Mouse Mammary Gland Cells Research Articles

LncRNA EPR regulates intestinal mucus production and protects against inflammation and tumorigenesis.

The long non-coding RNA EPR is expressed in epithelial tissues, binds to chromatin and controls distinct biological activities in mouse mammary gland cells. Because of its high expression in the intestine, in this study we have generated a colon-specific conditional targeted deletion (EPR cKO) to evaluate EPR in vivo functions in mice. EPR cKO mice display epithelium hyperproliferation, impaired mucus production and secretion, as well as inflammatory infiltration in the proximal portion of the large intestine. RNA sequencing analysis reveals a rearrangement of the colon crypt transcriptome with strong reduction of goblet cell-specific factors including those involved in the synthesis, assembly, transport and control of mucus proteins. Further, colon mucosa integrity and permeability are impaired in EPR cKO mice, and this results in higher susceptibility to dextran sodium sulfate (DSS)-induced colitis and tumor formation. Human EPR is down-regulated in human cancer cell lines as well as in human cancers, and overexpression of EPR in a colon cancer cell line results in enhanced expression of pro-apoptotic genes. Mechanistically, we show that EPR directly interacts with select genes involved in mucus metabolism whose expression is reduced in EPR cKO mice and that EPR deletion causes tridimensional chromatin organization changes.

Systemic Deficiency of PTEN Accelerates Breast Cancer Growth and Metastasis.

Mutation or loss of the tumor suppressor gene PTEN or its functional status in tumor stromal cells may affect tumor occurrence, development, invasion, and metastasis, in which, however, the role of overall low PTEN expression, mutation, or deletion in the tumor-bearing host has rarely been reported. Breast cancer is a common highly invasive metastatic tumor. We therefore treated mouse breast cancer 4T1 cells with the specific PTEN inhibitor VO-OHpic to study the effects of PTEN suppression or deletion on malignant behavior in vivo and in vitro. VO-OHpic effectively inhibited PTEN gene/protein expression in 4T1 cells, accelerated cell proliferation, and enhanced cell migration and invasion. We also transplanted 4T1 cells with VO-OHpic-inhibited PTEN into mice to create orthotopic and metastatic breast cancer models. The proliferation of 4T1 cells in mouse mammary gland was increased and distant metastasis was enhanced, with metastatic foci in the lung, liver, and intestinal tract. In addition, injection of mice with VO-OHpic to inhibit PTEN in the overall microenvironment accelerated the proliferation of transplanted 4T1 cells and enhanced distant metastasis and the formation of metastatic tumors. Metastatic foci formed in the lung, liver, intestine, thymus, and brain, and PTEN levels in the organ/tissues were negatively associated with the formation of metastatic foci. Similarly, inoculation of PTEN-deficient 4T1 cells into systemic PTEN-inhibited mice further enhanced the orthotopic growth and distant metastasis of 4T1 breast cancer. VO-OHpic inhibition of PTEN in 4T1 cells was also associated with significantly increased phosphorylation of Akt and phosphoinositide 3-kinase (PI3K), suggesting that inhibition of PTEN could activate the PI3K-Akt pathway, as a key signaling pathway regulating cell proliferation and death. These results confirmed that functional loss or deletion of the tumor suppressor gene PTEN significantly enhanced the proliferation, invasion, and metastasis of 4T1 cells. Systemic decrease or deletion of PTEN in the organism or organ/tissue microenvironment was conducive to the proliferation of breast cancer cells in situ and distant metastasis. These results suggest that, as well the PTEN in cancer cells the systemic microenvironment PTEN intensely mediates the proliferation, invasion and metastasis of mouse breast cancer cells via regulating the PI3K-Akt signaling pathway.

The Expression and Activity of Rhodanese, 3-Mercaptopyruvate Sulfurtransferase, Cystathionine γ-Lyase in the Most Frequently Chosen Cellular Research Models.

This paper provides information concerning the activity and expression levels of three sulfurtransferases (STRs): rhodanese (TST, EC: 2.8.1.1), 3-mercaptopyruvate sulfurtransferase (MPST, EC: 2.8.1.2) and cystathionine γ-lyase (CTH, EC: 4.4.1.1) in various cell lines. Since very limited data are available in the scientific literature on this subject, the available data are included in this paper. These shortages often force the researchers to carry out their own screening tests that allow them to choose an appropriate model for their further studies. This work supplements the existing deficiencies in this area and presents the activity and expression of STRs in the eight most frequently chosen cell lines: the mouse mammary gland cell line (NMuNG, ATCC: CRL-1636), mouse mammary gland tumor (4T1, ATCC: CRL-2539), mouse fibroblast (MEF, ATCC: SCRC-1008), mouse melanoma (B16-F1, ATCC: CRL-6323), human colorectal adenocarcinoma (Caco-2, ATCC: HTB-37), human embryonic kidney (HEK-293, ATCC: CRL-1573), human osteosarcoma (MG-63, ATCC: CRL-1427) and rat myocardium (H9c2, ATCC: CRL-1446). Changes in STRs activity are directly related to the bioavailability of cysteine and the sulfane sulfur level, and thus the present authors also measured these parameters, as well as the level of glutathione (its reduced (GSH) and oxidized (GSSG) form) and the [GSH]/[GSSG] ratio that determines the antioxidant capacity of the cells. STRs demonstrate diverse functionality and clinical relevance; therefore, we also performed an analysis of genetic variation of STRs genes that revealed a large number of polymorphisms. Although STRs still provide challenges in several fields, responding to them could not only improve the understanding of various diseases, but may also provide a way to treat them.

Apoptosis and metastasis inhibitory potential of pineapple vinegar against mouse mammary gland cells in vitro and in vivo

BackgroundPlant-based food medicine and functional foods have been consumed extensively due to their bioactive substances and health-beneficial effects. Vinegar is one of them due to its bioactivities, which confers benefits on human body. Our previous study has produced pineapple vinegar that is rich in gallic acid and caffeic acid via 2 steps fermentation. There are many evidences that show the effectiveness of these resources in inhibiting the proliferation and metastasis of the cancer cells through several mechanisms.MethodsFreeze-dried pineapple vinegar was evaluated for its in vitro apoptosis and metastasis inhibitory potential using MTT, cell cycle, Annexin V and scratch assays. The in vivo test using BALB/c mice challenged with 4 T1 cells was further investigated by pre-treating the mice with 0.08 or 2 ml/kg body weight of freshly-prepared pineapple vinegar for 28 days. The tumor weight, apoptotic state of cells in tumor, metastasis and immune response of the untreated and pineapple vinegar treatment group were evaluated and compared.ResultsFrom the in vitro study, an IC50 value of 0.25 mg/mL after 48 h of treatment was established. Annexin V/PI and scratch closure assays showed that pineapple vinegar induced 70% of cell population to undergo apoptosis and inhibited 30% of wound closure of 4 T1 cells. High concentration of pineapple vinegar (2 ml/kg body weight) led to the reduction of tumor weight and volume by 45%as compared to the untreated 4 T1-challenged mice. This effect might have been contributed by the increase of T cell and NK cells population associated with the overexpression of IL-2 andIFN-γ cytokines and splenocyte cytotoxicity. Furthermore, fewer instances of metastasis events were recorded in the pineapple vinegar treatment group and this could be explained by the downregulation of inflammation related genes (iNOS, NF-kB and COX2), metastasis related genes (iCAM, VEGF and MMP9) and angeogenesis related genes (CD26, TIMP1, HGF, MMP3, IGFBP-1 and IGFBP-2).ConclusionThe ability of pineapple vinegar to delay cancer progression portrayed its potential as chemopreventive dietry intervention for cancer therapy.

Abstract 5110: Runx knockout in breast luminal stem/progenitor cells induced precancerous lesion via robust expression of ERα

Abstract In breast cancer, 83% of patients were diagnosed as hormone receptor positive, luminal-type, breast cancer (Anderson et al., 2014). Although primary luminal-type breast cancer can be controlled by endocrine therapy, more than 20% of patients have recurrence in 20 years (Pan et al., 2017). Understanding of luminal-type breast cancer would provide benefits to a large number of patients. However, cell of origin and genes responsible for luminal-type breast cancer have not been fully understood. Luminal epithelial cells are considered as origin of breast cancer (Anderson et al., 2014), although not fully proven yet. In addition, extensive genomic analysis of breast cancer patients in TCGA study showed that RUNX genes were downregulated in luminal-type breast cancer: RUNX1 mutation was found in multiple patients, and RUNX3 gene was highly methylated. We previously reported that 270 bp Runx1 enhancer element (eR1) marks hematopoietic stem cell and stomach stem cell (Ng et al., 2010; Matsuo et al., 2017). We also found that this eR1 marks a subset of luminal epithelial cells in mouse mammary gland. Lineage-tracing experiment showed that the eR1+ cells mainly differentiated into luminal cells, but not to basal cells in vivo. These eR1+ cells also contribute to differentiation of alveolar cells in pregnant mice. Interestingly, in vitro lineage tracing by using organoid culture showed that eR1+ cells differentiated into both liminal and basal cells. Runx1 and Runx3 knockout in luminal stem cells showed increase of cell growth, and histology was similar to precancerous lesion such as hyperplasia and ductal carcinoma in situ (DCIS). These lesions showed robust expression of ERα compared with control mice. These results suggested that depletion of Runx in luminal stem cells induces precancerous lesion via robust expression of ERα.

PO-272 Optimisation of hypoxia regulated timed systems for genetic ablation and tracking of hypoxic breast cancer cells to study therapy resistance

IntroductionHypoxia plays a role in many types of cancer, and is linked to metastasis, genetic instability, resistance to therapy and poor prognosis. Hypoxic modification has been tested in clinical trials, but results have been inconclusive, in part due to a knowledge gap into the characterisation and behaviour of hypoxic tumour cells. In this project, hypoxic cells will be lineage traced to investigate their dissemination within the primary tumour and for their presence in circulating tumour cells and distant metastasis. To investigate the contribution of the hypoxic fraction to the tumour resistance, hypoxic cells will be specifically ablated, and the growth and response to treatment of tumours with and without this hypoxic population investigated.Material and methodsTo accomplish these aims, two complementary systems will be generated in the 4 T1 mouse mammary gland cell line, a model for stage IV metastatic human breast cancer. The first consists of a HIF-1α-CRE-ERTAM ‘hypoxia sensing’ construct coupled with a loxp DsRed loxp DTR-EGFP cassette (Diphtheria Toxin Receptor: DTR). Under normal conditions the HIF-1α-CRE-ERTAM fusion protein is expressed, but immediately degraded, but under hypoxia HIF-1α degradation is inhibited leading to accumulation. 4-OHT Administration translocates HIF-1α-CRE-ERTAM to the nucleus mediating loxp recombination in the coupled cassette. This allows lineage tracing of hypoxic cells and their progeny and selective ablation throught the DTR using Diphthteria toxin. Secondly, a DTR-UnaG-ODDD-HA construct (Oxygen-dependant degradation domain: ODDD), driven by the hypoxia dependent 5XHRE promoter will be used. This system allows ablation of hypoxic cells through the DTR and identification of hypoxic cells through the UnaG fluorescent protein. Only cells that are hypoxic at the time of administration/analysis will be labelled. Both cell lines will be transduced with luciferase to allow identification of distant metastasis.Results and discussionsThe two systems for this project have been generated and transduced into the 4 T1 cell line. Lineage tracing and selective ablation have been shown to work in vitro and clones are being characterised. In vitro experiments have shown the HIF-1α-CRE-ERTAM system to be extremely tightly regulated in an oxygen and tamoxifen dependant manner.ConclusionOnce optimised, both systems will be used in an in vivo orthotopic mouse model to investigate the role of the hypoxic cells in breast cancer, metastasis and response to radiotherapy.

Does the Mouse Mammary Gland Arise from Unipotent or Multipotent Mammary Stem/Progenitor Cells?

The presence of long-lived lineage restricted progenitor and multipotent progenitor cells in adult mouse mammary gland for cancer development is compelling. Mammary cancers are phenotypically diverse This might be explained by transformation of long-lived, lineage-limited progenitor subpopulations. Mammary multipotent epithelial stem cells and their environmental niches must be considered, since their niche(s), once empty might be occupied by lineage-limited progenitors that are proximal. The existence of premalignant mammary populationst that manifest characteristics of lineage limitation argues strongly for this proposition.

Low-dose diethylhexyl phthalate exposure does not impair the expressive patterns of epigenetics-related genes and DNA methylation of breast cancer-related genes in mouse mammary glands

Di-(2-ethylhexyl) phthalate (DEHP), as an endocrine-disrupting chemical (EDC), is widely used in plasticizer and other productions. Ubiquitous human exposure to DEHP has been proposed to be a potential risk to public health. Developmental exposure to DEHP could alter epigenetic programming and result in adult-onset disease. In this study, we investigated whether DEHP exposure to pregnant mice affected epigenetic changes as a result of increase in breast cancer incidence. Our results showed that the expression of total 143 epigenetics-related genes in mammary gland cells, have no significantly altered after short time and low-dose treated with DEHP from 0.5 days post-coitum (dpc) to 3.5 dpc of pregnant mice. DNA methylation status of some neoplastic development genes, such as EGFr, Esr1, Pgr, Fos and Rassf5 also had no obvious change. These finding showed no impact of DEHP on the expressive patterns of epigenetics-related genes and DNA methylation of breast cancer-related genes in pregnant mouse mammary gland cells.

Abstract B22: Analysis of Lgr5-positive cells in mouse mammary gland

Abstract Lgr5 has been identified as a stem cell marker in multiple adult tissues, and individual Lgr5+ mammary epithelial cells are capable of regenerating mouse mammary glands in transplantation assays. Here we investigated the proliferation and differentiation capabilities of murine mammary Lgr5+ cells in vitro, and their distribution in mouse mammary glands in vivo. Equal numbers of sorted Lgr5+ and Lgr5- epithelial cells were cultured in Matrigel to evaluate their stem-like properties in vitro. Compared with Lgr5- cells, Lgr5+ cells were more efficient at generating organoids in 3D culture, and the Lgr5+ cell-derived organoids grew to a larger size. Moreover, Lgr5+ cell-derived organoids survived repeated passaging and contained cells of both luminal and basal lineages, as indicated by keratin marker expression. Incidence of Lgr5+ cells in organoids was confirmed by in situ hybridization. These studies indicate that Lgr5+ cells retain the self-renewal and differentiation capabilities of stem cells ex vivo. The in vivo distribution of Lgr5+ cells was investigated using X-Gal-stained mammary glands from Lgr5-lacZ reporter mice ranging from 5 to 32 weeks old. In both adolescent and adult mice, we observed a graded distribution of Lgr5+ cells with greater abundance in the nipple-proximal (ventral) ducts than in the lymph-node proximal central region, and fewest Lgr5+ cells in the distal (dorsal) region of the glands. Lgr5+ cells from all mammary gland regions appeared capable of yielding organoids in culture. These results corroborate in organoid culture the known stem-like properties of Lgr5+ cells defined in intact mouse mammary gland. [Supported by NIH grant CA186981 and the China Scholarship Council] Citation Format: Lixing Zhang, Louise Howe, Richard Kolesnick, Anthony Brown. Analysis of Lgr5-positive cells in mouse mammary gland. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B22.

PIK3CA(H1047R) induces multipotency and multi-lineage mammary tumours.

The adult mouse mammary epithelium contains self-sustained cell lineages that form the inner luminal and outer basal cell layers, with stem and progenitor cells contributing to its proliferative and regenerative potential. A key issue in breast cancer biology is the effect of genomic lesions in specific mammary cell lineages on tumour heterogeneity and progression. The impact of transforming events on fate conversion in cancer cells of origin and thus their contribution to tumour heterogeneity remains largely elusive. Using in situ genetic lineage tracing and limiting dilution transplantation, we have unravelled the potential of PIK3CA(H1047R), one of the most frequent mutations occurring in human breast cancer, to induce multipotency during tumorigenesis in the mammary gland. Here we show that expression of PIK3CA(H1047R) in lineage-committed basal Lgr5-positive and luminal keratin-8-positive cells of the adult mouse mammary gland evokes cell dedifferentiation into a multipotent stem-like state, suggesting this to be a mechanism involved in the formation of heterogeneous, multi-lineage mammary tumours. Moreover, we show that the tumour cell of origin influences the frequency of malignant mammary tumours. Our results define a key effect of PIK3CA(H1047R) on mammary cell fate in the pre-neoplastic mammary gland and show that the cell of origin of PIK3CA(H1047R) tumours dictates their malignancy, thus revealing a mechanism underlying tumour heterogeneity and aggressiveness.

Minireview: Progesterone Regulation of Proliferation in the Normal Human Breast and in Breast Cancer: A Tale of Two Scenarios?

Progesterone (P), which signals through the P receptor (PR), is critical in normal development of the breast, but its signaling axis is also a major driver of breast cancer risk. Here we review recent advances in the understanding of P signaling in the normal human breast, with a focus on the importance of the balance between autocrine and paracrine signaling. To date, most data (which derive largely from mouse models or human breast cancer cell line studies) have demonstrated that the vast majority of PR+ cells appear to act as "sensor" cells, which respond to P stimulation by translating these hormonal cues into paracrine signals. However, growing evidence suggests that, dependent on the cellular context, P may also signal in an autocrine manner in a subset of cells in the normal mouse mammary gland and human breast. It has been suggested that it may be dysregulation of this autocrine signaling, resulting in a "switch" from a predominance of paracrine signaling to autocrine signaling in PR+ cells, which is an early event during breast tumorigenesis. This review summarizes current evidence in the literature that demonstrates the mechanisms through which P acts in the normal human breast, as well as highlighting the important questions that remain unanswered.

Enhancer cooperativity as a novel mechanism underlying the transcriptional regulation of E-cadherin during mesenchymal to epithelial transition

Epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) highlight crucial steps during embryogenesis and tumorigenesis. Induction of dramatic changes in gene expression and cell features is reflected by modulation of Cdh1 (E-cadherin) expression. We show that Cdh1 activity during MET is governed by two enhancers at +7.8kb and at +11.5kb within intron 2 that are activated by binding of Grhl3 and Hnf4α, respectively. Recruitment of Grhl3 and Hnf4α to the enhancers is crucial for activating Cdh1 and accomplishing MET in non-tumorigenic mouse mammary gland cells (NMuMG). Moreover, the two enhancers cooperate via Grhl3 and Hnf4α binding, induction of DNA-looping and clustering at the promoter to orchestrate E-cadherin re-expression. Our results provide novel insights into the cellular mechanisms whereby cells respond to MET signals and re-establish an epithelial phenotype by enhancer cooperativity. A general importance of our findings including MET-mediated colonization of metastasizing tumor cells is suggested.

Aquaporin 5 expression in mouse mammary gland cells is not driven by promoter methylation.

Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5), a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex) to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs) in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type.

CARM1 automethylation is controlled at the level of alternative splicing

Co-activator-associated arginine methyltransferase 1 (CARM1) is subjected to multiple post-translational modifications. Our previous finding that automethylation of CARM1 is essential for regulation of transcription and pre-mRNA splicing prompted us to investigate how automethylation is regulated. Here, we report that automethylation is regulated by alternative splicing of CARM1 mRNA to remove exon 15, containing the automethylation site. Specifically, we find that two major alternative transcripts encoding full-length CARM1 (CARM1FL) and CARM1 with exon 15 deleted (CARM1ΔE15) exist in cells, and each transcript produces the expected protein. Further biochemical characterizations of the automethylation-defective mutant and CARM1ΔE15 reveal overlapping yet different properties. Interestingly, other arginine methylation substrates also have missing exons encompassing the site(s) of methylation, suggesting that protein arginine methylation level may, in general, be controlled by the alternative splicing mechanism. Finally, we observed differential distribution of CARM1FL and CARM1ΔE15 in epithelial and stromal cells in normal mouse mammary gland. Thus, alternative splicing not only serves as the determinant for CARM1 automethylation but also generates cell type-specific isoforms that might regulate normal ERα biology in the mammary gland.

Combinatorial Effects of Thymoquinone on the Anticancer Activity and Hepatotoxicity of the Prodrug CB 1954

Background:One of the major causes of clinical trial termination is the liver toxicity induced by chemotherapeutic agents. Treatment with anticancer drugs like CB 1954 (5-(Aziridin-1-yl)-2,4-dinitrobenzamide) is associated with significant hepatotoxicity. Thymoquinone (TQ), extracted from Nigella sativa, is reported to possess anticancer and hepatoprotective effects. The aims of the present study were to use TQ to reduce hepatotoxicity associated with CB 1954 and to augment its anticancer activity against the resistant mouse mammary gland cell line (66 cl-4-GFP).Method:Balb/C mice were transplanted with the 66cl-4-GFP cell line and in vivo antitumor activity was assessed for CB 1954 (141 mg/kg), TQ (10 mg/kg), and a combination of CB 1954 and TQ. Changes in tumor size and body weight were measured for each treatment. Histological examination of tumors and liver tissue samples was performed using the standard hematoxylin/eosin staining protocol, and serum levels of the liver enzymes AST and ALT were used as biomarkers of hepatotoxicity.Results:Severe liver damage and elevated plasma levels of AST and ALT were observed in the group treated with CB 1954. Treatment of tumor-bearing mice with a combination of CB 1954 and TQ caused a significant regression in tumor size and induced extensive necrosis in these tumors. The combination also protected the liver from drug-induced damage and reduced the plasma levels of AST and ALT to their normal ranges.Conclusion:These results suggest that the use of TQ with CB 1954 can reduce CB 1954-induced hepatotoxicity and enhance its anticancer activity, indicating the potential use of this combination in clinical studies.

Abstract 3275: Development of a new ex-vivo orthotopic model-Human breast cancer cells in mouse mammary gland organ culture

Abstract Mouse mammary organ culture (MMOC) has been classically employed for evaluating the efficacy of chemopreventive agents against development of carcinogen-induced preneoplastic lesions. Efficacy of chemopreventive agents observed in MMOC correlates well with that observed in in-vivo carcinogenesis models. In the present study, we developed a new ex-vivo Human in Mouse organ culture model which mimics in-vivo orthotopic breast cancer model. Since we introduced human breast cancer cells in mouse mammary gland, this model is termed as human Breast cancer (BCA) in Mouse Mammary Organ Culture (BCa-in-MMOC)). Three to four week old female BALB/c mice were sensitized with estradiol (1μg) + progesterone (1mg) for 9 days. On the 10th day animals were sacrificed and 2.5x104 T47Dparental or T47D aromatase overexpressing cells were injected into the fourth pair of thoracic mammary glands. The glands were excised then cultured at 37°C under 95% O2 / 5% CO2 in hMEM medium containing 10% charcoal stripped FBS/supplemented with Testosterone (1nM) and progesterone (1uM) and growth promoting hormones (5 µg insulin, 5 μg prolactin per ml medium). At the end of the experiment, the glands were fixed in formalin. The paraffin embedded sections (longitudinal) of entire glands were processed for histopathological examination (H and E stain) and immnohistochemical staining of various proteins. Mammary glands were evaluated for the presence of T47D cells, their growth pattern and their molecular responsiveness to estradiol. T47D cells (both types) injected into mammary glands were easily identified against mouse cells by intense human specific Ck-18 immunofluorescence staining. Histopathological observation of mammary gland sections showed that growth pattern of injected cancer cells was identical to that observed of breast cancer cells injected in vivo in athymic mice. Interestingly, clusters of cancer cells in the mammary gland stroma appear similar to those observed in breast tumors in women. Cancer cells injected into glands survived and continued to grow (as evident from Ki-67 immunostaining) after 15 days in culture. Cancer cells maintained their original characteristics (ER+, PR+, EGFR+, and aromatase). T47D cells with enhanced aromatase expression growing in the MMOC could metabolize testosterone to estrogen, which resulted in enhanced cell proliferation and induction of estrogen target genes such as ER and PR-B. Mouse mammary glands with T47D aromatase overexpressing cells also showed changes typical to estrogenic milieu. In summary this model provides a novel, inexpensive ex-vivo model, which could be used to study effects of therapeutic agents on the cancer cells growing in orthotopic micromilieu. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3275. doi:1538-7445.AM2012-3275

Combination of Ononis hirta and Bifidobacterium longum decreases syngeneic mouse mammary tumor burden and enhances immune response

The resistance of solid tumors to conventional therapies has prompted the need for alternative therapies. To evaluate in vitro and in vivo effect of extracts from Ononis hirta against resistant mouse mammary gland cell line (66 cl-4-GFP) and to use a combination of Ononis hirta extract with Bifidobacterium longum to target resistant solid tumors in mice. Different solvent extracts of Ononis hirta were prepared and their in vitro antiproliferative activity was tested against 66 cl-4-GFP cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used to identify the active extracts. Balb/C mice were transplanted with 66 cl-4-GFP cell line and in vivo antitumor activity was assessed for the plant extract, Bifidobacterium longum, and a combination of plant extract and Bifidobacterium longum. Histological examination of tumors was performed using standard hematoxylin/eosin staining protocol while gram stain was used to detect the presence of anaerobic bacteria in these sections. A combination of Ononis hirta methanol extract and Bifidobacterium longum showed high ability in targeting solid mammary gland tumors in mice. It also induced extensive necrosis in these tumors. Thirty percent of mice treated with such combination were cured of their cancers. The mechanism underlying this anticancer activity involves immune system activation exemplified by the observed rejection of reinoculated tumors by cured mice. Chemical TLC analysis of the active methanol extract showed the presence of flavonoids, terpenoids, and alkaloids. HPLC analysis confirmed the presence of flavonoids and alkaloids in Ononis hirta methanol extract. The complete regression of the tumor is encouraging and shows that plant extracts in combination with Bifidobacterium longum is an inviting option to treat solid tumors.

Insulin-like growth factors 1 and 2 regulate expression of β-casein in vitro in mouse mammary epithelial cells

We studied the role of insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) in functional differentiation of HC11 mouse mammary gland cells. It was found that both IGF-1 and IGF-2 activate the expression of milk protein β-casein in the presence of prolactin and hydrocortisone. It was found that β-casein expression is accompanied by cyclin D1 coexpression.

BRCA1 Basal-like Breast Cancers Originate from Luminal Epithelial Progenitors and Not from Basal Stem Cells

Breast cancers in BRCA1 mutation carriers frequently have a distinctive basal-like phenotype. It has been suggested that this results from an origin in basal breast epithelial stem cells. Here, we demonstrate that deleting Brca1 in mouse mammary epithelial luminal progenitors produces tumors that phenocopy human BRCA1 breast cancers. They also resemble the majority of sporadic basal-like breast tumors. However, directing Brca1 deficiency to basal cells generates tumors that express molecular markers of basal breast cancers but do not histologically resemble either human BRCA1 or the majority of sporadic basal-like breast tumors. These findings support a derivation of the majority of human BRCA1-associated and sporadic basal-like tumors from luminal progenitors rather than from basal stem cells. They also demonstrate that when target cells for transformation have the potential for phenotypic plasticity, tumor phenotypes may not directly reflect histogenesis. This has important implications for cancer prevention strategies.

Mechanical Interactions of Mouse Mammary Gland Cells with Collagen in a Three-Dimensional Construct

An effort to understand the development of breast cancer motivates the study of mammary gland cells and their interactions with the extracellular matrix. A mixture of mammary gland epithelial cells (normal murine mammary gland), collagen, and fluorescent beads was loaded into microchannels and observed via four-dimensional imaging. Collagen concentrations of 1.3, 2, and 3 mg/mL were used. The displacements of the beads were used to calculate strains in the 3D matrix. To ensure physiologically relevant materials properties for analysis, the collagen was characterized using independent tensile testing with strain rates in the range of those measured in the cell-gel constructs. 3D elastic theory for an isotropic material was employed to calculate the stress. The technique presented adds to the field of measuring cell-generated stresses by providing the capability of measuring 3D stresses locally around a single cell and using physiologically relevant materials properties for analysis. The highest strains were observed in the most compliant matrix. Additionally, the stresses fluctuated over time due to the cells' interaction with the collagen matrix.