Tumor macrophage functional heterogeneity can inform the development of novel cancer therapies.

Macrophages represent a key component of the tumor microenvironment (TME) and are largely associated with poor prognosis. Therapeutic targeting of macrophages has historically focused on inhibiting their recruitment or reprogramming their phenotype from a protumor (M2-like) to an antitumor (M1-like) one. Unfortunately, this approach has not provided clinical breakthroughs that have changed practice. Emerging studies utilizing single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics have improved our understanding of the ontogeny, phenotype, and functional plasticity of macrophages. Overlaying the wealth of current information regarding macrophage molecular subtypes and functions has also identified novel therapeutic vulnerabilities that might drive better control of tumor-associated macrophages (TAMs). Here, we discuss the functional profiling of macrophages and provide an update of novel macrophage-targeted therapies in development.

Estrogen receptor beta expression in triple negative breast cancers is not associated with recurrence or survival.

BACKGROUND: Triple negative BCa (TNBC) is defined by a lack of expression of estrogen (ERα), progesterone (PgR) receptors and human epidermal growth factor receptor 2 (HER2) as assessed by protein expression and/or gene amplification. It makes up ~ 15% of all BCa and often has a poor prognosis. TNBC is not treated with endocrine therapies as ERα and PR negative tumors in general do not show benefit. However, a small fraction of the true TNBC tumors do show tamoxifen sensitivity, with those expressing the most common isoform of ERß1 having the most benefit. Recently, the antibodies commonly used to assess ERß1 in TNBC have been found to lack specificity, which calls into question available data regarding the proportion of TNBC that express ERß1 and any relationship to clinical outcome. METHODS: To confirm the true frequency of ERß1 in TNBC we performed robust ERß1 immunohistochemistry using the specific antibody CWK-F12 ERß1 on 156 primary TNBC cancers from patients with a median of 78 months (range 0.2-155 months) follow up. RESULTS: We found that high expression of ERß1 was not associated with increased recurrence or survival when assessed as percentage of ERß1 positive tumor cells or as Allred > 5. In contrast, the non-specific PPG5-10 antibody did show an association with recurrence and survival. CONCLUSIONS: Our data indicate that ERß1 expression in TNBC tumours does not associate with prognosis.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Parity reduces mammary repopulating activity but does not affect mammary stem cells defined as CD24 + CD29/CD49fhi in mice.

BACKGROUND: Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive (ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data. METHODS: We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24midCD49fhi)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity. RESULTS: Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24+CD49fhi) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity. CONCLUSIONS: Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.

High mammographic density in women is associated with protumor inflammation.

BACKGROUND: Epidemiological studies have consistently shown that increased mammographic density (MD) is a strong risk factor for breast cancer. We previously observed an elevated number of vimentin+/CD45+ leukocytes in high MD (HMD) epithelium. In the present study, we aimed to investigate the subtypes of immune cell infiltrates in HMD and low MD (LMD) breast tissue. METHODS: Fifty-four women undergoing prophylactic mastectomy at Peter MacCallum Cancer Centre or St. Vincent's Hospital were enrolled. Upon completion of mastectomy, HMD and LMD areas were resected under radiological guidance in collaboration with BreastScreen Victoria and were subsequently fixed, processed, and sectioned. Fifteen paired HMD and LMD specimens were further selected according to their fibroglandular characteristics (reasonable amount [> 20%] of tissue per block on H&E stains) for subsequent IHC analysis of immune cell infiltration. RESULTS: Overall, immune cell infiltrates were predominantly present in breast ducts and lobules rather than in the stroma, with CD68+ macrophages and CD20+ B lymphocytes also surrounding the vasculature. Macrophages, dendritic cells (DCs), B lymphocytes, and programmed cell death protein 1 (PD-1) expression were significantly increased in HMD epithelium compared with LMD. Moreover, significantly higher levels of DCs, CD4+ T cells, and PD-1 were also observed in HMD stroma than in LMD stroma. The increased expression of interleukin (IL)-6 and IL-4, with unaltered interferon-γ, indicate a proinflammatory microenvironment. CONCLUSIONS: Our work indicates that the immune system may be activated very early in breast cancer development and may in part underpin the breast cancer risk associated with HMD.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Scribble modulates the MAPK/Fra1 pathway to disrupt luminal and ductal integrity and suppress tumour formation in the mammary gland.

Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression.

Mammographically dense human breast tissue stimulates MCF10DCIS.com progression to invasive lesions and metastasis.

BACKGROUND: High mammographic density (HMD) not only confers a significantly increased risk of breast cancer (BC) but also is associated with BCs of more advanced stages. However, it is unclear whether BC progression and metastasis are stimulated by HMD. We investigated whether patient-derived HMD breast tissue could stimulate the progression of MCF10DCIS.com cells compared with patient-matched low mammographic density (LMD) tissue. METHODS: Sterile breast specimens were obtained immediately after prophylactic mastectomy from high-risk women (n = 10). HMD and LMD regions of each specimen were resected under radiological guidance. Human MCF10DCIS.com cells, a model of ductal carcinoma in situ (DCIS), were implanted into silicone biochambers in the groins of severe combined immunodeficiency mice, either alone or with matched LMD or HMD tissue (1:1), and maintained for 6 weeks. We assessed biochamber weight as a measure of primary tumour growth, histological grade of the biochamber material, circulating tumour cells and metastatic burden by luciferase and histology. All statistical tests were two-sided. RESULTS: HMD breast tissue led to increased primary tumour take, increased biochamber weight and increased proportions of high-grade DCIS and grade 3 invasive BCs compared with LMD. This correlated with an increased metastatic burden in the mice co-implanted with HMD tissue. CONCLUSIONS: Our study is the first to explore the direct effect of HMD and LMD human breast tissue on the progression and dissemination of BC cells in vivo. The results suggest that HMD status should be a consideration in decision-making for management of patients with DCIS lesions.

Human glandular organoid formation in murine engineering chambers after collagenase digestion and flow cytometry isolation of normal human breast tissue single cells.

Women with high mammographic density (MD) are at increased risk of breast cancer (BC) after adjustment for age and body mass index. We have developed a murine biochamber model in which both high MD (HMD) and low MD (LMD) tissue can be propagated. Here, we tested whether cells isolated by collagenase digestion and fluorescence-activated cell sorting (FACS) from normal breast can be reconstituted in our biochamber model, which would allow cell-specific manipulations to be tested. Fresh breast tissue was collected from women (n = 7) undergoing prophylactic mastectomy. The tissue underwent collagenase digestion overnight and, in some cases, additional FACS enrichment to obtain mature epithelial, luminal progenitor, mammary stem, and stromal cells. Cells were then transferred bilaterally into biochambers in SCID mice (n = 5-7) and incubated for 6 weeks, before harvesting for histological analyses, and immunohistochemical staining for cytokeratins (CK), vimentin, Ki-67, murine macrophages, and Cleaved Caspase-3. Biochambers inoculated with single cells after collagenase digestion or with flow cytometry contained glandular structures of human origin (human vimentin-positive), which expressed CK-14 and pan-CK, and were proliferating (Ki-67-positive). Glandular structures from the digested tissues were smaller than those in chambers seeded with finely chopped intact mammary tissue. Mouse macrophage infiltration was higher in the chambers arising from digested tissues. Pooled single cells and FACS fractionated cells were viable in the murine biochambers and formed proliferating glandular organoids of human origin. This is among the first report to demonstrate the success of formed human glandular organoids from isolated primary mammary cells in the murine biochamber model.

Estrogen receptor regulation of the immune microenvironment in breast cancer.

Breast cancer (BCa) is the most common cancer in women and the estrogen receptor (ER)+ subtype is increasing in incidence. There are numerous therapy options available for patients that target the ER, however issues such as innate and acquired treatment resistance, and treatment related side effects justify research into alternative therapeutic options for these patients. Patients of many solid tumour types have benefitted from immunotherapy, however response rates have been generally low in ER+ BCa. We summarise the recent work assessing CDK4/6 inhibitors for ER+ BCa and how they have been shown to prime anti-tumour immune cells and achieve impressive results in preclinical models. A great example of how the immune system might be activated against ER+ BCa. We review the role of estrogen signalling in immune cells, and explore recent data highlighting the hormonal regulation of the immune microenvironment of normal breast, BCa and immune disorders. As recent data has indicated that macrophages are particularly susceptible to estrogen signalling, we highlight macrophage phagocytosis as a key potential target for priming the tumour immune microenvironment. We challenge the generally accepted paradigm that ER+ BCa are "immune-cold" - advocating instead for research into therapies that could be used in combination with targeted therapies and/or immune checkpoint blockade to achieve durable antitumour responses in ER+ BCa.

Immune Regulation of Mammary Fibroblasts and the Impact of Mammographic Density.

Mammographic density is associated with a 4-6-fold increase in breast cancer risk independent of age and BMI. High mammographic density is characterized by breast tissue with high proportions of stroma comprised of fibroblasts, collagen, and immune cells. This study sought to investigate whether stromal fibroblasts from high mammographic density breast tissue contributes to increased extracellular matrix deposition and pro-tumorigenic signaling. Mammary fibroblasts were isolated from women with high and low mammographic density and exposed to immune factors myeloperoxidase (MPO), eosinophil peroxidase (EPO), transforming growth factor beta 1 (TGFB1) and tumour necrosis factor alpha (TNFA) for 72 h and profiled for expression of cancer-associated fibroblast and extracellular matrix regulation markers. No differences in gene expression profiles or collagen production were observed between fibroblasts with high or low mammographic density, and they did not have a differential response to immune mediators. MPO and EPO significantly increased the production of collagen 1. TGFB and TNFA induced variable changes in gene expression. Fibroblasts cultured in vitro from women with high mammographic density do not appear to be inherently different to those from women with low mammographic density. The function of fibroblasts in mammographic density-associated breast cancer risk is likely to be regulated by immune signals from surrounding cells in the microenvironment.

PTPN2 elicits cell autonomous and non-cell autonomous effects on antitumor immunity in triple-negative breast cancer.

The tumor-suppressor PTPN2 is diminished in a subset of triple-negative breast cancers (TNBCs). Paradoxically, PTPN2-deficiency in tumors or T cells in mice can facilitate T cell recruitment and/or activation to promote antitumor immunity. Here, we explored the therapeutic potential of targeting PTPN2 in tumor cells and T cells. PTPN2-deficiency in TNBC associated with T cell infiltrates and PD-L1 expression, whereas low PTPN2 associated with improved survival. PTPN2 deletion in murine mammary epithelial cells TNBC models, did not promote tumorigenicity but increased STAT-1-dependent T cell recruitment and PD-L1 expression to repress tumor growth and enhance the efficacy of anti-PD-1. Furthermore, the combined deletion of PTPN2 in tumors and T cells facilitated T cell recruitment and activation and further repressed tumor growth or ablated tumors already predominated by exhausted T cells. Thus, PTPN2-targeting in tumors and/or T cells facilitates T cell recruitment and/or alleviates inhibitory constraints on T cells to combat TNBC.

Estrogen-induced immune changes within the normal mammary gland.

Breast cancer (BCa) incidence increases following aberrant hormone exposure, which has been linked to direct effects on estrogen receptor (ER)+ mammary epithelium. While estrogen exposure during mammary involution has been shown to drive tumour growth via neutrophils, the potential for the ER + immune microenvironment to mediate part (in addition to mammary epithelial cells) of hormonally controlled BCa risk during normal development has not been assessed. We collected mammary tissue, lymph nodes and blood from tumour naïve mice treated with, oophorectomy, estrogen (17ß estradiol) or Fulvestrant. Flow cytometry was used to examine the impact on the frequency of innate and adaptive immune cells. Oophorectomy and fulvestrant decreased the proportion of macrophages, particularly pro-tumour polarized M2 macrophages and neutrophils. Conversely, dendritic cells were increased by these therapies, as were eosinophils. Estrogen increased the proportion of M2 macrophages and to a lesser extent CD4-CD8- double negative and FoxP3+ regulatory T cells but decreased CD8 + T cells and B cells. Excluding eosinophils, these changes were restricted to the mammary tissue. This suggests that inhibiting estrogen action lowers the immune suppressive myeloid cells, increases in antigen presentation and eosinophil-mediated direct or indirect cytotoxic effects. In contrast, estrogen exposure, which drives BCa risk, increases the suppressive myeloid cells and reduces anti-tumour cytotoxic T cells. The impact of hormonal exposure on BCa risk, may in part be linked to its immune modulatory activity.

RASSF1A Suppression as a Potential Regulator of Mechano-Pathobiology Associated with Mammographic Density in BRCA Mutation Carriers.

High mammographic density (MD) increases breast cancer (BC) risk and creates a stiff tissue environment. BC risk is also increased in BRCA1/2 gene mutation carriers, which may be in part due to genetic disruption of the tumour suppressor gene Ras association domain family member 1 (RASSF1A), a gene that is also directly regulated by tissue stiffness. High MD combined with BRCA1/2 mutations further increase breast cancer risk, yet BRCA1/2 mutations alone or in combination do not increase MD. The molecular basis for this additive effect therefore remains unclear. We studied the interplay between MD, stiffness, and BRCA1/2 mutation status in human mammary tissue obtained after prophylactic mastectomy from women at risk of developing BC. Our results demonstrate that RASSF1A expression increased in MCF10DCIS.com cell cultures with matrix stiffness up until ranges corresponding with BiRADs 4 stiffnesses (~16 kPa), but decreased in higher stiffnesses approaching malignancy levels (>50 kPa). Similarly, higher RASSF1A protein was seen in these cells when co-cultivated with high MD tissue in murine biochambers. Conversely, local stiffness, as measured by collagen I versus III abundance, repressed RASSF1A protein expression in BRCA1, but not BRCA2 gene mutated tissues; regional density as measured radiographically repressed RASSF1A in both BRCA1/2 mutated tissues. The combinatory effect of high MD and BRCA mutations on breast cancer risk may be due to RASSF1A gene repression in regions of increased tissue stiffness.

Annexin A1 Is Required for Efficient Tumor Initiation and Cancer Stem Cell Maintenance in a Model of Human Breast Cancer.

Triple-negative breast cancer (TNBC) has a poor outcome compared to other breast cancer subtypes, and new therapies that target the molecular alterations driving tumor progression are needed. Annexin A1 is an abundant multi-functional Ca2+ binding and membrane-associated protein. Reported roles of Annexin A1 in breast cancer progression and metastasis are contradictory. Here, we sought to clarify the functions of Annexin A1 in the development and progression of TNBC. The association of Annexin A1 expression with patient prognosis in subtypes of TNBC was examined. Annexin A1 was stably knocked down in a panel of human and murine TNBC cell lines with high endogenous Annexin A1 expression that were then evaluated for orthotopic growth and spontaneous metastasis in vivo and for alterations in cell morphology in vitro. The impact of Annexin A1 knockdown on the expression of genes involved in mammary epithelial cell differentia tion and epithelial to mesenchymal transition was also determined. Annexin A1 mRNA levels correlated with poor patient prognosis in basal-like breast tumors and also in the basal-like 2 subset of TNBCs. Unexpectedly, loss of Annexin A1 expression had no effect on either primary tumor growth or spontaneous metastasis of MDA-MB-231_HM xenografts, but abrogated the growth rate of SUM149 orthotopic tumors. In an MMTV-PyMT driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation in both immuno-competent and immuno-deficient mice and induced epithelial to mesenchymal transition and upregulation of basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1- population containing putative tumor initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in a model of human breast cancer and suggest that some basal-like TNBCs may require high endogenous tumor cell Annexin A1 expression for continued growth.

Lineage enforcement by inductive mesenchyme on adult epithelial stem cells across developmental germ layers.

During development, cell differentiation is accompanied by the progressive loss of pluripotent gene expression and developmental potential, although de-differentiation in specialized cells can be induced by reprogramming strategies, indicating that transdifferentiation potential is retained in adult cells. The stromal niche provides differentiating cues to epithelial stem cells (SCs), but current evidence is restricted to tissue types within the same developmental germ layer lineage. Anticipating the use of adult SCs for tissue regeneration, we examined if stroma can enforce lineage commitment across germ layer boundaries and promote transdifferentiation of adult epithelial SCs. Here, we report tissue-specific mesenchyme instructing epithelial cells from a different germ layer origin to express dual phenotypes. Prostatic stroma induced mammary epithelia (or enriched Lin(-)CD29(HI)CD24(+/MOD) mammary SCs) to generate glandular epithelia expressing both prostatic and mammary markers such as steroid hormone receptors and transcription factors including Foxa1, Nkx3.1, and GATA-3. Array data implicated Hh and Wnt pathways in mediating stromal-epithelial interactions (validated by increased Cyclin D1 expression). Other recombinants of prostatic mesenchyme and skin epithelia, or preputial gland mesenchyme and bladder or esophageal epithelia, showed foci expressing new markers adjacent to the original epithelial differentiation (e.g., sebaceous cells within bladder urothelium), confirming altered lineage specification induced by stroma and evidence of cross-germ layer transdifferentiation. Thus, stromal cell niche is critical in maintaining (or redirecting) differentiation in adult epithelia. In order to use adult epithelial SCs in regenerative medicine, we must additionally regulate their intrinsic properties to prevent (or enable) transdifferentiation in specified SC niches.

Key steps for effective breast cancer prevention.

Despite decades of laboratory, epidemiological and clinical research, breast cancer incidence continues to rise. Breast cancer remains the leading cancer-related cause of disease burden for women, affecting one in 20 globally and as many as one in eight in high-income countries. Reducing breast cancer incidence will likely require both a population-based approach of reducing exposure to modifiable risk factors and a precision-prevention approach of identifying women at increased risk and targeting them for specific interventions, such as risk-reducing medication. We already have the capacity to estimate an individual woman's breast cancer risk using validated risk assessment models, and the accuracy of these models is likely to continue to improve over time, particularly with inclusion of newer risk factors, such as polygenic risk and mammographic density. Evidence-based risk-reducing medications are cheap, widely available and recommended by professional health bodies; however, widespread implementation of these has proven challenging. The barriers to uptake of, and adherence to, current medications will need to be considered as we deepen our understanding of breast cancer initiation and begin developing and testing novel preventives.

Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells.

INTRODUCTION: In humans, an early full-term pregnancy reduces lifetime breast cancer risk by up to 50% whereas a later pregnancy (>35 years old) can increase lifetime risk. Several mechanisms have been suggested, including changes in levels of circulating hormones, changes in the way the breast responds to these hormones, changes in gene expression programmes which may alter susceptibility to transformation and changes to mammary stem cell numbers or behaviour. Previous studies have shown that the mammary tissue isolated from both virgin and parous mice has the ability to repopulate a cleared mammary fat pad in transplant experiments. Limited dilution transplant assays have demonstrated that early pregnancy (at 5 weeks of age) reduces stem/progenitor cell numbers in the mouse mammary epithelium by twofold. However, the effects on stem/progenitor cell numbers in the mammary epithelium of a pregnancy in older animals have not yet been tested. METHODS: Mice were put through a full-term pregnancy at 9 weeks of age, when the mammary epithelium is mature. The total mammary epithelium was purified from parous 7-week post-lactation and age-matched virgin mice and analysed by flow cytometry and limiting dilution cleared fat pad transplants. RESULTS: There were no significant differences in the proportions of different mammary epithelial cell populations or numbers of CD24(+/Low) Sca-1- CD49f(High) cells (stem cell enriched basal mammary epithelial compartment). There was no significant difference in stem/progenitor cell frequency based on limiting dilution transplants between the parous and age-matched virgin epithelium. CONCLUSIONS: Although differences between parous and virgin mammary epithelium at later time points post lactation or following multiple pregnancies cannot be ruled out, there are no differences in stem/progenitor cell numbers between mammary epithelium isolated from parous animals which were mated at 9 weeks old and virgin animals. However, a recent report has suggested that animals that were mated at 5 weeks old have a twofold reduction in stem/progenitor cell numbers. This is of interest given the association between early, but not late, pregnancy and breast cancer risk reduction in humans. However, a mechanistic connection between stem cell numbers and breast cancer risk remains to be established.

Estrogen receptor subtypes dictate the proliferative nature of the mammary gland.

Estrogen induces proliferation of breast epithelial cells and is responsible for breast development at puberty. This tightly regulated control is lost in estrogen-receptor-positive (ER+) breast cancers, which comprise over 70% of all breast cancers. Currently, breast cancer diagnosis and treatment considers only the α isoform of ER; however, there is a second ER, ERß. Whilst ERα mediates estrogen-driven proliferation of the normal breast in puberty and breast cancers, ERß has been shown to exert an anti-proliferative effect on the normal breast. It is not known how the expression of each ER (alone or in combination) correlates with the ability of estrogen to induce proliferation in the breast. We assessed the levels of each ER in normal mouse mammary glands subdivided into proliferative and non-proliferative regions. ERα was most abundant in the proliferative regions of younger mice, with ERß expressed most abundantly in old mice. We correlated this expression profile with function by showing that the ability of estrogen to induce proliferation was reduced in older mice. To show that the ER profile associated with breast cancer risk, we assessed ER expression in parous mice which are known to have a reduced risk of developing ERα breast cancer. ERα expression was significantly decreased yet co-localization analysis revealed ERß expression increased with parity. Parous mice had less unopposed nuclear ERα expression and increased levels of ERß. These changes suggest that the nuclear expression of ERs dictates the proliferative nature of the breast and may explain the decreased breast cancer risk with parity.

Mammographic density: a potential monitoring biomarker for adjuvant and preventative breast cancer endocrine therapies.

Increased mammographic density (MD) has been shown beyond doubt to be a marker for increased breast cancer risk, though the underpinning pathobiology is yet to be fully elucidated. Estrogenic activity exerts a strong influence over MD, which consequently has been observed to change predictably in response to tamoxifen anti-estrogen therapy, although results for other selective estrogen receptor modulators and aromatase inhibitors are less consistent. In both primary and secondary prevention settings, tamoxifen-associated MD changes correlate with successful modulation of risk or outcome, particularly among pre-menopausal women; an observation that supports the potential use of MD change as a surrogate marker where short-term MD changes reflect longer-term anti-estrogen efficacy. Here we summarize endocrine therapy-induced MD changes and attendant outcomes and discuss both the need for outcome surrogates in such therapy, as well as make a case for MD as such a monitoring marker. We then discuss the process and steps required to validate and introduce MD into practice as a predictor or surrogate for endocrine therapy efficacy in preventive and adjuvant breast cancer treatment settings.