Holly A. Martinson

Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution

Mammary gland development is dependent on macrophages, as demonstrated by their requirement during the expansion phases of puberty and pregnancy. Equally dramatic tissue restructuring occurs following lactation, when the gland regresses to a state that histologically resembles pre-pregnancy through massive programmed epithelial cell death and stromal repopulation. Postpartum involution is characterized by wound healing-like events, including an influx of macrophages with M2 characteristics. Macrophage levels peak after the initial wave of epithelial cell death, suggesting that initiation and execution of cell death are macrophage independent. To address the role of macrophages during weaning-induced mammary gland involution, conditional systemic deletion of macrophages expressing colony stimulating factor 1 receptor (CSF1R) was initiated just prior to weaning in the Mafia mouse model. Depletion of CSF1R+ macrophages resulted in delayed mammary involution as evidenced by loss of lysosomal-mediated and apoptotic epithelial cell death, lack of alveolar regression and absence of adipocyte repopulation 7 days post-weaning. Failure to execute involution occurred in the presence of milk stasis and STAT3 activation, indicating that neither is sufficient to initiate involution in the absence of CSF1R+ macrophages. Injection of wild-type bone marrow-derived macrophages (BMDMs) or M2-differentiated macrophages into macrophage-depleted mammary glands was sufficient to rescue involution, including apoptosis, alveolar regression and adipocyte repopulation. BMDMs exposed to the postpartum mammary involution environment upregulated the M2 markers arginase 1 and mannose receptor. These data demonstrate the necessity of macrophages, and implicate M2-polarized macrophages, for epithelial cell death during normal postpartum mammary gland involution.

Extracellular Matrix Composition Reveals Complex and Dynamic Stromal-Epithelial Interactions in the Mammary Gland

The mammary gland is an excellent model system to study the interplay between stroma and epithelial cells because of the gland’s unique postnatal development and its distinct functional states. This review focuses on the contribution of the extracellular matrix (ECM) to stromal-epithelial interactions in the mammary gland. We describe how ECM physical properties, protein composition, and proteolytic state impact mammary gland architecture as well as provide instructive cues that influence the function of mammary epithelial cells during pubertal gland development and throughout adulthood. Further, based on recent proteomic analyses of mammary ECM, we describe known mammary ECM proteins and their potential functions, as well as describe several ECM proteins not previously recognized in this organ. ECM proteins are discussed in the context of the morphologically-distinct stromal subcompartments: the basal lamina, the intra- and interlobular stroma, and the fibrous connective tissue. Future studies aimed at in-depth qualitative and quantitative characterization of mammary ECM within these various subcompartments is required to better elucidate the function of ECM in normal as well as in pathological breast tissue.

Cyclooxygenase-2-dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer.

Breast involution following pregnancy has been implicated in the high rates of metastasis observed in postpartum breast cancers; however, it is not clear how this remodeling process promotes metastasis. Here, we demonstrate that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. Moreover, lymphatic vessel density was increased in normal postpartum breast tissue compared with tissue from nulliparous women. In rodents, mammary lymphangiogenesis was upregulated during weaning-induced mammary gland involution. Furthermore, breast cancer cells exposed to the involuting mammary microenvironment acquired prolymphangiogenic properties that contributed to peritumor lymphatic expansion, tumor size, invasion, and distant metastases. Finally, in rodent models of postpartum breast cancer, cyclooxygenase-2 (COX-2) inhibition during the involution window decreased normal mammary gland lymphangiogenesis, mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Our data indicate that physiologic COX-2-dependent lymphangiogenesis occurs in the postpartum mammary gland and suggest that tumors within this mammary microenvironment acquire enhanced prolymphangiogenic activity. Further, our results suggest that the prolymphangiogenic microenvironment of the postpartum mammary gland has potential as a target to inhibit metastasis and suggest that further study of the therapeutic efficacy of COX-2 inhibitors in postpartum breast cancer is warranted.

Wound healing‐like immune program facilitates postpartum mammary gland involution and tumor progression

Women diagnosed with breast cancer within 5 years postpartum have poor survival rates. The process of postpartum mammary gland involution, whereby the lactating gland remodels to its prepregnant state, promotes breast cancer progression in xenograft models. Macrophage influx occurs during mammary gland involution, implicating immune modulation in the promotion of postpartum breast cancer. Herein, we characterize the postpartum murine mammary gland and find an orchestrated influx of immune cells similar to that which occurs during wound healing. Further, the normal involuting gland may be in an immunosuppressed state as discerned by the transient presence of Foxp3+ regulatory T cells and IL‐10+ macrophages with T cell suppressive function. To determine the influence of the postpartum immune microenvironment on mammary tumor promotion, we developed an immune‐competent model. In this model, mammary tumors in the involution group are sixfold larger than nulliparous group tumors, have decreased CD4+ and CD8+ T cell infiltrates and contain a greater number of macrophages with the ability to inhibit T cell activation. Targeting involution with a neutralizing antibody against the immunosuppressive cytokine IL‐10 reduces tumor growth in involution group mice but not in nulliparous mice, implicating the involution microenvironment as the primary target of αIL‐10 treatment. Relevance to women is implicated, as we find postlactational human breast tissue has transient high IL‐10+ and Foxp3+ immune cell infiltrate. These data show an immune modulated microenvironment within the normal involuting mammary gland suggestive of immunosuppression, that when targeted reduces tumor promotion, revealing possible immune‐based strategies for postpartum breast cancer.

Developmental windows of breast cancer risk provide opportunities for targeted chemoprevention

The magnitude of the breast cancer problem implores researchers to aggressively investigate prevention strategies. However, several barriers currently reduce the feasibility of breast cancer prevention. These barriers include the inability to accurately predict future breast cancer diagnosis at the individual level, the need for improved understanding of when to implement interventions, uncertainty with respect to optimal duration of treatment, and negative side effects associated with currently approved chemoprevention therapies. None-the-less, the unique biology of the mammary gland, with its postnatal development and conditional terminal differentiation, may permit the resolution of many of these barriers. Specifically, lifecycle-specific windows of breast cancer risk have been identified that may be amenable to risk-reducing strategies. Here, we argue for prevention research focused on two of these lifecycle windows of risk: postpartum mammary gland involution and peri-menopause. We provide evidence that these windows are highly amenable to targeted, limited duration treatments. Such approaches could result in the prevention of postpartum and postmenopausal breast cancers, correspondingly.

Emerging targets for the prevention of pregnancy-associated breast cancer

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

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 B099: Postpartum mammary gland involution promotes COX-2 dependent tumor cell invasion of lymphatics

Although tumor cell dissemination via the lymphatic vasculature is thought to be a common pathway of metastasis for solid human cancers, the mechanisms of lymphatic mediated metastasis remain poorly understood1. For breast cancer patients, lymph node involvement remains a very important indicator of patient prognosis and is utilized clinically for therapeutic choices2,3. A recent study has shown that the incidence of being diagnosed with stage IV breast cancer, i.e. breast cancer that has spread outside the breast, is increasing in young women4. Furthermore, young women9s breast cancers diagnosed within 5 years postpartum are more likely to be metastatic5-7. Given that 35-45% of young women9s breast cancers are likely to be diagnosed within 5 years of giving birth, we predict that postpartum cases may be driving the observed increase in breast cancers with distant involvement in young women. Here, we hypothesized that the postpartum period promotes lymphangiogenesis, which leads to breast tumor metastasis. To test this hypothesis we have generated xenograft and isograft mouse models of postpartum breast cancer that show increased metastasis in postpartum animals8,9. We utilized these rodent models and a cohort of young women9s breast tissues to investigate lymphatic mediated metastasis in the postpartum period. In our rodent models, we show that 1) lymphangiogenesis is enhanced during normal postpartum involution, 2) tumor cells can utilize the lymphatic vasculature to escape the mammary gland during postpartum involution, 3) postpartum tumors display increased lymphatic vessel density and lymphatic vessel invasion in the tumor periphery, and 4) that postpartum tumor cells promote lymphangiogenesis and express high levels of pro-lymphangiogenic molecules VEGF-C and Sem7a ex vivo. Importantly, we show that both normal and tumor associated lymphangiogenesis in vivo are dependent upon COX-2 and that COX-2 inhibitors can block metastasis of postpartum tumors. In our young women9s breast cancer cohort we also report increased mammary lymphangiogenesis tissues from women within one year of childbirth, increased lymphatic vessel density at the tumor periphery of postpartum breast cancers, as well as a positive correlation between lymphatic vessel density and lymphatic vessel invasion. Thus, we suggest that lymphatic mediated metastasis in young women may be decreased by COX-2 inhibitor or NSAID use. 1. Stacker, S.A., Baldwin, M.E. & Achen, M.G. The role of tumor lymphangiogenesis in metastatic spread. Faseb J 16, 922-934 (2002). 2. Alitalo, K. & Carmeliet, P. Molecular mechanisms of lymphangiogenesis in health and disease. Cancer cell 1, 219-227 (2002). 3. Pepper, M.S., Tille, J.C., Nisato, R. & Skobe, M. Lymphangiogenesis and tumor metastasis. Cell Tissue Res 314, 167-177 (2003). 4. Johnson, R.H., Chien, F.L. & Bleyer, A. Incidence of breast cancer with distant involvement among women in the United States, 1976 to 2009. Jama 309, 800-805 (2013). 5. Callihan, E.B., et al. Postpartum diagnosis demonstrates a high risk for metastasis and merits an expanded definition of pregnancy-associated breast cancer. Breast Cancer Res Treat (2013). 6. Johansson, A.L., Andersson, T.M., Hsieh, C.C., Cnattingius, S. & Lambe, M. Increased Mortality in Women with Breast Cancer Detected during Pregnancy and Different Periods Postpartum. Cancer Epidemiol Biomarkers Prev 20, 1865-1872 (2011). 7. Stensheim, H., Moller, B., van Dijk, T. & Fossa, S.D. Cause-specific survival for women diagnosed with cancer during pregnancy or lactation: a registry-based cohort study. J Clin Oncol 27, 45-51 (2009). 8. McDaniel, S.M., et al. Remodeling of the mammary microenvironment after lactation promotes breast tumor cell metastasis. The American journal of pathology 168, 608-620 (2006). 9. Lyons, T.R., et al. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nat Med 17, 1109-1115 (2011). Citation Format: Traci R. Lyons, Virginia F. Borges, Courtney B. Betts, Puja Kapoor, Holly A. Martinson, Sonali Jindal, Pepper Schedin. Postpartum mammary gland involution promotes COX-2 dependent tumor cell invasion of lymphatics. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B099.

Abstract A31: Immune cell influx during postpartum mammary gland involution reveals immunosuppression and tumor promotion

Young women with breast cancer diagnosed in the postpartum period, defined as up to 5 years from last child delivery, have a poorer prognosis compared to women who have never been pregnant. Our lab has proposed that the poor prognosis associated with postpartum breast cancers is due to gland remodeling following pregnancy, known as postpartum involution. Using the mouse mammary gland as a model, our understanding of postnatal involution has greatly improved, however our understanding of the immune systems role during involution is limited. Recently M2 macrophages, that have tumor promotional characteristics, have been identified as essential for epithelial cell death during normal postpartum involution, demonstrating involution requires assistance from the immune system. We hypothesize that immune cells within the normal involuting gland set up an immunosuppressive environment that favors the growth and metastasis of breast cancer cells. To characterize the role of immune cells during involution and in the promotion of postpartum breast cancer, we created an immunocompetent mouse model using the Balb/c mouse. Using flow cytometry, we have determined that dendritic cells, GR1+ monocytic immature myeloid cells (iMCs), and T cells increase exponentially in the mammary gland during involution. Functional assays using myeloid cells isolated from the mammary gland indicate the iMCs isolated from involuting glands are capable of suppressing T cell activation ex vivo. The ability of iIMCs to suppress T cell activation indicates they are capable of immune suppression. To address whether iMCs are able to promote tumor progression in the postpartum breast cancer setting, D2A1 a triple negative mammary carcinoma cell line and Balb/c mouse were utilized. D2A1 cells injected into the involuting mammary fat pad have a significant growth advantage over D2A1 cells injected into the nulliparous gland, demonstrating that mammary gland involution promotes the growth of mammary carcinoma cells in this immune competent model. T cells identified by flow cytometry are significantly lower in number systemically and within the tumors of involution-injected mammary glands than T cells in nulliparous mice, indicating systemic immunosuppression. To demonstrate immunosuppressive function, iMCs were isolated from tumors and incubated with T cells ex vivo. iMCs from involution tumors were significantly more suppressive when compared to iMCs from nulliparous mice. These data suggest that transient exposure of latent cancer cells to the immune suppressed microenvironment of involution confers a prolonged poor prognostic signature. Further, we report evidence that postpartum involution can persistently alter breast cancer in young women. CD45+ immune cell infiltrate was elevated in mammary lobules during postpartum breast involution and in breast cancers diagnosed within 2 years of a completed pregnancy, suggesting an immunosuppressed population of immune cells is enriched in women diagnosed with postpartum breast cancer. While it is unknown if the immune cell infiltrate in normal involuting breast tissue or in postpartum breast cancers has immunosuppressive function, this may be expected based on our rodent data that shows postpartum breast cancers are characterized by high monocyte population with immune suppressive function. Our study demonstrates monocytes infiltrating the involuting mammary gland are capable of suppressing T cell function supporting the notion that immune suppression can promote a more aggressive tumor microenvironment, providing justification for targeting the immune-suppressive monocytes as a potential therapy for postpartum breast cancer patients. Citation Format: Holly Martinson, Sonail Jindal, Virginia Borges, Pepper Schedin. Immune cell influx during postpartum mammary gland involution reveals immunosuppression and tumor 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 A31.

Abstract A09: Advancements in understanding NSAID-based chemoprevention of postpartum breast cancer

Young women, within 5 years from last childbirth, have increased incidence of breast cancer as well as poorer prognosis compared to women who have never been pregnant. We refer to these breast cancers as postpartum. Our lab has proposed that the poor prognosis and possibly increased incidence of postpartum breast cancers are due to mammary gland remodeling following pregnancy, known as postpartum involution. Using rodent models and human breast tissue, characterization of postpartum involution has identified activated wound healing programs utilized to remodel the lactation competent gland back to a non-secretory state. For example, fibrillar collagen, matrix metalloproteinases, COX-2 expression, M2-like macrophages, T helper 2 (Th2)-associated cytokines IL-4, IL-13, TGF-β, and gene expression consistent with immune cell infiltrate are all increased within the mammary gland during postpartum involution. Here we report the pattern of immune cell influx into the postpartum murine mammary gland is very similar to that of classical wound healing, and importantly, we find infiltration of regulatory T cells and immunosuppressive IL-10+ macrophages that establish an immunosuppressive mammary microenvironment. Based on these studies, we have proposed that postpartum breast involution is a unique window of risk for breast cancer incidence and progression and may be an optimal target for chemoprevention. Previously we have shown in an immune deficient model of postpartum breast cancer that NSAID therapy, restricted to postpartum involution, is sufficient to decrease tumor number, growth and metastasis to levels observed in nulliparous controls. However, given that we have found pro-tumorigenic immune modulation during postpartum involution, it becomes important to determine if NSAIDs are cancer-protective in an immune competent model. We report that immune competent Balb/c mice treated with ibuprofen during postpartum involution have decreased tumor growth due in part to reduced tumor cell proliferation and increased apoptosis. Since the tumor cells are COX-2 negative, the tumor suppressive effect of ibuprofen appears to be mediated through changes in the microenvironment. Consistent with a stromal mechanism, ibuprofen treated postpartum tumors had a decrease in total CD45 leukocytes, the ratio of cytokines IL-10/IL-12 was also decreased, and T cell presence was increased. These data suggest that ibuprofen is capable of improving T cell anti-tumor activity within the context of the immune suppressed microenvironment that is present during involution. In summary, in the context of an immune competent, postpartum breast cancer model, ibuprofen decreases the tumor promotional attributes of the involution microenvironment. The question of how NSAIDs, classically considered to be immune suppressive, reverse the immune suppressive microenvironment of postpartum mammary cancers is under investigation. Potential relevance to chemoprevention in young women at high risk of developing postpartum breast cancer is evident; as ibuprofen is safe in the general population and further, treatment would be limited to the window of postpartum involution. However, caution is urged until the interactions of NSAIDs with the unique biology of the postpartum involving gland are more fully investigated. Citation Format: Holly A. Martinson, Michelle Borakove, Adriana Jones, Virginia F. Borges, Pepper Schedin. Advancements in understanding NSAID-based chemoprevention of postpartum breast cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A09.