CBA/J × DBA/2) and normal pregnant mouse model (CBA/J × BALB/c) were established. The female mice were checked for a mucus plug twice daily to identify a potential pregnancy. When a mucus plug was found, conception was considered to have occurred 12 h previously. The pregnant mice were divided randomly into a normal pregnancy control group, an RSA model group, and an RSA + LMWH experimental group (n = 10 mice in each group). Halfway through the 12th day of pregnancy, the embryonic loss of the mice was observed; a real-time quantitative polymerase chain reaction was used to detect the messenger ribonucleic acid (mRNA) expressions of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) in the decidua of the mice. Additionally, the decidual tissues of patients with RSA and those of normal women in early pregnancy who required artificial abortion were collected and divided into an RSA group and a control group. Decidual stromal cells were isolated and cultured to compare cell proliferation between the two groups, and cellular migration and invasion were detected by membrane stromal cells. Western blotting was used to detect the protein expressions of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase- (MMP) 2, and MMP-7 in stromal cells treated with LMWH. Results Compared with the RSA group, LMWH significantly reduced the pregnancy loss rate in the RSA mice (p < 0.05). Compared with the RSA group, the LMWH + RSA group had significantly higher expression levels of PRL and IGFBP1 mRNA (p < 0.01). LMWH promoted the proliferation, migration, and invasion of human decidual stromal cells; compared with the control group, the expression levels of MMP-2, MMP-7, cyclin D1, and PCNA proteins in the decidual stromal cells of the LMWH group increased (p < 0.05). Conclusions The use of LMWH can improve pregnancy outcomes by enhancing the proliferation and migration of stromal cells in early pregnancy and the decidualization of stromal cells.

Interleukin-8 gene and protein expression are up-regulated by interleukin-1β in normal human ovarian cells and a granulosa tumor cell line

The present study assessed the diagnostic and prognostic significance of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for suspected intrathoracic metastasis after HNC treatment. A retrospective analysis was conducted on 75 patients with a prior history of head and neck cancer treatment who underwent EBUS-TBNA for suspected intrathoracic metastases between March 2012 and December 2021. A total of 126 targeted lesions, including 107 mediastinal/hilar lymph nodes and 19 intrapulmonary/mediastinal masses, were sampled. The metastatic head and neck cancer (HNC) cases detected by EBUS-TBNA consisted of nasopharyngeal carcinoma (n = 24), oropharyngeal carcinoma (n = 3), hypopharynx carcinoma (n = 6), laryngeal carcinoma (n = 6), and oral cavity carcinoma (n = 6). Cases with negative EBUS-TBNA results consisted of tuberculosis (n = 9), sarcoidosis (n = 3), anthracosis (n = 9), and reactive lymphadenitis (n = 9). Six false-negative cases were found among the 75 patients with suspected intrathoracic metastases. The diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the EBUS-TBNA procedure for metastatic HNC were 88.2, 100.0, 100.0, 80, and 92.0%, respectively. The diagnosis of HNC intrathoracic metastasis by EBUS-TBNA correlated with an adverse prognosis in terms of overall survival (OS) (P = .008). The log-rank univariate analysis and Cox regression multivariate analysis results indicated that the detection of metastatic HNC through EBUS-TBNA was a significant independent prognostic factor for patients with HNC who had received prior treatment. Endobronchial ultrasound-guided transbronchial needle aspiration is a safe, effective, and minimally invasive procedure for assessing suspected intrathoracic metastasis in HNC patients after treatment. The intrathoracic metastasis detected by EBUS-TBNA has crucial prognostic significance in previously treated HNC patients.

Enhanced invasion of stromal cells from adenomyosis in a three-dimensional coculture model is augmented by the presence of myocytes from affected uteri

Assays to examine lymphocyte invasion.

Analysis of stromal gene expression for the identification of prognostic and predictive molecular markers in cancer therapy

Pyrvinium pamoate inhibits proliferation and invasion of human endometriotic stromal cells.

ObjectiveEndometriosis is an estrogen-dependent chronic disease. The abnormal proliferation and invasion of ectopic stromal cells (ESCs) are important manifestations of endometriosis, and it is necessary to find safer and more effective treatments. Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (UC-MSCs) have been shown to be promising for the treatment of many diseases, except endometriosis. The main purpose of this study was to explore the effect of EVs derived from UC-MSCs on ESCs and evaluate the therapeutic value of EVs on endometriosis.Study DesignFollowing the successful culture and identification of UC-MSCs, we collected the medium of UC-MSCs and extracted EVs by ultracentrifugation. Then, 120 μg/mL EVs were used to stimulate ESCs, which were collected to evaluate cell proliferation and invasion and expression of the estrogen-related proteins steroidogenic factor-1 (SF-1), estrogen receptors β (ERβ), and aromatase.ResultsCompared with the control group treated with isodose phosphate buffered saline (PBS), 120 μg/mL EVs exposure significantly decreased the expression of cyclin D1 (mRNA: n = 6, P = 0.02; protein: n = 6, P = 0.000) and matrix metalloproteinase (MMP) 9 (mRNA: n = 6, P = 0.04; protein: n = 6, P = 0.000) of ESCs, which were consistent with Cell Counting Kit-8(CCK-8) results (day 0: NC: 0.29 ± 0.04, 120 μg/mL EVs: 0.28 ± 0.04; day 1: NC: 0.42 ± 0.08, 120 μg/mL EVs: 0.32 ± 0.01; day 2: NC: 0.64 ± 0.07, 120 μg/mL EVs: 0.50 ± 0.05, P = 0.000; day 3: NC: 0.82 ± 0.09, 120 μg/mL EVs: 0.65 ± 0.07, P = 0.000; day 4: NC: 0.95 ± 0.11, 120 μg/mL EVs: 0.76 ± 0.07, P = 0.012; n = 6) and Transwell experiments (n = 6, P = 0.000). In addition, the expression of SF-1 (encoded by NR5A1; mRNA: n = 6, P = 0.000; protein: n = 6, P = 0.000), ERβ (encoded by ESR2; mRNA: n = 6, P = 0.000; protein: n = 6, P = 0.000), and aromatase (encoded by CYP19A1; mRNA: n = 6, P = 0.04; protein: n = 6, P = 0.000) in ESCs decreased significantly.ConclusionTaken together, the results show that 120 μg/mL EVs derived from UC-MSCs can effectively inhibit the proliferation and invasion of ESCs, as well as their expression of SF-1, ERβ and aromatase, and thus may lead to the alleviation of endometriosis.

Endometriosis (EM) is a multifactorial and debilitating chronic benign gynecological disease, but the pathogenesis of the disease is not completely understood. Dysregulated expression of microRNAs (miRNA/miR) is associated with the etiology of EM due to their role in regulating endometrial stromal cell proliferation and invasion. The present study aimed to identify the functions and mechanisms underlying miR-143-3p in EM. To explore the role of miR-143-3p in EM, functional miRNAs were analyzed via bioinformatics analysis. miR-143-3p expression levels in endometriotic stromal cells (ESCs) and normal endometrial stromal cells (NESCs) were measured via reverse transcription-quantitative PCR. The role of miR-143-3p in regulating ESC proliferation and invasion was assessed by performing Cell Counting Kit-8 and Transwell assays, respectively. miR-143-3p expression was significantly upregulated in ESCs compared with NESCs. Functionally, miR-143-3p overexpression inhibited ESC proliferation and invasion, whereas miR-143-3p knockdown promoted ESC proliferation and invasion. Moreover, miR-143-3p inhibited autophagy activation in ESCs, as indicated by decreased green puncta, which represented autophagic vacuoles, decreased microtubule associated protein 1 light chain 3α expression and increased p62 expression in the miR-143-4p mimic group compared with the control group. Moreover, compared with the control group, miR-143-3p overexpression significantly decreased the expression levels of autophagy-related 2B (ATG2B), a newly identified target gene of miR-143-3p, in ESCs. ATG2B overexpression reversed miR-143-3p overexpression-mediated inhibition of ESC proliferation and invasion. Collectively, the results of the present study suggested that miR-143-3p inhibited EM progression, thus providing a novel target for the development of therapeutic agents against EM.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Cold exposure triggers neogenesis in classic interscapular brown adipose tissue (iBAT) that involves activation of β1-adrenergic receptors, proliferation of PDGFRA+ adipose tissue stromal cells (ASCs), and recruitment of immune cells whose phenotypes are presently unknown. Single-cell RNA-sequencing (scRNA-seq) in mice identified three ASC subpopulations that occupied distinct tissue locations. Of these, interstitial ASC1 were found to be direct precursors of new brown adipocytes (BAs). Surprisingly, knockout of β1-adrenergic receptors in ASCs did not prevent cold-induced neogenesis, whereas pharmacological activation of the β3-adrenergic receptor on BAs was sufficient, suggesting that signals derived from mature BAs indirectly trigger ASC proliferation and differentiation. In this regard, cold exposure induced the delayed appearance of multiple macrophage and dendritic cell populations whose recruitment strongly correlated with the onset and magnitude of neogenesis across diverse experimental conditions. High-resolution immunofluorescence and single-molecule fluorescence in situ hybridization demonstrated that cold-induced neogenesis involves dynamic interactions between ASC1 and recruited immune cells that occur on the micrometer scale in distinct tissue regions. Our results indicate that neogenesis is not a reflexive response of progenitors to β-adrenergic signaling, but rather is a complex adaptive response to elevated metabolic demand within brown adipocytes. Editor's evaluation This study elucidates transcriptional profiles of the stromal vascular fraction of murine brown adipose tissue in the context of thermogenic stimulation. The authors combine systems and reductionist approaches to show the reliance of mature brown adipocytes on adrenergic activation to indirectly stimulate progenitor proliferation and differentiation. This timely work will provide beneficial data for public use and further resolve the complexities underlying brown adipose physiology. https://doi.org/10.7554/eLife.80167.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Brown adipose tissue (BAT) is a specialized organ that is the dominant site of non-shivering thermogenesis in neonatal mammals (Dawkins and Hull, 1964). While critical for regulation of neonatal body temperature, adult mammals, including humans, also have metabolically active BAT (Cypess et al., 2009; Lee et al., 2013b; Sacks and Symonds, 2013). Importantly, adult BAT mass and function are associated with a healthier metabolic profile in both rodents and humans (Becher et al., 2021; Wibmer et al., 2021; Herz et al., 2021). Although the direct mechanisms have not been fully elucidated, BAT abundance and activity is associated with higher energy expenditure, altered secretion of hormones (Chondronikola et al., 2016; Géloën et al., 1988; Hanssen et al., 2015; Lee et al., 2015; Villarroya et al., 2017; Villarroya et al., 2019), and reduced visceral fat mass (Herz et al., 2021). Therefore, a more thorough understanding of the processes that regulate the activation and physiological expansion of BAT could have important therapeutic implications for obesity-related metabolic disease. Cold exposure triggers BAT neogenesis and is an important adaptive means to increase the capacity for non-shivering thermogenesis in rodents (Bukowiecki et al., 1982; Bukowiecki et al., 1986; Foster and Frydman, 1978; Nedergaard, 1982; Nedergaard et al., 2019). Fate mapping studies in rats by Bukowiecki et al. strongly indicated that brown adipocytes (BAs) arise from interstitial cells that proliferate and differentiate during the first few days of cold exposure (Bukowiecki et al., 1986; Géloën et al., 1988). More recently, genetic lineage-tracing experiments from our lab demonstrated that most, if not all, BAs induced by cold arise from stromal cells that express the surface marker platelet-derived growth factor receptor alpha (PDGFRA). In addition to effects on brown adipogenesis, cold exposure also remodels the vasculature, recruits monocytes/macrophages, and triggers proliferation of uncharacterized population(s) of cells that are potentially important for proper BAT expansion and remodeling (Lee et al., 2015; Géloën et al., 1988). The complete cellular complexity of BAT is presently unknown. In this regard, recent single-cell transcriptional analysis indicates the existence of multiple stromal cell types in white adipose tissue (WAT) depots that express PDGFRA (Burl et al., 2018; Hepler et al., 2018; Merrick et al., 2019; Schwalie et al., 2018; Emont et al., 2022; Rondini and Granneman, 2020; Sárvári et al., 2021; Rondini et al., 2021), and that recruitment of brown/beige adipocytes in response to β3-adrenergic activation involves specific subsets of stromal cells and immune cells. In addition, we previously observed that cold triggers proliferation of cells expressing the myeloid cell surface marker F4/80, as well as uncharacterized population(s) of stromal cells (Lee et al., 2015). The simultaneous proliferation of multiple cell types within BAT suggests that cold-induced neogenesis involves the functional interaction of numerous cell types, as has been demonstrated for BA neogenesis in WAT (Lee and Granneman, 2012; Lee et al., 2016; Lee et al., 2013a; Lee et al., 2014). Curiously, cold-induced mitogenesis in rodent BAT only commences after more than 2 days of cold exposure (Bukowiecki et al., 1982; Bukowiecki et al., 1986; Hunt and Hunt, 1967; Lee et al., 2015) and is concentrated within specific regions of the tissue (Lee et al., 2015). The basis of the timing and location of cold-induced brown adipogenesis is not known, nor is the relationship, if any, to proliferating immune cells. Cold-induced neogenesis in BAT requires intact sympathetic activity that can be mimicked by systemic norepinephrine (NE) infusion in warm-adapted mice (Lee et al., 2015). Moreover, the effects of NE infusion are prevented by global knockout (KO) of the β1-adrenergic receptor (ADRB1) (Lee et al., 2015). ADRB1, but not β3-adrenergic receptors (ADRB3), are present on ASC, and in vitro experiments suggested that activation of preadipocyte ADRB1 mediates cold-induced BA neogenesis (Bronnikov et al., 1999; Bronnikov et al., 1992). However, ADRB1 is expressed on additional cell types, including mature BAs. Therefore, whether the effects of cold on ASC proliferation are directly mediated by ADRB1 signaling in ASCs or indirectly by metabolic activation of BAs remains unclear. To address these unresolved questions, we profiled global gene transcription in BAT to identify the duration of cold exposure that captures peak cellular proliferation and differentiation. We observed that the timing and magnitude of cold-induced neogenesis varied among individual mice, but was nonetheless strongly predicted by immune cell recruitment, independent of time in the cold. We then performed comprehensive single-cell RNA-sequencing (scRNA-seq) of mouse interscapular BAT (iBAT) stromal cells under control conditions, and at the peak of cold-induced proliferation and differentiation. scRNA-seq identified three major Pdgfra-expressing subtypes, one of which, termed adipose tissue stromal cell 1 (ASC1), was highly responsive to cold exposure and appears to exclusively contribute to preadipocyte proliferation and BA differentiation in vivo. scRNA-seq also revealed that cold exposure recruits several immune cell types to iBAT, including specialized lipid-handling macrophages and diverse populations of dendritic cells. Immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH) analysis demonstrated that cold exposure recruits immune cells to distinct tissue locations near the periphery of the iBAT and sites within the central parenchyma where BAs undergo efferocytosis. Importantly, we demonstrate by high resolution confocal imaging of genetic and chemical tracers that cold-induced adipogenic niches are created and resolved by the dynamic interactions of ASC1 and recruited immune cells. Results RNA-seq analysis of mouse iBAT during a cold exposure time course reveals activation of immune cells that correlates with proliferation To gain insight into the timing of cold-induced iBAT neogenesis, we first sequenced total tissue RNA to establish the time course and profile individual variation of cold-induced gene expression (Figure 1). K-means clustering of the top differentially expressed genes (DEGs) identified three distinct patterns of upregulated gene expression (Figure 1A). Acutely upregulated genes were induced within 6 hours of cold exposure, but then returned to control levels after chronic cold acclimation. Genes in this cluster are well-known targets of protein kinase A (PKA), including peroxisome proliferative activated receptor gamma coactivator 1 alpha (Ppargc1a), uncoupling protein 1 (Ucp1), nuclear receptor subfamily 4 group A member 1 (Nr4a1), and iodothyronine deiodinase 2 (Dio2) (Figure 1A). Chronically upregulated genes were induced following 1–2 days of cold exposure and remained elevated for the duration of the time in cold. This cluster included genes involved in lipid synthesis and oxidation, such as elongation of very long chain fatty acids-like 3 (Elovl3), fatty acid synthase (Fasn), and pyruvate dehydrogenase kinase isoenzyme 4 (Pdk4). The third cluster was interesting, as the gene upregulation was delayed and variable among individual mice. Genes in this cluster included proliferation markers (Birc5, Top2a), as well as various genes indicating innate immune activation and macrophage recruitment (Lgals3, Gpnmb, Trem2) (Figure 1A). Importantly, these variables were highly correlated and this relationship was largely independent of time spent in the cold (Figure 1B; p<0.001). These data confirm that cell proliferation and immune cell recruitment are connected and peak around the fourth day of cold exposure. Figure 1 Download asset Open asset Whole tissue RNA-sequencing analysis reveals proliferation correlates with immune cell recruitment during cold exposure. (A) Heatmap of K-means clustering of whole tissue RNA-sequencing data. Rows of the heatmap are genes, and columns are individual RNA-seq libraries. Red and green colors represent upregulation and downregulation, respectively. Analysis includes five replicates (individual mice) each from seven different cold exposure durations: room temperature controls, and 6 hr, 1 day, 2 days, 3 days, 4 days, or 5 days of cold exposure for a total of 35 RNA-seq libraries. (B) Correlation of specific variables in the RNA-seq data with Top2a expression by individual library. r2 values are displayed on the plot. iBAT scRNA-seq of total stromal cells identifies multiple stromal cell subtypes To investigate heterogeneity of iBAT stromal and immune cells, as well as gain insight into adipogenic differentiation in vivo, we performed scRNA-seq analysis of stromal cells isolated from iBAT (Figure 2—figure supplement 1A). Mice were adapted to room temperature (RT; 22–23 °C) or exposed to 6 °C for 4 days to induce iBAT neogenesis and capture the peak in cold-induced proliferation. iBAT stromal cells were isolated and cells were split into immune and non-immune cell populations by magnetic bead cell separation (MACS) with a lineage marker cocktail (Figure 2—figure supplement 1A). Individual single-cell libraries were prepared from two independent experiments of RT control and cold-exposed mice, yielding a total of eight single-cell libraries (Figure 2—figure supplement 1C). Sequencing data from these independent cohorts were merged and integrated, as detailed in Materials and methods (Figure 2—figure supplement 1B). Lineage marker negative (Lin-) cell libraries contained adipose stromal cells (Pdgfra+ ASCs), vascular cells, and proliferating/newly differentiating adipocytes (Figure 2A). Clustering of scRNA-seq data from control and cold-exposed mice identified eight major clusters, ranging from ~500 to 6,500 cells per cluster (Figure 2A, Supplementary file 1). Three of these clusters were defined as ASCs based on their expression of common mesenchymal stem cell markers Pdgfra, Cd34, and lymphocyte antigen 6 complex locus A (Ly6a, a.k.a. Sca1) (Figure 2—figure supplement 2A) that are often used for identification of adipocyte progenitors (Burl et al., 2018; Hepler et al., 2018; Merrick et al., 2019; Schwalie et al., 2018). ASCs clustered together at low resolution (resolution < or = 0.04), indicating they are more similar to each other than the other cell types present in the libraries (Figure 2—figure supplement 2B). scRNA-seq also identified a cluster of proliferating and differentiating ASCs (Prolif/Diff) (Figure 2A). The remaining clusters appeared to be a mixture of vascular endothelial cells (VEC), vascular smooth muscle cells (VSMC), Schwann cells, and a small (~5%) mixture of immune cells that were not excluded by MACs separation (Figure 2A). Separating data by treatment revealed that two cell clusters were unique to cold-exposed mice (circled; Figure 2B). One cluster retained numerous ASC markers like Pdgfra, whereas the other was largely Pdgfra negative and expressed high levels of markers of proliferation (e.g. Birc5) and adipocyte differentiation (e.g. Car3) (Figure 2—figure supplement 2A, Supplementary file 1). Figure 2 with 2 supplements see all Download asset Open asset scRNA-seq reveals ASC heterogeneity and maps adipogenic trajectories in mouse iBAT. (A) t-SNE plot of 28,691 lineage marker negative (Lin-) cells from iBAT of control mice and mice exposed to cold for four days. Clustering identified eight major clusters, highlighted in different colors. ASC, adipose tissue stromal cell; VEC, vascular endothelial cell; VSMC, vascular smooth muscle cells; Prolif/Diff, proliferating/differentiating cells. DEGs that define these clusters are in Supplementary file 1. (B) t-SNE plot from (A) split into cells from the separate treatments (CONTROL and COLD). Circles highlight cold-induced clusters. (C) t-SNE plot of 19,659 re-clustered ASC and Prolif/Diff cells from (A). The t-SNE plot and clustering identified six clusters. Prolif/Non-diff, proliferating/non-differentiating; Prolif/Diff, proliferating/differentiating. DEGs that define these clusters are in Supplementary file 2. (D) Violin plots of log2 expression levels of select marker genes for individual clusters from the CONTROL and COLD data presented in (C). (E) t-SNE plots displaying the log2 expression levels for genes involved in adipogenic differentiation from the CONTROL and COLD data presented in (C). To gain greater insight into the relationships among the various ASCs, we reclustered the ASC and Prolif/Diff populations at a higher resolution (Figure 2C, Supplementary file 2). This clustering resolved three distinct ASC cell types prominent in iBAT controls: ASC1-3 (Figure 2C, left). Genes that define these clusters were similar to the expression profiles of mouse PDGFRA+ ASC subtypes recently identified in various mouse fat depots (Burl et al., 2018; Dong et al., 2022; Hepler et al., 2018; Merrick et al., 2019; Rondini and Granneman, 2020; Schwalie et al., 2018). In control mice, ASC subtypes were distinguished by genes that encode extracellular matrix (ECM) and matrix remodeling proteins, and paracrine signaling proteins of the transforming growth factor beta superfamily. Thus, ASC1 selectively expressed collagen type V alpha 3 chain (Col5a3), C-X-C motif chemokine ligand 14 (Cxcl14), and the bone morphogenic protein (BMP)-binding endothelial regulator (Bmper) (Figure 2D). Cells in ASC2 expressed secreted protease inhibitor peptidase inhibitor 16 (Pi16), surface glycoprotein dipeptidyl peptidase 4 (Dpp4), and the ECM component fibronectin (Fbn1). ASC3 cells selectively expressed secreted ligand growth differentiation factor 10 (Gdf10), C-type lectin domain containing 11a (Clec11a), and fibulin1 (Fbln1) (Figure 2D). High-resolution clustering identified three additional cell clusters in cold-exposed mice (Figure 2C, right). All of these clusters expressed ASC1-specific markers; however, two of the clusters were primarily defined by genes for proliferation (Top2a, Birc5, Stmn1), and/or adipogenic differentiation (Pparg, Lpl, Nnat) (Figure 2D). This dramatic change in the expression profile of cells expressing ASC1 markers (e.g. Col5a3, Cxcl14, and Bmper) (Figure 2—figure supplement 2C) allowed for the characterization of these clusters as distinct expression states of the ASC1 subtype. For the purpose of exposition, we referred to these clusters as 'quiescent' and 'cold-activated' ASC1 from control and cold-exposed libraries, respectively (Figure 2C). Cold exposure had comparatively little impact on the profiles of ASC2 or ASC3 (Figure 2—figure supplement 2C). Examination of the two ASC1 expression states indicated that cold activation greatly reduced expression of genes involved in cholesterol biosynthesis (sterol biosynthetic process; GO:0016126, p=2.8E-10), cell adhesion (GO:0007155, p=4.9E-2) and extracellular matrix organization (GO:0030198, p=7.7E-7), and strongly induced expression of genes involved in immune system process (GO:0002376, p=2.2E-6), chemokine activity (GO:0008009, p=5.0E-3) and cell migration (GO:0016477, p=3.6E-4). In addition, ASC1 cells appeared highly poised for adipogenesis, expressing higher levels of the master adipocyte transcriptional regulator peroxisome proliferator activated receptor gamma (Pparg) and its target genes, such as lipoprotein lipase (Lpl) (Figure 2D). Notably, cells in the differentiating cluster selectively expressed the imprinted gene neuronatin (Nnat) that was transiently upregulated during differentiation and silenced in mature BAs (Figure 2D). iBAT scRNA-seq identifies cells undergoing cold-induced adipogenic differentiation Numerous ASC1-specific marker genes were co-expressed in the proliferating and differentiating clusters (Figure 2D), indicating that expression of these genes persists as cell differentiated into BAs. In contrast, none of the aforementioned ASC2 and ASC3 markers were expressed in these proliferating/differentiating clusters (Figure 2D). From these data, we concluded that cold-activated ASC1 are the immediate progenitors of new BAs induced by cold exposure. In addition, differentiating cells exhibited a clear trajectory along t-SNE2 that included loss of ASC marker expression (Pdgfra), transient proliferation (Birc5), and sequential upregulation of early (Cebpa) and late (Adipoq, Ucp1) markers of brown adipogenesis (Figure 2E). In contrast, the proliferating, non-differentiating cells that retained ASC1 marker expression did not appear to contribute to adipogenesis and might function to replenish the ASC1 population, as suggested by previous fate mapping studies (Lee et al., 2015). In summary, analysis of BAT ASCs and total stromal cell populations indicates that interstitial ASC1 cells are highly responsive to cold exposure and comprise most, if not all, BA progenitors during acute cold-induced neogenesis. Localizing ASC subtypes and adipogenic niches within the tissue microenvironment scRNA-seq does not retain the tissue architecture and spatial relationships among cell subtypes, yet previous work suggested that cold-induced neogenesis occurs in specific tissue regions (Lee et al., 2015). To address this issue, we used scRNA-seq data to identify subtype-specific mRNAs for spatial analysis by smFISH. scRNA-seq data indicate that ASC1-3 are distinguished by the differential expression of ECM proteins and paracrine signaling factors, suggesting that these cells have distinct functions in the tissue microenvironment. Therefore, we examined the spatial distribution of ASCs by smFISH using the subtype-specific markers Bmper (ASC1), Pi16 (ASC2), and Gdf10 (ASC3) (Figure 3A). Using smFISH in combination with Pdgfra-CreERT2 x LSL-tdTomato genetic tracing, we note that PDGFRA+ cells are found throughout the tissue, including the parenchyma and fascia. Using smFISH, we found that while ASC1 comprised the majority of the PDGFRA+ parenchymal interstitial cells, ASC2 were localized to the tissue fascia and surrounding large vessels, whereas ASC3 were predominately localized to areas surrounding vessels, but not capillaries (Figure 3B–C). Importantly, the interstitial location of ASC1 is consistent with the work of Bukowiecki et al., 1986 who, using electron microscopy, concluded that cold-induced brown adipocytes are derived from interstitial stromal cells. Figure 3 Download asset Open asset Pdgfra+ ASC subtypes occupy distinct areas of the tissue. (A) t-SNE plot of log2 gene expression from Pdgfra genetic tracing and smFISH probes in scRNA-seq data. t-SNE plot is ASCs from iBAT of control and cold-exposed mice, as in Figure 2C. (B) Representative image of fixed frozen iBAT from Pdgfra-CreERT2 x LSL-tdTomato reporter mice. (Left) Brightfield image shows gross tissue structures, including the tissue fascia, parenchyma, and large vessels. (Center) TdTomato (red) native fluorescence. (Right) Tissue was bleached and stained with smFISH probes Bmper (green), Pi16 (red), and Gdf10 (pink). Bmper distinguishes ASC1, Pi16 ASC2, and Gdf10 ASC3. Scale bar, 100 μm. (C) Representative images of control fixed frozen iBAT stained with smFISH probes Bmper (green), Pi16 (red), and Gdf10 (pink) taken at higher resolution. Associated brightfield image shows gross tissue structures. Nuclei were counterstained with DAPI. Scale bar, 100 μm. Mapping an adipogenic trajectory in situ scRNA-seq data indicated an adipogenic trajectory in ASC1 cells expression of stromal markers and while proliferation (Top2a, Birc5) and early differentiation markers and and the differentiation marker genes expressed within the adipogenic we found that was induced during early then silenced differentiation (Figure Thus, expression the transient of early differentiation. To whether we could an adipogenic trajectory in we used smFISH to for ASC1 cells, proliferating ASC1 cells, and early differentiating cells (Figure imaging a distribution of ASCs throughout control iBAT, with little of proliferation or active differentiation In contrast, cold exposure the appearance of numerous cells, as well as clusters of cells with and of Top2a (Figure Immunofluorescence analysis of protein cells containing lipid (Figure supplement 1A). Figure 4 with 1 supplement see all Download asset Open asset smFISH maps adipogenic trajectories and ASC1 as the direct precursors of new (A) t-SNE plot of log2 gene expression of smFISH probes in scRNA-seq data. t-SNE plot ASCs from iBAT of control and cold-exposed mice. (B) Representative low images of fixed frozen iBAT stained with smFISH probes for (green), Top2a (red), and (pink). Tissue is from control and cold-exposed mice, as Associated brightfield image shows gross tissue Scale bar, 100 μm. (C) image of fixed frozen cold-exposed mouse iBAT stained with smFISH probes (green), Top2a (red), and (pink). Nuclei were counterstained with DAPI. Scale bar, 10 μm. (D) image of fixed frozen cold-exposed mouse iBAT stained with smFISH probes Bmper (green), Pi16 (red), and (pink). Nuclei were counterstained with DAPI. Scale bar, μm. High-resolution confocal imaging for an adipogenic trajectory within a tissue Thus, we observed proliferating ASC1 to proliferating/differentiating ASC1, and more to differentiating ASC1 expression of proliferation the loss of the ASC marker as cells undergo early differentiation (Nnat) (Figure from scRNA-seq data, the majority of cells co-expressed the ASC1 marker but not the ASC2 marker Pi16 (Figure and Figure supplement 1B). ASC is indirectly adrenergic activation of BAs Cold-induced neogenesis in BAT requires intact sympathetic and can be mimicked by infusion of NE et al., Lee et al., 2015). global knockout of ADRB1 neogenesis induced by systemic NE infusion (Lee et al., 2015). In our iBAT single-cell data, is expressed in ASC1, proliferating/differentiating ASCs, and in immune cells (Figure supplement 1A). on these and previous we and that ADRB1 on mediates cold-induced proliferation. To this we used Pdgfra-CreERT2 to knockout in PDGFRA+ cells from mice and performed scRNA-seq analysis (Figure and Supplementary file mice were mice with Although scRNA-seq data indicates that is expressed in ASC1, but not in ASC2 or ASC3 (Figure treatment reduced expression in ASC1 by more than Figure Surprisingly, we found that of in ASC1 had on the of cold exposure to increase ASC1 in and mice) Supplementary file and Figure Figure 5 with 1 supplement see all Download asset Open asset is for cold-induced brown adipocyte to (A) t-SNE plot of cells from iBAT of or mice, at room temperature or exposed to cold for 4 days. Clustering identified cell ASC, adipose tissue stromal cell; VEC, vascular endothelial cell; VSMC, vascular smooth muscle cells; Prolif/Diff, proliferating/differentiating cells. DEGs that define these clusters are in Supplementary file (B) t-SNE plot from split into cells from or cells have expression (C) ASCs and Prolif/Diff cells from (A) and split into cells from control or cold-exposed libraries. The shows the of each cell type in the in the individual libraries. DEGs that define these clusters are in Supplementary file (D) analysis of proliferation and immune cell activation genes in iBAT of or mice at room temperature or exposed to cold for four days per are from analysis of data. (C) Correlation of specific genes in the data with Top2a expression by individual r2 values are displayed on the plot. also Figure data 1. Figure data 1 analysis of proliferation and immune cell activation genes in iBAT of or mice at room temperature or exposed to cold for 4 days per Figure supplement data 1. of the of in cells between and Download 1 Analysis of β-adrenergic receptor by (A) and for proliferating/differentiating cells (Prolif/Diff) and ASCs in the single-cell libraries. were by analysis between CONTROL and COLD libraries for the two (B) and for Prolif/Diff cells and ASCs in the CONTROL and single-cell libraries. were by analysis between CONTROL and libraries. of Prolif/Diff Cells of ASCs of of Prolif/Diff Cells of ASCs In a independent of mice, we found that knockout in ASCs had on cold-induced expression of proliferation at the whole tissue (Figure In addition, smFISH demonstrated that cold induced the appearance of differentiating in the of following Figure supplement these data demonstrate that ASC expression is not for cold-induced BA neogenesis. However, while expression did not impact neogenesis, of the total individual variation in proliferation marker expression (Birc5, across treatment was for by variation in markers of immune cell recruitment Figure BAs also express ADRB1 to its higher for is a major receptor for

Uteri from immature (21-day-old) and adult mice show different patterns of cell division in response to a physiological dose of 17 beta-estradiol. In the immature mouse uterus, estradiol increased stromal and epithelial cell proliferation by shortening the cell generation time (Tc). The epithelial Tc was reduced from 100 to 14.5 h; the stromal Tc was reduced to 16 h. In both cell types, the reduction in Tc was primarily due to a reduction of the G1 phase of the cell cycle. In the adult mouse uterus, estradiol stimulated epithelial but not stromal cell proliferation. Here, estradiol reduced the epithelial Tc from 86 to 13 h, mostly by reducing the G1 phase of the cell cycle. Therefore, estrogens stimulate uterine hyperplasia by selectively decreasing the G1 phase of the cell cycle in specific cell populations. At some point during reproductive tract maturation, uterine stromal cells lose their ability to divide in response to estrogen stimulation. A developmental study showed that in the intact mouse, the stromal cell population gradually lost its ability to divide in response to estrogen stimulation during days 22-52 after birth. In prepubertally ovariectomized mice, the stromal cell population showed a very low mitotic response to estrogen stimulation at all ages. Thus, an ovarian mechanism may regulate the change in stromal responsiveness to estrogen stimulation.