Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas.

Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.

Factors Associated With Perioperative Complications in the Treatment of Pediatric Retroperitoneal Teratoma.

BACKGROUND: Perioperative complications are common during the surgical treatment of pediatric retroperitoneal teratoma (RPT). Some clinical and radiographic features could be associated with perioperative complications. This study was designed to identify the factors associated with such complications. MATERIALS AND METHODS: We retrospectively analyzed the clinical data of RPT patients who underwent surgical treatment at the Department of Pediatric Surgery of The Affiliated Hospital of Qingdao University between January 2008 and January 2020, including demographics, imaging data, intraoperative findings, perioperative complications, pathological data, and outcomes. RESULTS: A total of 91 patients were included in this study, including 30 boys and 61 girls. Of these, 71 patients (78%) were 1 y old or younger. Thirty-eight patients (41%) had perioperative complications (44 intraoperative and 7 postoperative). Preoperative imaging studies showed that the tumor distorted adjacent arteries, veins, and organs in all patients. More veins and organs were displaced and distorted by the tumor in patients who had perioperative complications. Multivariate analysis showed that the number of organs compressed and distorted by the tumor was significantly related to perioperative complications (odds ratio 1.69, 95% confidence interval 1.19-2.41). CONCLUSIONS: Surgical treatment of RPT is complex and challenging. As majority are benign, a complete excision is usually curative. The number of organs compressed and distorted by the tumor is positively related to perioperative complications.

Clinical Monitoring of Sacrococcygeal Teratoma.

BACKGROUND: Sacrococcygeal teratomas (SCT) are often highly vascularized and may result in high-output cardiac failure, polyhydramnios, fetal hydrops, and demise. Delivery is guided by the SCT to fetus volume ratio (SCTratio), SCT growth rate, and cardiac output indexed for weight (CCOi). METHODS: We compared measurements and outcome in 12 consecutive fetuses referred with SCT. Adverse outcomes were: fetal surgery, delivery < 32 gestational weeks or neonatal demise. Only SCTratio and CCOi were used to manage the cases. SCT vascularization index (VI%) was derived from the 3D virtual organ computer-aided analysis (VOCAL) software. The SCTModel (modified from acardiac twins) calculated a hypothetical SCT draining vein size and derived a risk line, using diameters of the superior and inferior vena cava, the azygous and umbilical veins. VI% and a model of systemic and umbilical venous volumes (SCTModel) were tested as indicators for outcome in SCT. RESULTS: Fetuses were monitored from 20.1 to 36.4 gestational weeks and 5/12 had adverse outcomes: 1 had successful open fetal surgery at 23.8 weeks and delivered at term, 4 delivered at < 32 weeks with 3/4 having neonatal demise between 25 and 29 weeks. VI% was significantly higher in cases with adverse outcomes (mean 10.3 [8.9-11.6] vs. 4.4 [3.4-5.3], p < 0.0001). The additional fraction of the fetal cardiac output required to perfuse the SCT-draining vein (XSCO%) (p = 0.46), SCTratio (p = 0.08), and CCOi (p = 0.64) were not significant. All cases with adverse outcome had VI% > 8%. The SCTModel risk line predicted nonadverse outcomes well but lacked data in 2/5 cases with adverse outcomes. CONCLUSIONS: VI% is a significant indicator of SCT cases with adverse outcomes and combined with SCTratio may guide timing of delivery better than current measures.

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis.

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.

Bioluminescence reporter gene imaging characterize human embryonic stem cell-derived teratoma formation.

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However, teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore, tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study, hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology, we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover, FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4, and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency, retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers.

Preoperative giant sacrococcygeal teratoma embolization in a newborn - A case report and a review. / Embolización preoperatoria de teratoma sacrococcigéo gigante en un recién nacido. Reporte de un caso y revisión.

Sacrococcygeal teratoma (SCT) is the most frequent congenital germ cell tumor. Patients have a higher risk of perinatal complications and death, with bleeding and cardiac decompensation being the most common causes of neonatal mortality. This is the case of a 35-week preterm newborn with a large SCT diagnosed at ultrasound screening in the second trimester. Preoperative selective embolization of the middle sacral artery and total surgical resection were performed postnatally with minimal blood loss. The patient was discharged at 25 days of life with a normal physical examination. Selective embolization prior to giant SCT resection is feasible and appears as a safe and useful technique in the control of perioperative bleeding.

El teratoma sacrococcígeo (TSC) es el tumor congénito de células germinales más frecuente. Los pacientes afectados tienen un mayor riesgo de complicaciones perinatales y muerte, siendo la hemorragia y la descompensación cardiaca las causas más comunes de mortalidad neonatal. Presentamos el caso de un recién nacido pretérmino de 35 semanas con un TSC de gran tamaño diagnosticado por ecografía en el segundo trimestre. La embolización selectiva preoperatoria de la arteria sacra media y la resección quirúrgica total postnatal se realizaron con una mínima pérdida de sangre. El paciente fue dado de alta a los 25 días de vida con un examen físico normal. La embolización selectiva antes de la cirugía de resección del TSC gigante es factible y aparece como una técnica segura y útil en el control del sangrado perioperatorio.

Beta 1 integrin is essential for teratoma growth and angiogenesis.

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of beta 1 integrin during teratoma formation, we compared teratomas induced by normal and beta1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, beta 1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of beta 1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in beta1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface. Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell-derived endothelial cells. In contrast, beta 1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although beta 1- deficient endothelial cells were absent in teratomas, beta 1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in beta1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in beta 1-null embryoid bodies.

Contribution of host-derived tissue factor to tumor neovascularization.

OBJECTIVE: The role of host-derived tissue factor (TF) in tumor growth, angiogenesis, and metastasis has hitherto been unclear and was investigated in this study. METHODS AND RESULTS: We compared tumor growth, vascularity, and responses to cyclophosphamide (CTX) of tumors in wild-type (wt) mice, or in animals with TF levels reduced by 99% (low-TF mice). Global growth rate of 3 different types of transplantable tumors (LLC, B16F1, and ES teratoma) or metastasis were unchanged in low-TF mice. However, several unexpected tumor/context-specific alterations were observed in these mice, including: (1) reduced tumor blood vessel size in B16F1 tumors; (2) larger spleen size and greater tolerance to CTX toxicity in the LLC model; (3) aborted tumor growth after inoculation of TF-deficient tumor cells (ES TF(-/-)) in low-TF mice. TF-deficient tumor cells grew readily in mice with normal TF levels and attracted exclusively host-related blood vessels (without vasculogenic mimicry). We postulate that this complementarity may result from tumor-vascular transfer of TF-containing microvesicles, as we observed such transfer using human cancer cells (A431) and mouse endothelial cells, both in vitro and in vivo. CONCLUSIONS: Our study points to an important but context-dependent role of host TF in tumor formation, angiogenesis and therapy.

Survival after an antiangiogenetic therapy and surgery in a wide spread growing teratoma originating from a testicular mixed malignant germ cell tumor.

Growing teratoma is still an often unsolved problem especially in male with mixed malignant GCTs of the testis or the mediastinum. This specific situation with progressive tumor growth and simultaneous normalization of tumor markers during or after treatment of malignant GCTs with teratomatous elements is judged as a fatal situation if this situation can not be controlled by extensive surgery, as teratoma are not sensible to chemotherapy or irradiation. Here, we report the case history of a 17-year old male patient with a testicular malignant GCT and wide spread lymph node metastases, who developed a rapidly progressive growing teratoma within the lymph node metastases. Within the molecular profile of the tumor we could find a cytogenetic picture typically found in malignant adult GCTs. In view of the bulky abdominal, thoracic and cervical metastases and the uncontrolled tumor progression, the situation was considered incurable. However, following an individual treatment attempt, this patient was treated with a four-agent combination of drugs with antiangiogenetic potential as well as low-dose cyclic chemotherapy. This approach resulted in a sustained disease stabilization followed by extensive surgical resection of the metastases. We therefore would like to highlight this treatment approach in unresectable growing teratoma and would like to stimulate further research and collaboration to come to an optimized treatment suggestion for this group of poor prognostic patients.

Florid vascular proliferation in mature cystic teratoma of the ovary: case report and review of the literature.

A case of mature cystic teratoma that contained florid vascular proliferation is reported. The ovarian tumor occurred in a 9-year-old girl; symptoms consisted of vomiting, abdominal pain and a palpable mass. The microscopic findings were mostly typical of a mature cystic teratoma, but also abundant vascular proliferation mimicking hemangioma in association with mature neural tissue was observed. There was a disorganized arrangement of medium- and large-sized spaces lined by cuboidal endothelial cells. Immunohistochemical staining for vascular proliferation showed immunoreactivity for CD31 and smooth muscle actin. Florid vascular proliferation may be seen in association with neural tissue of ovarian teratomas and should not be misdiagnosed as immature teratoma or a vascular neoplasm.

Three-dimensional power Doppler sonography in the diagnosis of a cystic sacrococcygeal teratoma mimicking a meningomyelocele: A case report.

Sacrococcygeal teratomas have been diagnosed prenatally on sonograms as masses of cystic, solid, or mixed echogenicity from the sacral area and protruding through the perineum or buttocks. However, a cystic sacrococcygeal teratoma may be misdiagnosed as an anterior sacral meningomyelocele, especially when presenting as a posterior cystic mass. We report a case in which three-dimensional power Doppler imaging was helpful for making a correct prenatal diagnosis of a type 1 cystic sacrococcygeal teratoma, which mimicked a meningomyelocele.

Preoperative selective embolization with vascular coiling of giant sacrococcygeal teratoma.

Sacrococcygeal teratoma is one of the most common congenital tumors. Its optimal management requires interdisciplinary care by obstetricians, radiologists, pediatric surgeons, and neonatologists. Early surgery entailing complete tumor excision is the main therapy aim, but a substantial risk of life-threatening complications remains, especially uncontrollable intraoperative hemorrhage. To reduce the risk of bleeding in a female neonate with a giant sacrococcygeal teratoma, we successfully coil-embolized the tumor's main feeding arteries. Her subsequent complete surgical resection was uneventful, and the child is well with favorable reconstructive and functional status of all involved and adjacent organ systems.

Laparoscopic Division of Median Sacral Artery and Dissection of Types III and IV Sacrococcygeal Teratomas to Decrease Intraoperative Hemorrhagic Complications: Case Series and Review of the Literature.

INTRODUCTION: Sacrococcygeal teratoma (SCT) is the most common teratoma presenting at birth. Life-threatening bleeding is a major complication during tumor excision in children. In this study we demonstrate our technique for laparoscopic division of median sacral artery (MSA) during dissection of SCT in 2 pediatric patients as a safe technique to minimize risk of hemorrhage. METHODS: Two female infants diagnosed with types III and IV SCTs underwent preoperative evaluation in the postnatal period. The first patient was an 18-month-old girl who presented with metastatic type IV teratoma, resected after neoadjuvant therapy, and the second patient was a 6-day-old girl with prenatal diagnosis of cystic type III teratoma. Using laparoscopy in both patients, the presacral space was reached by opening the peritoneal reflection with blunt dissection and the MSA was identified. Then it was carefully isolated and divided with 3 or 5 mm sealing device. The pelvic components of the tumors were partially dissected using laparoscopy. The first patient's tumor resection was completed using a posterior sagittal approach and the second patient required a standard Chevron incision. Along with the description of our technique, a review of the current literature for the management of SCT and MSA was performed. RESULTS: Both patients underwent successful laparoscopic division of the MSA and resection of the SCTs without complications. CONCLUSION: Laparoscopic MSA division before SCT excision offers a safe approach that can reduce the risk of hemorrhage during surgery.

Platelet-derived growth factor receptor-beta constitutive activity promotes angiogenesis in vivo and in vitro.

OBJECTIVE: Knockout studies have demonstrated crucial roles for the platelet-derived growth factor-B and its cognate receptor, platelet-derived growth factor receptor-beta (PDGFR-beta), in blood vessel maturation, that is, the coverage of newly formed vessels with mural cells/pericytes. This study describes the consequences of a constitutively activating mutation of the PDGFR-beta (Pdgfrb(D849V)) introduced into embryonic stem cells with respect to vasculogenesis/angiogenesis in vitro and in vivo. METHODS AND RESULTS: Embryonic stem cells were induced to either form teratomas in vivo or embryoid bodies, an in vitro model for mouse embryogenesis. Western blotting studies on embryoid bodies showed that expression of a single allele of the mutant Pdgfrb led to increased levels of PDGFR-beta tyrosine phosphorylation and augmented downstream signal transduction. This was accompanied by enhanced vascular development, followed by exaggerated angiogenic sprouting with abundant pericyte coating as shown by immunohistochemistry/immunofluorescence. Pdgfrb(D849V/+) embryoid bodies were characterized by increased expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor-2; neutralizing antibodies against VEGF-A/VEGF receptor-2 blocked vasculogenesis and angiogenesis in mutant embryoid bodies. Moreover, Pdgfrb(D849V/+) embryonic stem cell-derived teratomas in nude mice were more densely vascularized than wild-type teratomas. CONCLUSIONS: Increased PDGFR-beta kinase activity is associated with elevated expression of VEGF-A and VEGF receptor-2, acting directly on endothelial cells and resulting in increased vessel formation.

Ovarian mature cystic teratoma with florid vascular proliferation and Wagner-Meissner--like corpuscles.

An ovarian mature cystic teratoma featuring florid vascular proliferation and Wagner-Meissner-like corpuscles is presented. The vascular proliferation is analogous to that seen in other tumors having a prominent neural component. The Wagner-Meissner-like corpuscles are viewed as evidence of a specialized type of differentiation of this neural component. To the best of our knowledge, they had not been previously reported in this setting.

The influence of a human embryonic stem cell-derived microenvironment on targeting of human solid tumor xenografts.

The awareness of the important role that the surrounding tissue microenvironment and stromal response play in the process of tumorigenesis has grown as a result of in vivo models of tumor xenograft growth in immunocompromised mice. In the current study, we used human embryonic stem cells in order to study the interactions of tumor cells with the surrounding microenvironment of differentiated human cell tissues and structures. Several cancer cell types stably expressing an H2A-green fluorescence protein fusion protein, which allowed tracking of tumor cells, were injected into mature teratomas and developed into tumors. The salient findings were: (a) the observation of growth of tumor cells with high proliferative capacity within the differentiated microenvironment of the teratoma, (b) the identification of invasion by tumor cells into surrounding differentiated teratoma structures, and (c) the identification of blood vessels of human teratoma origin, growing adjacent to and within the cancer cell-derived tumor. Mouse embryonic stem cell-derived teratomas also supported cancer cell growth, but provided a less suitable model for human tumorigenesis studies. Anticancer immunotherapy treatment directed against A431 epidermoid carcinoma cell-related epitopes induced the complete regression of A431-derived tumor xenografts following direct i.m. injection in immunocompromised mice, as opposed to corresponding tumors growing within a human embryonic stem cell-derived microenvironment, wherein remnant foci of viable tumor cells were detected and resulted in tumor recurrence. We propose using this novel experimental model as a preclinical platform for investigating and manipulating the stromal response in tumor cell growth as an additional tool in cancer research.

TIMP-3 deficiency in the host, but not in the tumor, enhances tumor growth and angiogenesis.

Tumor cells, stromal cell compartment and the extracellular matrix (ECM) together generate a multifaceted tumor microenvironment. Matrix metalloproteinases and their tissue inhibitors (TIMPs) provide a means for tumor-stromal interaction during tumorigenesis. Among TIMPs, TIMP-3 is uniquely localized to the ECM and is frequently silenced in human cancers. Here, we asked whether the absence of TIMP-3 in the tumor cell or the host affects the process of tumorigenesis. Timp-3(-/-) ES-cell clones were generated and used to develop teratomas in nude mice. Timp-3(-/-) teratomas showed similar tumor take, growth, and angiogenesis compared to timp-3(+/+) teratomas. To study the effect of TIMP-3 ablation in the host stroma, we measured the growth kinetics of subcutaneous B16F10 melanomas in timp-3(-/-) and wild-type littermates. Tumors grew significantly faster in timp-3(-/-) than in wild-type mice and their CD31 content was significantly higher indicating increased angiogenesis. Augmented angiogenesis in timp-3(-/-) mice was directly tested using Matrigel plug and Gelfoam assays. In response to FGF-2, timp-3(-/-) endothelial cells invaded more efficiently, leading to enhanced formation of functional blood vessels. Thus, TIMP-3 deficiency in the host, but not in the tumor per se, leads to enhanced tumor growth and angiogenesis. TIMP-3 located within the tumor microenvironment inhibits tumorigenesis.

Targeted replacement of hypoxia-inducible factor-1alpha by a hypoxia-inducible factor-2alpha knock-in allele promotes tumor growth.

Hypoxia-inducible factors (HIF) are essential transcriptional regulators that mediate adaptation to hypoxic stress in rapidly growing tissues such as tumors. HIF activity is regulated by hypoxic stabilization of the related HIF-1alpha and HIF-2alpha subunits, which are frequently overexpressed in cancer cells. To assess the relative tumor-promoting functions of HIF-1alpha and HIF-2alpha directly, we replaced HIF-1alpha expression with HIF-2alpha by creating a novel "knock-in" allele at the Hif-1alpha locus through homologous recombination in primary murine embryonic stem cells. Compared with controls, s.c. teratomas derived from knock-in embryonic stem cells were larger and more proliferative, had increased microvessel density, and exhibited increased expression of vascular endothelial growth factor, transforming growth factor-alpha, and cyclin D1. These and other data indicate that HIF-2alpha promotes tumor growth more effectively than HIF-1alpha in multiple contexts.

Three-dimensional power Doppler (3DPD) ultrasound in the diagnosis and follow-up of fetal vascular anomalies.

OBJECTIVE: This study was undertaken to examine the value of 3-dimensional power Doppler (3DPD) ultrasound imaging in diagnosis and follow-up of fetal vascular anomalies. STUDY DESIGN: In 174 women undergoing early second-trimester targeted organ scanning, followed by a midtrimester second scan in a university hospital setting, 3DPD was applied to the fetal intra-abdominal and intrathoracic vessels. RESULTS: In 137 of 174 fetuses (75%) in the earlier scan, and in 164 of 174 fetuses (95%) in the later scan, 3DPD ultrasound successfully visualized the fetal vessels. In an additional 9 cases, anomalous vascularity was identified: fetal intra-abdominal umbilical vein varix (2), persistent right umbilical vein (1), agenesis of ductus venosus (2), eventration of diaphragm (1), parenchymal and vascular lung anomaly (1), sacrococcygeal teratoma (1), and chorioangioma (1). 3DPD improved diagnostic precision, aided our understanding of anomalous structure, and added information on the vascular volume of lesions in some cases. CONCLUSION: 3DPD improved ultrasound visualization of the fetal vessels of the abdomen and thorax in normal and anomalous cases.

Fibroblast growth factor receptor-1 expression is required for hematopoietic but not endothelial cell development.

OBJECTIVE: The purpose of this study was to clarify the role of fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in hematopoietic/endothelial development. METHODS AND RESULTS: Using several different FGFR-1-specific antibodies and FGFR-1 promoter-driven LacZ activity, we show that FGFR-1 is expressed and active as a tyrosine kinase in a subpopulation of endothelial cells (approximately 20% of the endothelial pool) during development in embryoid bodies. In agreement, in stem cell-derived teratomas, expression of FGFR-1 was detected in some but not all vessels. The FGFR-1 expressing endothelial cells were mitogenically active in the absence and presence of vascular endothelial growth factor (VEGF). Expression of FGFR-1 in endothelial cell precursors was not required for vascular development, and vascularization was enhanced in FGFR-1-deficient embryoid bodies compared with wild-type stem cells. In contrast, hematopoietic development was severely disturbed, with reduced expression of markers for primitive and definitive hematopoiesis. CONCLUSIONS: Our data show that FGFR-1 is expressed in early hematopoietic/endothelial precursor cells, as well as in a subpool of endothelial cells in tumor vessels, and that it is critical for hematopoietic but not for vascular development.