Early Events in Xenograft Development from the Human Embryonic Stem Cell Line HS181 - Resemblance with an Initial Multiple Epiblast Formation

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart

Enhanced Platelet Activation Mediates the Accelerated Angiogenic Switch in Mice Lacking Histidine-Rich Glycoprotein

BACKGROUND: The heparin-binding plasma protein histidine-rich glycoprotein (HRG; alternatively, HRGP/HPRG) can suppress tumor angiogenesis and growth in vitro and in vivo. Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times. In addition, the angiogenic switch is significantly enhanced in HRG-deficient mice. METHODOLOGY/PRINCIPAL FINDINGS: To address whether HRG deficiency affects tumor development, we have crossed HRG knockout mice with the RIP1-Tag2 mouse, a well established orthotopic model of multistage carcinogenesis. RIP1-Tag2 HRG(-/-) mice display significantly larger tumor volume compared to their RIP1-Tag2 HRG(+/+) littermates, supporting a role for HRG as an endogenous regulator of tumor growth. In the present study we also demonstrate that platelet activation is increased in mice lacking HRG. To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα. Interestingly, this treatment suppressed the increase in angiogenic neoplasias seen in HRG knockout mice. However, if GP1bα treatment was initiated at a later stage, after the onset of the angiogenic switch, no suppression of tumor growth was detected in HRG-deficient mice. CONCLUSIONS: Our data show that increased platelet activation mediates the accelerated angiogenic switch in HRG-deficient mice. Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated

Optimization and validation of a species-specific in vivo approach for studies on growth and progression of neural crest derived tumours

Background: Despite great advantages in cancer therapeutics during the last decades, therapy-resistance remains a problem in the majority of malignancies. Hence, development of more effective therapeutics is critical to enhance survival of patients with cancer. A big challenge for therapy development is low efficacy upon clinical translation; therapies proven highly effective in animal trials often exhibit a poor effect or even fail in the clinic. This indicates the need for more relevant preclinical in vivo model systems for cancer. Malignant melanoma and pediatric neuroblastoma, occurring in derivatives of the neural crest, are both exhibiting a high degree of therapy-resistance, with a high number of deaths as a consequence. These malignancies are characterized by a significant plastic developmental capacity with a tumour phenotype extensively depending on the surrounding microenvironment. Thus, they provide excellent opportunity for studies on interactions between the tumour and adjacent supportive tissue. Objective: The overall aim of this thesis is to optimize and validate a human in vivo model system, based on human embryonic stem cell derived teratomas (hEST-model), for growth and progression of neural crest derived malignancies. Further on, I aim to elucidate whether human tumours grafted in such system are having a higher resemblance to clinical tumours, as compared to conventionally used xenografts. Results: Based on a thorough kinetic study of development in hESC derived teratoma from the cell line HS181, we chose day 45 as the time point for tumour inoculation. At this time, mature embryonic tissues are formed and human vascularisation is initiated. Also, this allows further grafting of the tumour cells without affecting the animals well-being and occurrence of necrotic areas. The capacity for teratoma formation was also explored using a subline of HS181 with altered karyotype, i.e. trisomic for chromosome 12. Results revealed that these cells are capable of forming teratomas, however with a skewed tissue contribution with higher frequency of renal formation. Cell lines from malignant melanoma and neuroblastomas were grafted in the hEST-model and compared to the corresponding xenograft. For the melanomas, striking differences in expression of markers related to melanocytic differentiation was seen, indicative of induced differentiation from the xenograft-environment. Also, a dedifferentiated and invasive subpopulation of melanoma cells was detected, but not present in the xenograft, indicating species-specific interactions upon migration and invasion into surrounding stroma. Grafting of three different neuroblastoma cell lines in the hEST-model resulted in a general finding of higher histological heterogeneity and more resemblance to clinical neuroblastomas, as compared to the xenografts. Grafting of both tumour types in the hEST-model induced an extensive neo-vascularisation of human origin in the surrounding mesenchyme indicating species-specific effects. Conclusions: The results presented in this thesis indicate that a human in vivo model system for cancer based on hESC-derived teratomas add significant importance for preclinical cancer studies. The embryonic environment of the teratoma is probably most relevant for grafting of embryonic tumours, indicated also by our results using pediatric neuroblastoma. Altogether, the hEST-model provides unique possibilities to study human tumours in a species-specific environment, and is therefore suggested a well-needed complement to current preclinical in vivo models

The pro-inflammatory role of platelets in cancer

Thrombosis is a frequent issue in cancer patients. Tumor-induced platelet activation and coagulation does not only constitute a significant risk for thrombosis, but also contribute to tumor progression by promoting critical processes such as angiogenesis and metastasis. In addition to their role in hemostasis, platelets are increasingly recognized as regulators of inflammation. By modulating the immune system, platelets regulate several aspects of cancer-associated pathology. Platelets influence the inflammatory response in cancer by affecting the activation status of the endothelium and by recruiting leukocytes to primary and metastatic tumor sites, as well as to distant organs unaffected by tumor growth. Furthermore, platelets participate in the formation of neutrophil extracellular traps, which can promote metastasis, thrombosis, and contribute to organ failure. In this review, we discuss the role of platelets as coordinators of the immune system during malignant disease and the potential of targeting platelets to prevent cancer-associated pathology

NETosis in Cancer - Platelet-Neutrophil Crosstalk Promotes Tumor-Associated Pathology

It has become increasingly clear that circulating immune cells in the body have a major impact on cancer development, progression, and outcome. The role of both platelets and neutrophils as independent regulators of various processes in cancer has been known for long, but it has quite recently emerged that the platelet-neutrophil interplay is yet a critical component to take into account during malignant disease. It was reported a few years ago that neutrophils in mice with cancer have increased propensity to form neutrophil extracellular traps (NETs) - web-like structures formed by externalized chromatin and secreted proteases. The initial finding describing this as a cell death-associated process has been followed by reports of additional mechanisms for NET formation (NETosis), and it has been shown that similar structures can be formed also without lysis and neutrophil cell death as a consequence. Furthermore, presence of NETs in humans with cancer has been verified in a few recent studies, indicating that tumor-induced NETosis is clinically relevant. Several reports have also described that NETs contribute to cancer-associated pathology, by promoting processes responsible for cancer-related death such as thrombosis, systemic inflammation, and relapse of the disease. This review summarizes current knowledge about NETosis in cancer, including the role of platelets as regulators of tumor-induced NETosis. It has been shown that platelets can serve as inducers of NETosis, and the platelet-neutrophil interface can therefore be an important issue to consider when designing therapies targeting cancer-associated pathology in the future

Tumor-Induced Local and Systemic Impact on Blood Vessel Function

Endothelial dysfunction plays a role in several processes that contribute to cancer-associated mortality. The vessel wall serves as a barrier for metastatic tumor cells, and the integrity and activation status of the endothelium serves as an important defense mechanism against metastasis. In addition, leukocytes, such as cytotoxic T-cells, have to travel across the vessel wall to enter the tumor tissue where they contribute to killing of cancer cells. Tumor cells can alter the characteristics of the endothelium by recruitment of leukocytes such as neutrophils andmacrophages, which further stimulate inflammation and promote tumorigenesis. Recent findings also suggest that leukocyte-mediated effects on vascular function are not limited to the primary tumor or tissues that represent metastatic sites. Peripheral organs, such as kidney and heart, also display impaired vascular function in tumor-bearing individuals, potentially contributing to organ failure. Here, we discuss how vascular function is altered in malignant tissue and distant organs in individuals with cancer and how leukocytes function as potent mediators of these tumor-induced effects

Tumor-Induced Local and Systemic Impact on Blood Vessel Function

Endothelial dysfunction plays a role in several processes that contribute to cancer-associated mortality. The vessel wall serves as a barrier for metastatic tumor cells, and the integrity and activation status of the endothelium serves as an important defense mechanism against metastasis. In addition, leukocytes, such as cytotoxic T-cells, have to travel across the vessel wall to enter the tumor tissue where they contribute to killing of cancer cells. Tumor cells can alter the characteristics of the endothelium by recruitment of leukocytes such as neutrophils andmacrophages, which further stimulate inflammation and promote tumorigenesis. Recent findings also suggest that leukocyte-mediated effects on vascular function are not limited to the primary tumor or tissues that represent metastatic sites. Peripheral organs, such as kidney and heart, also display impaired vascular function in tumor-bearing individuals, potentially contributing to organ failure. Here, we discuss how vascular function is altered in malignant tissue and distant organs in individuals with cancer and how leukocytes function as potent mediators of these tumor-induced effects