Multicellular tumor spheroids are widely used models in tumor research. Because of their three dimensional organization they can simulate avascular tumor areas comprising proliferative and necrotic cells. Nonetheless, protocols for spheroid generation are still inconsistent. Therefore, in this study the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3 have been used to compare different spheroid generation models including hanging drop, liquid overlay and suspension culture techniques, each under several conditions. Experimental approaches differed in cell numbers (400-10,000), media and additives (25 % methocel, 25 % methocel plus 1 % Matrigel, 3.5 % Matrigel). In total, 42 different experimental setups have been tested. Generation of spheroids was evaluated by light microscopy and the structural composition was assessed immunohistochemically by means of Ki-67, cleaved poly (ADP-ribose) polymerase (cPARP) and mucin-1 (MUC-1) expression. Although the tested cell lines diverged widely in their capacity of forming spheroids we recommend hanging drops supplemented with 25 % methocel as the most reliable and efficient method with regard to success of generation of uniform spheroids, costs, experimental complexity and time expenditure in the different cell lines. MCF-7 cells formed spheroids under almost all analyzed conditions, and MDA-MB-231 cells under only one protocol (liquid overlay technique, 3.5 % Matrigel), while SK-BR-3 did not under neither condition. Therefore, we outline specific methods and recommend the use of adapted and standardized spheroid generation protocols for each cell line.

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Breast cancer is one of the most common malignancies diagnosed in pregnancy. Although the tumor is often detected at an advanced stage, placental metastases are rare. Here, we describe the case of a woman with breast cancer recurrence during pregnancy and subsequent metastases. The focus of this study is the large amount of placenta metastases, which have been analyzed immunohistochemically. Staining with trophoblast markers (placenta alkaline phosphatase, beta human chorionic gonadotropin and human placental lactogen) showed the strict localization of metastases in the intervillous space without invasion into fetal tissue. They have a large spheroidal shape and are free of blood vessels. Staining with Ki-67 revealed an outer proliferative shell and inner necrotic core. At week 28, a healthy newborn was born by elective cesarean section. A few weeks later, after surgery and FEC60 (fluorouracil, epirubicin, cyclophosphamide) cycles, the patient died. Breast cancer metastases in the placenta are rarely described. The special immunological environment in pregnancy may influence phenotype, growth, and behavior of tumor and metastases.

Although male–female differences in placental structure and function have been observed, little is understood about their molecular underpinnings. Here, we present a mega-analysis of 14 publicly available placenta DNA methylation (DNAm) microarray datasets to identify individual CpGs and regions associated with fetal sex. In the discovery dataset of placentas from full term pregnancies (N = 532 samples), 5212 CpGs met genome-wide significance (p < 1E−8) and were enriched in pathways such as keratinization (FDR p-value = 7.37E−14), chemokine activity (FDR p-value = 1.56E−2), and eosinophil migration (FDR p-value = 1.83E−2). Nine differentially methylated regions were identified (fwerArea < 0.1) including a region in the promoter of ZNF300 that showed consistent differential DNAm in samples from earlier timepoints in pregnancy and appeared to be driven predominately by effects in the trophoblast cell type. We describe the largest study of fetal sex differences in placenta DNAm performed to date, revealing genes and pathways characterizing sex-specific placenta function and health outcomes later in life.

Although male-female differences in placental structure and function have been observed, little is understood about their molecular underpinnings. Here, we present a mega-analysis of 14 publicly available placenta DNA methylation (DNAm) microarray datasets to identify individual CpGs and regions associated with fetal sex. In the discovery dataset of placentas from full term pregnancies (N = 532 samples), 5,212 CpGs met genome-wide significance (p < 1E-8) and were enriched in pathways such as keratinization (FDR p-value = 7.37E-14), chemokine activity (FDR p-value = 1.56E-2), and eosinophil migration (FDR p-value = 1.83E-2). Nine DMRs were identified (fwerArea < 0.1) including a region in the promoter of ZNF300 that showed consistent differential DNAm in samples from earlier timepoints in pregnancy and appeared to be driven predominately by effects in the trophoblast cell type. We describe the largest study of fetal sex differences in placenta DNAm performed to date, revealing genes and pathways characterizing sex-specific placenta function and health outcomes later in life.

Although male-female differences in placental structure and function have been observed, little is understood about their molecular underpinnings. Here, we present a mega-analysis of 14 publicly available placenta DNA methylation (DNAm) microarray datasets to identify individual CpGs and regions associated with fetal sex. In the discovery dataset of placentas from full term pregnancies (N = 532 samples), 5,212 CpGs met genome-wide significance (p < 1E-8) and were enriched in pathways such as keratinization (FDR p-value = 7.37E-14), chemokine activity (FDR p-value = 1.56E-2), and eosinophil migration (FDR p-value = 1.83E-2). Nine differentially methylated regions were identified (fwerArea < 0.1) including a region in the promoter of ZNF300 that showed consistent differential DNAm in samples from earlier timepoints in pregnancy and appeared to be driven predominately by effects in the trophoblast cell type. We describe the largest study of fetal sex differences in placenta DNAm performed to date, revealing genes and pathways characterizing sex-specific placenta function and health outcomes later in life.

Introduction Women benefit from higher levels of protection from cardiovascular diseases until menopause, after which they gradually lose their privileged status. Pivotal role of sex hormones, primarily estrogens were the focus of interest in explaining this clinical observation. Estradiol (E2) was a prime target of these investigations showing promising results. Nonetheless the potential influence of other estrogens and numerous estrogen metabolites have so far been neglected. Purpose The aim of our study was to investigate the influence of circulating steroid hormones on early functional and proteomic changes following repeated ischemic periods in female rats by applying the highly unbiased methods of in vivo pressure-volume analysis and mass spectrometry based proteomics. Methods Diffuse subendocardial ischemia was induced in female (F-Isch) Wistar rats with sc. injection of isoproterenol (ISO, 85mg/kg) daily for two consecutive days, while the control group (F-Co) received an equivalent volume of sc. saline solution. 48 hours after the first injection pressure-volume analysis (P-V) was carried out to assess left ventricular function. FFPE tissue slides were scanned and analyzed digitally, while peptides from the snap frozen left ventricular myocardium were measured by liquid chromatography-tandem mass spectrometry using isobaric labeling (TMT11plex). Serum and plasma samples were taken to measure circulating steroid hormone levels with mass spectrometry. Results Two day induction of ischemia resulted in 17% mortality in F-Isch. ISO treatment resulted in significant myocardial tissue damage compared to controls as assessed by histology. Ischemia led to a prominent impairment of diastolic aspects (active relaxation and myocardial stiffness) of left ventricular function (Tau: 11.1±0.7 vs. 16.1±1.1 F-Co vs. F-Isch; EDPVR: 0.05±0.005 vs. 0.131±0.016 F-Co vs. F-Isch.). Unsupervised hierarchical clustering performed on P-V parameters identified two distinct subgroups of F-Isch with severe or mild functional impairment. Supervised PLS-DA analysis of P-V and hormone datasets found three estrogens that might play a role in determining functional outcomes (2-Hydroxyestrone: 2-OHE1, 4-Hydroxyestrone: 4-OHE1, 4-Methoxyestrone: 4-MeE2). PLS analysis followed by gene ontology enrichment associated E2 and 2-OHE1 concentrations with mitochondrial protein expressions, while 4-OHE1 seemed to influence the reassembly of contractile structures after ischemia. Conclusions Our study has highlighted 2-OHE1 and 4-OHE1 as well as E2 as potentially influential estrogens on early post-ischemic recovery both on functional and on proteomic level. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): New National Excellence Program of the Ministry of Human Capacities of Hungary (ÚNKP-20-3-I-SE-1 to BA. B.) National Research, Development and Innovation Office (NKFIH) of Hungary (K134939 to T. R.)