Ovarian cycle stage critically affects 21-gene recurrence scores in Mmtv-Pymt mouse mammary tumours.

BACKGROUND: The Oncotype DX 21-gene Recurrence Score is predictive of adjuvant chemotherapy benefit for women with early-stage, estrogen receptor (ER)-positive, HER2-negative breast cancer. In premenopausal women, fluctuations in estrogen and progesterone during the menstrual cycle impact gene expression in hormone-responsive cancers. However, the extent to which menstrual cycling affects the Oncotype DX 21-gene signature remains unclear. Here, we investigate the impact of ovarian cycle stage on the 21-gene signature using a naturally cycling mouse model of breast cancer. METHODS: ER-positive mammary tumours were dissected from naturally cycling Mmtv-Pymt mice at either the estrus or diestrus phase of the ovarian cycle. The Oncotype DX 21-gene signature was assessed through quantitative real time-PCR, and a 21-gene experimental recurrence score analogous to the Oncotype DX Recurrence Score was calculated. RESULTS: Tumours collected at diestrus exhibited significant differences in expression of 6 Oncotype DX signature genes (Ki67, Ccnb1, Esr1, Erbb2, Grb7, Bag1; p ≤ 0.05) and a significant increase in 21-gene recurrence score (21.8 ± 2.4; mean ± SEM) compared to tumours dissected at estrus (15.5 ± 1.9; p = 0.03). Clustering analysis revealed a subgroup of tumours collected at diestrus characterised by increased expression of proliferation- (p < 0.001) and invasion-group (p = 0.01) genes, and increased 21-gene recurrence score (p = 0.01). No correlation between ER, PR, HER2, and KI67 protein abundance measured by Western blot and abundance of mRNA for the corresponding gene was observed, suggesting that gene expression is more susceptible to hormone-induced fluctuation compared to protein expression. CONCLUSIONS: Ovarian cycle stage at the time of tissue collection critically affects the 21-gene signature in Mmtv-Pymt murine mammary tumours. Further studies are required to determine whether Oncotype DX Recurrence Scores in women are similarly affected by menstrual cycle stage.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

PURPOSE: Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. METHODS: Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. RESULTS: In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. CONCLUSIONS: Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

Attenuated TGFB signalling in macrophages decreases susceptibility to DMBA-induced mammary cancer in mice.

BACKGROUND: Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates mammary gland development and cancer progression through endocrine, paracrine and autocrine mechanisms. TGFB1 also plays roles in tumour development and progression, and its increased expression is associated with an increased breast cancer risk. Macrophages are key target cells for TGFB1 action, also playing crucial roles in tumourigenesis. However, the precise role of TGFB-regulated macrophages in the mammary gland is unclear. This study investigated the effect of attenuated TGFB signalling in macrophages on mammary gland development and mammary cancer susceptibility in mice. METHODS: A transgenic mouse model was generated, wherein a dominant negative TGFB receptor is activated in macrophages, in turn attenuating the TGFB signalling pathway specifically in the macrophage population. The mammary glands were assessed for morphological changes through wholemount and H&E analysis, and the abundance and phenotype of macrophages were analysed through immunohistochemistry. Another cohort of mice received carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), and tumour development was monitored weekly. Human non-neoplastic breast tissue was also immunohistochemically assessed for latent TGFB1 and macrophage marker CD68. RESULTS: Attenuation of TGFB signalling resulted in an increase in the percentage of alveolar epithelium in the mammary gland at dioestrus and an increase in macrophage abundance. The phenotype of macrophages was also altered, with inflammatory macrophage markers iNOS and CCR7 increased by 110% and 40%, respectively. A significant decrease in DMBA-induced mammary tumour incidence and prolonged tumour-free survival in mice with attenuated TGFB signalling were observed. In human non-neoplastic breast tissue, there was a significant inverse relationship between latent TGFB1 protein and CD68-positive macrophages. CONCLUSIONS: TGFB acts on macrophage populations in the mammary gland to reduce their abundance and dampen the inflammatory phenotype. TGFB signalling in macrophages increases mammary cancer susceptibility potentially through suppression of immune surveillance activities of macrophages.

Interferon-gamma inhibits seminal plasma induction of colony-stimulating factor 2 in mouse and human reproductive tract epithelial cells.

Seminal fluid interacts with the female reproductive tract to initiate a permissive immune response that facilitates embryo implantation and pregnancy success. The immune-regulatory cytokine interferon-γ (IFNG), which can be elevated in seminal plasma, is associated with reduced fertility. Here, we investigated how IFNG influences the female immune response to seminal fluid. In human Ect1 cervical epithelial cells, IFNG added at physiologically relevant concentrations substantially impaired seminal plasma-induced synthesis of key cytokines colony-stimulating factor 2 (CSF2) and interleukin-6 (IL6). Seminal fluid-induced CSF2 synthesis was also suppressed in the uterus of mice in vivo, when IFNG was delivered transcervically 12 h after mating. Transforming growth factor B1 (TGFB1) is the major seminal fluid signaling factor which elicits CSF2 induction, and IFNG exhibited potent dose-dependent suppression of CSF2 synthesis induced by TGFB1 in murine uterine epithelial cells in vitro. Similarly, IFNG suppressed TGFB1-mediated CSF2 induction in Ect1 cells and human primary cervical epithelial cells; however, IL6 regulation by IFNG was independent of TGFB1. Quantitative PCR confirmed that CSF2 regulation by IFNG in Ect1 cells occurs at the gene transcription level, secondary to IFNG suppression of TGFBR2 encoding TGFB receptor 2. Conversely, TGFB1 suppressed IFNG receptor 1 and 2 genes IFNGR1 and IFNGR2. These data identify IFNG as a potent inhibitor of the TGFB-mediated seminal fluid interaction with relevant reproductive tract epithelia in mice and human. These findings raise the prospect that IFNG in the male partner's seminal fluid impairs immune adaptation for pregnancy following coitus in women.

Male Seminal Relaxin Contributes to Induction of the Post-mating Cytokine Response in the Female Mouse Uterus.

The hormone relaxin is important in female reproduction for embryo implantation, cardiovascular function, and during labor and lactation. Relaxin is also synthesized in males by organs of the male tract. We hypothesized that relaxin might be one component of seminal plasma responsible for eliciting the female cytokine response induced in the uterus at mating. When recombinant relaxin was injected into the uterus of wild-type (Rln+/+) mice at estrus, it evoked the production of Cxcl1 mRNA and its secreted protein product CXCL1 in four of eight animals. Mating experiments were then conducted using mice with a null mutation in the relaxin gene (Rln-/- mice). qRT-PCR analysis of mRNA expression in wild-type females showed diminished uterine expression of several cytokine and chemokine genes in the absence of male relaxin. Similar differences were also noted comparing Rln-/- and Rln+/+ females mated to wild-type males. Quantification of uterine luminal fluid cytokine content confirmed that male relaxin provokes the production of CXCL10 and CSF3 in Rln+/+ females. Differences were also seen comparing Rln-/- and Rln+/+ females mated with Rln-/- males for CXCL1, CSF3, and CCL5, implying that endogenous relaxin in females might prime the uterus to respond appropriately to seminal fluid at coitus. Finally, pan-leukocyte CD45 mRNA was increased in wild-type matings compared to other combinations, implying that male and female relaxin may trigger leukocyte expansion in the uterus. We conclude that male and/or female relaxin may be important in activating the uterine cytokine/chemokine network required to initiate maternal immune adaptation to pregnancy.

CCL2-driven inflammation increases mammary gland stromal density and cancer susceptibility in a transgenic mouse model.

BACKGROUND: Macrophages play diverse roles in mammary gland development and breast cancer. CC-chemokine ligand 2 (CCL2) is an inflammatory cytokine that recruits macrophages to sites of injury. Although CCL2 has been detected in human and mouse mammary epithelium, its role in regulating mammary gland development and cancer risk has not been explored. METHODS: Transgenic mice were generated wherein CCL2 is driven by the mammary epithelial cell-specific mouse mammary tumour virus 206 (MMTV) promoter. Estrous cycles were tracked in adult transgenic and non-transgenic FVB mice, and mammary glands collected at the four different stages of the cycle. Dissected mammary glands were assessed for cyclical morphological changes, proliferation and apoptosis of epithelium, macrophage abundance and collagen deposition, and mRNA encoding matrix remodelling enzymes. Another cohort of control and transgenic mice received carcinogen 7,12-Dimethylbenz(a)anthracene (DMBA) and tumour development was monitored weekly. CCL2 protein was also quantified in paired samples of human breast tissue with high and low mammographic density. RESULTS: Overexpression of CCL2 in the mammary epithelium resulted in an increased number of macrophages, increased density of stroma and collagen and elevated mRNA encoding matrix remodelling enzymes lysyl oxidase (LOX) and tissue inhibitor of matrix metalloproteinases (TIMP)3 compared to non-transgenic controls. Transgenic mice also exhibited increased susceptibility to development of DMBA-induced mammary tumours. In a paired sample cohort of human breast tissue, abundance of epithelial-cell-associated CCL2 was higher in breast tissue of high mammographic density compared to tissue of low mammographic density. CONCLUSIONS: Constitutive expression of CCL2 by the mouse mammary epithelium induces a state of low level chronic inflammation that increases stromal density and elevates cancer risk. We propose that CCL2-driven inflammation contributes to the increased risk of breast cancer observed in women with high mammographic density.

Human glandular organoid formation in murine engineering chambers after collagenase digestion and flow cytometry isolation of normal human breast tissue single cells.

Women with high mammographic density (MD) are at increased risk of breast cancer (BC) after adjustment for age and body mass index. We have developed a murine biochamber model in which both high MD (HMD) and low MD (LMD) tissue can be propagated. Here, we tested whether cells isolated by collagenase digestion and fluorescence-activated cell sorting (FACS) from normal breast can be reconstituted in our biochamber model, which would allow cell-specific manipulations to be tested. Fresh breast tissue was collected from women (n = 7) undergoing prophylactic mastectomy. The tissue underwent collagenase digestion overnight and, in some cases, additional FACS enrichment to obtain mature epithelial, luminal progenitor, mammary stem, and stromal cells. Cells were then transferred bilaterally into biochambers in SCID mice (n = 5-7) and incubated for 6 weeks, before harvesting for histological analyses, and immunohistochemical staining for cytokeratins (CK), vimentin, Ki-67, murine macrophages, and Cleaved Caspase-3. Biochambers inoculated with single cells after collagenase digestion or with flow cytometry contained glandular structures of human origin (human vimentin-positive), which expressed CK-14 and pan-CK, and were proliferating (Ki-67-positive). Glandular structures from the digested tissues were smaller than those in chambers seeded with finely chopped intact mammary tissue. Mouse macrophage infiltration was higher in the chambers arising from digested tissues. Pooled single cells and FACS fractionated cells were viable in the murine biochambers and formed proliferating glandular organoids of human origin. This is among the first report to demonstrate the success of formed human glandular organoids from isolated primary mammary cells in the murine biochamber model.

TLR4 Signaling Is a Major Mediator of the Female Tract Response to Seminal Fluid in Mice.

Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce expression of proinflammatory cytokines and chemokines, initiating immune tolerance mechanisms to facilitate pregnancy. TGFB cytokines are key signaling agents in seminal plasma but do not fully account for the female response to seminal fluid. We hypothesized that additional molecular pathways are utilized in seminal fluid signaling. Affymetrix microarray was employed to compare gene expression in the endometrium of mice 8 h after mating with either intact males or seminal fluid deficient (SVX/VAS) males. Bioinformatics analysis revealed TLR4 signaling as a strongly predicted upstream regulator activated by the differentially expressed genes and implicated TGFB signaling as a second key pathway. Quantitative PCR and microbead data confirmed that seminal fluid induces endometrial synthesis of several TLR4-regulated cytokines and chemokines, including CSF3, CXCL1, CXCL2, IL1A, IL6, LIF, and TNF. In primary uterine epithelial cells, CSF3, CXCL1, and CXCL2 were strongly induced by the TLR4 ligand LPS but suppressed by TGFB, while IL1A, TNF, and CSF2 were induced by both ligands. TLR4 was confirmed as essential for the full endometrial cytokine response using mice with a null mutation in Tlr4, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 contributes to seminal fluid modulation of the periconception immune environment. Activation of TLR4 signaling by microbial or endogenous components of seminal fluid is thus implicated as a key element of the female tract response to seminal fluid at the outset of pregnancy in mice.

Mouse models of mastitis - how physiological are they?

Lactation mastitis is a common, but poorly understood, inflammatory breast disease that is a significant health burden. A better understanding of the aetiology of mastitis is urgently required, and will assist in the development of improved prevention and treatment strategies in both human and animal species. Studies in mice have the potential to greatly assist in identifying new drug candidates for clinical trials, and in developing a better understanding of the disease. Mouse models of mastitis involve administration of a mastitis-inducing agent to the mammary gland usually during lactation to examine the host immune response, and progression through to resolution of the disease. There are important variations in the protocols of these mouse models that critically affect the conclusions that can be drawn from the research. Some protocols involve weaning of offspring at the time of mastitis induction, and there are variations in the mastitis-inducing agent and its carrier. Induction of mammary gland involution through weaning of offspring limits the capacity to study the disease in the context of a lactating mammary gland. Administration of live bacteria in an aqueous carrier can cause sepsis, restricting the physiological relevance of the model. Mouse model research should employ appropriately designed controls and closely monitor the health of the mice. In this commentary, we discuss the advantages and study design limitations of each mouse model, and highlight the potential for further development of physiologically relevant mouse models of mastitis.

Hormonal regulation of the cytokine microenvironment in the mammary gland.

The mammary gland is a unique organ that undergoes hormone-driven developmental changes over the course of the ovarian cycle during adult life. Macrophages play a role in regulating cellular turnover in the mammary gland and may affect cancer susceptibility. However, the immune microenvironment that regulates macrophage function has not been described. Hormonal regulation of the cytokine microenvironment across the ovarian cycle was explored using microbead multiplex assay for 15 cytokines in mammary glands from C57Bl/6 mice at different stages of the oestrous cycle, and in ovariectomised mice administered oestradiol and progesterone. The cytokines that were found to fluctuate over the course of the oestrous cycle were colony-stimulating factor (CSF)1, CSF2, interferon gamma (IFNG) and tumour necrosis factor alpha (TNFA), all of which were significantly elevated at oestrus compared with other phases. The concentration of serum progesterone during the oestrus phase negatively correlated with the abundance of cytokines CSF3, IL12p40, IFNG and leukaemia inhibitory factor (LIF). In ovariectomised mice, exogenous oestradiol administration increased mammary gland CSF1, CSF2, IFNG and LIF, compared with ovariectomised control mice. Progesterone administration together with oestradiol resulted in reduced CSF1, CSF3 and IFNG compared with oestradiol administration alone. This study suggests that the cytokine microenvironment in the mammary gland at the oestrus phase of the ovarian cycle is relatively pro-inflammatory compared with other stages of the cycle, and that the oestradiol-induced cytokine microenvironment is significantly attenuated by progesterone. A continuously fluctuating cytokine microenvironment in the mammary gland presumably regulates the phenotypes of resident leukocytes and may affect mammary gland cancer susceptibility.

Inflammatory mediators in mastitis and lactation insufficiency.

Mastitis is a common inflammatory disease during lactation that causes reduced milk supply. A growing body of evidence challenges the central role of pathogenic bacteria in mastitis, with disease severity associated with markers of inflammation rather than infection. Inflammation in the mammary gland may be triggered by microbe-associated molecular patterns (MAMPs) as well as danger-associated molecular patterns (DAMPs) binding to pattern recognition receptors such as the toll-like receptors (TLRs) on the surface of mammary epithelial cells and local immune cell populations. Activation of the TLR4 signalling pathway and downstream nuclear factor kappa B (NFkB) is critical to mediating local mammary gland inflammation and systemic immune responses in mouse models of mastitis. However, activation of NFkB also induces epithelial cell apoptosis and reduced milk protein synthesis, suggesting that inflammatory mediators activated during mastitis promote partial involution. Perturbed milk flow, maternal stress and genetic predisposition are significant risk factors for mastitis, and could lead to a heightened TLR4-mediated inflammatory response, resulting in increased susceptibility and severity of mastitis disease in the context of low MAMP abundance. Therefore, heightened host inflammatory signalling may act in concert with pathogenic or commensal bacterial species to cause both the inflammation associated with mastitis and lactation insufficiency. Here, we present an alternate paradigm to the widely held notion that breast inflammation is driven principally by infectious bacterial pathogens, and suggest there may be other therapeutic strategies, apart from the currently utilised antimicrobial agents, that could be employed to prevent and treat mastitis in women.

Toll-like receptor 4 regulates lipopolysaccharide-induced inflammation and lactation insufficiency in a mouse model of mastitis.

Lactation mastitis is a debilitating inflammatory breast disease in postpartum women. Disease severity is associated with markers of inflammation rather than bacterial load, suggesting that immune-signaling pathways activated in the host are important in the disease pathology. The role of the innate pattern recognition receptor toll-like receptor 4 (TLR4) in progression and resolution of mastitislike disease was investigated in a mouse model. Lipopolysaccharide in Matrigel (10 µg/10 µl) was administered into the teat canal of lactating Tlr4 null mutant and wild-type mice to induce a localized area of inflammation. Mastitis induction resulted in a marked influx of RB6-positive neutrophils and F4/80-positive macrophages, which was higher in Tlr4(-/-) mice compared to wild-type mice. Tlr4 null mutation resulted in an altered immune-signaling fingerprint following induction of mastitis, with attenuated serum cytokines, including CXCL1, CCL2, interleukin 1 beta, and tumor necrosis factor alpha compared to wild-type mice. In both genotypes, the localized area of inflammation had resolved after 7 days, and milk protein was evident. However, the mammary glands of wild-type mice exhibited reduced capacity for milk production, with decreased percent area populated with glandular epithelium and decreased abundance of nuclear phosphorylated signal transducer and activator of transcription 5 compared to Tlr4 null mice. This study demonstrates that inflammatory pathways activated in the host are critically important in mastitis disease progression and suggests that lactation insufficiency associated with mastitis may be a consequence of TLR4-mediated inflammation, rather than the bacterial infection itself.

Peri-conceptual cytokines--setting the trajectory for embryo implantation, pregnancy and beyond.

PROBLEM: The peri-conceptual environment influences the early embryo to impart long-term consequences for the fetus and neonate; however, the underlying mechanisms are not well defined. METHOD OF STUDY: We argue that the cytokine network acting in the female reproductive tract during the pre- and peri-implantation period integrates environmental information to program the embryo and fine-tune the maternal immune response and endometrial remodelling to determine implantation success. RESULTS: As well as sex steroid hormones and male seminal fluid factors, female tract cytokines are influenced by agents signalling via the Toll-like receptors including the microbiome and a plethora of metabolic, chemical and other stressors. In mouse models, an altered peri-conceptual cytokine environment induced by cytokine deficiency, inflammatory insults or dysregulated seminal fluid signalling is associated with adverse effects on embryo development, pregnancy viability and reproductive outcome. CONCLUSION: The cytokine network provides a pivotal mechanism through which environmental factors influence both embryo development and receptivity of the uterus.