Potential of green-synthesized ZnO NPs against human ovarian teratocarcinoma: an in vitro study.

BACKGROUND: Ovarian cancer leads to devastating outcomes, and its treatment is highly challenging. At present, there is a lack of clinical symptoms, well-known sensitivity biomarkers, and patients are diagnosed at an advanced stage. Currently, available therapeutics against ovarian cancer are inefficient, costly, and associated with severe side effects. The present study evaluated the anticancer potential of zinc oxide nanoparticles (ZnO NPs) that were successfully biosynthesized in an ecofriendly mode using pumpkin seed extracts. METHODS AND RESULTS: The anticancer potential of the biosynthesized ZnO NPs was assessed using an in vitro human ovarian teratocarcinoma cell line (PA-1) by well-known assays such as MTT assay, morphological alterations, induction of apoptosis, measurement of reactive oxygen species (ROS) production, and inhibition of cell adhesion/migration. The biogenic ZnO NPs exerted a high level of cytotoxicity against PA-1 cells. Furthermore, the ZnO NPs inhibited cellular adhesion and migration but induced ROS production and cell death through programmed cell death. CONCLUSION: The aforementioned anticancer properties highlight the therapeutic utility of ZnO NPs in ovarian cancer treatment. However, further research is recommended to envisage their mechanism of action in different cancer models and validation in a suitable in vivo system.

Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells.

BACKGROUND: One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. METHODS: EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. RESULTS: EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3ζ endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. CONCLUSIONS: These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.

MAPK activation drives male and female mouse teratocarcinomas from late primordial germ cells.

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.

Selective elimination of pluripotent stem cells by PIKfyve specific inhibitors.

Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.

The foundational framework of tumors: Gametogenesis, p53, and cancer.

The completion-of-tumor hypothesis involved in the dynamic interplay between the initiating oncogenic event and progression is essential to better recognize the foundational framework of tumors. Here we review and extend the gametogenesis-related hypothesis of tumors, because high embryonic/germ cell traits are common in tumors. The century-old gametogenesis-related hypothesis of tumors postulated that tumors arise from displaced/activated trophoblasts, displaced (lost) germ cells, and the reprogramming/reactivation of gametogenic program in somatic cells. Early primordial germ cells (PGCs), embryonic stem (ES) cells, embryonic germ cells (EGCs), and pre-implantation embryos at the stage from two-cell stage to blastocysts originating from fertilization or parthenogenesis have the potential to develop teratomas/teratocarcinomas. In addition, the teratomas/teratocarcinomas/germ cells occur in gonads and extra-gonads. Undoubtedly, the findings provide strong support for the hypothesis. However, it was thought that these tumor types were an exception rather than verification. In fact, there are extensive similarities between somatic tumor types and embryonic/germ cell development, such as antigens, migration, invasion, and immune escape. It was documented that embryonic/germ cell genes play crucial roles in tumor behaviors, e.g. tumor initiation and metastasis. Of note, embryonic/germ cell-like tumor cells at different developmental stages including PGC and oocyte to the early embryo-like stage were identified in diverse tumor types by our group. These embryonic/germ cell-like cancer cells resemble the natural embryonic/germ cells in morphology, gene expression, the capability of teratoma formation, and the ability to undergo the process of oocyte maturation and parthenogenesis. These embryonic/germ cell-like cancer cells are derived from somatic cells and contribute to tumor formation, metastasis, and drug resistance, establishing asexual meiotic embryonic life cycle. p53 inhibits the reactivation of embryonic/germ cell state in somatic cells and oocyte-like cell maturation. Based on earlier and our recent studies, we propose a novel model to complete the gametogenesis-related hypothesis of tumors, which can be applied to certain somatic tumors. That is, tumors tend to establish a somatic asexual meiotic embryonic cycle through the activation of somatic female gametogenesis and parthenogenesis in somatic tumor cells during the tumor progression, thus passing on corresponding embryonic/germ cell traits leading to the malignant behaviors and enhancing the cells' independence. This concept may be instrumental to better understand the nature and evolution of tumors. We rationalize that targeting the key events of somatic pregnancy is likely a better therapeutic strategy for cancer treatment than directly targeting cell mitotic proliferation, especially for those tumors with p53 inactivation.

A Comprehensive Analysis of Human Endogenous Retroviruses HERV-K (HML.2) from Teratocarcinoma Cell Lines and Detection of Viral Cargo in Microvesicles.

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles-MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC.

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

Murine Teratocarcinoma-Derived Neuronal Cultures.

This chapter describes the culture and propagation of murine embryonic stem cells, F9 and P19, and strategies for differentiation of these stem cells into neurons. Additional techniques are described for obtaining enriched populations of mature neurons from P19 cells and differentiation of F9 cells into serotonergic or catecholaminergic neurons. The protocols described herein can be used for dissection of the pathways such as gliogenesis and neurogenesis that are involved in differentiation of pluripotent stem cells such as F9 and P19 into glial cells or terminally differentiated neurons.

Impact of Reck expression and promoter activity in neuronal in vitro differentiation.

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth.

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.

LINC00324 facilitates cell proliferation through competing for miR­214­5p in immature ovarian teratocarcinoma.

Immature ovarian teratocarcinoma (IOT) is a rare and malignant type of ovarian teratoma, and the molecular mechanisms underlying the pathogenesis and malignant phenotype of IOT remain uncharacterized. The present study examined a long non­coding RNA (lncRNA), long­chain intergenic non­coding RNA324 (LINC00324), which may serve a crucial role in pathogenesis of IOT. According to the results, LINC00324 was upregulated in IOT tissues and cells, as determined by reverse transcription­quantitative PCR, and its depletion impaired cell proliferation ability and improved cell apoptosis ability in IOT. Furthermore, LINC00324 acted as a miR­214­5p sponge to derepress cyclin dependent kinase 6 (CDK6), cyclin D1 (CCND1), murine double minute homolog 2 (MDM2), and mouse double minute 4 (MDM4) expression, thus increasing IOT cell proliferation and repressing apoptosis. Taken together, these results demonstrated that LINC00324 could serve as a competing endogenous RNA to facilitate IOT cell proliferation by regulation of miR­214­5p­CDK6/CCND1/MDM2/MDM4 network, which possibly provide a novel therapeutic target for IOT.

Testicular Teratocarcinoma in a Ferret (Mustela putorius furo).

A 7-month-old male domestic ferret (Mustela putorius furo) was presented for evaluation of unilateral testicular enlargement. Microscopic examination of the left testicle revealed a neoplasm with differentiation along multiple cell lines (ectoderm, endoderm, mesoderm) including respiratory epithelium, bone and haired skin. A poorly differentiated epithelial component was dispersed throughout the neoplasm with invasion of testicular lymphatics. The animal developed progressive dysuria and was euthanized. At necropsy, metastasis of the poorly differentiated epithelial component was present in the urinary bladder, ureters, prostate gland, pelvic fat, abdominal and thoracic lymph nodes, kidney and lung. This is the first report of a malignant testicular teratoma with widespread metastasis in this species.

Metastatic Ovarian Teratocarcinoma in a Horse.

A 3-year-old Quarter Horse mare presented with an approximate 1-month history of progressive weight loss, anorexia and lethargy that abruptly worsened 48 h before death. Post-mortem examination revealed free flocculent fluid and a large mass within the ventral abdomen that dorsally displaced the caecum and large intestine. An ovarian teratocarcinoma with metastasis to regional lymph nodes was diagnosed histologically. Although benign teratomas are the second most common ovarian neoplasm in equids, reports of malignant teratomas in horses are rare. This report documents an unusual presentation of a rarely reported malignant neoplasm of the reproductive tract in horses.

Necrosis pulmonar masiva por micosis / Massive pulmonary necrosis due to mycosis

Introducción: La aspergilosis es una infección micótica oportunista que se presenta fundamentalmente en pacientes inmunodeprimidos y su principal fuente de transmisión lo constituyen las esporas presentes en el aire de salones de operaciones y unidades de cuidados intensivos. Objetivo: Presentar un caso de una micosis pulmonar masiva por una variante angioinvasiva de Aspergillus. Caso clínico: Se presenta un paciente con aspergilosis pulmonar grave, diagnosticada después de la resección de un tumor mediastinal. Se describen las características de la primera intervención, la evolución postoperatoria que condujo a la segunda, se muestran las imágenes tomográficas, quirúrgicas, microbiológicas y anátomo-patológicas que permitieron definir el diagnóstico. Conclusiones: La posibilidad de una micosis pulmonar debe tenerse en cuenta, aun cuando sea una afección rara y de manejo difícil, en pacientes inmunodeprimidos, con condensación pulmonar rebelde al tratamiento(AU)

Introduction: Aspergillosis is an opportunistic fungal infection that occurs mainly in immunosuppressed patients and its main source of transmission is the spores present in the air of operating rooms and intensive care units. Objective: To present a case of a massive pulmonary mycosis due to an angioinvasive variant of Aspergillus. Clinical case: A patient with severe pulmonary aspergillosis, diagnosed after resection of a mediastinal tumor, is presented. The characteristics of the first intervention are described, the postoperative evolution that led to the second one, the tomographic, surgical, microbiological and anatomo-pathological images that allowed to define the diagnosis are shown. Conclusions: The possibility of a pulmonary mycosis should be taken into account, even when it is a rare and difficult-to-handle condition, in immunocompromised patients, with pulmonary condensation that is rebellious to treatment. Aspergillosis is an opportunistic fungal infection that occurs mainly in immunosuppressed patients and its main source of transmission is the spores present in the air of operating rooms and intensive care units(AU)

Teratocarcinoma-derived growth factor-1 (Cripto-1) is overexpressed in epithelial odontogenic lesions displaying more aggressive behaviour.

PURPOSE: Cripto-1 also known as teratoma-derived growth factor 1 (TDGF-1) belongs to the EGF-CFC family of growth factor-like molecules. Cripto-1 is involved with embryonic development and not expressed in adult tissue, but some tumours are accompanied by reactivation. METHODS: The aim of this study was to evaluate the immunohistochemical expression of Cripto-1 in most common odontogenic cysts and tumours. Thirty ameloblastomas, 30 keratocysts, 30 dentigerous cysts and two ameloblastic carcinomas were evaluated using the polymeric immunoperoxidase technique. Immunohistochemical expressions were analysed by the IRS (immunoreactive score). Statistical analyses were performed by the Kruskal-Wallis and Mann-Whitney tests (p ≤ 0.05). RESULTS: Age ranged from 9 to 75 years old, with a prevalence of females (n = 49/53.3%). The mandible was the most affected anatomical site (n = 69/75.0%). Cripto-1 immunoexpression was observed in all ameloblastoma, keratocyst and ameloblastic carcinoma cases, although nine dentigerous cyst cases (30%) were negative. Expression scores were higher in ameloblastoma, keratocyst and ameloblastic carcinoma cases (median ranging from 8 to 11) when compared with dentigerous cyst cases (median of 2), with a statistically significant difference (p < 0.001). CONCLUSIONS: Cripto-1 is critically important in the progression of several tumours since it is related to significant cell survival and differentiation pathways. The high expression of Cripto-1 in more aggressive odontogenic lesions suggests that this molecule may be involved in the activation of important pathways related to the etiopathogenesis of these lesions.

Evaluating the effect of siRNA on SOX2OT expression in the human neuron-committed teratocarcinoma NT2 cell line.

Non-coding RNA elongated (lncRNAs) have recently attracted as molecules that regulate gene expression of the pluripotent properties (pluripotency) of stem cells. Recently our colleagues examined the role of one of these RNAs called SOX2OT in esophageal squamous cell carcinoma, and found a concomitant increase in its expression with some regulatory genes of cell proliferation. In the present study, using the design of suitable primers from SOX2OT gene, we investigated the effect of siRNA on expression of SOX2OT.

Other Rare Sinonasal Malignant Tumours Involving the Anterior Skull Base.

Sinonasal tumours are rare, and among these there exist a small number of histologic subtypes that are infrequently encountered and rarely mentioned in the literature. These have been presented as either case reports or small case series, and their very low incidence makes prospective studies practically impossible. This review analyses the available literature, including our own experience and endeavours to outline management strategies, which involve a high index of suspicion and counselling of patients. In most instances, these tumours require aggressive multimodal treatment to improve survival outcomes. The overall prognosis remains dismal.

TGFß Family Signaling Pathways in Pluripotent and Teratocarcinoma Stem Cells' Fate Decisions: Balancing Between Self-Renewal, Differentiation, and Cancer.

The transforming growth factor-ß (TGFß) family factors induce pleiotropic effects and are involved in the regulation of most normal and pathological cellular processes. The activity of different branches of the TGFß family signaling pathways and their interplay with other signaling pathways govern the fine regulation of the self-renewal, differentiation onset and specialization of pluripotent stem cells in various cell derivatives. TGFß family signaling pathways play a pivotal role in balancing basic cellular processes in pluripotent stem cells and their derivatives, although disturbances in their genome integrity induce the rearrangements of signaling pathways and lead to functional impairments and malignant transformation into cancer stem cells. Therefore, the identification of critical nodes and targets in the regulatory cascades of TGFß family factors and other signaling pathways, and analysis of the rearrangements of the signal regulatory network during stem cell state transitions and interconversions, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression, as well as for clinical applications. This review summarizes recent advances in our understanding of TGFß family functions in naїve and primed pluripotent stem cells and discusses how these pathways are involved in perturbations in the signaling network of malignant teratocarcinoma stem cells with impaired differentiation potential.

Analysis of Human Stem Cell Transcription Factors.

Transcription factors NANOG, OCT4, SOX2, and NESTIN are expressed in both human embryonic stem cells (hESCs) and cancer stem cells and they play a crucial role in maintaining characteristics of stemness such as self-renewal and pluripotency. This article evaluates the expression of variants of the main stem cell-specific transcription factors NANOG and OCT4 critically and accurately with specific primers designed for identifying the most important variants that maintain stemness. We have examined four variants of NANOG along with a processed pseudogene and seven variants of OCT4 in human teratocarcinoma cell lines (NTERA2D1, SuSa, GCT-27, and 833KE), hESCs, and ovarian cancer cells by reverse transcriptase-polymerase chain reaction. In addition, we have examined their expression in NTERA2D1 cells on differentiation with all-trans-retinoic-acid. We show that NANOG1 is expressed in all teratocarcinoma cells and can be distinguished from NANOGP8, which is an expressed pseudogene. NANOG2 was not expressed in any of the cell lines, including ESCs. OCT4A was expressed in all cells, whereas the variant OCT4B-variant 3 was expressed only in NTERA2D1 cells. On differentiation of NTERA2D1 with retinoic acid, only NANOGP8 and OCT4A were expressed. In ovarian cancer cells, only 3/6 expressed NANOG1 and OCT4A. All malignant cells from patients with ovarian cancer (N = 6) expressed NANOG1 and OCT4A. These results demonstrate the necessity to precisely evaluate the expression of stem cell transcription factors when defining stemness.

The HERV-K accessory protein Np9 controls viability and migration of teratocarcinoma cells.

Human endogenous retroviruses are remnants of ancient germline infections that make up approximately 8% of the modern human genome. The HERV-K (HML-2) family is one of the most recent entrants into the human germline, these viruses appear to be transcriptionally active, and HERV-K viral like particles (VLPs) are found in cell lines from a number of human malignancies. HERV-K VLPs were first found to be produced in teratocarcinoma cell lines, and since then teratocarcinoma has been thought of as the classical model for HERV-Ks, with the NCCIT teratocarcinoma cell line particularly known to produce VLPs. Treatment for teratocarcinoma has progressed since its discovery, with improved prognosis for patients. Since the introduction of platinum based therapy, first year survival has greatly improved even with disseminated disease; however, it is estimated that 20% to 30% of patients present with metastatic germ cell tumor relapse following initial treatments. Also, the toxicity associated with the use of chemotherapeutic agents used to treat germ cell tumors is still a major concern. In this study, we show that the depletion of the HERV-K accessory protein Np9 increases the sensitivity of NCCIT teratocarcinoma cells to bleomycin and cisplatin. While decreasing the expression of Np9 had only a modest effect on the baseline viability of the cells, the reduced expression of Np9 increased the sensitivity of the teratocarcinoma cells to environmental (serum starvation) and chemical (chemotherapeutic) stresses. Np9 is also essential to the migration of NCCIT teratocarcinoma cells: in a wound closure assay, reduced expression of Np9 resulted in cells migrating into the wound at a slower rate, whereas reintroduction of Np9 resulted in NCCIT cells migrating back into the wound in a manner similar to the control. These findings support the implication that the HERV-K accessory protein Np9 has oncogenic potential.