Postpartum Involution Research Articles

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

Abstract 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.

Rat Mammary Extracellular Matrix Composition and Response to Ibuprofen Treatment During Postpartum Involution by Differential GeLC–MS/MS Analysis

Breast cancer patients diagnosed within five years following pregnancy have increased metastasis and decreased survival. A hallmark of postpartum biology that may contribute to this poor prognosis is mammary gland involution, involving massive epithelial cell death and dramatic stromal remodeling. Previous studies show pro-tumorigenic properties of extracellular matrix (ECM) isolated from rodent mammary glands undergoing postpartum involution. More recent work demonstrates systemic ibuprofen treatment during involution decreases its tumor-promotional nature. Utilizing a proteomics approach, we identified relative differences in the composition of mammary ECM isolated from nulliparous rats and those undergoing postpartum involution, with and without ibuprofen treatment. GeLC-MS/MS experiments resulted in 20327 peptide identifications that mapped to 884 proteins with a <0.02% false discovery rate. Label-free quantification yielded several ECM differences between nulliparous and involuting glands related to collagen-fiber organization, cell motility and attachment, and cytokine regulation. Increases in known pro-tumorigenic ECM proteins osteopontin, tenascin-C, and laminin-α1 and pro-inflammatory proteins STAT3 and CD68 further identify candidate mediators of breast cancer progression specific to the involution window. With postpartum ibuprofen treatment, decreases in tenascin-C and three laminin chains were revealed. Our data suggest novel ECM mediators of breast cancer progression and demonstrate a protective influence of ibuprofen on mammary ECM composition.

Immunohistochemical localization of beta defensins in the endometrium of rat uterus during the postpartum involution period

β-Defensins are small cationic molecules that have antimicrobial actions against bacteria, fungi and viruses and contribute to mucosal immune responses at epithelial sites. The female reproductive tract is an important site of defensin production. This study was conducted to determine the possible changes in proportions and localization of β-defensin 1-4 in the rat uterus at the 1st, 3th, 5th, 10th and 15th days of postpartum and at the period of diestrus using immunohistochemical techniques. In the present study, it was determined that β-defensin 1-4 were generally found in all structural components of the endometrium (luminal and glandular epithelium, stromal cells and blood vessels) in both the nucleus and the cytoplasm of cells during the involution period and diestrus. Suprisingly, immunoreaction of β-defensin 2 was also observed in the lateral membrane of the luminal and glandular epithelial cells on the 10th day of involution and immunostaining of β-defensin 4 was also localized in the apical membrane of the luminal and glandular epithelial cells. The current study demonstrated β-defensin 1-4 immunoreactivities in the endothelium of blood vessels were stronger throughout the involution period. Although β-defensins 2 and 3 were localized in both the nuclei and the cytoplasm of endothelial cells, β-defensins 1 and 4 were present in only cytoplasm. These results show that the most component of rat endometrium expresses human β-defensin 1-4 in a involution-dependent manner. Therefore it may be asserted that these molecules constitute a organised protection to prevent uterus from probable infections during the involution process.

Development of the maternal anal canal during pregnancy and the postpartum period: a longitudinal and functional ultrasound study

Normal anatomical and physiological development of the maternal anal canal during and after pregnancy has been documented scarcely. We aimed to study the position and volume of the anal canal, during and after pregnancy, in women without previous delivery. This was a longitudinal study in which transvaginal three-dimensional ultrasound was used to measure anatomical structures in the anal canal during rest and squeeze in 23 nulliparous women. The total anal canal volume (ACV), anorectal curvature (ARC), anovaginal angle (AVA) and anal canal length were determined at 18, 28 and 36 weeks of pregnancy and at 3 months postpartum. Total ACV at rest increased from a mean of 10.17 cm(3) at 18 weeks to 12.37 cm(3) and 12.21 cm(3) at 28 and 36 weeks, respectively (P = 0.001 and P = 0.010 vs. first measurement). For anal canal length, the corresponding mean measurements were 3.91 cm, 4.07 cm (P = 0.13) and 4.21 cm (P = 0.017). Postpartum, the mean total ACV was 10.86 cm(3) and length was 3.90 cm (P = 0.10 and P = 0.70 vs. first measurement). No significant changes were observed in ARC and AVA during or after pregnancy. Compared to at-rest status, the anal length significantly increased on voluntary squeeze (P = 0.007, 0.007, 0.022 and 0.004 at the four time points), while no differences in total ACV were observed. In mid-pregnancy AVA significantly increased during squeeze (P = 0.006 and 0.002 at weeks 18 and 28, respectively). Anal canal length and total ACV increase during pregnancy in women without previous delivery. Voluntary squeezing elongates the anal canal and increases the angle formed with respect to the direction of the vagina. During postpartum involution, the characteristics of the anal canal revert to those observed at 18 weeks of pregnancy.

Decidual and myometrial myeloid cells contribute to term and preterm parturition, and post-partum uterine involution in mice

Decidual and myometrial myeloid cells contribute to term and preterm parturition, and post-partum uterine involution in mice

Could NSAIDs become a preventative therapy in pregnancy-associated breast cancer?

SUMMARY Pregnancy-associated breast cancer (PABC) is a unique type of young women’s breast cancer that includes two biologically distinct conditions: those diagnosed during pregnancy and those diagnosed postpartum. It is the dominant subset of postpartum PABC that is more consistently associated with higher breast cancer mortality. Preclinical work has identified the normal event of postpartum involution as a wound-healing milieu rich in immune cells. We have shown that the involution environment drives tumor growth, proliferation and metastasis. Moreover, we have demonstrated in animal models that this ‘involution effect’ can be abrogated with drug therapy, namely NSAIDs, which target normal involution pathways implicated in PABC tumor promotion. In this perspective, we review the contemporary understanding of PABC, our preclinical modeling and its implications and the unmet research needs required for future translation of these preclinical studies into rational and safe human clinical trials.

P2-01-04: The Post-Partum Diagnosis of Pregnancy Associated Breast Cancer Confers an Increased Risk for Metastasis without Increased Incidence of Poorer Prognosis Biologic Subtype.

Abstract Background: All women who complete a childbirth have an increased risk for the subsequent breast cancer regardless of age at their first birth. In younger women, this period truncates 10 years later and pregnancy then becomes protective, whereas in older mothers the risk persists. Moreover, breast cancers diagnosed subsequent to a recent childbirth have an increased risk for metastasis and death. However much of the existing data is confounded by inclusion of cases diagnosed during pregnancy. Hypothesis: Our preclinical models of pregnancy associated breast cancer (PABC) has identified the “involution hypothesis”, where the normal physiologic post-partum breast involution is the pregnancy-related event promotional of breast cancer growth, invasion and metastasis. We have performed a retrospective cohort study, restricted to postpartum PABC and controls, to fully characterize post-partum PABC by biologic subtype and known predictors for metastasis. Also we identify if the specific period of post-partum PABC has a higher risk for metastasis then non-PABC, as predicted by our animal model studies. Methods: We performed a retrospective cohort of 527 cases of breast cancer diagnosed at age 45 or younger from 1981–2011. Pregnancy status was identified from medical records and defined as nulliparous (n=107), PABC (up to 5 years post-partum of last childbirth, n=114), and Later Parous (&amp;gt;10 years post-partum, n=239). Clinicopathologic characteristics were obtained from pathology records. Outcomes data obtained through the University of Colorado Tumor Registrar. Hazard ratios of risk of metastatic recurrence of nulliparous and PABC groups were analyzed using a Cox Proportional Hazards Model and time to metastatic recurrence was analyzed using standard Kaplan Meier curves. Results: Compared to nulliparous cases, PABC cases were more frequently Stage IV(11.4% v. 4.9%), T3 tumor size(17.3% v. 13.1%), poorly differentiated(56.6% v. 49.0%), lobular histology(4.5% v.1.0%), with lymphovascular invasion(30.4% v. 24.5%) and with involved lymphnodes(57.6% v. 48.6%). No differences were seen between Luminal A, Luminal B, Her 2 and Triple Negative subtypes. Average follow-up was 3.4 years for nulliparous, 3.5 for PABC and 4.6 for Later Parous. Survival analysis revealed a cumulative 5-year metastatic recurrence free probability of 80.6% for nulliparous, 68.5% for PABC and 80.5% for Later Parous. Compared to nulliparous, PABC had greater than a three times higher risk for subsequent metastatic recurrence (HR 3.29, 95% CI: 1.25−8.63). No significant difference in the risk of metastatic recurrence for Later Parous compared to nulliparous was observed (HR: 1.079, 95% CI: 0.46−2.55). Conclusion: Post-partum PABC is characterized by higher stage at diagnosis but without enrichment for poorer prognosis biologic subtypes. Post-Partum PABC has an increased risk for metastasis within five years of diagnosis, with an overall 3.29 times higher risk of a metastatic recurrence. These data support our hypothesis that the post-partum window, and the associated role of naturally occurring involution, may be promotional of tumor growth, invasion and metastasis as previously identified in our animal models. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-04.

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.

Abstract B61: Postpartum mammary gland involution drives DCIS progression through collagen and COX-2, identifying a target for intervention

Abstract Prognosis of young women's breast cancer is influenced by reproductive history. Women diagnosed within five years postpartum have worse prognosis than nulliparous women or women diagnosed during pregnancy. We propose that breast involution following pregnancy accounts for the poor prognosis of breast cancers diagnosed postpartum. Our ‘involution hypothesis’ states that the physiologically normal microenvironment of the postpartum involuting gland is tumor promotional; however, testing the involution hypothesis has been limited by lack of pre-clinical models. We developed a mouse xenograft model of PABC that isolates postpartum mammary gland involution as a driving force for breast cancer progression and metastasis. In this model, human breast cancer cells are injected into actively involuting mouse mammary glands and nulliparous controls. Results from this model indicate that tumor cells exposed to the collagen rich involuting mammary microenvironment form larger tumors characterized by abundant fibrillar collagen, high COX-2 expression, and an invasive phenotype. In culture, these tumor cells are invasive in a collagen/COX-2-dependent manner. Importantly, in the involuting mammary gland, inhibition of COX-2 with celecoxib or ibuprofen reduced the collagen fibrillogenesis associated with involution, as well as tumor growth and lung infiltration. These anti-tumor effects were observed without blocking apoptosis of secretory mammary epithelial cells, which is required to remodel the gland to a non-secretory state, and is a requisite for advancing this strategy to clinical trial. The question of whether an NSAID based intervention study could be aimed at recently pregnant women at high risk for breast cancer remains to be determined, but is an extremely desirable objective given that there are more than 6 million pregnancies in the US per year. In summary, these data support further research to determine whether women at high-risk for postpartum breast cancer would benefit from treatment with NSAIDs during postpartum involution. This work was funded by DOD, Komen, and ACS. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B61.

Tissue States Provide Novel Insights into Attributes that Drive Metastasis

Recently, in Nature Medicine, Schedin and colleagues define attributes within the involuting postpartum breast microenvironment that promote breast cancer metastasis. Cell invasiveness is controlled by a positive feedback loop involving COX-2 and fibrillar collagen. NSAIDs suppress tumor progression during postpartum involution, potentially providing effective agents for intervention in pregnant women.

The role of collagen and COX-2 in post-partum breast involution on the progression of pregnancy-associated breast cancer and its inhibition by NSAIDs.

e11117 Background: Prognosis of young women’s breast cancer is influenced by reproductive history with worse prognosis when diagnosis is within five years postpartum. We propose that breast involution accounts for the poor prognosis of postpartum breast cancers (BC), as involution is identified by tissue remodeling programs that share similarities with tumor promoting microenvironments including elevated fibrillar collagen deposition and COX-2 expression. Methods: We developed murine models of postpartum BC. To determine if targeting COX-2 may decrease progression of postpartum BC, mice were treated with NSAID ibuprofen for two weeks during the window of involution. Further, the collagen/COX-2 pathway in human postpartum BC was investigated in breast tissue and DCIS from young women. Results: The normal murine involuting gland showed high levels of radiating collagen fibers organized similar to collagen from invasive tumors. Mammary tumors arising in this collagen-rich microenvironment have increased levels of COX-2. In vitro, tumor cells isolated from PABCs are motile and invasive in a collagen and COX-2 dependent manner. Surprisingly inhibition of COX-2 during involution decreased collagen levels and frequency of radial fibers. Ibuprofen treatment reduced tumor size, collagen architecture, COX-2 expression, and lung cell seeding. In humans, comparison of breast tissue from nulliparous women to involution shows increased levels of collagen and >2x the frequency of radially aligned fibers. In our DCIS study, analysis of COX-2 expression reveals a trend towards increased COX-2 expression in the postpartum DCIS lesions. Conclusions: Our results confirm involution as a driver of postpartum BC and indicate two roles for COX-2 in tumor cell invasion in the postpartum setting: 1) COX-2 activity in the tumor is required for motility and 2) COX-2 activity in the host promotes collagen fibrillogenesis, which promotes metastasis. The human correlative results implicate collagen and COX-2 in the poor prognosis of postpartum BC. Our data preliminarily support the idea of an NSAID intervention study for recently pregnant women at high risk for breast cancer.

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

Abstract 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

Postpartum factors and natural fibroid regression

Many fibroids regress with pregnancy or postpartum involution. We sought to identify factors that might inhibit or enhance this natural regression. We used a prospective cohort of women with fibroids (n = 494) determined by ultrasound screening during the early first trimester identified from the Right from the Start study. Ultrasounds were repeated 3-6 months postpartum (n = 279). Logistic regression analyses were used to identify factors associated with fibroid regression (>50% reduction in volume). Postpartum progestin users had significantly less fibroid regression (P = .01), whereas there was no association for combined estrogen-progestin use. Cesarean delivery and fever (hypothesized to inhibit regression) and breast feeding (hypothesized to enhance regression) were not associated with fibroid regression. Progestin use in the postpartum period may limit regression of fibroids, consistent with prior literature on progesterone's role in fibroid development. Research into progestin-only treatments in critical reproductive periods is needed.

Non-steroidal anti-inflammatory drugs target the pro-tumorigenic extracellular matrix of the postpartum mammary gland

Breast cancer patients diagnosed postpartum have poor prognosis. The postpartum mammary gland undergoes tissue regression to return to the pre-pregnant state. This involution is characterized by wound healing programs known to be tumor promotional in other contexts. Previous studies have shown that mammary extracellular matrix (ECM) from nulliparous rats has tumor suppressive attributes, while mammary ECM from involuting mammary glands is promotional. In models of pregnancy-associated breast cancer, non-steroidal anti-inflammatory drug (NSAID) treatment targeted to postpartum involution inhibits tumor progression, in part by suppressing COX-2 dependent collagen deposition. Because mammary ECM proteins are coordinately regulated, NSAID treatment is anticipated to result in additional protective changes in the mammary extracellular matrix. Here, systemic NSAID treatment was utilized during postpartum involution to reduce mammary COX-2 activity. ECM was isolated from actively involuting glands of rats treated with NSAIDs and compared to ECM isolated from control-involution and nulliparous rats in 3D cell culture and xenograft assays. Compositional changes in ECM between groups were identified by proteomics. In four distinct 3D culture assays, normal and transformed mammary epithelial cells plated in NSAID-involution ECM, phenocopied cells plated in ECM from nulliparous rats rather than ECM from control-involution rats. Tumor cells mixed with NSAID-involution ECM and injected orthotopically in mice formed smaller tumors than cells mixed with control-involution ECM. Proteomic analyses identified and 3D culture assays implicated the ECM protein tenascin-C as a potential mediator of tumor progression during involution that is decreased by NSAID treatment. In summary, NSAID treatment decreases tumor-promotional attributes of postpartum involution mammary ECM.

Abstract CN10-03: Stromal signals from the involuting microenvironment that are important in postpartum breast cancer

Abstract Prognosis of young women's breast cancer is influenced by pregnancy. Women diagnosed within 5 years of childbirth have worse prognosis than age-matched nulliparous women or women diagnosed while pregnant. These data indicate that pregnancy-associated breast cancer (PABC) exists as two subtypes, with poor prognosis associated with cases diagnosed in the postpartum setting rather than during pregnancy. We propose that breast involution following pregnancy accounts for the poor prognosis of breast cancers diagnosed within 5 years of childbirth. Characterization of the involuting mammary gland has identified tissue remodeling programs associated with wound healing and inflammatory responses that share similarities with cancer microenvironments known to be tumor promotional. Based on these results, we proposed the Involution Hypothesis, which states that the physiologically normal microenvironment of the postpartum involuting gland is tumor promotional. We estimate a high proportion of young women with breast cancer actually have a “recent” pregnancy as a negative prognostic feature. Specifically, with an upper limit of 40 years of age for reproductive potential and a recent pregnancy defined as within 5 years of diagnosis, an estimated 20,000 cases of breast cancer per year in the U.S. may qualify as postpartum. Targeting the postpartum window with interventions designed to reduce the tumor promotional attributes of involution is highly attractive, as this window of risk is restricted in time and the population is readily identifiable. Testing the Involution Hypothesis has been limited by lack of preclinical models. We describe here a mouse xenograft model of PABC that isolates postpartum mammary gland involution as a driving force for breast cancer progression and metastasis. In this model, human mammary MCFDCIS.com tumor cells injected into the postpartum, involuting mammary gland form more proliferative and invasive tumors than tumor cells injected into the nulliparous gland. The involuting gland is characterized by high levels of radiating collagen fibers organized similar to collagen associated with invasive tumors across species. In our model of postpartum breast cancer, fibrillar collagen is identified as a primary mediator of tumor progression. Mammary tumors arising in the collagen-rich, involuting microenvironment are invasive and have increased levels of COX-2. In vitro, primary tumor cells isolated from PABC tumors are motile and invasive in a COX-2 dependent manner. Evidence that COX-2 induced motility is dependent on collagen signaling is demonstrated with functional blocking antibodies against β1-integrin, 2β1-integrin, and integrin associated protein CD47. These data suggest that treatment targeting COX-2 during postpartum involution may decrease progression of postpartum breast cancer. To test this hypothesis, mice were treated with the NSAID ibuprofen for two weeks coinciding with postpartum mammary gland involution. NSAID treatment was found to reduce tumor size, COX-2 expression, and lung cell seeding in this model. These antitumor effects were observed without blocking apoptosis of secretory mammary epithelial cells, which is required to remodel the gland to a nonsecretory state, and is a requisite for advancing this strategy to clinical trial. The question of whether an NSAID based intervention study could be aimed at recently pregnant women at high risk for breast cancer remains to be determined, but is an extremely desirable objective given that there are more than 6 million pregnancies in the U.S. per year. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN10-03.

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

Abstract 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.

Structural Manifestations of Mechanisms of Myometrium Involution after Repeated Pregnancies in Mice

Myocyte hypertrophy and proliferation in the myometrium were observed in mice during the first pregnancy and involution of the myometrium was completed by day 10 postpartum. Parameters of all structures returned to values observed in intact uterus. During the third pregnancy, the processes of myometrium hypertrophy were similar to those during the first pregnancy, but the intensity of proliferation was higher against the background of poorer vascularization, which led to destruction of some myocytes. Postpartum involution of the myometrium in these mice was not completed by day 10 after delivery. We observed hypertrophy and proliferation of some myocytes followed by their destruction, decrease in vessel number, and many-fold decrease in collagen content in the myometrial interstitium. The number of fetuses in the litter decreased after multiple pregnancies.

Hans Strahl’s pioneering studies in comparative placentation

Hans Strahl, a contemporary of Duval and Hubrecht, made many important contributions to comparative placentation. Despite this he is not well known and some of his original observations tend to be attributed to later authors. Strahl published a classification of placental types based on their shape and relationship to maternal tissues. This greatly influenced the work of Otto Grosser, who became better known in part because his work was more accessible to other scientists and clinicians. Strahl described the development of the fetal membranes across a broad range of mammalian orders extending his observations beyond parturition to the post partum involution of the uterus. He paid close attention to structures designed for histotrophic nutrition including the areolae of moles, haemophagous organs of carnivores and tenrecs and chorionic vesicles of lemurs and lorises. We here provide a summary of some of the most important findings made by Strahl including work on placentation in carnivores and higher primates that remains unsurpassed.

Effect of prostaglandin F2α on uterine contractile activity and hormonal and biochemical status of cows

The stimulating effect of PGF2α on uterine contractile function of cows after calving and reaction of endocrine, bioenergetic, and amino acid systems and nitrogen oxide system to its injection are determined. The current notion about the molecular and biochemical mechanisms of the effect of prostaglandins on processes of postpartum involution of genital organs is broadened.

Abstract CN13-03: Postnatal breast involution as a highly targeted window for breast cancer prevention

Abstract Epidemiologic studies demonstrate a dual effect of pregnancy on breast cancer risk. Women with early age first pregnancy experience reduced breast cancer risk in their post-menopausal years compared to nulliparous women; hence the protective effect of pregnancy. However, pregnancy transiently increases risk of breast cancer for at least ten years following parturition, thus young parous women have higher risk of breast cancer than age-matched nulliparous women. In addition to increasing breast cancer incidence in young women, pregnancy can also negatively impact prognosis, with relative risk for death reported between 1.0–2.2, depending on age of first pregnancy. We recently proposed two distinct subtypes of PABC; breast cancer diagnosed during pregnancy and breast cancer diagnosed postpartum. This distinction is important because emerging epidemiologic data indicates worsened outcomes specific to postpartum cases. We hypothesize that postpartum PABC has poor prognosis because mammary gland involution promotes progression and metastasis of preexisting disease . Characterization of postpartum gland involution has identified tissue remodeling programs associated with wound healing, inflammation and immunosuppression including fibrillar collagen deposition, high MMP−2, −3 and−9 activities, ECM matrix proteolysis, infiltration of alternatively activated macrophages similar to tumor associated macrophages, and high levels of the M2/immunosuppressive cytokines IL-4 and IL-13. Thus, the postpartum involuting microenvironment, while physiologically normal, shares similarities with cancer microenvironments known to be tumor promotional. Using mammary ECM isolated from actively involuting or quiescent nulliparous glands as substratum in cell culture studies, ECM from involuting glands was found to promote breast cancer cell motility and invasion. Using two orthotopic murine xenograft models of pregnancy associated breast cancer (PABC), we demonstrate that MCF10A.DCIS cells form tumors that are larger and more metastatic when transplanted into an actively involuting mammary gland then into quiescent mammary glands of nulliparous mice. Tumors that develop in the involuting microenvironment are more likely to express the proliferative marker Ki67, to overexpress the inflammatory enzyme COX-2 and the basal tumor cell marker p63, and to be infiltrated with thick taut collagen fibers. These data demonstrate that the promotional effects of involution on PABC can be separated from the anticipated tumor promotional effects of gestation; independently identifying the microenvironment of the involuting gland as a key determinant of tumor growth and invasiveness. To address whether targeting involution with non-steroidal anti-inflammatory agents aspirin or ibuprofen could reduce the tumor promotional attributes of postpartum involution, rats were treated with 200 mg/kg dose of aspirin or 30 mg/kg dose of ibuprofen starting on the day of weaning and continuing for 6 days post weaning. Drug treatments did not inhibit normal alveolar regression that occurs with involution. However, mammary ECM isolated from involuting glands of rats treated with aspirin or ibuprofen was found to phenocopy tumor-suppressive nulliparous mammary ECM in 3D culture of transformed mammary epithelial cells. These data suggest that aspirin and ibuprofen reduce the pro-inflammatory microenvironment of postpartum involution consistent with tumor suppression. To evaluate whether targeting involution could reduce the tumor promotional effects of involution in vivo, mice were treated with ibuprofen starting on the day of weaning and continued for 2 weeks, such that treatment was limited to the involution window. MCF10A.DCIS cells were injected into the involuting glands one day post weaning and tumor growth characterized for 6 weeks. Average tumor growth was decreased in mice with ibuprofen treatment during involution compared to the control-involution group. Further, tumor size in the ibuprofen-involution group was reduced to similar tumor size as observed in the control-nulliparous group. These data demonstrate that postpartum mammary gland involution is characterized by a pro-inflammatory microenvironment, implicating physiologic tissue inflammation in the poor prognosis of PABC. Likewise, in two new murine models for PABC, involution is associated with increased tumor invasion and metastasis. Finally, these data identify the window of postpartum gland involution as a potential target for chemoprevention. Citation Information: Cancer Prev Res 2010;3(1 Suppl):CN13-03.