Open-source Software Sustainability Models: Initial White Paper From the Informatics Technology for Cancer Research Sustainability and Industry Partnership Working Group.

BACKGROUND: The National Cancer Institute Informatics Technology for Cancer Research (ITCR) program provides a series of funding mechanisms to create an ecosystem of open-source software (OSS) that serves the needs of cancer research. As the ITCR ecosystem substantially grows, it faces the challenge of the long-term sustainability of the software being developed by ITCR grantees. To address this challenge, the ITCR sustainability and industry partnership working group (SIP-WG) was convened in 2019. OBJECTIVE: The charter of the SIP-WG is to investigate options to enhance the long-term sustainability of the OSS being developed by ITCR, in part by developing a collection of business model archetypes that can serve as sustainability plans for ITCR OSS development initiatives. The working group assembled models from the ITCR program, from other studies, and from the engagement of its extensive network of relationships with other organizations (eg, Chan Zuckerberg Initiative, Open Source Initiative, and Software Sustainability Institute) in support of this objective. METHODS: This paper reviews the existing sustainability models and describes 10 OSS use cases disseminated by the SIP-WG and others, including 3D Slicer, Bioconductor, Cytoscape, Globus, i2b2 (Informatics for Integrating Biology and the Bedside) and tranSMART, Insight Toolkit, Linux, Observational Health Data Sciences and Informatics tools, R, and REDCap (Research Electronic Data Capture), in 10 sustainability aspects: governance, documentation, code quality, support, ecosystem collaboration, security, legal, finance, marketing, and dependency hygiene. RESULTS: Information available to the public reveals that all 10 OSS have effective governance, comprehensive documentation, high code quality, reliable dependency hygiene, strong user and developer support, and active marketing. These OSS include a variety of licensing models (eg, general public license version 2, general public license version 3, Berkeley Software Distribution, and Apache 3) and financial models (eg, federal research funding, industry and membership support, and commercial support). However, detailed information on ecosystem collaboration and security is not publicly provided by most OSS. CONCLUSIONS: We recommend 6 essential attributes for research software: alignment with unmet scientific needs, a dedicated development team, a vibrant user community, a feasible licensing model, a sustainable financial model, and effective product management. We also stress important actions to be considered in future ITCR activities that involve the discussion of the sustainability and licensing models for ITCR OSS, the establishment of a central library, the allocation of consulting resources to code quality control, ecosystem collaboration, security, and dependency hygiene.

Review: Annexin-A5 and cell membrane repair.

Annexins are soluble proteins that bind to biological membranes containing negatively charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. Annexin-A5 (AnxA5), the smallest member of the annexin family, presents unique properties of membrane binding and self-assembly into ordered two-dimensional (2D) arrays on membrane surfaces. We have previously reported that AnxA5 plays a central role in the machinery of membrane repair by enabling rapid resealing of plasma membrane disruption in murine perivascular cells. AnxA5 promotes membrane repair via the formation of a protective 2D bandage at membrane damaged site. Here, we review current knowledge on cell membrane repair and present recent findings on the role of AnxA5 in membrane resealing of human trophoblasts.

Review: hCGs: different sources of production, different glycoforms and functions.

Human chorionic gonadotropin (hCG) is the first hormonal message from the placenta to the mother. It is detectable in maternal blood two days after implantation and behaves like an agonist of LH stimulating progesterone secretion by the corpus luteum. hCG has also a role in quiescence of the myometrium and local immune tolerance. Specific to humans, hCG is a complex glycoprotein composed of two glycosylated subunits. The α-subunit is identical to the pituitary gonadotropin hormones (LH, FSH, TSH), contains two N-glycosylation sites, and is encoded by a single gene (CGA). By contrast the ß-subunits are distinct in each of the hormones and confer receptor and biological specificity. The hCG ß-subunit contains two sites of N-glycosylation and four sites of O-glycosylation and is encoded by a cluster of genes (CGB). In this review, we will stress the importance of hCG glycosylation state, which varies with the stage of pregnancy, its source of production and in the pathology. It is well established that hCG is mainly secreted by the syncytiotrophoblast into maternal blood where it peaks around 8-10 weeks of gestation (WG). The invasive extravillous trophoblast also secretes hCG, and in particular like choriocarcinoma cells, hyperglycosylated forms of hCG (hCG-H). In maternal blood hCG-H is high during early first trimester. In addition to its endocrine role, hCG has autocrine and paracrine roles. It promotes formation of the syncytiotrophoblast and angiogenesis through LHCG receptor. In contrast, hCG-H stimulates trophoblast invasion and angiogenesis by interacting with the TGFß receptor 2. hCG is largely used in antenatal screening and hCG-H represents a serum marker of early trophoblast invasion. Other abnormally glycosylated hCG are described in aneuploidies. In conclusion, hCG is the major pregnancy glycoprotein hormone, whose maternal concentration and glycan structure change all along pregnancy. Depending on its source of production, glycoforms of hCG display different biological activities and functions that are essential for pregnancy outcome.

Drug transporter expression during in vitro differentiation of first-trimester and term human villous trophoblasts.

Drug transporters interfere with drug disposition during pregnancy by actively transporting drugs from mother to fetus, and vice versa. Data on their placental expression are scarce, especially during the first trimester of pregnancy. The aim of our study was to assess mRNA expression of more than 80 drug transporters by using an RT-qPCR array in primary cytotrophoblastic cells isolated from first-trimester and term human placentas and cultured for 72 h to form syncytiotrophoblasts. This original expression panel of human placental drug transporters should help to understand transplacental drug transfer and to ensure more rational drug use during pregnancy.

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Homeobox genes and down-stream transcription factor PPARγ in normal and pathological human placental development.

The placenta provides critical transport functions between the maternal and fetal circulations during intrauterine development. Formation of this interface is controlled by nuclear transcription factors including homeobox genes. Here we summarize current knowledge regarding the expression and function of homeobox genes in the placenta. We also describe the identification of target transcription factors including PPARγ, biological pathways regulated by homeobox genes and their role in placental development. The role of the nuclear receptor PPARγ, ligands and target genes in human placental development is also discussed. A better understanding of these pathways will improve our knowledge of placental cell biology and has the potential to reveal new molecular targets for the early detection and diagnosis of pregnancy complications including human fetal growth restriction.

Iodide transporters expression in early human invasive trophoblast.

CONTEXT: The placenta plays an essential role in the fetomaternal exchanges of iodine and thyroid hormones. Propylthiouracil (PTU) is presently considered to be the treatment of choice for hyperthyroidism during the first trimester of pregnancy. Little is known on the expression of iodide transporters in invasive human trophoblast and the possible effect of PTU on this early phase of human placental development. OBJECTIVE: To analyze during early pregnancy expression of sodium/iodide symporter (NIS) and pendrin at the feto-maternal interface in situ in first trimester placentas, in vitro during human trophoblastic cell differentiation in presence or not of PTU. DESIGN: NIS and pendrin immunodetection were performed on 8-10 WG placental tissue sections and in primary cultures of first trimester placenta trophoblastic cells, which differentiate in vitro into syncytiotrophoblast or invasive extravillous cytotrophoblasts (EVCT). The effect of PTU (1 mM) was tested in EVCT on iodide transporters expression, cell invasion, and hCG secretion. RESULTS: NIS and pendrin were present in early human trophoblast at the maternofetal interface. Their expression was modulated with in vitro trophoblast differentiation. Early invasive EVCT were characterized by higher expression of NIS than pendrin. In vitro PTU did modify significantly neither EVCT iodide transporters expression nor EVCT biological functions: i.e. invasive properties and hCG secretion. CONCLUSION: This study reveals that NIS is highly expressed in early human trophoblast at the feto-maternal interface. PTU has no effect on early human trophoblast invasion.

Quantification of utero-placental vascularization in a rabbit model of IUGR with three-dimensional power Doppler angiography.

OBJECTIVES: Our objective was to evaluate the 3D power Doppler angiography (PDA) in terms of feasibility and ability to detect placental hypo-perfusion in an experimental rabbit model of intrauterine growth restriction (IUGR). STUDY DESIGN: 14 pregnant females were treated with NG-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase inhibitor, from day 24 to day 28 of gestation, to induce an IUGR. Concomitantly, 8 pregnant rabbits were used as controls. On day 28, 3D power Doppler indices were quantified in each utero-placental unit. Morphological examination of the placentas for the control group (n = 4) and the L-NAME group (500 mg/day, n = 4) were performed with immunohistochemical staining to discriminate the fetal capillaries in the labyrinthine area. RESULTS: A total of 180 live fetuses were obtained, 108 from the L-NAME group and 72 from the control group. G28 fetal weight was significantly lower in the L-NAME group than in the control group (27.40 ± 0.55 g vs 33.14 ± 0.62 g, p < 0.0001). In the L-NAME group the vascularization index (VI), flow index (FI) and vascularization flow index (VFI) were significantly lower than in the control group (2.6 [1.4; 6.0] vs 7.6 [3.5; 12.6], p < 0.05; 28.7 [26.5; 31.3] vs 32.9 [28.3; 38.1], p < 0.05; 0.8 [0.4; 1.8] vs 2.5 [1.1; 4.1], p < 0.05, for VI, FI and VFI, respectively). Morphological examinations revealed a substantial disorganization of the placental vascular architecture in the L-NAME group. CONCLUSION: This experimental study demonstrates that quantitative 3D PDA indices are sensitive enough to detect placental vascular insufficiency in an experimental rabbit model of IUGR.

Comparative expression of hCG ß-genes in human trophoblast from early and late first-trimester placentas.

Human chorionic gonadotropin (hCG) displays a major role in pregnancy initiation and progression and is involved in trophoblast differentiation and fusion. However, the site and the type of dimeric hCG production during the first trimester of pregnancy is poorly known. At that time, trophoblastic plugs present in the uterine arteries disappear, allowing unrestricted flow of maternal blood to the intervillous space. The consequence is an important modification of the trophoblast environment, including a rise of oxygen levels from about 2.5% before 10 wk of amenorrhea (WA) to ∼8% after 12 WA. Two specific ß-hCG proteins that differ from three amino acids have been described: type 1 (CGB7) and type 2 (CGB3, -5, and -8). Here, we demonstrated in situ and ex vivo on placental villi and in vitro in primary cultures of human cytotrophoblasts that type 1 and 2 ß-hCG RNAs and proteins were expressed by trophoblasts and that these expressions were higher before blood enters in the intervillous space (8-9 vs. 12-14 WA). hCG was immunodetected in villous mononucleated cytotrophoblasts (VCT) and syncytiotrophoblast (ST) at 8-9 WA but only in ST at 12-14 WA. Furthermore, hCG secretion was fourfold higher in VCT cultures from 8-9 WA compared with 12-14 WA. Interestingly, VCT from 8-9 WA placentas were found to exhibit more fusion features. Taken together, we showed that type 1 and type 2 ß-hCG are highly expressed by VCT in the early first trimester, contributing to the high levels of hCG found in maternal serum at this term.

Review: Human trophoblast fusion and differentiation: lessons from trisomy 21 placenta.

The syncytiotrophoblast layer plays a major role throughout pregnancy, since it is the site of numerous placental functions, including ion and nutrient exchange and the synthesis of steroid and peptide hormones required for fetal growth and development. Inadequate formation and regeneration of this tissue contributes to several pathologies of pregnancy such as intrauterine growth restriction and preeclampsia, which may lead to iatrogenic preterm delivery in order to prevent fetal death and maternal complications. Syncytiotrophoblast formation can be reproduced in vitro using different models. For the last ten years we have routinely purified villous cytotrophoblastic cells (CT) from normal first, second and third trimester placentas and from gestational age-matched Trisomy 21 placentas. We cultured villous CT on plastic dishes to follow the molecular and biochemical aspects of their morphological and functional differentiation. Taking advantage of this unique collection of samples, we here discuss the concept that trophoblast fusion and functional differentiation may be two differentially regulated processes, which are linked but quite distinct. We highlight the major role of mesenchymal-trophoblast cross talk in regulating trophoblast cell fusion. We suggest that the oxidative status of the trophoblast may regulate glycosylation of proteins, including hCG, and thereby modulate major trophoblast cell functions.

Expression and localization of DLX3, PPARG and SP1 in bovine trophoblast during binucleated cell differentiation.

The differentiation of the trophoblast is marked in ruminants by the formation of binucleated cells (BNC). They appear from pre-implantation onwards but the molecular mechanisms underlying their differentiation remain largely unexplored. Taking advantage of our recent data, we analyzed the expression pattern of DLX3 and PPARG that are known regulators of early placenta formation and extended our analysis to one of their potential regulators, SP1. Our study is the first to demonstrate the co-expression of DLX3, PPARG and SP1 in bovine BNC nuclei. This suggests a possible role of these transcription factors through BNC specific genes at the time of pre-placental differentiation.

Hyperglycosylated hCG is a marker of early human trophoblast invasion.

CONTEXT: Human chorionic gonadotropin (hCG) is the major pregnancy glycoprotein hormone whose maternal concentration and glycan structure change all along pregnancy. hCG is mainly secreted by the syncytiotrophoblast covering the chorionic villi, but little is known about the source of hyperglycosylated hCG (hCG-H) production. OBJECTIVE: The objective of the study was to analyze expression and secretion of hCG and hCG-H in vitro during human trophoblastic cell differentiation, in situ in first-trimester placentas, and in maternal sera during early pregnancy. DESIGN: hCG and hCG-H were measured in cell supernatants from primary cultures of first-trimester placenta trophoblastic cells, which differentiate in vitro into syncytiotrophoblast or invasive extravillous cytotrophoblasts (evct). hCG-H immunodetection were performed on 9 wk gestation (WG) placental tissue sections. Total hCG and hCG-H were quantified by chemiluminometric assay in 539 maternal sera collected between 9 and 19 WG during normal pregnancies. RESULTS: In vitro, hCG secretion reached 37 ng/ml per µg DNA during syncytiotrophoblast formation but contained few hCG-H (2-5% of total hCG). In contrast, hCG secretion (20 ng/ml per µg DNA) in evct supernatants contained 10-20% hCG-H. In situ, hCG-H immunostaining was strong in invasive and endovascular evct, weaker in mononucleated villous cytotrophoblasts, but negative in the syncytiotrophoblast. In maternal sera, hCG-H concentrations continuously decreased during pregnancy from 406 ± 222 ng/ml at 9 WG to 8 ± 6 ng/ml at 19 WG, whereas total hCG picked up at 11 WG and then decreased. CONCLUSIONS: This study suggests that the high levels of hCG-H observed in first-trimester maternal sera are mainly from invasive evct origin, reflecting the early trophoblast invasion process.

Correlation between uteroplacental three-dimensional power Doppler indices and true uterine blood flow: evaluation in a pregnant sheep model.

OBJECTIVES: Three-dimensional (3D) Doppler quantification within the uteroplacental unit could be of great help in understanding and screening for pre-eclampsia and intrauterine growth restriction. Yet the correlation between 3D Doppler indices and true blood flow has not been confirmed in vivo. The aim of this study was to evaluate this correlation in a pregnant sheep model. METHODS: A blood flow quantitative sensor and a controllable vascular occlusion system were placed around the common uterine artery in seven sheep in late pregnancy, while all the other arterial supplies were ligated. Several occlusion levels were applied, from 0 to 100%, simultaneously with 3D Doppler acquisitions of several placentomes, using standardized settings. Each placentome was analyzed using VOCAL™ (Virtual Organ Computer-aided AnaLysis) software. The correlation between true blood flow and Doppler indices (vascularization index (VI), flow index (FI) and vascularization flow index (VFI)) was evaluated, together with measurement reproducibility. RESULTS: Forty-eight acquisitions were analyzed. All 3D Doppler indices were significantly correlated with true blood flow. Higher correlations were observed for VI and VFI (r = 0.81 (0.74-0.87), P < 0.0001 and r = 0.75 (0.67-0.82), P < 0.0001) compared with FI (r = 0.53 (0.38-0.64) P < 0.0001). Both intra- and interobserver reproducibility were high, with intraclass correlation coefficients of at least 0.799. CONCLUSION: This is the first in-vivo experimental study confirming a significant correlation between true blood perfusion and quantitative 3D Doppler indices measured within the uteroplacental unit. These results confirm the potential usefulness of 3D Doppler ultrasound for the assessment of placental vascular insufficiency both in clinical cases and in a research setting.

Homeobox gene distal-less 3 is expressed in proliferating and differentiating cells of the human placenta.

DLX3, a member of the large homeobox gene family of transcription factors, is necessary for normal placentation. Targeted deletion of dlx3 in mouse resulted in embryonic death due to placental failure. This study demonstrates the presence of DLX3 mRNA expression in human first trimester and term placental tissue, cultured trophoblast-like cell lines and in isolated primary villous and extravillous trophoblast cells. Using an ovine polyclonal antibody, the spatial distribution was identified for DLX3 in human placental tissues, trophoblast cell lines and in freshly isolated primary trophoblast cells. A 50 kDa immunoreactive DLX3 protein was detected in the human placenta, in trophoblast cell lines and in primary trophoblast cells. Nuclear expression for DLX3 was observed in villous cytotrophoblasts, syncytiotrophoblast and extravillous cytotrophoblast in the proximal regions of the cytotrophoblast cell columns in first trimester placental tissues. Immunoreactivity was also detected in few stromal cells and microvascular endothelial cells surrounding the fetal capillaries. In the first trimester placental bed, DLX3 expression was predominantly observed in the cytoplasm of the endovascular and interstitial trophoblasts. We conclude that the cellular expression of DLX3 was extensive in the human placenta and propose that DLX3 may play an important role in normal placental development.

Expression and regulation by PPARgamma of hCG alpha- and beta-subunits: comparison between villous and invasive extravillous trophoblastic cells.

During human placental development trophoblast follows two differentiation pathways: the extravillous (EVCT) and the villous cytotrophoblasts (VCT) that display different phenotypes and functions. It is well established that human chorionic gonadotropin hormone (hCG) is mainly secreted by the endocrine VCT (syncytiotrophoblast) into the maternal compartment and stimulates the formation of the syncytiotrophoblast (ST) in an autocrine manner. We recently reported that the invasive EVCT also produces hCG that promotes trophoblast invasion in vitro. Herein, we compared hCG gene expression in primary culture of villous and extravillous trophoblasts obtained from the same first trimester human chorionic villi and differentiated in vitro into ST and invasive EVCT, respectively. Total hCG, free alpha and free beta subunits were quantified in cell supernatants by immunometric assays and normalized to DNA content. alpha and beta transcript levels were quantified by Q-PCR and normalized to cytokeratin 7. We show that free alpha-, free beta-subunits and total hCG are differently expressed and secreted by the two trophoblast subtypes during their differentiation in vitro. We found an alpha/beta ratio 100 times lower in invasive EVCT in comparison to the ST suggesting that beta subunit may not be step limiting for hCG production in EVCT. Finally we investigated the regulation of hCG gene expression by PPARgamma, a nuclear receptor that controls trophoblast differentiation and invasion. Interestingly, activation of PPARgamma by the agonist rosiglitazone gave opposite results in the endocrine VCT and invasive EVCT: alpha and beta subunit transcript levels and protein secretions were up regulated in VCT, whereas they were down regulated in EVCT. Our results demonstrated that hCG gene expression is differentially regulated in the two trophoblast lineages during their in vitro differentiation and modulated in an opposite way by PPARgamma.

Clinical and biochemical improvement in acromegaly during pregnancy.

Numerous case reports of pregnancy in acromegaly exist, however detailed descriptions of changes in placental and pituitary GH and IGF-I throughout gestation are rare. A 19-yr-old female presented to this institution with signs and symptoms of a GH-secreting pituitary adenoma. Following transphenoidal hypophysectomy, she had 3 unplanned pregnancies, despite ongoing active disease. No pregnancy was complicated by glucose intolerance or hypertension and 3 healthy newborns were delivered near or at term. Clinical improvement was observed during each pregnancy, accompanied by IGF-I levels lower than in the non-pregnant state, the majority lying within the normal range. This was despite increasing placental GH levels, and was not consistent with previous reports in the literature. Further surgical and medical therapies for acromegaly failed to normalize nonpregnant GH or IGF-I levels in this woman. Estrogen is known to alter GH signaling via its interaction with Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways. We hypothesize that increasing concentrations of estrogen or other pregnancy-related hormones resulted in her clinical and biochemical improvement during pregnancy. This may be used for future therapeutic benefit.

Involvement of PPARgamma in human trophoblast invasion.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily that controls the expression of a large array of genes in a ligand-dependent manner. In the human placenta, PPARgamma is specifically expressed in the villous cytotrophoblast and syncytiotrophoblast as well as in the extravillous cytotrophoblastic cells (EVCT) along their invasive pathway. The present study used two cellular models, primary cultures of trophoblastic cells differentiated in vitro in extravillous trophoblastic cells and a cell line (HIPEC65), which was established from a primary culture of EVCT transformed by T-SV40. We observed that natural (15d-PGJ2) or synthetic ligands of PPARgamma (rosiglitazone) inhibit cell invasion in a concentration-dependent manner, with no effect on cell proliferation. This is associated with a modulation of the expression of trophoblastic genes described to be directly involved in the control of EVCT invasiveness, such as GH-V (-20%), TGFbeta2 (-30%), PAPP-A (-60%) and IL1beta (+300%.). In order to identify PPARgamma potential ligands at the fetomaternal interface, we purified LDL (low density lipoprotein) from human sera and oxidized them in vitro in the presence of copper. OxLDL inhibit in vitro extravillous trophoblast cell invasion, whereas native LDL have no effect. In situ OxLDL and their LOX-1 receptor, as well as PPARgamma are immunodetected in trophoblasts at the maternofetal interface.