Jaime Fornetti

An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.

Physiological COX-2 Expression in Breast Epithelium Associates with COX-2 Levels in Ductal Carcinoma in Situ and Invasive Breast Cancer in Young Women

Cyclooxygenase-2 (COX-2) overexpression is implicated in increased risk and poorer outcomes in breast cancer in young women. We investigated COX-2 regulation in normal premenopausal breast tissue and its relationship to malignancy in young women. Quantitative COX-2 immunohistochemistry was performed on adjacent normal and breast cancer tissues from 96 premenopausal women with known clinical reproductive histories, and on rat mammary glands with distinct ovarian hormone exposures. COX-2 expression in the normal breast epithelium varied more than 40-fold between women and was associated with COX-2 expression levels in ductal carcinoma in situ and invasive cancer. Normal breast COX-2 expression was independent of known breast cancer prognostic indicators, including tumor stage and clinical subtype, indicating that factors regulating physiological COX-2 expression may be the primary drivers of COX-2 expression in breast cancer. Ovarian hormones, particularly at pregnancy levels, were identified as modulators of COX-2 in normal mammary epithelium. However, serial breast biopsy analysis in nonpregnant premenopausal women suggested relatively stable baseline levels of COX-2 expression, which persisted independent of menstrual cycling. These data provide impetus to investigate how baseline COX-2 expression is regulated in premenopausal breast tissue because COX-2 levels in normal breast epithelium may prove to be an indicator of breast cancer risk in young women, and predict the chemopreventive and therapeutic efficacy of COX-2 inhibitors in this population.

Emerging targets for the prevention of pregnancy-associated breast cancer

Comment on: Lyons TR, et al. Nat Med 2011; 17:1109–15

Isolation of Mammary-Specific Extracellular Matrix to Assess Acute Cell-ECM Interactions in 3D Culture

Studies of mammary epithelial cells (MECs) cultured with reconstituted basement membrane proteins derived from EHS tumors have contributed greatly to the understanding of both normal physiology and transformation. Only when plated on such biologically relevant substratum are MECs able to form morphologically correct, differentiated structures, highlighting a critical role for extracellular matrix (ECM) proteins in MEC organization and function. Here, we describe methods modified from the original EHS matrix protocol for isolating tissue-specific ECM from rat mammary glands, and for subsequent use in short-term 3D cell culture models designed to assess acute cell-ECM interactions. Using this protocol, the final matrix is enriched up to 58-fold for ECM proteins such as fibronectin and laminin, while cellular proteins such as GAPDH are reduced 98-fold. We have previously shown that MECs plated in mammary-specific ECM form more elaborate duct-like and alveolar-like structures compared to MECs plated in Matrigel™, demonstrating the biological relevance of tissue-specific ECM. Use of mammary-specific ECM in 3D cell culture models will further our ability to study the intricate interplay between a cell and its microenvironment, and permit identification of modifying factors.

Mammary Gland Involution as an Immunotherapeutic Target for Postpartum Breast Cancer

Postpartum mammary gland involution has been identified as tumor-promotional and is proposed to contribute to the increased rates of metastasis and poor survival observed in postpartum breast cancer patients. In rodent models, the involuting mammary gland microenvironment is sufficient to induce enhanced tumor cell growth, local invasion, and metastasis. Postpartum involution shares many attributes with wound healing, including upregulation of genes involved in immune responsiveness and infiltration of tissue by immune cells. In rodent models, treatment with non-steroidal anti-inflammatory drugs (NSAIDs) ameliorates the tumor-promotional effects of involution, consistent with the immune milieu of the involuting gland contributing to tumor promotion. Currently, immunotherapy is being investigated as a means of breast cancer treatment with the purpose of identifying ways to enhance anti-tumor immune responses. Here we review evidence for postpartum mammary gland involution being a uniquely defined ‘hot-spot’ of pro-tumorigenic immune cell infiltration, and propose that immunotherapy should be explored for prevention and treatment of breast cancers that arise in this environment.

Abstract A12: A novel in vitro model to study the role of phagocytic mammary epithelial cells in postpartum breast cancer promotion

Women diagnosed with breast cancer within 5-10 years of pregnancy have significantly poorer prognosis than age-matched nulliparous patients. This poor prognosis is specific to postpartum cases, suggesting that an event related to the completion of pregnancy may drive the poor prognosis of postpartum breast cancer. We have identified postpartum mammary gland involution as a tumor-promotional window following pregnancy/lactation. During postpartum involution, 50-80% of the milk-producing mammary epithelial cells (MECs) undergo apoptosis. Additionally, during involution, the MECs themselves become phagocytic and clear the gland of their dying neighbors. Peak phagocytosis precedes an influx of M2 activated macrophages with immunosuppressive characteristics which are thought to directly contribute to the tumor-promotional microenvironment of the postpartum gland. We hypothesize that clearance of apoptotic cells during involution induces secretion of immune-suppressive signals by phagocytic MECs, promoting M2 macrophage maturation and local immune suppression. These host events are anticipated to facilitate breast tumor cell immune evasion and tumor progression. This hypothesis is based on data that demonstrate clearance of apoptotic cells by professional phagocytes promotes M2 macrophages and immune suppression through TGF-β and COX-2. Here, we characterize the phenotype of phagocytic MECs and determine if phagocytic MECs induce tumor-promotional, M2 macrophage activation. To study the role of phagocytic MECs in promoting M2 macrophages, we developed an in vitro model in which MECs are grown to monolayers with high electrical resistance, modeling the tight cell-cell junctions unique to the lactating epithelium. To mimic involution, these monolayers are then treated with TGF-β3, which disrupts cell junctions and induces phagocytosis. We provide evidence that disruption of cell junctions is required for MECs to switch from a secretory to a phagocytic phenotype. We observe reorganization of cell junctions and E-cadherin cleavage in our in vitro model, and the extent of junction disruption positively correlates with phagocytosis. Similar adherens junction reorganization, E-cadherin cleavage, and β-catenin nuclear localization are observed upon the switch from lactation to involution in vivo in rodents and humans, consistent with a role for junction disruption in the phagocytic switch. To assess whether phagocytic MECs may contribute to M2 macrophage activation and local immune suppression during involution, bone marrow-derived macrophages (BMDM) were treated with conditioned media from phagocytic or control MEC cultures and expression of the M2 marker arginase-1 (Arg-1) was evaluated. Phagocytic cell conditioned media induced Arg-1, indicating M2 macrophage activation. Importantly, Arg-1 expression in BMDM correlated with COX-2 expression by phagocytic MECs, which suggests phagocytic MECs may promote M2 macrophages through a COX-2 dependent mechanism, as observed in the promotion of M2 macrophages by professional phagocytes. In vivo, COX-2 expression detected by IHC increases in MECs during involution, consistent with a role for phagocytic MEC COX-2 in promoting M2 macrophages. Additionally, CD86, a co-stimulatory molecule that promotes T cell activation, was decreased in BMDM treated with phagocytic MEC conditioned media, further supporting a role for phagocytic MECs in promotion of an immune-suppressive microenvironment. By further understanding the role of phagocytic MECs in regulating the immune environment in the breast during involution, we hope to identify novel pathways of metastasis, as well as new targets for postpartum breast cancer therapy. Citation Format: Jaime Fornetti, Peter Henson, Virginia Borges, Pepper Schedin. A novel in vitro model to study the role of phagocytic mammary epithelial cells in postpartum breast cancer promotion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A12.

Abstract P1-05-02: COX-2 expression in normal mammary epithelium is estrogen responsive and predicts expression in DCIS and invasive ductal carcinoma

Background: Cyclooxygenase-2 (COX-2) overexpression is implicated in the poor prognosis of young women9s breast cancer. Understanding the regulation of COX-2 expression in normal premenopausal breast tissue and its relationship to malignancy is anticipated to provide insight into COX-2 as a potential therapeutic target for young breast cancer patients. Methods: To investigate COX-2 regulation and expression, quantitative COX-2 immunohistochemistry was performed on adjacent normal and breast cancer tissues from 83 premenopausal women with known clinical reproductive histories as well as on rat mammary glands with distinct estradiol and progesterone exposures. To determine the stability of COX-2 expression over time, a small cohort of women with serial breast biopsies 2-3 weeks apart was evaluated. Results: COX-2 expression in normal adjacent breast tissue was predominantly epithelial, varied >40-fold between individual women, and was independent of proximity to tumor and tumor biology. COX-2 upregulation occurred in alveoli during pregnancy and with estradiol and progesterone exposure in the rat. Though these data demonstrate ovarian hormone regulation, COX-2 expression was relatively stable for each individual premenopausal woman over a 2-3 week assessment window. Furthermore, COX-2 expression levels in normal epithelium independently predicted COX-2 expression levels in tumors. Conclusions : COX-2 expression in the adjacent normal breast epithelium is highly variable across premenopausal women, and may be further modified by ovarian hormone exposure. Moreover, COX-2 expression in the normal breast independently predicts COX-2 expression in tumors, implicating women with high COX-2 expression in the normal breast epithelium as high risk patients who may benefit from COX-2 inhibitor therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-05-02.

Abstract B29: A novel mammary intraductal delivery model that permits study of human ductal carcinoma in situ progression.

Young African American women have an increased risk of developing aggressive forms of breast cancer (i.e. triple negative/basal-like) than young non-Hispanic white women. Recent epidemiological data show increased risk of basal-like breast cancer with increased childbearing in African American women (Millikan et al 2008; Palmer et al 2011). Breast cancers associated with a recent pregnancy (pregnancy-associated breast cancer) are more likely to be metastatic (Lyons et al 2011). We predict that the triple negative/basal-like breast cancer subtype is promoted by a recent pregnancy, accounting in part, for the poor prognosis of young African American breast cancer patients. We have developed a murine intraductal mammary model to examine the effect of host reproductive status on the progression of early stage human breast cancer. Our model delivers human mammary tumor cells directly through the intact mouse teat into the correct anatomical location for ductal carcinoma in situ (DCIS) without surgical manipulations. MCF10ADCIS.com (triple negative/basal cell line) or HCC70 (triple negative cell line derived from a young African American woman) cells were delivered into the mammary gland via intraductal injection to assess influence of host reproductive state (nulliparity, pregnancy, active post-partum involution) on tumor progression. Lesions from both cell lines displayed the full representation of human DCIS histologic subtypes, which progressed to DCIS through an atypical ductal hyperplasia stage. Although the MCF10ADCIS.com cell line is typically triple negative in vitro and other in vivo models, established tumors in the intraductal model were observed to re-express estrogen receptor. HCC70 tumors maintained the triple negative phenotype in our model. Using the MCF10ADCIS.com model to assess the effects of host reproductive status on DCIS progression, we found tumor burden in the pregnancy group was not significantly different than nulliparous controls. Within the pregnant group, tumors appear to be less proliferative and have slightly lower ER expression than nulliparous control tumors. Tumor burden in the postpartum involution group is significantly greater than the respective nulliparous control group; however, the Ki67 proliferative index is lower 4 weeks post injection in comparison to nulliparous controls. DCIS progression to locally invasive disease occurred with progressive loss of myoepithelial cell differentiation markers. In both cell line models, the loss of p63 was identified as an early indicator of compromised myoepithelium. Further, our data suggest that the protective myoepithelial cell layer may be preferentially compromised by tumors formed in postpartum involuting mammary glands. Our murine mammary intraductal model of human breast cancer provides a rigorous approach to study early stage-tumor progression, and is well suited to study the effect of the host reproductive state on DCIS progression. Since occult tumors in women develop within ducts, we propose that this teat injection model will aid research of early disease progression, a requisite for research focused on breast cancer prevention and inhibition of local invasion. Further, this model may provide a unique opportunity to address and study tumor growth disparities among African American and non-Hispanic white women. Citation Format: Tanya D. Russell, Samiat Agunbiade, Sonali Jindal, Jaime Fornetti, Virginia Borges, Pepper Schedin. A novel mammary intraductal delivery model that permits study of human ductal carcinoma in situ progression. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B29.

Abstract 1590: A preclinical mammary intraductal model to study early stage human breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Our goal is to develop a murine intraductal mammary model to examine the progression of early stage human breast cancer. Current models include injection of human mammary epithelial cell lines into the mammary fat pad (MFP) of immunocompromised mice or intraductal injection via cleaved nipple. With the MFP model, these cells are injected into the correct anatomical location for primary invasive tumors but do not form in the location of initiation, i.e., the mammary ducts. Intraductal models allow direct study of tumor cell formation and dissemination from mammary ducts; however, surgical manipulations performed to expose the teat canal are confounded by wound healing and inflammatory programs that are reported to be tumor promotional in other contexts. Our intraductal model of human breast cancer delivers human mammary tumor cells directly through the mouse teat into the correct anatomical location for ductal in situ carcinoma (DCIS) without surgical manipulations. Further, our model provides co-evolution of tumor progression with stromal changes versus the MFP model where the stroma responds to tumor cells at time of injection. Using the bi-potential progenitor MCF10ADCIS.com cell line and harvesting tissue 4 weeks post-injection, we corroborate reported studies showing DCIS lesions with distinct characteristics of the main human subtypes and progression to invasive disease. Immunohistochemical (IHC) analysis shows that these cells incorporate into the mouse mammary ducts and form E-cadherin-based junctional complexes with neighboring cells, including other human tumor cells or normal mouse epithelial cells. Although these cells have been characterized as estrogen receptor (ER) negative both in vitro and in vivo, IHC revealed that some tumors developed within our model were ER positive according to the Allred scoring method. Our model permits a rigorous evaluation of the effects that physiologic-induced changes in mammary tissue remodeling (i.e. postpartum involution) has on tumor progression. We investigated tumor incidence and burden in the postpartum involution setting and found much greater increases in the involution group compared to the respective nulliparous control group. Interestingly, the Ki67 proliferative index is lower in the involution compared to the respective nulliparous control, suggesting a mechanism other than proliferation is driving tumor promotion in the postpartum setting. In summary, our murine intraductal model of human breast cancer provides a rigorous approach to studying early stage tumor progression. This model is particularly suited to studying host effects on tumor progression and future studies will determine the effects of physiological endocrine status on tumor cell dissemination from mammary ducts. Since mammary ducts are the primary sites of occult tumors in women, we propose that this teat injection model will be a more relevant model for all stages of human disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1590. doi:10.1158/1538-7445.AM2011-1590

Abstract B54: Pregnancy and postpartum involution promote tumorigenesis in a preclinical model for triple negative/basal breast cancer

Young African American women have an increased risk of developing and dying from “triple negative/basal breast cancer” compared to young Caucasian women, but the reason for this is unknown. Insight into this problem may be elucidated by the fact that breast cancers associated with a recent pregnancy are more likely to be metastatic. For all women, having had a recent pregnancy is an independent risk factor for poor prognosis. Epidemiologic data shows increased risk of triple negative/basal breast cancer with increased childbearing in African American women (Millikan et al 2008). We speculate that the hormones of pregnancy combined with the tissue-remodeling of the post-partum breast back to its pre-pregnant state (a normal process called postpartum involution that occurs after childbirth in the absence of breastfeeding, or at weaning in women who breastfeed) provides a two-hit tumor promotional tissue environment that results in increased metastasis of these tumors. Using an innovative, intraductal preclinical model developed in our laboratory, we tested whether a triple negative/basal breast cancer cell line (MCF10ADCIS.com) is promoted by pregnancy or postpartum involution. When these cells are injected into mammary ducts of nulliparous female mice, there is evidence of tumor progression from hyperplastic alveolar nodules to ductal carcinoma in situ to local invasion that is accompanied by progressive loss of the protective myoepithelial cell layer. Further, tumor burden in pregnancy and postpartum involution is much greater than their respective nulliparous controls. Importantly, with pregnancy and postpartum involution the protective myoepithelial cell layer surrounding tumors is compromised at earlier stages than in nulliparous controls. Physiologic changes to adherens junctions and myoepithelial cells occur during normal pregnancy and post-partum involution, respectively. These data suggest that the physiological environments of pregnant and involuting glands promote tumor progression and begin to unfold the biological mechanisms for the effects of parity on aggressive breast cancers. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B54.