Mammary Duct System Research Articles

Phosphotungstic acid-enhanced microcomputed tomography for quantitative visualization of mouse mammary gland morphology.

Even though current techniques provide two-dimensional (2D) imaging of the mouse mammary gland, they fail to achieve high-resolution three-dimensional (3D) reconstruction and quantification. The objective of this study is to establish and evaluate quantitative visualization of the mouse mammary epithelium through microcomputed tomography (microCT) using phosphotungstic acid (PTA) as a contrast agent. Ex vivo microCT scan images of the mouse mammary glands were obtained following staining by PTA, whereas for quantification we adapted volumetric parameters that are used for assessing trabecular bone morphometry and can be structurally applicable in the mammary ductal system. The proposed method was validated in distinct developmental stages and upon short-term treatment with synthetic progesterone, using the carmine alum staining for comparison. We demonstrate a simple PTA staining procedure that allows high contrast 3D imaging of mammary glands and quantitation of mammary duct structures using microCT. We validated the proposed method in distinct developmental stages, such as at puberty, adult mice, pregnancy as well as upon progesterone treatment. Compared with carmine alum staining, the microCT analysis provided higher resolution 2D and 3D images of the mammary gland morphology, with lower background that enabled the detection of subtle changes. This work is the first study that employs PTA-enhanced microCT for 3D imaging and volumetric analysis of mouse mammary glands. Our results establish PTA-enhanced microCT as a useful tool for comparative studies of the mouse mammary gland morphology that can apply in mutant mice and for the preclinical evaluation of pharmaceuticals in breast cancer models.

Intraductal Therapy in Breast Cancer: Current Status and Future Prospective.

With our improved understanding of the biological behavior of breast cancer, minimally invasive intervention is urgently needed for personalized treatment of early disease. Intraductal therapy is one such minimally invasive approach. With the help of appropriate tools, technologies using the intraductal means of entering the ducts may be used both to diagnose and treat lesions in the mammary duct system with less trauma and at the same time avoid systemic toxicity. Traditional agents such as those targeting pathways, endocrine therapy, immunotherapy, or gene therapy can be used alone or combined with other new technologies, such as nanomaterials, through the intraductal route. Additionally, relevant mammary tumor models in rodents which reflect changes in the tumor microenvironment will help deepen our understanding of their biological behavior and heterogeneity. This article reviews the current status and future prospects of intraductal therapy in breast cancer, with emphasis on ductal carcinoma in situ.

Abstract P3-03-07: TNFR-signaling in breast cancer development

Abstract Introduction: Breast cancer (BC) is the 2nd leading cause of cancer death among women in the US. Known risk factors for the development of BC include thoracic radiotherapy during puberty and obesity with the latter turning into a nation-wide health crisis. The underlying mechanisms that lead to increased BC incidences in these two scenarios are incompletely understood. However, exposure to ionizing radiation (IR) or obesity generate pro-inflammatory conditions that have been linked to mammary carcinogenesis. As inflammation involves signaling through TNF receptors (TNFR), we have therefore employed crosses between mice that spontaneously develop BC (MMTV-Wnt1) and TNFR KO mice to study the impact of TNFR downstream signaling on BC development. Material and Methods: MMTV-Wnt1 were crossed with TNFR1 or TNFR2 KO mice to explore the BC incidence during the first year post-partum in both genders. Mammary gland area and ductal outgrowth were evaluated by whole mount histological staining with 6-week-old female (end of puberty) and 14-week-old mice (adult). Tumor cells were harvested from tumor-developed MMTV-Wnt1 and MMTV-Wnt1 x TNFR2 KO female mice, the population of tumor initiating cells (EpCAMlow/CD49fhi) was analyzed by FACS. Vasculature formation (CD31), cell proliferation (Ki67) and immune system (CD4, CD8, F4/80) were evaluated within tumors by immunohistochemistry staining. Tumor cells were digested out from primary tumors and cultured in spheres condition. In-vitro sphere formation assay was performed w/o IR or TNFα treatment. All quantitative results were treated by ANOVA and Bonferroni analysis, upon verification of normal data distribution with α value at 0.05. Results: TNFR2KO drastically accelerates BC in female animals but not in males. In contrary, TNFR1 KOhad no effect on female BC development but completely prevented BC in males. TNFR2 KO led to elongation of mammary gland duct system, expansion of mammary epithelial stem cell pool and increase of BC initiating cells, as well as more vasculatures and proliferating cells in developed tumors. T-cellsand macrophage content in established tumors don’t correlate with differences in BC incidence between the crosses. MMTV-Wnt1 x TNFR2 KO mammospheres were more resistant to IR compared to Wnt1 x TNFR1 KO and MMTV-Wnt1. Pro-inflammatory TNFα ligand increased the sphere formation in a dose-dependent manner within Wnt1 x TNFR1 KO and MMTV-Wnt1 spheres, but not in Wnt1 x TNFR2 KO ones. Conclusion: Heterozygous TNFR2 KO in female MMTV-Wnt1 Tg mice increases the BC incidence, along with more resistance to IR, suggesting the downstream of TNFR2 may potentially suppress breast carcinogenesis. Also TNFR2 KO leads to more mammary gland branching and expansion of epithelial stem cells during development, which imply the potential use of TNFR2 agonist in thoracic radiotherapy and obese scenarios to reduce the BC incidence. Citation Format: Ling He, Kruttika Bhat, Paul Medina, Claudia Alli, Mohammad Saki, Fei Cheng, Frank Pajonk. TNFR-signaling in breast cancer development [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-07.

Long-label-retaining mammary epithelial cells are created early in ductal development and distributed throughout the branching ducts

Long-label retention has been used by many to prove Cairns' immortal strand hypothesis and to identify potential stem cells. Here, we describe two strategies using 5-ethynl-2′-deoxyuridine (EdU) to identify and understand the distribution of long-label-retaining mammary epithelial cells during formation of the mouse mammary ductal system. First, EdU was given upon two consecutive days per week during weeks 4 through 10 and analyzed for label retention at 13 weeks of age. Alternatively, EdU was given for 14 consecutive days beginning at 28 days of age and ending at 42 days of age. Analyses were conducted at >91 days of age (13 weeks). Many more LREC were detected following the second labeling method and their distribution among the subsequently developed ducts. This finding indicated that the early-labeled cells that retained their label were distributed into portions of the gland that developed after the ending of EdU treatment (i.e. 42–>91 days). These observations may have important meaning with respect to the previously demonstrated retention of regenerative capacity throughout the mouse mammary gland despite age or reproductive history. These results suggest LREC may represent long-lived progenitor cells that are responsible for mammary gland homeostasis. Additionally, these cells may act as multipotent stem cells capable of mammary gland regeneration upon random fragment transplantation into epithelium-denuded mammary fat pads.

DNA promoter hypermethylation in nipple fluid: a potential tool for early breast cancer detection.

IntroductionNipple fluid aspiration provides direct non-invasive sampling of fluid from the mammary ductal system, where the majority of breast cancers originate. DNA promoter hypermethylation (“methylation”) occurs early and at high frequency in breast carcinogenesis, bearing the potential as a biomarker for cancer detection at its earliest stages. We assessed methylation in nipple fluid from breasts of healthy women, of women with sporadic breast cancer and of their contralateral breasts. Our goal was to investigate whether nipple fluid can be used as a reliable methylation biomarker source.MethodsMethylation levels of 13 genes were analysed by quantitative multiplex-methylation specific PCR (QM-MSP) in nipple fluid samples from breasts of healthy women, and from the affected and contralateral breasts of breast cancer patients.ResultsMethylation analysis of the low-volume nipple fluid samples was feasible. Despite the generally low methylation levels, cancerous and healthy breasts nipple fluid could be discriminated with an area under the receiver operating characteristic curve (AUC) of 0.64 (p<0.01) based on a multivariate model including AKR1B1, ALX1, RASSF1A and TM6SF1. Within-patient differences between cancerous and contralateral nipple fluid samples were less prominent.ConclusionsCancerous nipple fluid contains increased levels of methylation of tumor suppressor genes that potentially could serve as a biomarker for early breast cancer detection.

Repeated nipple fluid aspiration: compliance and feasibility results from a prospective multicenter study.

BackgroundDespite intensive surveillance, a high rate of interval malignancies is still seen in women at increased breast cancer risk. Therefore, novel screening modalities aiming at early detection remain needed. The intraductal approach offers the possibility to directly sample fluid containing cells, DNA and proteins from the mammary ductal system where, in the majority of cases, breast cancer originates. Fluid from the breast can non-invasively be obtained by oxytocin-assisted vacuum aspiration, called nipple fluid aspiration (NFA). The goal of this feasibility study was to evaluate the potential of repeated NFA, which is a critical and essential step to evaluate its possible value as a breast cancer screening method.MethodsIn this multicenter, prospective study, we annually collected nipple fluid for up to 5 consecutive years from women at increased breast cancer risk, and performed a questionnaire-based survey regarding discomfort of the aspiration. Endpoints of the current interim analyses were the feasibility and results of 994 NFA procedures in 451 women with total follow-up of 560 person years of observation.ResultsIn this large group of women at increased risk of breast cancer, repetitive NFA appeared to be feasible and safe. In 66.4% of aspirated breasts, nipple fluid was successfully obtained. Independent predictive factors for successful NFA were premenopausal status, spontaneous nipple discharge, smaller breast size, bilateral oophorectomy and previous use of hormone replacement therapy or anti-hormonal treatment. The procedure was well tolerated with low discomfort. Drop-out rate was 20%, which was mainly due to repeated unsuccessful aspiration attempts. Only 1.6% of women prematurely declined further participation because of side effects.ConclusionsRepeated NFA in women at increased breast cancer risk is feasible and safe. Therefore, NFA is a promising method to non-invasively obtain a valuable source of potential breast cancer specific biomarkers.

Triple-negative vimentin-positive heterogeneous feline mammary carcinomas as a potential comparative model for breast cancer.

BackgroundHuman breast cancer is a heterogeneous disease classified by molecular subtyping into luminal A, luminal B, HER2-overexpressing, basal-like, claudin-low and normal-breast like. The routinely applied and standardized immunohistochemical-based surrogates of this classification group together the last three entities as triple-negative breast cancer (TNBCs) that show the most diverse and complex heterogeneity and represent a therapeutic challenge.In the present work 156 feline mammary lesions consisting of feline mammary carcinomas (FMCs), benign neoplasms, and hyperplastic/dysplastic tissues were evaluated histologically and by immunohistochemistry for expression of basal and luminal cytokeratins (CK), vimentin, alpha-smooth muscle actin, calponin, estrogen receptor (ER) alpha (a), and progesterone receptor (PR). Thirty-seven FMCs with 27 matched non-neoplastic controls were also investigated for gene expression of ERa, ER beta, PR, and HER2.ResultsA large group of hormone receptors (HRs)-negative aggressive carcinomas - that did not overexpress HER2 - could be distinguished from the less aggressive (10.8%) and benign (8%) HRs + tumors, that showed bilineage (luminal and myoepithelial) differentiation. Immunohistochemical evaluations of cytoplasmic filaments indicated that HRs- FMCs are vimentin+, CK14+, and CK5_6+ carcinomas that may resemble the TNBCs (basal like/claudin low) described in women. The identification of luminal and myoepithelial progenitors within the mammary ductal system suggested potential cells/sites of origin of these tumors. A diffuse and never previously described CKs/vimentin luminal cell co-expression was detected in the non-neoplastic ducts, indicating a potential bilineage progenitor.ConclusionsThese results indicate and potentially explain the high incidence of triple-negative, vimentin + aggressive tumors in cats that may used to elucidate some of the challenging features of TNBCs in women.

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus)

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERβ. Yet throughout pregnancy, ERα and ERβ levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.

The potential role of ductoscopy in modern breast diagnostics and therapy

Mammary ductoscopy is a modern, minimally invasive procedure that enables direct, in vivo observation of the mammary ductal system, primarily by nipple discharge. The rapidly developing device is suitable for aimed biopsy for further cytological or molecular examinations. High-tech equipments facilitate polypectomy or laser vaporization of certain intraluminal lesions, and play an important role in the direct surgical excision of the duct or the so-called terminal duct-lobular unit. The above listed facilitate the early diagnosis of malignancies even before imaging could detect them, and the control of high risk patients. Ductoscopy can foster surgical removal of ductal in situ tumors as anatomical units, thus enabling the optimization of radicality of breast conserving surgeries. Authors give a detailed description of the surgical techniques, and provide a wide review of the literature, for the first time in Hungarian language. Orv. Hetil., 2011, 152, 1284–1293.

The Phenotypic Spectrum of Basal-like Breast Cancers: A Critical Appraisal

There are 2 well-recognized cell populations lining the mammary duct system: the epithelial cells lining the lumen and the myoepithelial cells surrounding them. The mammary stem cell, a putative third cell type, has not yet been well characterized. It is not established whether the putative stem cell expresses the full complement, a subset, or none of the markers of normal epithelial and/or myoepithelial cells. However, it is likely that they would have distinctive markers of their own; whether these are retained or lost in their neoplastic progeny is unknown. All 3 cell types may theoretically undergo malignant transformation. Until recently, however, nearly all attention has been focused on carcinomas of epithelial derivation/differentiation. The advent of oligonucleotide and cDNA microarrays has facilitated gene expression profiling of breast cancers, revealing molecular subclasses that may be prognostically relevant. One such subclass, the basal-like breast carcinomas, has been found in numerous independent datasets to be associated with a comparatively worse overall and disease-free survival. These cancers show expression of molecules characteristic of the normal myoepithelial cell, such as basal cytokeratins, and reduced expression of estrogen receptor-related and Erb-B2-related genes and proteins. The classifier genes that formed the basis for the delineation of basal-like carcinomas were derived from datasets that were composed predominantly of ductal type cancers. Therefore, the clinical significance of a basal-like gene expression or immunohistochemical profile in the other breast cancer subtypes is presently unknown. Herein, we evaluate in detail the current state of knowledge on the pathologic features of breast carcinomas classified as basal-like by immunohistochemical and/or gene expression profiling criteria, with an emphasis on their full phenotypic spectrum and also previously underemphasized areas of heterogeneity and ambiguity where present. There seems to be a phenotypic and biologic spectrum of basal-like or myoepithelial-type carcinomas, just as there is a wide range among tumors of luminal epithelial derivation/differentiation. It is critical to promote lucid morphologic definitions of the molecular subtypes, if this information is intended for use in targeted therapies and patient management.

Stromal and Epithelial Caveolin-1 Both Confer a Protective Effect Against Mammary Hyperplasia and Tumorigenesis: Caveolin-1 Antagonizes Cyclin D1 Function in Mammary Epithelial Cells

Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1-/- mice and performed a series of mammary transplant studies, using both wild-type and Cav-1-/- mammary fat pads. Cav-1-/- mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1-/- mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis.

Breast duct micro-endoscopy: A study of technique and a morphological classification of endo-luminal lesions

Endoscopic visualisation of the human mammary ductal system has been sporadically reported over the last decade. Recent rapid and groundbreaking developments in the field of optics have made the previously unseen labyrinth of mammary ducts more easily accessible to direct visualisation and examination. The emphasis so far has been on visualisation of ectatic ducts with pathological nipple discharge. The purpose of this study was to assess the feasibility of mammary duct epithelium in patients with a range of other pathologies. Based on our findings we have developed a morphological classification of endo-luminal lesions seen on endoscopy. We successfully conducted ex vivo mammary duct micro-endoscopy on 115 ducts in 35 mastectomy specimens. Visualisation of mammary duct epithelium was achieved using a solid rod depth of field imaging micro-minimally invasive (DOFI MMI, Acueity Inc., USA) and more recently the LaDuScope (PolyDiagnost GmbH, Germany) system. Both these systems consist of 0.9 mm maximum outer diameter micro-endoscope, with working channels 0.35 and 0.45 mm, respectively. Saline or air insufflation was used to keep the mammary ducts from collapsing. An average of 3.3 (median 3) mammary ducts could be identified and cannulated in all 35 mastectomy specimens (total of 115 ducts). Visualisation beyond 2 cm of the ductal system was possible in 23/35 (66%) of specimens. Abnormalities were visualised in 40% of the ducts. The maximum depth we could negotiate to was 8.9 cm and in doing so manoeuvred past eight duct divisions. In 34% of ducts cannulated, we were able to navigate the scope beyond at least one bifurcation of the principal duct and in 16% of cases extensive intra-ductal navigation was possible. Peripheral ducts were visualised in 16% of cases. False passages were created in 16% of cases. Previous history of smoking, parity, breastfeeding and radiotherapy offered neither significant advantages nor disadvantages for the technique nor did they increase or decrease the number of normal ducts visualised per specimen. This study showed that mammary duct micro-endoscopy is a practical and technically feasible procedure even in the absence of nipple discharge, in normal non-ectatic milk ducts. A simple morphological classification of endoscopically visualised intra-ductal abnormalities is suggested.

Mammary ductoscopy: past, present, and future

Mammary ductoscopy (MD) allows direct visual access to the mammary ducts, using fiberoptic microendoscopes inserted through the ductal opening onto the nipple surface. Therefore it has a potential role in the diagnosis and treatment of intraductal breast disease. This article describes the anatomy of the mammary ductal system, the early beginnings of MD, its ongoing evolution, and the need for further development for its future usage in increasing clinical indications. MD is a useful diagnostic adjunct in patients with pathological nipple discharge (PND) and can guide duct excision surgery. However, its potential use in the early detection of breast cancer, in guiding breast-conserving surgery (BCS) for cancer, and in the therapeutic ablation of intraductal disease, as well as in guiding risk-reducing strategies among high-risk women, requires further research and evaluation. The development of a biopsy kit that obtains adequate microbiopsy samples for histological diagnosis under direct visualization will enhance the use of this technique by breast surgeons and radiologists. Future developments also include combining MD with molecular diagnostic markers and optical biopsy systems for the diagnosis of premalignant and early malignant disease, and combining MD with radiofrequency for curative ablation of intraductal lesions.

The Evolving Role of Mammary Ductoscopy

SummaryMammary ductoscopy (MD) is an emerging technique that allows direct visualisation of the mammary duct system, and that produces sharp and clear video images and ductal washings for cytological analysis. There is a growing body of evidence that MD may have a role in the management of women with pathological nipple discharge, the guiding of breast conserving surgery for cancer, and the screening of high risk women. Further research is required to confirm these potential applications and the feasibility of its use in the rapid intervention and outpatient setting under local anaesthesia. Furthermore, the addition of molecular and genetic analysis of cells obtained by MD and the emergence of newer generations of microendoscopes are likely to enhance the use of this technique.

β-Adrenergic mechanisms modulate central nervous system effects of prolactin on milk ejection

It is known that prolactin (PRL) is produced within the brain and numerous central actions of the hormone have been reported. In anesthetized lactating rats, central administration of PRL, i.e., intracerebroventricular (icv) or intrathecally (it), facilitated milk ejection (ME) by depressing the sympathetically mediated facilitatory tone of the mammary ductal system. However, it is not known whether or not the same effects and similar mechanisms take place in conscious rats after PRL administration. In the present study, the effects of centrally administered PRL, i.e., icv or it, on ME was determined in both conscious and anesthetized rats. In conscious rats, the rate of ME was determined by applying a 15-min period of suckling by the litter, following a 6-h period of isolation. In anesthetized rats, intramammary pressure (IMP) responses of the mammary glands to exogenous oxytocin (OT) were recorded. The results showed that, whereas in anesthetized rats, increased responsiveness of the mammary glands to OT were observed after PRL administration, an intense inhibition of ME occurred in conscious rats. Because, in conscious and anesthetized rats, these effects were prevented by prior administration of the β-adrenergic blocker propranolol (PROP) to the mothers, this suggests that the PRL effects on ME are modulated through sympathomimetic and sympatholytic actions in conscious and anesthetized rats, respectively. Thus, as shown by ductal tone measurements, in conscious, but not in anesthetized rats, the effect of PRL was associated with increased ductal constriction within the mammary glands; an effect that was mimicked by icv administration of the β-adrenergic agonist isoproterenol (ISOP) and that was prevented by PROP. Further, the sympatholytic action of icv-PRL in anesthetized rats prevented the effect on ductal tone of both icv-PRL in conscious rats and of ISOP in anesthetized rats. Taken together, these results clearly suggest that the central effects of PRL on ME are modulated by adrenergic mechanisms.

Feasibility and Technical Considerations of Mammary Ductoscopy in Human Mastectomy Specimens.

Recent advances in endoscopic technology have made visualization of human mammary ducts possible. The purpose of this study was to assess the feasibility and technical factors influencing the ability to successfully visualize the epithelium of the human mammary ductal system. Lacrimal duct probes were used to dilate nipple orifices to 1.2 mm on 42 mastectomy specimens. The Depth of Field Imaging Micro-Minimally Invasive (DOFI(R) MMI) system consisting of a 1.2 mm rigid ductoscope with a 350 &mgr;m working channel was introduced into mammary ducts under air insufflation or saline irrigation. At least one major duct could be dilated and cannulated in all 42 specimens. Visualization of the proximal duct was accomplished in 34 of 42 (81%) specimens, whereas more extensive navigation through the distal subsegmental ducts was achieved in 22 of 42 (52%) specimens. Ductoscopy into the terminal ducts was accomplished in all patients with a previous history of nipple discharge or discharge at the time of the procedure (10 of 10). In three patients with no history of nipple discharge prior to ductoscopy, incidental papillomas were discovered and confirmed by the pathologist. In conclusion, mammary ductoscopy is technically feasible and may have an application as an additional diagnostic modality for patients with pathologic nipple discharge.

Serial transplants of DMBA-induced mammary tumors in Fischer rats as model system for human breast cancer: V.Myoepithelial-mesenchymal conversion during passaging as possible cause for modulation of pineal-tumor interaction

An elevation of melatonin secretion parallel to an enhanced production of macrophage-derived biopterin was observed in female F344 Fischer rats bearing passage 2 serial transplants derived from a malignant mammary tumor induced by 7,12-dimethylbenz[a]anthracene (DMBA). As opposed to that both parameters were depressed at passage 12. These results indicate the presence of divergent immunoneuroendocrine interactions during different phases of tumor growth. Since these biochemical events must have their common origin in changes taking place within these tumor transplants the current histopathological study was initiated. The primary tumor used for serial transplantation was a moderately differentiated adenocarcinoma of the mammary gland showing cytokeratin-positive epithelial components located in the inner epithelial tubule layer. In addition, bland-looking round or elongated actin-positive myoepithelial cells were detected which apart from epithelial cells are known to constitute the main cellular components of the mammary ductal system which resemble smooth muscle cells both morphologically and functionally. The tumor of passage 1 showed glandular tubules, lined by an inner epithelial layer, and many nests of clear, bland-looking actin-positive myoepithelial cells lying around tubules as well as in the stroma between actin-negative epithelial elements. The tumor of passage 2 used for transplantation consisted of a chaotic mixture of epithelial carcinomatous cells, forming a few irregular small tubules or solid nests, and, predominantly, of elongated plump or spindle-shaped, "myoid" atypical myoepithelial cells with a strong actin-positive reaction and some of these cells showed a focal vimentin expression. The tumor was characterized as a carcinosarcoma. At passage 12 epithelial cells were not identified. The tumor displayed features of a pleomorphic sarcoma consisting mainly of giant cells with bizarre nuclei being cytokeratin- and desmin-negative, weakly vimentin-positive but strongly actin-positive. These results indicate that DMBA-induced mammary tumor cells in female F344 Fischer rats undergo dramatic morphological changes during serial transplantation characterized by a total loss of malignant epithelial (carcinomatous) cells and the emergence and subsequent predominance of malignant (sarcomatous) mesenchymal cells. It appears that these sarcomatous cells develop out of myoepithelial cells since atypical myoepithelial cells with a strong actin-positive reaction showed a focal vimentin expression at passage 2 indicating myofibroblastic differentiation as part of mesenchymal transition. The loss of epithelial cell elements as well as a parallel transition of myoepithelial to mesenchymal cell elements during passaging could lead to a lack of immunological recognition of these tumor transplants and to depression of melatonin. Possible mechanisms involved in these phenomena as well as the relevance of these findings for a better understanding of the role of melatonin in human mammary cancer are discussed.

Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development.

Parathyroid hormone-related protein (PTHrP) was originally discovered as a tumor product that causes humoral hypercalcemia of malignancy. PTHrP is now known to be widely expressed in normal tissues and growing evidence suggests that it is an important developmental regulatory molecule. We had previously reported that overexpression of PTHrP in the mammary glands of transgenic mice impaired branching morphogenesis during sexual maturity and early pregnancy. We now demonstrate that PTHrP plays a critical role in the epithelial-mesenchymal communications that guide the initial round of branching morphogenesis that occurs during the embryonic development of the mammary gland. We have rescued the PTHrP-knockout mice from neonatal death by transgenic expression of PTHrP targeted to chondrocytes. These rescued mice are devoid of mammary epithelial ducts. We show that disruption of the PTHrP gene leads to a failure of the initial round of branching growth that is responsible for transforming the mammary bud into the rudimentary mammary duct system. In the absence of PTHrP, the mammary epithelial cells degenerate and disappear. The ability of PTHrP to support embryonic mammary development is a function of amino-terminal PTHrP, acting via the PTH/PTHrP receptor, for ablation of the PTH/PTHrP receptor gene recapitulates the phenotype of PTHrP gene ablation. We have localized PTHrP expression to the embryonic mammary epithelial cells and PTH/PTHrP receptor expression to the mammary mesenchyme using in situ hybridization histochemistry. Finally, we have rescued mammary gland development in PTHrP-null animals by transgenic expression of PTHrP in embryonic mammary epithelial cells. We conclude that PTHrP is a critical epithelial signal received by the mammary mesenchyme and involved in supporting the initiation of branching morphogenesis.

Overexpression of parathyroid hormone-related protein or parathyroid hormone in transgenic mice impairs branching morphogenesis during mammary gland development.

Parathyroid hormone-related protein (PTHrP) was originally discovered as the tumor product that causes humoral hypercalcemia of malignancy. PTHrP is now known to be widely expressed in many normal fetal tissues where it may participate in the regulation of organogenesis. In this report, we document that overexpression of PTHrP in myoepithelial cells in the mammary glands of transgenic mice resulted in a form of breast hypoplasia characterized by a profound defect in branching morphogenesis of the developing mammary duct system. In addition, transgenic mice manifested a defect in lobuloalveolar development during pregnancy that seemed to be, in part, the consequence of an impaired ability to form terminal ducts in response to estrogen and progesterone stimulation. The effects of PTHrP on branching morphogenesis during breast development appeared to be the result of amino-terminal PTH-like sequences that signal through the PTH/PTHrP receptor, since overexpression of parathyroid hormone itself in the mammary glands of transgenic mice caused a similar development phenotype, and delivery of PTHrP (1-36) via locally implanted slow-release pellets impaired breast development in normal mice. These results suggest that PTHrP, which is a native product of mammary epithelial and myoepithelial cells may participate in normal breast development, perhaps as a locally secreted growth inhibitor.

Morphofunctional Features of Intraductal Hyperplasia, Atypical Intraductal Hyperplasia, and Various Grades of Intraductal Carcinoma

Abstract: The spectrum of epithelial proliferations within the mammary duct system includes lobular, ductal, and papillary patterns of proliferation. Among those with a ductal growth pattern, the highest proportion qualifies as ordinary intraductal hyperplasia (IDH), a small proportion shows atypical intraductal hyperplasia (AIDH), and a steadily increasing percentage is composed of intraductal carcinomas (IDCA). AIDH and some variants of IDCA share many characteristics and their distinction can be quite troublesome, in some cases relying completely on an arbitrary quantitative criterion. IDH, however, is quite distinctive from both a morphologic and biologic standpoint and should not be confused with AIDH. This review discusses our criteria for diagnosing IDH, AIDH, and IDCA, followed by a review of the common subtypes of IDCA and a presentation of our approach to grading IDCA. The clinical significance and expression of estrogen and progesterone receptor (ER and PR), as well as expression of Ki67 and p53 in IDH, AIDH, and various grades of IDCA are presented.