Antiepileptic/teratogen valproate (VPA) is a histone deacetylase inhibitor/ epigenetic drug proposed for the antitumor therapy where it is generally crucial to target poorly or undifferentiated cells to prevent a recurrence. Transplanted rodent gastrulating embryos-proper (primitive streak and three germ layers) are the source of teratoma/teratocarcinoma tumors. Human primitive-streak remnants develop sacrococcygeal teratomas that may recur even when benign (well differentiated). To screen for unknown VPA impact on teratoma-type tumors, we used original 2-week embryoderived teratoma in vitro biological system completed by a spent media metabolome analysis. Gastrulating 9.5-day-old rat embryos-proper were cultivated in Eagle's minimal essential medium (MEM) with 50% rat serum (controls) or with the addition of 2 mM VPA. Spent media metabolomes were analyzed by FTIR. Compared to controls, VPA acetylated histones; significantly diminished overall teratoma growth, impaired survival, increased the apoptotic index, and decreased proliferation index and incidence of differentiated tissues (e.g., neural tissue). Control teratomas continued to grow and differentiate for 14 days in isotransplants in vivo, but in vitro VPA-treated teratomas resorbed. Principal component analysis of FTIR results showed that spent media metabolomes formed well-separated clusters reflecting the treatment and day of cultivation. In metabolomes of VPA-treated teratomas, we found elevation of previously described histone acetylation biomarkers [amide I a-helix and A(CH 3)/A(CH 2)]) with apoptotic biomarkers within the amide I region for b-sheets, and unordered and CH 2 vibrations of lipids. VPA may be proposed for therapy of the undifferentiated component of teratoma tumors and this biological system Abbreviations ASD, autism spectrum disorder; EC, embryonal carcinoma; GTS, growing teratoma syndrome; HDACi, histone deacetylase inhibitor; MEM, minimal essential medium; PCA, principal component analysis; PCNA, proliferating cell nuclear antigen; RMSEC, root mean square error of calibration; RMSECV, root mean square error of cross-validation; SAHA, suberoylanilidehydroxamic acid; VPA, valproate; WEC, whole rat embryo.

Graphical AbstractDepiction of the experimental design.

The spin-trap free radical scavenger N -tert-butyl-α-phenylnitron (PBN) ameliorated effects of several teratogens involving reactive oxygen species (ROS). We investigated for the first time whether PBN could ameliorate teratogenesis induced by a DNA hypomethylating hematological therapeutic 5-azacytidine (5azaC). At days 12 and 13 of gestation, Fisher rat dams were pretreated by an i.v. injection of PBN (40 mg/kg) and 1 h later by an i.p. injection of 5azaC (5mg/kg). Development was analyzed at gestation day 15 in embryos and day 20 in fetuses. PBN alone did not significantly affect development. PBN pretreatment restored survival of 5azaC-treated dams' embryos to the control level, restored weight of embryos and partially of fetuses, and partially restored crown-rump lengths. PBN pretreatment converted limb adactyly to less severe oligodactyly. PBN pretreatment restored global DNA methylation level in the limb buds to the control level. Cell proliferation in limb buds of all 5azaC-treated dams remained significantly lower than in controls. In the embryonic liver, PBN pretreatment normalized proliferation diminished significantly by 5azaC; whereas in embryonic vertebral cartilage, proliferation of all 5azaC-treated dams was significantly higher than in PBN-treated dams or controls. Apoptotic indices significantly enhanced by 5azaC in liver and cartilage were not influenced by PBN pretreatment. However, PBN significantly diminished ROS or reactive nitrogen species markers nitrotyrosine and 8-hydroxy-2′deoxyguanosine elevated by 5azaC in embryonic tissues, and, therefore, activity of this DNA hypomethylating agent was associated to the activation of free radicals. That pretreatment with PBN enhanced proliferation in the liver and not in immature tissue is interesting for the treatment of 5azaC-induced hepatotoxicity and liver regeneration.

Testicular germ cell tumors (TGCTs) are ever more affecting the young male population. Germ cell neoplasia in situ (GCNIS) is the origin of TGCTs, namely, seminomas (SE) and a heterogeneous group of nonseminomas (NS) comprising embryonal carcinoma, teratoma, yolk sac tumor, and choriocarcinoma. Response to the treatment and prognosis, especially of NS, depend on precise diagnosis with a necessity for discovery of new biomarkers. We aimed to perform comprehensive in silico analysis at the DNA, RNA, and protein levels of six prospective (HOXA9, MGMT, CFC1, PRSS21, RASSF1A, and MAGEC2) and six known TGCT biomarkers (OCT4, SOX17, SOX2, SALL4, NANOG, and KIT) and assess its congruence with histopathological analysis in all forms of TGCTs. Cancer Hallmarks Analytics Tool, the Search Tool for the Retrieval of Interacting Genes/Proteins database, and UALCAN, an interactive web resource for analyzing cancer OMICS data, were used. In 108 TGCT and 48 tumor-free testicular samples, the immunoreactivity score (IRS) was calculated. SE showed higher frequency in DNA alteration, while DNA methylation was significantly higher for all prospective biomarkers in NS. In GCNIS, we assessed the clinical positivity of RASSF1 and PRSS21 in 52% and 62% of samples, respectively, in contrast to low or nil positivity in healthy seminiferous tubules, TGTCs as a group, SE, NS, or all NS components. Although present in approximately 80% of healthy seminiferous tubules (HT) and GCNIS, HOXA9 was diagnostically positive in 64% of TGCTs, while it was positive in 82% of NS versus 29% of SE. Results at the DNA, mRNA, and protein levels on putative and already known biomarkers were included in the suggested panels that may prove to be important for better diagnostics of various forms of TGCTs.

Among testicular germ cell tumors, teratomas may often be very aggressive and therapy-resistant. Our aim was to investigate the impact of histone deacetylase inhibitors (HDACi) on the in vitro growth of experimental mouse teratoma by treating their embryonic source, the embryo-proper, composed only of the three germ layers. The growth of teratomas was measured for seven days, and histopathological analysis, IHC/morphometry quantification, gene enrichment analysis, and qPCR analysis on a selected panel of pluripotency and early differentiation genes followed. For the first time, within teratomas, we histopathologically assessed the undifferentiated component containing cancer stem cell-like cells (CSCLCs) and differentiated components containing numerous lymphocytes. Mitotic indices were higher than apoptotic indices in both components. Both HDACi treatments of the embryos-proper significantly reduced teratoma growth, although this could be related neither to apoptosis nor proliferation. Trichostatin A increased the amount of CSCLCs, and upregulated the mRNA expression of pluripotency/stemness genes as well as differentiation genes, e.g., T and Eomes. Valproate decreased the amount of CSCLCs, and downregulated the expressions of pluripotency/stemness and differentiation genes. In conclusion, both HDACi treatments diminished the inherent tumorigenic growth potential of the tumor embryonal source, although Trichostatin A did not diminish the potentially dangerous expression of cancer-related genes and the amount of CSCLC.

Background: Testicular germ cell tumors (TGCTs) are the most common young male malignancy with a steadily rising incidence. Standard clinical practice is radical orchidectomy of suspicious lumps followed by histopathological diagnosis and tumor subtyping. This practice can lead to complications and quality of life issues for the patients. Liquid biopsies, especially cell-free DNA (cfDNA), promised to be true surrogates for tissue biopsies, which are considered dangerous to perform in cases of testicular tumors. In this study, we have performed a systematic review on the potential of cfDNA in TGCT patient management, its potential challenges in translation to clinical application and possible approaches in further research. Materials & Methods: The review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines on EuropePMC and PUBMED electronic databases, with the last update being on October 21, 2021. Due to the high heterogeneity in identified research articles, we have performed an overview of their efficacy. Results: Eight original articles have been identified on cfDNA in TGCT patients published from 2004 to 2021, of which six had more than one TGCT patient enrolled and were included in the final analysis. Three studies investigated cfDNA methylation, one has investigated mutations in cfDNA, two have investigated cfDNA amount, and one has investigated cfDNA integrity in TGCT. The sensitivity of cfDNA for TGCT was found to be higher than in serum tumor markers and lower than miR-371a-3p, with comparable specificity. cfDNA methylation analysis has managed to accurately detect teratoma in TGCT patients. Conclusion: Potential challenges in cfDNA application to TGCT patient management were identified. The challenges relating to the biology of TGCT with its low mutational burden and low cfDNA amounts in blood plasma make next-generation sequencing (NGS) methods especially challenging. We have also proposed possible approaches to help find clinical application, including a focus on cfDNA methylation analysis, and potentially solving the challenge of teratoma detection.

therapies. The relationship between acquisition of drug resistance and invasive potential is poorly understood. Currently, invasive behaviour is thought to be driven mainly by epithelial to mesenchymal transition. Material and methods MCF7 cell line and derived resistant clones were used for this study. MCF7 Tamoxifen Resistant (MCF7TR) and LTED (Long Term Oestrogen Deprivation) were derived from MCF7 upon one-year Tamoxifen or oestrogen deprivation, respectively. LTED combination treatments were also used (LTEDT and LTEDF). Additionally, we used T47D and T47D-LTED. Stable cell lines were generated for both KRT80 over-expression and knockdown. 3D organoids invasion assay, immunofluorescence, confocal microscopy, RNA-seq, ChIP-seq, RT-qPCR and Western blot were performed. Seventy-five human breast specimens and ten metastatic lymph nodes were selected with the approval of Imperial College Healthcare NHS Trust Tissue Bank. Twenty women with suspected breast cancer were prospectively recruited and radiological exam using shear wave ultrasound was used to determine tissue stiffness in the normal and peritumoral stroma, and suspected lesion. Results and discussions In this study, we show that cells that acquire resistance to aromatase inhibitors (AI) undergo active cytoskeleton re-organisation via Keratin 80 (KRT80) and FActin remodelling. These features directly drive the invasive phenotype. Mechanistically, we show that this process is driven by epigenetic reprogramming at the type II keratin locus (chromosome 12) leading to Keratin 80 (KRT80) up-regulation. Reprogramming is dependent on de novo SREBP1 binding to a single enhancer that is activated upon chronic AI treatment. AI-treated patients show KRT80 cytoskeletal reorganisation and an increased number of KRT80 positive cells at relapse. We find that KRT80 activation and redeployment leads to increased F-actin deposition and focal adhesion. Additionally, we show that KRT80 manipulation directly contributes to changes in cellular stiffness and invasive potential. In agreement, shear-wave elasticity imaging of prospective patients show that KRT80 levels correlate with stiffer tumours in vivo. Conclusion Collectively, our data uncover an unexpected and potentially targetable link between epigenetic reprogramming and cytoskeletal changes promoting cell invasion.

Background: The aim of this study was to compare for the first time IL-6 (Interleukin 6), testosterone (T) and estradiol (E) levels, their ratio (E/T), micronucleus (MN), and nuclear bridge (NB) frequency between newborns with regard to their mother’s residency and diet. Our results should enable an assessment of the possible environmental endocrine effects and interaction between biomarkers, pointing to possible associated health risks. Methods: Fifty full-term newborns of both sexes, whose mothers were healthy and not occupationally exposed to any known carcinogen, were analyzed. All of the mothers filled in a detailed questionnaire. Results: The results showed significantly higher levels of E in newborns of mothers with agricultural residency than those born by mothers with urban residency. Significantly, lower levels of E were measured in newborns of mothers who drank milk and carbonated beverages more frequently. Testosterone was significantly higher in boys of mothers with agricultural residency than from mothers with urban residency. Residence and other parameters had no impact on the difference in MN frequency. IL-6 levels were higher in newborns of mothers with agricultural residency. NB levels were significantly associated with E. A significant association between E levels and IL-6 was found. Conclusion: Our results were the first to show a significant impact of the mother’s agricultural residency and diet on their newborns’ sex hormone and IL-6 levels and their association.