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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., Funding Agencies|Knut and Alice Wallenberg Foundation (KAW) [2016.0231, 2021.0186]; European Research Council [101044665]; Carl Tryggers Stiftelse

BackgroundBreast 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. MethodsWomen 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. ResultsWe 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. ConclusionsMetabolic 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.

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-alpha), Funding: This work was supported by grants to C.D. from the Swedish Cancer Society (2018/464), the Swedish Research Council (2018-02584), LiU-Cancer, and ALF of Linköping University Hospital.

Context: High mammographic density in postmenopausal women is an independent risk factor for breast cancer by undetermined mechanisms. No preventive therapy for this risk group is available. Activated platelets release growth factors that modulate the microenvironment into a protumorigenic state. Estrogens may affect the risk of breast cancer and platelet function. Whether platelets are activated in situ in breast cancer or in normal breast tissue at high risk of breast cancer and the association to estradiol remains elusive. Objective: To investigate whether platelets are activated in situ in breast cancers and in dense breast tissue of postmenopausal women and explore correlations between estradiol, released platelet factors, and inflammatory proteins. Setting and design: Sampling of in vivo proteins was performed using microdialysis in a total of 71 women: 10 with breast cancer, 42 healthy postmenopausal women with different breast densities, and 19 premenopausal women. Results: Our data demonstrate increased levels of coagulation factors in dense breast tissue similar to that found in breast cancers, indicating excessive platelet activation. Premenopausal breasts exhibited similar levels of coagulation factors as postmenopausal dense breasts. Out of 13 coagulations factors that were upregulated in dense breasts, 5 exhibited significant correlations with estradiol, both locally in the breast and systemically. In breast tissue, positive correlations between coagulation factors and key inflammatory proteins and matrix metalloproteinases were detected. Conclusions: Breast density, not estradiol, is the major determinant of local platelet activation. Inactivation of platelets may be a therapeutic strategy for cancer prevention in postmenopausal women with dense breasts., Funding Agencies|Swedish Cancer SocietySwedish Cancer Society [2018/464]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2018-02584]; LiU-Cancer; ALF of Linkoping University Hospital

Context: Dense breast tissue is associated with 4 to 6 times higher risk of breast cancer by poorly understood mechanisms. No preventive therapy for this high-risk group is available. After menopause, breast density decreases due to involution of the mammary gland. In dense breast tissue, this process is haltered by undetermined biological actions. Growth hormone (GH) and insulin-like binding proteins (IGFBPs) play major roles in normal mammary gland development, but their roles in maintaining breast density are unknown. Objective: To reveal in vivo levels of GH, IGFBPs, and other pro-tumorigenic proteins in the extracellular microenvironment in breast cancer, in normal breast tissue with various breast density in postmenopausal women, and premenopausal breasts. We also sought to determine possible correlations between these determinants. Setting and Design: Microdialysis was used to collect extracellular in vivo proteins intratumorally from breast cancers before surgery and from normal human breast tissue from premenopausal women and postmenopausal women with mammographic dense or nondense breasts. Results: Estrogen receptor positive breast cancers exhibited increased extracellular GH (P <.01). Dense breasts of postmenopausal women exhibited similar levels of GH as premenopausal breasts and significantly higher levels than in nondense breasts (P <.001). Similar results were found for IGFBP-1, -2, -3, and -7 (P <.01) and for IGFBP-6 (P <.05). Strong positive correlations were revealed between GH and IGFBPs and pro-tumorigenic matrix metalloproteinases, urokinase-type plasminogen activator, Interleukin 6, Interleukin 8, and vascular endothelial growth factor in normal breast tissue. Conclusions: GH pathways may be targetable for cancer prevention therapeutics in postmenopausal women with dense breast tissue., Funding Agencies|Swedish Cancer SocietySwedish Cancer Society [2018/464]; Swedish Research CouncilSwedish Research Council [2018-02584]; LiU-Cancer; ALF of Linkoping University Hospital

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 ERthorn breast cancer and evaluate its effects under physiologic levels of estradiol, represent, Funding Agencies|Swedish Cancer SocietySwedish Cancer Society [2018/464]; Swedish Research CouncilSwedish Research Council [2018-02584]; LiU-Cancer; ALF of Linkoping University Hospital

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 TGFβ1 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 TGFβ1 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 TGFβ1, 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 TGFβ1 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 TGFβ1 is a relevant target in human breast cancer., Funding agencies: Swedish Cancer Society [2015/309]; Swedish Research Council [2013-2457]; LiU-Cancer; Research Funds of Linkoping University Hospital

The estrogen receptor-alpha (ER alpha) is used as a predictive marker for antiestrogen therapy in breast cancer patients. In addition to aromatase inhibitors, ER alpha 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 (ER beta), as a therapeutic target or prognostic marker in breast cancer is still elusive. Hitherto, it is not known if ER alpha+/ER beta+ breast cancers would benefit from a treatment strategy combining tamoxifen and fulvestrant or if fulvestrant exert any therapeutic effects in ER alpha-/ER beta+ breast cancer. Here, we report that fulvestrant up-regulated ER beta in ER alpha+/ER beta+ breast cancer and in triple negative ER beta+ breast cancers (ER alpha-/ER beta+). In ER alpha+/ER beta+ 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 ER alpha-/ER beta+ 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 ER beta. The role of ER beta was further confirmed in cells where ER beta was knocked-in or knocked-down. Inhibition of DNA methyltransferase (DNMT) increased the levels of ER beta 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 ER alpha+/ER beta+ breast cancer where fulvestrant synergizes with tamoxifen and ii) in triple negative/ER beta+ breast cancer patients, a subgroup of breast cancer patients with poor prognosis., Funding Agencies|Swedish Cancer Society; Swedish Research Council [2013-2457]; LiU-Cancer; Research Funds of Linkoping University Hospital

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-1 was decreased in dense breasts. No differences were found in levels of IL-1, 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., Funding Agencies|Swedish Cancer Society [2015/309]; Swedish Research Council [2013-2457]; LiU-Cancer; Research Funds of Linkoping University Hospital

Introduction: Events in the microenvironment are important for carcinogenesis of the breast. Adipocytes, which produce adipokines with paracrine effects, are the most abundant cell type in breast tissue. Exposure to sex steroids affects the risk of breast cancer. It has previously been shown that estrogen regulates the extracellular levels of leptin, adiponectin, IL-1β, and VEGF in normal human breast tissue in vivo. Objective: We aimed to determine if there were any relationships between leptin, adiponectin, IL-1β, and/or VEGF in normal human breast tissue in vivo and to elucidate the role of adipocytes in the regulation of these factors. Design and methods: Microdialysis was used to sample proteins of normal human breast tissue and abdominal subcutaneous (s.c.) fat in situ in pre-and postmenopausal women. An in vitro co-culture model of breast cancer cells and primary mature human adipocytes was used. Results: In vivo, in normal breast tissue, significant positive correlations between VEGF and leptin, and VEGF and leptin/adiponectin ratio were detected. No correlations were found in s.c. abdominal 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. In breast tissue, significant correlations between IL-1β and leptin and VEGF were revealed. Conclusions: Our results suggest that VEGF regulates leptin in normal human breast tissue. Moreover, physical contact between adipocytes and breast cancer cells, induces phenotypic changes and enhances the effects of estradiol. These mechanisms may be involved in breast cancer progression.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broadspectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address s, Funding Agencies|Terry Fox Foundation Grant [TF-13-20]; UAEU Program for Advanced Research (UPAR) [31S118]; NIH [AR47901, R21CA188818, R15 CA137499-01, F32CA177139, P20RR016477, P20GM103434, R01CA170378, U54CA149145, U54CA143907, R01-HL107652, R01CA166348, R01GM071725, R01 CA109335-04A1, 109511R01CA151304CA168997 A11106131R03CA1711326 1P01AT003961RO1 CA100816P01AG034906 R01AG020642P01AG034906-01A1R01HL108006]; NIH NRSA Grant [F31CA154080]; NIH (NIAID) R01: Combination therapies for chronic HBV, liver disease, and cancer [AI076535]; Sky Foundation Inc. Michigan; University of Glasgow; Beatson Oncology Centre Fund; Spanish Ministry of Economy and Competitivity, ISCIII [PI12/00137, RTICC: RD12/0036/0028]; FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion [CTS-6844, CTS-1848]; Consejeria de Salud of the Junta de Andalucia [PI-0135-2010, PI-0306-2012]; ISCIII [PIE13/0004]; FEDER funds; United Soybean Board; NIH NCCAM Grant [K01AT007324]; NIH NCI Grant [R33 CA161873-02]; Michael Cuccione Childhood Cancer Foundation Graduate Studentship; Ovarian and Prostate Cancer Research Trust, UK; West Virginia Higher Education Policy Commission/Division of Science Research; National Institutes of Health; Italian Association for Cancer Research (AIRC) [IG10636, 15403]; GRACE Charity, UK; Breast Cancer Campaign, UK; Michael Cuccione Childhood Cancer Foundation Postdoctoral Fellowship; Connecticut State University; Swedish Research Council; Swedish Research Society; University of Texas Health Science Centre at Tyler, Elsa U. Pardee Foundation; CPRIT; Cancer Prevention and Research Institute of Texas; NIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); NIH National Institute on Alcohol Abuse and Alcoholism (NIAAA); Gilead and Shire Pharmaceuticals; NIH/NCI [1R01CA20009, 5R01CAl27258-05, R21CA184788, NIH P30 CA22453, NCI RO1 28704]; Scottish Governments Rural and Environment Science and Analytical Services Divis

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. (C)2015 AACR.

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., Funding Agencies|Swedish Cancer Society [2012/454]; Swedish Research Council [2013-2457]; Linkoping University

Breast cancer is one of the most common malignancies of women in the reproductive years. In the Western world there is a trend towards delaying pregnancy to later in life, and in combination with an increased incidence of breast cancer an increased number of women are diagnosed with breast cancer before they have completed their reproductive plans. In addition, breast cancer during pregnancy may affect an increased number of women as the childbearing years are delayed. The survival rate after breast cancer has improved during the last decades, and many young breast cancer survivors will consider a pregnancy subsequent to the completion of adjuvant breast cancer therapy. Traditionally, many women are advised against a pregnancy due to a fear of increased risk of recurrence, especially women with estrogen receptor-positive breast cancer. Due to feasibility issues, evidence from large prospective randomized trials is missing regarding the safety of pregnancy after breast cancer. Today guidelines are based on cohort studies and population-based registry evidence with its limitations. Overall, data suggest that pregnancy after breast cancer therapy is safe, and the current evidence is summarized in this overview.

Cancer is considered an inflammatory condition where immune cells play an important role in progression and metastasis. Neutrophils may be pro- or antitumorigenic, depending on their phenotype or the number of infiltrating neutrophils in the tumor microenvironment. Massive infiltration of neutrophils in cancer tissue may elicit a cytotoxic effect, leading to tumor regression, whereas a S139 low-grade neutrophil gradient is tumor progressive. Chemokines, cytokines, and growth factors present in the tumor microenvironment, as well as cell-cell interactions mediated by integrins have shown to be determinant steps for cancer cells to break through the endothelial wall and establish metastatic niches. In this work we evaluated the role of lymphocyte functionassociated antigen 1 (LFA-1) integrin in neutrophils-mediated metastasis of estrogen receptor positive breast cancer cells (MCF-7) cells in a tumor xenograft model in zebrafish and in neutrophil infiltration in MCF-7 mammospheres. The metastatic capability of MCF-7 cells was evaluated in presence or absence of human neutrophils and with/without estradiol treatment. Two days old zebrafish embryos were injected into the perivitelline space with labeled MCF-7 cells and human neutrophils, an anti-human LFA-1 antibody (CD11a) was included. We show that estradiol treatment significantly increased the infiltration of neutrophils into MCF-7 mammospheres and this infiltration was significantly reduced by the presence of an anti-human CD11a antibody. Co-injection of MCF-7 cells with neutrophils significantly increased the migration of MCF-7 cells to distant sites in zebrafish and this effect was inhibited by using an anti-human CD11a antibody. We conclude that neutrophils affect the dissemination of breast cancer cells via LFA-1 integrin. Although estradiol increased the number of infiltrating neutrophils into mammospheres exposure to estradiol seemed to have minor effects on the dissemination in the zebrafish.

Matrix metalloproteinases (MMPs) have been suggested as therapeutic targets in cancer treatment, but broad-spectrum MMP inhibitors have failed in clinical trials. Recent data suggest that several MMPs including MMP-9 exert both pro- and antitumorigenic properties. This is also the case of the natural inhibitors of MMPs, tissue inhibitor of metalloproteinases (TIMPs). The inhibitor of MMP-9 is TIMP-1, and high levels of this enzyme have been associated with decreased survival in breast cancer. Inflammation is one hallmark of cancer progression, and MMPs/TIMPs may be involved in the local immune regulation. We investigated the role of MMP-9/TIMP-1 in regulating innate antitumor immunity in breast cancer. Breast cancers were established in nude mice and treated with intratumoral injections of adenoviruses carrying the human TIMP-1 or MMP-9 gene (AdMMP-9). In vivo microdialysis for sampling of cancer cell-derived (human) and stroma-derived (murine) proteins, immunostainings, as well as cell cultures were performed. We report a dose-dependent decrease of tumor growth and angiogenesis after AdMMP-9 treatment. In addition to increased generation of endostatin, AdMMP-9 promoted an antitumor immune response by inducing massive neutrophil infiltration. Neutrophil depletion prior to gene transfer abolished the therapeutic effects of AdMMP-9. Additionally, AdMMP-9 activated tumor-infiltrating macrophages into a tumor-inhibiting phenotype both in vivo and in vitro. AdMMP-9 also inhibited tumor growth in immune-competent mice bearing breast cancers. Adenoviruses carrying the human TIMP-1 gene had no effect on tumor growth or the immune response. Our novel data identify MMP-9 as a potent player in modulating the innate immune response into antitumor activities.

Matrix metalloproteinases (MMPs) have been suggested as therapeutic targets in cancer treatment, but broad-spectrum MMP inhibitors have failed in clinical trials. Recent data suggest that several MMPs including MMP-9 exert both pro-and antitumorigenic properties. This is also the case of the natural inhibitors of MMPs, tissue inhibitor of metalloproteinases (TIMPs). The inhibitor of MMP-9 is TIMP-1, and high levels of this enzyme have been associated with decreased survival in breast cancer. Inflammation is one hallmark of cancer progression, and MMPs/TIMPs may be involved in the local immune regulation. We investigated the role of MMP-9/TIMP-1 in regulating innate antitumor immunity in breast cancer. Breast cancers were established in nude mice and treated with intratumoral injections of adenoviruses carrying the human TIMP-1 or MMP-9 gene (AdMMP-9). In vivo microdialysis for sampling of cancer cell-derived (human) and stroma-derived (murine) proteins, immunostainings, as well as cell cultures were performed. We report a dose-dependent decrease of tumor growth and angiogenesis after AdMMP-9 treatment. In addition to increased generation of endostatin, AdMMP-9 promoted an antitumor immune response by inducing massive neutrophil infiltration. Neutrophil depletion prior to gene transfer abolished the therapeutic effects of AdMMP-9. Additionally, AdMMP-9 activated tumor-infiltrating macrophages into a tumor-inhibiting phenotype both in vivo and in vitro. AdMMP-9 also inhibited tumor growth in immune-competent mice bearing breast cancers. Adenoviruses carrying the human TIMP-1 gene had no effect on tumor growth or the immune response. Our novel data identify MMP-9 as a potent player in modulating the innate immune response into antitumor activities. The Journal of Immunology, 2013, 190: 4420-4430., Funding Agencies|Swedish Cancer Society|2009/799|Swedish Research Council|2010-3458|Research Funds of Linkoping University Hospital

Introduction: Sex steroid exposure increases the risk of breast cancer by unclear mechanisms. Diet modifications may be one breast cancer prevention strategy. The proinflammatory cytokine family of IL-1 is implicated in cancer progression. IL-1Ra is an endogenous inhibitor of the proinflammatory IL-1 alpha and IL-1 beta. less thanbrgreater than less thanbrgreater thanObjective: The objective of this study was to elucidate whether estrogen, tamoxifen, and/or diet modification altered IL-1 levels in normal human breast tissue. less thanbrgreater than less thanbrgreater thanDesign and Methods: Microdialysis was performed in healthy women under various hormone exposures, tamoxifen therapy, and diet modifications and in breast cancers of women before surgery. Breast tissue biopsies from reduction mammoplasties were cultured. less thanbrgreater than less thanbrgreater thanResults: We show a significant positive correlation between estradiol and in vivo levels of IL-1 beta in breast tissue and abdominal sc fat, whereas IL-1Ra exhibited a significant negative correlation with estradiol in breast tissue. Tamoxifen or a dietary addition of 25 g flaxseed per day resulted in significantly increased levels of IL-1Ra in the breast. These results were confirmed in ex vivo culture of breast biopsies. Immunohistochemistry of the biopsies did not reveal any changes in cellular content of the IL-1s, suggesting that mainly the secreted levels were affected. In breast cancer patients, intratumoral levels of IL-1 beta were significantly higher compared with normal adjacent breast tissue. less thanbrgreater than less thanbrgreater thanConclusion: IL-1 may be under the control of estrogen in vivo and may be attenuated by antiestrogen therapy and diet modifications. The increased IL-1 beta in breast cancers of women strongly suggests IL-1 as a potential therapeutic target in breast cancer treatment and prevention., Funding Agencies|Swedish Cancer Society|2009/799|Swedish Research Council|2010-3458|Research Funds of Linkoping University Hospital

The incidence of breast cancer is increasing in the Western world and there is an urgent need for studies of the mechanisms of sex steroids in order to develop novel preventive strategies. Diet modifications may be among the means for breast cancer prevention. Angiogenesis, key in tumor progression, is regulated by the balance between pro-and anti-angiogenic factors, which are controlled in the extracellular space. Sampling of these molecules at their bioactive compartment is therefore needed. The aims of this study were to explore if tamoxifen, one of the most used anti-estrogen treatments for breast cancer affected some of the most important endogenous angiogenesis regulators, vascular endothelial growth factor (VEGF), angiogenin, and endostatin in normal breast tissue in vivo and if a diet supplementation with flaxseed had similar effects as tamoxifen in the breast. Microdialysis was used for in situ sampling of extracellular proteins in normal breast tissue of women before and after six weeks of tamoxifen treatment or before and after addition of 25 g/day of ground flaxseed to the diet or in control women. We show significant correlations between estradiol and levels of VEGF, angiogenin, and endostatin in vivo, which was verified in ex vivo breast tissue culture. Moreover, tamoxifen decreased the levels of VEGF and angiogenin in the breast whereas endostatin increased significantly. Flaxseed did not alter VEGF or angiogenin levels but similar to tamoxifen the levels of endostatin increased significantly. We conclude that one of the mechanisms of tamoxifen in normal breast tissue include tipping of the angiogenic balance into an anti-angiogenic state and that flaxseed has limited effects on the pro-angiogenic factors whereas the anti-angiogenic endostatin may be modified by diet. Further studies of diet modifications for breast cancer prevention are warranted., Funding Agencies|Swedish Cancer Society||Swedish Research Counsil||Ekhaga Foundation||Academy of Finland|114526115459

The proinflammatory cytokines IL-1 alpha and IL-1 beta promote tumor angiogenesis that might be counteracted by the IL-1 receptor antagonist (IL-1Ra), anakinra, a clinically approved agent. A diet with high amounts of phytoestrogens, such as flaxseed (Flax), genistein (GEN), and the mammalian lignan enterolactone (ENL), may affect breast cancer progression in a similar fashion as the antiestrogen tamoxifen. Both cancer cells and tumor stroma may be targets for cancer therapy. By using microdialysis in a model of human breast cancers in nude mice, we could perform species-specific analyses of released proteins in the microenvironment. We show that tumors treated with tamoxifen and fed Flax or ENL exhibited decreased in vivo release of IL-1 beta derived from the murine stroma and decreased microvessel density whereas dietary GEN had no effects. Cancer cell-released IL-1Ra were approximately 5 times higher than stroma-derived IL-1Ra. Tamoxifen, Flax, and ENL increased IL-1Ra levels significantly whereas GEN did not. The tumor stroma contained macrophages, which expressed the estrogen receptor. In vitro, estradiol decreased IL-1Ra released from breast cancer cells and from cultured macrophages. IL-1Ra decreased endothelial cell proliferation significantly in vitro whereas breast cancer cell proliferation was unaffected in presence of estradiol. Finally, IL-1Ra therapy of tumor-bearing mice opposed estrogen-dependent breast cancer growth and decreased angiogenesis. We conclude that the release of IL-1s both by cancer cells and the stroma, where macrophages are a key component, may offer feasible targets for antiestrogen therapy and dietary interventions against breast cancer.

Purpose: Angiogenin, a 14.2-kDa polypeptide member of the RNase A superfamily, has potent angiogenic effects. Nuclear accumulation of angiogenin is essential for its angiogenic activity. Increased angiogenin expression has been associated with the transition of normal breast tissue into invasive breast carcinoma. In this article, we investigated whether estradiol (E-2) affected angiogenin in breast tissue. Experimental Design: We used microdialysis for sampling of extracellular angiogenin in vivo. In vitro cultures of whole normal breast tissue, breast cancer cells, and endothelial cells were used. Results: We show that extracellular angiogenin correlated significantly with E-2 in normal human breast tissue in vivo and that exposure of normal breast tissue biopsies to E-2 stimulated angiogenin secretion. In breast cancer patients, the in vivo angiogenin levels were significantly higher in tumors compared with the adjacent normal breast tissue. In estrogen receptor-positive breast cancer cells, E-2 increased and tamoxifen decreased angiogenin secretion. Moreover, E-2-induced angiogenin derived from cancer cells significantly increased endothelial cell proliferation. Tamoxifen reversed this increase as well as inhibited nuclear translocation of angiogenin. In vivo, in experimental breast cancer, tamoxifen decreased angiogenin levels and decreased angiogenesis. Additionally, treating tumor-bearing mice with an antiangiogenin antibody resulted in tumor stasis, suggesting a role for angiogenin in estrogen-dependent breast cancer growth. Conclusion: Our results suggest previously unknown mechanisms by which estrogen and antiestrogen regulate angiogenesis in normal human breast tissue and breast cancer. This may be important for estrogen-driven breast cancer progression and a molecular target for therapeutic interventions.