Increased matrix stiffness enhances pro-tumorigenic traits in a physiologically relevant breast tissue- monocyte 3D model.

High mammographic density, associated with increased tissue stiffness, is a strong risk factor for breast cancer per se. In postmenopausal women there is no differences in the occurrence of ductal carcinoma in situ (DCIS) depending on breast density. Preliminary data suggest that dense breast tissue is associated with a pro-inflammatory microenvironment including infiltrating monocytes. However, the underlying mechanism(s) remains largely unknown. A major roadblock to understanding this risk factor is the lack of relevant in vitro models. A biologically relevant 3D model with tunable stiffness was developed by cross-linking hyaluronic acid. Breast cancer cells were cultured with and without freshly isolated human monocytes. In a unique clinical setting, extracellular proteins were sampled using microdialysis in situ from women with various breast densities. We show that tissue stiffness resembling high mammographic density increases the attachment of monocytes to the cancer cells, increase the expression of adhesion molecules and epithelia-mesenchymal-transition proteins in estrogen receptor (ER) positive breast cancer. Increased tissue stiffness results in increased secretion of similar pro-tumorigenic proteins as those found in human dense breast tissue including inflammatory cytokines, proteases, and growth factors. ER negative breast cancer cells were mostly unaffected suggesting that diverse cancer cell phenotypes may respond differently to tissue stiffness. We introduce a biological relevant model with tunable stiffness that resembles the densities found in normal breast tissue in women. The model will be key for further mechanistic studies. Additionally, our data revealed several pro-tumorigenic pathways that may be exploited for prevention and therapy against breast cancer. STATEMENT OF SIGNIFICANCE: Women with mammographic high-density breasts have a 4-6-fold higher risk of breast cancer than low-density breasts. Biological mechanisms behind this increase are not fully understood and no preventive therapeutics are available. One major reason being a lack of suitable experimental models. Having such models available would greatly enhance the discovery of relevant targets for breast cancer prevention. We present a biologically relevant 3D-model for studies of human dense breasts, providing a platform for investigating both biophysical and biochemical properties that may affect cancer progression. This model will have a major scientific impact on studies for identification of novel targets for breast cancer prevention.

Zebrafish tumour xenograft models: a prognostic approach to epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the gynaecological malignancy with highest mortality. Although adjuvant treatment with carboplatin and paclitaxel leads to an objective response in ~80% of these patients, a majority will relapse within two years. Better methods for assessing long-term treatment outcomes are needed. To address this, we established safe and efficacious doses of carboplatin and paclitaxel using IGROV-1 zebrafish-CDX models. Then fluorescently-labelled cell suspensions from 83 tumour biopsies collected at exploratory laparotomy of women with suspected EOC were generated and 37 (45%) were successfully implanted in zebrafish larvae. Among these 19 of 27 pathology-confirmed EOC samples (70%) engrafted. These zebrafish patient-derived tumour xenograft (ZTX) models were treated with carboplatin or paclitaxel and tumour growth/regression and metastatic dissemination were recorded. In a subgroup of nine patients, four ZTX models regressed during carboplatin treatment. All four corresponding patients had >24 months PFS. Furthermore, both ZTX models established from two patients having <24 months PFS failed to regress during carboplatin treatment. Seven of eight models seeding <6 metastatic cells were established from patients having >24 months PFS. In eleven of fourteen patients, FIGO stage I + II or III tumours gave rise to ZTX models seeding <4 or >4 metastatic cells, respectively. In conclusion, ZTX models predicted patients having >24 or <24 months PFS, based on response/no response to carboplatin. Furthermore, high metastatic dissemination in ZTX models correlated to shorter PFS and more advanced disease at diagnosis. These preliminary results suggest that ZTX models could become a useful prognostic tool in EOC treatment planning.

Low-dose acetylsalicylic acid reduces local inflammation and tissue perfusion in dense breast tissue in postmenopausal women.

PURPOSE: One major risk factor for breast cancer is high mammographic density. It has been estimated that dense breast tissue contributes to ~ 30% of all breast cancer. Prevention targeting dense breast tissue has the potential to improve breast cancer mortality and morbidity. Anti-estrogens, which may be associated with severe side-effects, can be used for prevention of breast cancer in women with high risk of the disease per se. However, no preventive therapy targeting dense breasts is currently available. Inflammation is a hallmark of cancer. Although the biological mechanisms involved in the increased risk of cancer in dense breasts is not yet fully understood, high mammographic density has been associated with increased inflammation. We investigated whether low-dose acetylsalicylic acid (ASA) affects local breast tissue inflammation and/or structural and dynamic changes in dense breasts. METHODS: Postmenopausal women with mammographic dense breasts on their regular mammography screen were identified. A total of 53 women were randomized to receive ASA 160 mg/day or no treatment for 6 months. Magnetic resonance imaging (MRI) was performed before and after 6 months for a sophisticated and continuous measure breast density by calculating lean tissue fraction (LTF). Additionally, dynamic quantifications including tissue perfusion were performed. Microdialysis for sampling of proteins in vivo from breasts and abdominal subcutaneous fat, as a measure of systemic effects, before and after 6 months were performed. A panel of 92 inflammatory proteins were quantified in the microdialysates using proximity extension assay. RESULTS: After correction for false discovery rate, 20 of the 92 inflammatory proteins were significantly decreased in breast tissue after ASA treatment, whereas no systemic effects were detected. In the no-treatment group, protein levels were unaffected. Breast density, measured by LTF on MRI, were unaffected in both groups. ASA significantly decreased the perfusion rate. The perfusion rate correlated positively with local breast tissue concentration of VEGF. CONCLUSIONS: ASA may shape the local breast tissue microenvironment into an anti-tumorigenic state. Trials investigating the effects of low-dose ASA and risk of primary breast cancer among postmenopausal women with maintained high mammographic density are warranted. Trial registration EudraCT: 2017-000317-22.

Proteolytic remodeling of 3D bioprinted tumor microenvironments.

In native tissue, remodeling of the pericellular space is essential for cellular activities and is mediated by tightly regulated proteases. Protease activity is dysregulated in many diseases, including many forms of cancer. Increased proteolytic activity is directly linked to tumor invasion into stroma, metastasis, and angiogenesis as well as all other hallmarks of cancer. Here we show a strategy for 3D bioprinting of breast cancer models using well-defined protease degradable hydrogels that can facilitate exploration of the multifaceted roles of proteolytic extracellular matrix remodeling in tumor progression. We designed a set of bicyclo[6.1.0]nonyne functionalized hyaluronan (HA)-based bioinks cross-linked by azide-modified poly(ethylene glycol) (PEG) or matrix metalloproteinase (MMP) degradable azide-functionalized peptides. Bioprinted structures combining PEG and peptide-based hydrogels were proteolytically degraded with spatial selectivity, leaving non-degradable features intact. Bioprinting of tumor-mimicking microenvironments using bioinks comprising human breast cancer cells (MCF-7) and fibroblast in hydrogels with different susceptibilities to proteolytic degradation shows that MCF-7 proliferation and spheroid size were significantly increased in protease degradable hydrogel compartments, but only in the presence of fibroblasts. In the absence of fibroblasts in the stromal compartment, cancer cell proliferation was reduced and did not differ between degradable and nondegradable hydrogels. The interactions between spatially separated fibroblasts and MCF-7 cells consequently resulted in protease-mediated remodeling of the bioprinted structures and a significant increase in cancer cell spheroid size, highlighting the close interplay between cancer cells and stromal cells in the tumor microenvironment and the influence of proteases in tumor progression.

Breast density and estradiol are associated with distinct different expression patterns of metabolic proteins in normal human breast tissue in vivo.

Background: Breast density and exposure to sex steroids are major risk factors for breast cancer. The local microenvironment plays an essential role in progression of breast cancer. Metabolic adaption is a major hallmark of cancer. Whether proteins from the extracellular space regulating metabolism are affected in breast cancer, dense breasts or by estrogen exposure are not yet fully elucidated. Methods: Women with breast cancer, postmenopausal women with normal breast tissue with varying breast density or premenopausal women with breasts exposed to high levels of estradiol were included in the study. Microdialysis was used to collect proteins from the extracellular space in vivo in 73 women; 12 with breast cancer, 42 healthy postmenopausal women with different breast densities, and 19 healthy premenopausal women. Breast density was determined as lean tissue fraction (LTF) using magnetic resonance imaging. Data were evaluated in a murine breast cancer model. We quantified a panel of 92 key proteins regulating metabolism using proximity extension assay. Results: We report that 29 proteins were upregulated in human breast cancer. In dense breasts 37 proteins were upregulated and 17 of these were similarly regulated as in breast cancer. 32 proteins correlated with LTF. In premenopausal breasts 19 proteins were up-regulated and 9 down-regulated. Of these, 27 correlated to estradiol, a result that was confirmed for most proteins in experimental breast cancer. Only two proteins, pro-cathepsin H and galanin peptide, were similarly regulated in breast cancer, dense- and estrogen exposed breasts. Conclusions: Metabolic proteins may be targetable for breast cancer prevention. Depending on risk factor, this may, however, require different approaches as breast density and estradiol induce distinct different expression patterns in the breast. Additionally, metabolic proteins from the extracellular space may indeed be further explored as therapeutic targets for breast cancer treatment.

Breast Density and Estradiol Are Major Determinants for Soluble TNF-TNF-R Proteins in vivo in Human Breast Tissue.

High mammographic density and exposure to sex steroids are independent risk factors for breast cancer by yet unknown mechanisms. Inflammation is one hallmark of cancer and the tumor necrosis factor family of proteins (TNFSFs) and receptors (TNFRSFs) are key determinants of tissue inflammation. The relationship between TNFSFs/TNFRSFs and breast tissue density or local breast estradiol levels is unknown. We investigated whether TNFSFs and soluble TNFRSFs (sTNFRSFs) are dysregulated in vivo in human breast cancer and dense breast tissue of postmenopausal women. We explored TNFSF/TNFRSF correlations with breast density and estradiol, both locally in the breast and in abdominal subcutaneous (s.c.) fat as a measure of systemic effects. Microdialysis was used for local sampling of in vivo proteins and estradiol in a total of 73 women; 12 with breast cancer, 42 healthy postmenopausal women with different breast densities, and 19 healthy premenopausal women. Breast density was determined as lean tissue fraction (LTF) using magnetic resonance imaging. Microdialysis was also performed in estrogen receptor (ER) positive breast cancer in mice treated with the pure anti-estrogen fulvestrant and tumor tissue was subjected to immunohistochemistry. 23 members of the TNFSF/sTNFRSF families were quantified using proximity extension assay.Our data revealed upregulation of TNFSF10, 13 and 13B, TNFRSF6, 6B, 9, 11A, 11B, 13B, 14, and 19, and TNFR-1 and -2 in ER+ breast cancer in women. In dense breast tissue TNFSF10, 13, and 14, TNFRSF3, 6, 9, 10B, 13B, 14, 19, and TNFR-1 and -2 were upregulated. Certain TNFSFs/TNFRSFs were increased in premenopausal breasts relative to postmenopausal breasts. Furthermore, estradiol correlated with most of the TNFSF/sTNFRSF members, though LTF only correlated with some of the proteins. Several of these associations were breast tissue-specific, as very few correlated with estradiol in abdominal s.c. fat. Estrogen dependent regulations of TNFSF2 (TNF-α) and TNF-R2 were corroborated in ER+ breast cancer in mice. Taken together, our data indicate TNFSFs/sTNFRSFs may represent potential targetable pathways for treatment of breast cancer patients and in prevention of breast cancer development in women with dense breasts.

Lysine in Combination With Estradiol Promote Dissemination of Estrogen Receptor Positive Breast Cancer via Upregulation of U2AF1 and RPN2 Proteins.

The majority of estrogen receptor positive (ER+) breast cancer (BC) maintain the ER at metastatic sites. Despite anti-estrogen therapy, almost 30% of ER+ BC patients relapse. Thus, new therapeutic targets for ER+ BC are needed. Amino acids (AAs) may affect the metastatic capacity by affecting inflammatory cells. Essential AAs (EAAs) cannot be produced by human cells and might therefore be targetable as therapeutics. Here we sampled extracellular EAAs in vivo by microdialysis in human BC. Mass spectrometry-based proteomics was used to identify proteins affected after EAA and estradiol (E2) exposure to BC cells. Proteins relevant for patient survival were identified, knocked down in BC cells, and metastatic capability was determined in vivo in the transgenic zebrafish model. We found that lysine was the most utilized EAA in human ER+BC in vivo. In zebrafish, lysine in presence of E2 increased neutrophil-dependent dissemination of ER+ BC cells via upregulation of U2AF1 and RPN2 proteins, which both correlated with poor prognosis of ER+ BC patients in clinical databases. Knockdown of U2AF1 and RPN2 decreased the expression of several cell-adhesion molecules resulting in diminished dissemination. Dietary lysine or its related metabolic pathways may be useful therapeutic targets in ER+ BC.

Fulvestrant-Mediated Attenuation of the Innate Immune Response Decreases ER+ Breast Cancer Growth In Vivo More Effectively than Tamoxifen.

Although blocking estrogen-dependent signaling is a cornerstone of adjuvant treatment for breast cancer, 25% of patients experience recurrent disease. Stroma events including innate immune responses are key in cancer progression. How different estrogen receptor (ER)-targeting therapies, including the partial agonist tamoxifen and the pure antagonist fulvestrant, affect the tumor stroma has not yet been elucidated. Fulvestrant is used in only postmenopausal patients, and its effects in the presence of estradiol remain undetermined. Here we observe that fulvestrant decreases ER+ breast cancer growth compared with tamoxifen in the presence of physiologic levels of estradiol in human breast cancer in nude mice and in murine breast cancer in immune-competent mice. Fulvestrant significantly inhibited macrophage and neutrophil infiltration in both models. These effects were corroborated in a zebrafish model where fulvestrant inhibited neutrophil- and macrophage-dependent cancer cell dissemination more effectively than tamoxifen. A comprehensive analysis of 234 human proteins released into the cancer microenvironment by the cancer cells sampled via microdialysis in vivo revealed that 38 proteins were altered following both treatments; 25 of these proteins were associated with immune response and were altered by fulvestrant only. Compared with tamoxifen, fulvestrant significantly affected inflammatory proteins released by murine stroma cells. Importantly, in vivo microdialysis of human ER+ breast cancer revealed that the majority of affected proteins in murine models were upregulated in patients. Together, these results suggest that fulvestrant targets ER+ breast cancer more effectively than tamoxifen even in the presence of estradiol, mainly by attenuation of the innate immune response. SIGNIFICANCE: These findings demonstrate novel effects of the pure antiestrogen fulvestrant in ER+ breast cancer and evaluate its effects under physiologic levels of estradiol, representative of premenopausal patients.

Dietary flaxseed and tamoxifen affect the inflammatory microenvironment in vivo in normal human breast tissue of postmenopausal women.

BACKGROUND: Anti-oestrogens such as tamoxifen, decrease the risk of breast cancer but are unsuitable for prevention because of their side-effects. Diet modifications may be a breast cancer prevention strategy. Here, we investigated if a diet addition of flaxseed, which can be converted to the phytoestrogen enterolactone by the gut microbiota, exhibited similar effects as tamoxifen on normal human breast tissue in vivo, with special emphasis on inflammatory mediators implicated in cancer progression. SUBJECTS: A total of 28 postmenopausal women were included. Thirteen women added 25 g of ground flaxseed per day and 15 were treated with tamoxifen as an adjuvant for early breast cancer for 6 weeks. Microdialysis of normal breast tissue and, as a control, in subcutaneous abdominal fat was performed for sampling of extracellular proteins in vivo before and after exposures. RESULTS: Enterolactone levels increased significantly after flaxseed. IL-1Ra and IL-1Ra/IL-1ß ratio in the breast increased in a similar fashion after the two different treatments. Flaxseed also increased breast specific levels of IL-1RT2, IL-18 and sST2 and an overall increase of MMP-9. These changes correlated significantly with enterolactone levels. Tamoxifen decreased breast tissue levels of IL-8 and IL-18. None of the treatments induced any changes of IL-1ß, IL-1RT1, IL-18BP, IL-33, IL-6, IL-6RA, MMP-1, MMP-2 and MMP-3. CONCLUSIONS: We conclude that dietary flaxseed and tamoxifen exert both similar and different effects, as listed above, on normal breast tissue in vivo and that a relatively modest diet change can induce significant effects on the breast microenvironment.

Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8.

Fat is a major tissue component in human breast cancer (BC). Whether breast adipocytes (BAd) affect early stages of BC metastasis is yet unknown. BC progression is dependent on angiogenesis and inflammation, and interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) are key regulators of these events. Here, we show that BAd increased the dissemination of estrogen receptor positive BC cells (BCC) in vivo in the zebrafish model of metastasis, while dissemination of the more aggressive and metastatic BCC such as estrogen receptor negative was unaffected. While anti-VEGF and anti-IL-8 exhibited equal inhibition of angiogenesis at the primary tumor site, anti-IL-8 reduced BCC dissemination whereas anti-VEGF had minor effects on this early metastatic event. Mechanistically, overexpression of cell-adhesion molecules in BCC and neutrophils via IL-8 increased the dissemination of BCC. Importantly, the extracellular in vivo levels of IL-8 were 40-fold higher than those of VEGF in human BC. Our results suggest that IL-8 is a clinical relevant and promising therapeutic target for human BC.

Downregulation of tumor suppressive microRNAs in vivo in dense breast tissue of postmenopausal women.

Women with dense breast tissue on mammography are at higher risk of developing breast cancer but the underlying mechanisms are not well understood. De-regulation of microRNAs (miRNAs) has been associated with the onset of breast cancer. miRNAs in the extracellular space participate in the regulation of the local tissue microenvironment. Here, we recruited 39 healthy postmenopausal women attending their mammography-screen that were assessed having extreme dense or entirely fatty breasts (nondense). Microdialysis was performed in breast tissue and a reference catheter was inserted in abdominal subcutaneous fat for local sampling of extracellular compounds. Three miRNAs, associated with tumor suppression, miR-193b, miR-365a, and miR-452 were significantly down-regulated in dense breast tissue compared with nondense breast tissue. In addition, miR-452 exhibited significant negative correlations with several pro-inflammatory cytokines in vivo, which was confirmed in vitro by overexpression of miR-452 in breast cancer cells. No differences were found of miR-21, -29a, -30c, 146a, -148a, -203, or -451 in breast tissue and no miRs were different in plasma. Extracellular miRNAs may be among factors that should be included in studies of novel prevention strategies for breast cancer.

Estradiol Promotes Breast Cancer Cell Migration via Recruitment and Activation of Neutrophils.

Estradiol (E2) plays a key role in breast cancer progression. Most breast cancer recurrences express the estrogen receptor (ER), but nearly 50% of patients are resistant to antiestrogen therapy. Novel therapeutic targets of ER-positive breast cancers are needed. Protumoral neutrophils expressing the lymphocyte function-associated antigen 1 (LFA-1) integrin may mediate cancer metastasis, and TGFß1 is the major chemoattractant for neutrophils. The role of E2 in neutrophil-ER+ breast cancer cell interactions is unknown. We studied this in vivo using murine breast cancers in immunocompetent mice and human breast cancers in nude mice. Cell dissemination was evaluated in a zebrafish model, and microdialysis of breast cancer patients was performed. In vitro studies were done with mammosphere cultures of breast cancer cells and human neutrophils. We found that E2 increased the number of LFA-1+ neutrophils recruited to the invasive edge of mouse tumors, increased TGFß1 secretion and promoted neutrophil infiltration in mammospheres, and induced overexpression of LFA-1 in neutrophils. In zebrafish, in the presence of E2, neutrophils increased dissemination of ER+ breast cancer cells via LFA-1 and TGFß1, thus causing noninvasive cancer cells to be highly metastatic. Time-lapse imaging in zebrafish revealed close interactions of neutrophils with cancer cells, which drove breast cancer metastasis. We also found that extracellular TGFß1 was overproduced in human breast cancer tissue compared with adjacent normal breast tissue. Thus, E2 can regulate immune/cancer cell interactions in tumor microenvironments. Our results indicate that extracellular TGFß1 is a relevant target in human breast cancer. Cancer Immunol Res; 5(3); 234-47. ©2017 AACR.

Increased nutrient availability in dense breast tissue of postmenopausal women in vivo.

Metabolic reprogramming is a hallmark of cancer. Nutrient availability in the tissue microenvironment determines cellular events and may play a role in breast carcinogenesis. High mammographic density is an independent risk factor for breast cancer. Whether nutrient availability differs in normal breast tissues with various densities is unknown. Therefore we investigated whether breast tissues with various densities exhibited differences in nutrient availability. Healthy postmenopausal women from the regular mammographic screening program who had either predominantly fatty breast tissue (nondense), n = 18, or extremely dense breast tissue (dense), n = 20, were included. Microdialysis was performed for the in vivo sampling of amino acids (AAs), analyzed by ultra-high performance liquid chromatography with tandem mass spectroscopy, glucose, lactate and vascular endothelial growth factor (VEGF) in breast tissues and, as a control, in abdominal subcutaneous (s.c.) fat. We found that dense breast tissue exhibited significantly increased levels of 20 proteinogenic AAs and that 18 of these AAs correlated significantly with VEGF. No differences were found in the s.c. fat, except for one AA, suggesting tissue-specific alterations in the breast. Glucose and lactate were unaltered. Our findings provide novel insights into the biology of dense breast tissue that may be explored for breast cancer prevention strategies.

Equal Pro-inflammatory Profiles of CCLs, CXCLs, and Matrix Metalloproteinases in the Extracellular Microenvironment In Vivo in Human Dense Breast Tissue and Breast Cancer.

The inflammatory microenvironment affects breast cancer progression. Proteins that govern the inflammatory response are secreted into the extracellular space, but this compartment still needs to be characterized in human breast tissues in vivo. Dense breast tissue is a major risk factor for breast cancer by yet unknown mechanisms and no non-toxic prevention for these patients exists. Here, we used the minimal invasive technique of microdialysis for sampling of extracellular proteins in live tissues in situ in breast cancers of women before surgery and in healthy women having dense or non-dense breast tissue on mammography. Proteins were profiled using a proximity extension assay. Out of the 32 proteins assessed, 26 exhibited similar profiles in breast cancers and dense breast tissues; CCL-4, -7, -8, -11, -15, -16, -22, -23, and -25, CXCL-5, -8, -9, -16 as well as sIL-6R, IL-18, vascular endothelial growth factor, TGF-α, fibroblast growth factor 19, matrix metalloproteinase (MMP)-1, -2, -3, and urokinase-type plasminogen activator were all increased, whereas CCL-3, CX3CL1, hepatocyte growth factor, and MMP-9 were unaltered in the two tissues. CCL-19 and -24, CXCL-1 and -10, and IL-6 were increased in dense breast tissue only, whereas IL-18BP was increased in breast cancer only. Our results provide novel insights in the inflammatory microenvironment in human breast cancer in situ and define potential novel therapeutic targets. Additionally, we show previously unrecognized similarities of the pro-inflammatory microenvironment in dense breast tissue and breast cancer in vivo suggesting that anti-inflammatory breast cancer prevention trials for women with dense breast tissue may be feasible.

Dense breast tissue in postmenopausal women is associated with a pro-inflammatory microenvironment in vivo.

Inflammation is one of the hallmarks of carcinogenesis. High mammographic density has been associated with increased risk of breast cancer but the mechanisms behind are poorly understood. We evaluated whether breasts with different mammographic densities exhibited differences in the inflammatory microenvironment. Postmenopausal women attending the mammography-screening program were assessed having extreme dense, n = 20, or entirely fatty breasts (nondense), n = 19, on their regular mammograms. Thereafter, the women were invited for magnetic resonance imaging (MRI), microdialysis for the collection of extracellular molecules in situ and a core tissue biopsy for research purposes. On the MRI, lean tissue fraction (LTF) was calculated for a continuous measurement of breast density. LTF confirmed the selection from the mammograms and gave a continuous measurement of breast density. Microdialysis revealed significantly increased extracellular in vivo levels of IL-6, IL-8, vascular endothelial growth factor, and CCL5 in dense breast tissue as compared with nondense breasts. Moreover, the ratio IL-1Ra/IL-1ß was decreased in dense breasts. No differences were found in levels of IL-1ß, IL-1Ra, CCL2, leptin, adiponectin, or leptin:adiponectin ratio between the two breast tissue types. Significant positive correlations between LTF and the pro-inflammatory cytokines as well as between the cytokines were detected. Stainings of the core biopsies exhibited increased levels of immune cells in dense breast tissue. Our data show that dense breast tissue in postmenopausal women is associated with a pro-inflammatory microenvironment and, if confirmed in a larger cohort, suggests novel targets for prevention therapies for women with dense breast tissue.

Fulvestrant inhibits growth of triple negative breast cancer and synergizes with tamoxifen in ERα positive breast cancer by up-regulation of ERß.

The estrogen receptor-alpha (ERα) is used as a predictive marker for anti-estrogen therapy in breast cancer patients. In addition to aromatase inhibitors, ERα can be targeted at the receptor level using the receptor modulator tamoxifen or by the pure anti-estrogen fulvestrant. The role of the second ER, ER-beta (ERß), as a therapeutic target or prognostic marker in breast cancer is still elusive. Hitherto, it is not known if ERα+/ERß+ breast cancers would benefit from a treatment strategy combining tamoxifen and fulvestrant or if fulvestrant exert any therapeutic effects in ERα-/ERß+ breast cancer. Here, we report that fulvestrant up-regulated ERß in ERα+/ERß+ breast cancer and in triple negative ERß+ breast cancers (ERα-/ERß+). In ERα+/ERß+ breast cancer, a combination therapy of tamoxifen and fulvestrant significantly reduced tumor growth compared to either treatment alone both in vivo and in vitro. In ERα-/ERß+ breast cancer fulvestrant had potent effects on cancer growth, in vivo as well as in vitro, and this effect was dependent on intrinsically expressed levels of ERß. The role of ERß was further confirmed in cells where ERß was knocked-in or knocked-down. Inhibition of DNA methyltransferase (DNMT) increased the levels of ERß and fulvestrant exerted similar potency on DNMT activity as the DNMT inhibitor decitabine. We conclude that fulvestrant may have therapeutic potential in additional groups of breast cancer patients; i) in ERα+/ERß+ breast cancer where fulvestrant synergizes with tamoxifen and ii) in triple negative/ERß+ breast cancer patients, a subgroup of breast cancer patients with poor prognosis.

Adipokines and Vascular Endothelial Growth Factor in Normal Human Breast Tissue in Vivo - Correlations and Attenuation by Dietary Flaxseed.

Exposure to sex steroids increases the risk of breast cancer but the exact mechanisms are yet to be elucidated. Events in the microenvironment are important for carcinogenesis. Diet containing phytoestrogens can affect the breast microenvironment and alter the risk of breast cancer. It has previously been shown that estrogen regulates extracellular levels of leptin, adiponectin, and VEGF in normal breast tissue in vivo. Whether these proteins correlate in breast tissue in vivo or if diet addition of flaxseed, a major source of phytoestrogens in Western diets, alters adipokine levels in breast tissue are unknown. We used microdialysis to sample proteins of normal human breast tissue and abdominal subcutaneous fat in situ in 34 pre-and postmenopausal women. In vitro, co-culture of breast cancer cells and primary human adipocytes was used. In vivo, in normal breast tissue, a significant positive correlation between VEGF and leptin was detected. No correlations were found in fat tissue. Co-culture of adipocytes and breast cancer cells per se increased the secretion of VEGF and leptin and enhanced the effects of estradiol compared to culture of either cell type alone. In vitro, inhibition of VEGF diminished the release of leptin while inhibition of leptin had no influence on VEGF secretion. The levels of leptin decreased and adiponectin increased after a dietary addition of 25 g of flaxseed/day for one menstrual cycle. We conclude that VEGF and leptin correlate significantly in normal human breast tissue in vivo and that dietary addition of flaxseed affect adipokine levels in the breast.

Tissue specific expression of extracellular microRNA in human breast cancers and normal human breast tissue in vivo.

Extracellular circulating microRNAs (miRNAs) have been suggested to be biomarkers for disease monitoring but data are inconsistent, one reason being that blood miRNA is of heterogeneous origin. Here, we sampled extracellular microRNAs locally in situ using microdialysis. Three different cohorts of women were included; postmenopausal women with ongoing breast cancer investigated within the cancer and in normal adjacent breast tissue, postmenopausal women investigated in their normal healthy breast and subcutaneous fat before and after six weeks of tamoxifen therapy, premenopausal women during the menstrual cycle. Samples were initially screened using TaqMan array cards with subsequently absolute quantification. 124 miRNA were expressed in microdialysates. After absolute quantifications extracellular miRNA-21 was found to be significantly increased in breast cancer. In addition, the levels were significantly higher in pre-menopausal breast tissue compared with postmenopausal. In breast tissue of pre-menopausal women miRNA-21 exhibited a cyclic variation during the menstrual cycle and in postmenopausal women six weeks of tamoxifen treatment decreased miRNA-21 suggesting that this miRNA may be important for breast carcinogenesis. None of these changes were found in plasma or microdialysates from subcutaneous fat. Our data revealed tissue specific changes of extracellular circulating miRNAs that would be otherwise unraveled using blood samples.

CCL2 and CCL5 Are Novel Therapeutic Targets for Estrogen-Dependent Breast Cancer.

PURPOSE: Novel therapeutic targets of estrogen receptor (ER)-positive breast cancers are urgently needed because current antiestrogen therapy causes severe adverse effects, nearly 50% of patients are intrinsically resistant, and the majority of recurrences have maintained ER expression. We investigated the role of estrogen-dependent chemokine expression and subsequent cancer growth in human tissues and experimental breast cancer models. EXPERIMENTAL DESIGN: For in vivo sampling of human chemokines, microdialysis was used in breast cancers of women or normal human breast tissue before and after tamoxifen therapy. Estrogen exposure and targeted therapies were assessed in immune competent PyMT murine breast cancer, orthotopic human breast cancers in nude mice, cell culture of cancer cells, and freshly isolated human macrophages. Cancer cell dissemination was investigated using zebrafish. RESULTS: ER(+) cancers in women produced high levels of extracellular CCL2 and CCL5 in vivo, which was associated with infiltration of tumor-associated macrophages. In experimental breast cancer, estradiol enhanced macrophage influx and angiogenesis through increased release of CCL2, CCL5, and vascular endothelial growth factor. These effects were inhibited by anti-CCL2 or anti-CCL5 therapy, which resulted in potent inhibition of cancer growth. In addition, estradiol induced a protumorigenic activation of the macrophages. In a zebrafish model, macrophages increased cancer cell dissemination via CCL2 and CCL5 in the presence of estradiol, which was inhibited with anti-CCL2 and anti-CCL5 treatment. CONCLUSIONS: Our findings shed new light on the mechanisms underlying the progression of ER(+) breast cancer and indicate the potential of novel therapies targeting CCL2 and CCL5 pathways.

Estradiol affects extracellular leptin:adiponectin ratio in human breast tissue in vivo.

CONTEXT: Exposure to sex steroids is associated with increased breast cancer risk, and adipokines, leptin and adiponectin have been implicated in cancer progression. However, it is not known whether sex steroids affect adipokine secretion in breast tissue. OBJECTIVE: To elucidate the role of estrogen and tamoxifen on adipokine release in normal human breast tissue and breast cancer. SETTING AND DESIGN: Microdialysis sampling was used to collect extracellular in vivo leptin and adiponectin from normal human breast tissue in premenopausal healthy volunteers during the menstrual cycle and in postmenopausal women before tamoxifen treatment and after 6 weeks of treatment. In women with breast cancer, microdialysis was performed intratumorally before surgery. In addition, whole normal breast tissue biopsies were cultured ex vivo, and murine breast cancer models were evaluated. RESULTS: In normal breast tissue, plasma estradiol negatively correlated with local extracellular adiponectin levels (r = -0.34; P < .05) and positively correlated with leptin (r = 0.37; P < .05) and leptin:adiponectin ratio (r = 0.38; P < .05). In postmenopausal women, tamoxifen treatment increased adiponectin (P < 0.05) and decreased leptin (P < .01) and the leptin:adiponectin ratio (P < .01). These in vivo results were confirmed in breast tissue biopsies cultured ex vivo. In patients with breast cancer, extracellular leptin was higher (P < .01) and adiponectin lower (P < .05) in tumors than in normal adjacent breast tissue. In a murine model of breast cancer, estrogen exposure increased leptin secretion (P < .05). CONCLUSIONS: Estrogen exposure may have a critical role in the regulation of adipokines in human breast tissue and may serve as therapeutic targets for treatment and prevention.