Journal of Mammary Gland Biology and Neoplasia最新文献

The use of mouse derived mammary organoids can provide a unique strategy to study mammary gland development across a normal life cycle, as well as offering insights into how malignancies form and progress. Substantial cellular and epigenomic changes are triggered in response to pregnancy hormones, a reaction that engages molecular and cellular changes that transform the mammary epithelial cells into "milk producing machines". Such epigenomic alterations remain stable in post-involution mammary epithelial cells and control the reactivation of gene transcription in response to re-exposure to pregnancy hormones. Thus, a system that tightly controls exposure to pregnancy hormones, epigenomic alterations, and activation of transcription will allow for a better understanding of such molecular switches. Here, we describe the characterization of ex vivo cultures to mimic the response of mammary organoid cultures to pregnancy hormones and to understand gene regulation and epigenomic reprogramming on consecutive hormone exposure. Our findings suggest that this system yields similar epigenetic modifications to those reported in vivo, thus representing a suitable model to closely track epigenomic rearrangement and define unknown players of pregnancy-induced development.

The field of mammary gland biology and breast cancer research encompasses a wide range of topics and scientific questions, which span domains of molecular, cell and developmental biology, cancer research, and veterinary and human medicine, with interdisciplinary overlaps to non-biological domains. Accordingly, mammary gland and breast cancer researchers employ a wide range of molecular biology methods, in vitro techniques, in vivo approaches as well as in silico analyses. The list of techniques is ever-expanding; together with the refinement of established, staple techniques in the field, new technologies keep emerging thanks to technological advances and scientific creativity. This issue of the Journal of Mammary Gland Biology and Neoplasia represents a compilation of original articles and reviews focused on methods used in mammary gland biology and breast cancer research.

Over the past decade, the cellular content of human milk has been a focus in lactation research due to the benefit a potential non-invasive stem cell compartment could provide either to the infant or for therapeutic applications. Despite an increase in the number of studies in this field, fundamental knowledge in regard to milk cell identification and characterisation is still lacking. In this project, we investigated the nature, morphology and content of membrane enclosed structures (MESs) and explored different methods to enrich human milk cells (HMCs) whilst reducing milk fat globule (MFG) content. Using both flow cytometry and immunofluorescence imaging, we confirmed previous reports and showed that nucleated HMCs make up a minority of milk-isolated MESs and are indistinguishable from MFGs without the use of a nuclear stain. HMC heterogeneity was demonstrated by differential uptake of nuclear stains Hoechst 33258 and DRAQ5™ using a novel technique of imaging milk MESs (by embedding them in agar), that enabled examination of both extracellular and intracellular markers. We found that MESs often contain multiple lipid droplets of various sizes and for the first time report that late post-partum human milk contains secretory luminal binucleated cells found across a number of participants. After investigation of different techniques, we found that viably freezing milk cells is an easy and effective method to substantially reduce MFG content of samples. Alternatively, milk MESs can be filtered using a MACS® filter and return a highly viable, though reduced population of milk cells. Using the techniques and findings we've developed in this study; future research may focus on further characterising HMCs and the functional secretory mammary epithelium during lactation.

Branching morphogenesis of the murine mammary gland starts during late embryogenesis. It is regulated by the signals emanating both from the epithelium and the mesenchyme, yet the molecular mechanisms regulating this process remain poorly understood. We have previously developed a unique whole organ culture technique for embryonic mammary glands, which provides a powerful tool to monitor and manipulate branching morphogenesis ex vivo. Nowadays, RNA sequencing and other transcriptional profiling techniques provide robust methods to identify components of gene regulatory networks driving branching morphogenesis. However, validation of the candidate genes still mainly depends on the use of the transgenic mouse models, especially in mammary gland studies. By comparing different serotypes of recombinant adeno-associated virus (rAAVs), we found out that rAAVs provide sufficient efficiency for gene transfer with different tissue preferences depending on the serotypes of the virus. AAV-2 and AAV-8 preferentially target epithelial and mesenchymal compartments, respectively, while AAV-9 infects both tissues. Here, we describe a protocol for AAV-mediated gene transfer in ex vivo cultured murine embryonic mammary gland facilitating gene function studies on mammary gland branching morphogenesis.

Tumor organoids mimic the architecture and heterogeneity of in vivo tumors and enable studies of collective interactions between tumor cells as well as with their surrounding microenvironment. Although tumor organoids hold significant promise as cancer models, they are also more costly and labor-intensive to cultivate than traditional 2D cell culture. We sought to identify critical factors regulating organoid growth ex vivo, and to use these observations to develop a more efficient organoid expansion method. Using time-lapse imaging of mouse mammary tumor organoids in 3D culture, we observed that outgrowth potential varies non-linearly with initial organoid size. Maximal outgrowth occurred in organoids with a starting size between ~10 to 1000 cells. Based on these observations, we developed a suspension culture method that maintains organoids in the ideal size range, enabling expansion from 1 million to over 100 million cells in less than 2 weeks and less than 3 hours of hands-on time. Our method facilitates the rapid, cost-effective expansion of organoids for CRISPR based studies and other assays requiring a large amount of organoid starting material.

Insulin-like growth factor (IGF) signaling and control of local bioavailability of free IGF by the IGF binding proteins (IGFBP) are important regulators of both mammary development and breast cancer. A recent genome-wide association study (GWAS) identified small nucleotide polymorphisms that reduce the expression of IGFBP-5 as a risk factor of developing breast cancer. This observation suggests that genetic alterations leading to a decreased level of IGFBP-5 may also contribute to breast cancer. In the current review, we focus on Pregnancy-Associated Plasma Protein A (PAPP-A), a protease involved in the degradation of IGFBP-5. PAPP-A is overexpressed in the majority of breast cancers but its role in cancer has only begun to be explored. More specifically, this review aims at highlighting the role of post-partum involution in the oncogenic function of PAPP-A. Notably, we summarize recent studies indicating that PAPP-A plays a role not only in the degradation of IGFBP-5 but also in the deposition of collagen and activation of the collagen receptor discoidin 2 (DDR2) during post-partum involution. Finally, considering the immunosuppressive microenvironment of post-partum involution, we also discuss the unexpected finding made in Ewing Sarcoma that PAPP-A plays a role in immune evasion. While the immunosuppressive role of PAPP-A in breast cancer remains to be determined, collectively these studies highlight the multifaced role of PAPP-A in cancer that extends well beyond its effect on IGF-signaling.

Breast cancer is the most commonly diagnosed cancer in women and the second most common cause of cancer-related deaths in the United States. Although early detection has significantly decreased breast cancer mortality, patients diagnosed with distant metastasis still have a very poor prognosis. The most common site that breast cancer spreads to are local lymph nodes. Therefore, the presence of lymph node metastasis remains one of most important prognostic factors in breast cancer patients. Given its significant clinical implications, increased efforts have been dedicated to better understand the molecular mechanism governing lymph node metastasis in breast cancer. The identification of lymphatic-specific biomarkers, including podoplanin and LYVE-1, has propelled the field of lymphatic metastasis forward. In addition, several animal models such as cell line-derived xenografts, patient-derived xenografts, and spontaneous tumor models have been developed to recreate the process of lymphatic metastasis. Moreover, the incorporation of various -omic platforms have provided further insight into the genetic drivers facilitating lymphatic metastasis, as well as potential biomarkers and therapeutic targets. Here, we highlight various models of lymphatic metastasis, their potential pitfalls, and other tools available to study lymphatic metastasis including imaging modalities and -omic studies.

Deregulated STAT5 activity in the mammary gland of transgenic mice results in parity-dependent latent tumorigenesis. The trigger for cell transformation was previously associated with hyperactivation of the H2AX proximal promoter in a small basal cell population during pregnancy. The current study focuses on the latent activation of tumor development. H2AX was highly expressed in carcinoma and adenocarcinoma as compared to the multiparous mammary gland, whereas pSTAT5 expression decreased in a tumor type-dependent manner. In contrast to the pregnant gland, no positive correlation between H2AX and pSTAT5 expression could be defined in carcinoma and adenocarcinoma. Using targeted methylation analysis, the methylation profile of the H2AX promoter was characterized in the intact gland and tumors. Average H2AX promoter methylation in the tumors was relatively high (~90%), but did not exceed that of the multiparous gland; 5mC methylation was higher in the differentiated tumors and negatively correlated with its oxidative product 5hmC and H2AX expression. Individual analysis of 25 H2AX promoter-methylation sites revealed two consecutive CpGs at positions -77 and - 54 that were actively demethylated in the multiparous gland, but not in their age-matched virgin counterpart. The different methylation profiles at these sites distinguished tumor types and may assume a prognostic role. In-silico and ChIP analyses revealed overlapping methylation-independent SP1-binding and methylation-dependent p53-binding to these sites. We propose that interference with SP1-assisted p53-binding to these sites abrogates H2AX's ability to arrest the cell cycle upon DNA damage, and contributes to triggering latent development of STAT5-induced tumors in estrapausal multiparous mice.