Osteosarcoma: From Molecular Biology to Mesenchymal Stem Cells

Osteosarcoma is the most common primary malignant tumour of bone. Currently, despite treatment with multi-agent chemotherapy and limb salvage surgery, the five-year survival rate for osteosarcoma remains at 70%. The pathogenesis of osteosarcoma is complex and involves alterations in cellular apoptosis, adhesion, migration, invasion and molecular signalling. Research most recently has focused on the molecular basis of the disease with the goal of identifying novel therapeutic targets. To this end, mesenchymal stem cells (MSCs) have been identified to play a role in sarcomagenesis. MSC transformation may give rise to tumours, whereas interactions of MSCs with osteosarcoma cells in the tumour microenvironment may cause increased cell proliferation. This is in stark contrast to the role of MSCs as a promising source for tissue repair and regeneration. In order to utilize MSCs for biological reconstruction in the setting of osteosarcoma, further research is necessary to delineate the role of MSCs in osteosarcoma transformation and progression

The Role of Nodal in the Regulation of Bi-Potential Trophoblast Progenitor Cells

The human placenta develops from highly proliferative and phenotypically plastic cells called trophoblasts. Bi-potential trophoblast stem cells differentiate into the villous pathway to form the syncytiotrophoblast layer and the extravillous trophoblast (EVT). The HTR-8/SVneo cell line is widely used to study trophoblast biology. These cells variably express villous-specific or EVT-specific genes depending on conditions. Such phenotypic plasticity is indicative of a bi-potential cytotrophoblast progenitor. Preliminary work has shown that similar to progenitors in situ, a subpopulation of HTR-8/SVneo cells expresses a6b4 integrin. This a6b4high subset exhibits enhanced clonogenicity and differentiation capacity. This cell line also expresses Nodal, a stem cell-associated factor that sustains the pluripotency of embryonic stem cells and is re-expressed in certain cancers. I hypothesized that the a6b4high subset within HTR-8/SVneo is enriched with bi-potential cytotrophoblast progenitor-like cells that are maintained by Nodal signaling. Our results revealed that the a6b4high subset expresses greater amounts of Nodal protein relative to the a6b4low subset. To investigate the role of Nodal in regulating trophoblast progenitors, stable Nodal knock-down and over-expressing cells were analyzed. It was found that Nodal is required for clonogenicity, maintenance of enhanced a6b4 expression and endovascular differentiation along the EVT pathway. These results suggest that the a6b4high population may represent a villous cytotrophoblast progenitor cell in which Nodal regulates clonogenicity and capacity for differentiation along the EVT pathway

Role of the fibrinolytic system in experimental allergic encephalomyelitis.

The immunopathology of multiple sclerosis (MS), a disease of the central nervous system (CNS), is characterised by widespread inflammation, focal demyelination and axonal degeneration. As a result of early disturbances in the blood brain barrier (BBB), serum proteins, including fibrin(ogen) enter into the CNS. Up-regulation of components of the plasminogen activator (fibrinolytic) system correlates with onset of inflammation and migration of leucocytes into the brain parenchyma. Significant upregulation of plasminogen and plasminogen activator inhibitor-1 (PAI-1), and an accumulation of fibrin D-dimer was found during neuroinflammation, in the established mouse model of MS, chronic relapsing experimental allergic encephalomyelitis (CREAE) induced with spinal cord homogenate (SCH). Onset and progression of disease correlated with a reduction in dendritic markers, supporting evidence of early neuronal/axonal dysfunction. Furthermore an impairment of fibrinolysis in these mice ensured that fibrin entering the CNS was not effectively removed, suggesting a role for the PA system in the pathogenesis of CREAE. Initially, using mice deficient in tissue-type plasminogen activator (tPA"/") and myelin oligodendrocyte glycoprotein (MOG)-induced EAE, animals displayed an early and a more severe acute disease characterised by incomplete recovery when compared to wild-type controls, with significantly higher CNS levels of PAI-1. This correlated with fibrin accumulation, which co-localised with non-phosphorylated neurofilament on thickened axons in EAE tissue. In contrast, urokinase plasminogen activator receptor knockout mice (uPAR"A) had a delayed, less acute disease reflected in delayed infiltration of inflammatory cells. However, these animals developed chronic disease as a result of steadily increasing inflammation, high levels of urokinase-type plasminogen activator (uPA) and greater degree of demyelination. Due to low rates of EAE susceptibility, mice deficient for PAI-1 were backcrossed onto the ABH strain for 4 generations. Induction of SCH-CREAE in PAI-1"7" mice resulted in a lower incidence of disease, with mice developing clinical signs of EAE significantly later than WT littermates. A delay in cellular entry into the CNS accompanied by a higher capacity for fibrinolysis resulted in a milder disease in PAI-1"7" mice with no clinical relapses and less axonal damage. Thus, the plasminogen activator system can modulate both inflammatory and degenerative events in the CNS through the respective effects of tPA, PAI-1 and uPAR on fibrinolysis and cell adhesion/migration, manipulation of which may be of therapeutic importance in multiple sclerosis

Implication de la mélanotransferrine dans la progression tumorale : identification d'une nouvelle cible thérapeutique

La mélanotransferrine (MTf) a tout d'abord été identifiée comme étant un antigène majeur des mélanomes. Son expression a par la suite été rapportée dans plusieurs cellules néoplasiques et quelques tissus sains. Bien que la majorité de la MTf soit associée à la membrane plasmique, une faible portion est sécrétèe dans le milieu extracellulaire sous forme soluble. Notre équipe a récemment démontré que la MTf régulait l'activation du plasminogène et la motilité cellulaire in vitro. Les activateurs du plasminogène sécrétés par les cellules cancéreuses catalysent la conversion du plasminogène en protéase active. La plasmine joue un rôle central dans la progression tumorale en dégradant les protéines de la membrane basale et de la matrice extracellulaire (MEC). Ce remodelage tissulaire facilite grandement l'angiogenèse et l'invasion des cellules cancéreuses vers les tissus adjacents. Le premier objectif de cette thèse fut de démontrer que la MTf favorise l'activation du plasminogène par son activateur de type tissulaire (tPA). À cet effet, une forme recombinante tronquée de la MTf (sMTf) a été utilisée. En présence de plasminogène et de tPA, la sMTf stimule la formation de plasmine dans le milieu extracellulaire de cellules endothéliales (CE) et accroît la dégradation de la fibronectine, une des composantes de la MEC. La dégradation de la MEC conduit au détachement des CE et à leur mort par anoïkis. Ces données suggèrent que la sMTf possède des propriétés anti-angiogéniques en induisant l'anoïkis des CE. Nous avons ensuite évalué ses effets sur le développement angiogénique et sur la croissance tumorale in vivo. Les résultats montrent que la sMTf inhibe significativement la croissance de tumeurs sous-cutanées dérivées de glioblastome et de carcinome pulmonaire humains chez la souris immunosupprimée. Nous démontrons également que le traitement à la sMTf entraîne une réduction de l'expression du marqueur vasculaire d'endogline, de même qu'une diminution de la quantité d'hémoglobine au sein des tumeurs sous-cutanées de glioblastomes. L'ensemble de ces résultats démontre clairement que la sMTf réduit l'angiogenèse et la croissance tumorale in vivo. Tout comme la forme recombinante tronquée, la MTf membranaire (mMTf) interagit avec le système d'activation du plasminogène. La formation de plasmine à proximité de la membrane plasmique favorise l'invasion des cellules tumorales et le développement métastatique. La participation de la mMTf dans la formation de métastases cérébrales a donc été évaluée pour une lignée cellulaire de mélanome humain. Les résultats indiquent la présence de cellules exprimant la MTf humaine dans les cerveaux de souris ayant reçu une injection intraveineuse de cellules de mélanome humain. De même, l'administration d'un anticorps monoclonal dirigé contre la MTf humaine (L235) a réduit de moitié le développement de métastases cérébrales de mélanomes chez la souris. Ces résultats démontrent que l'implication de la mMTf dans l'activation du plasminogène faciliterait la migration des cellules cancéreuses à travers la barière hémato-encéphalique (BHE) et l'invasion du système nerveux central. Les principales contributions de ce travail sont d'avoir démontré d'une part que la sMTf induit le détachement des cellules endothéliales par stimulation de la cascade d'activation du plasminogène. D'autre part, la sMTf entraîne l'inhibition du développement angiogénique et de la croissance tumorale chez la souris. Puis, l'expression de la MTf à la surface des cellules cancéreuses facilite leur migration à travers la BHE dans le but de produire des métastases cérébrales. Cette étude identifie la MTf clairement comme une cible intéressante dans la progression tumorale, tout en suggérant des outils thérapeutiques comme la sMTf recombinante ou une forme humanisée du L235. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Croissance tumorale, Angiogenèse, Métastases cérébrales, Barrière hémato-encéphalique, Mélanotransferrine, Mélanome, Plasminogène

Endothelial colony-­‐forming cells and transforming growth factor-­‐β superfamily signalling in idiopathic pulmonary arterial hypertension

Circulating endothelial progenitor cells may be important in the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) and give rise to endothelial colony-­‐forming cells (ECFCs) in culture. These cells represent an accessible surrogate population to investigate endothelial dysfunction in IPAH. Peripheral blood mononuclear cells were cultured from healthy volunteers (n=25, 72% female; age range 23-­‐57 yr) and IPAH patients (n=30, 60% female; age range 22-­‐56 yr). Older IPAH patients (n=6, 50% female; age range 62-­‐82 yr) were also sampled. Distinct colonies appeared after 13-­‐35 days and exhibited a typical cobblestone morphology. The average frequency of colonies and clonal growth of isolated cells was similar in healthy volunteers and IPAH patients. Age-­‐dependent differences were observed however in the number and frequency of colonies, which declines with age in the control but not in IPAH subjects. The endothelial phenotype was confirmed by immunostaining and flow cytometry, exhibiting endothelial and progenitor markers, but not hematopoietic markers. Endothelial cell functions, including proliferation, angiogenesis, migration and responses to apoptotic stimuli, were compared in cells between passages 4 to 7. IPAH cells showed enhanced angiogenic capacity (tube formation on Matrigel), significantly less apoptosis (lower caspase-­‐3/7 activity) in response to serum and growth factor deprivation, and impaired migratory capacity compared with control ECFCs. Dysfunctional transforming growth factor (TGF)-­‐β and bone morphogenetic protein receptor expression and signalling are implicated in IPAH and were assessed by RT-­‐PCR and western blotting. IPAH and control ECFCs differed in their TGF-­‐β type I (ALK5) receptor expression/signalling and in the expression of other regulatory proteins (e.g. chloride-­‐like intracellular channel-­‐4). Blood-­‐derived ECFCs display an endothelial lineage similar to that of mature endothelial cells. ECFCs from IPAH patients have a distinct functional phenotype and exhibit differences in TGF-­‐β receptor superfamily expression/signalling, which may contribute to endothelial dysfunction and vascular remodelling in IPAH

Colorado State University, College of Veterinary Medicine and Biomedical Sciences, 9th annual CVMBS research day scientific proceedings

Includes the Pfizer Research Award winner and abstracts only of the oral sessions and posters

A Human Hepatocyte-Bearing Mouse: An Animal Model to Predict Drug Metabolism and Effectiveness in Humans

Preclinical studies to predict the efficacy and safety of drugs have conventionally been conducted almost exclusively in mice and rats as rodents, despite the differences in drug metabolism between humans and rodents. Furthermore, human (h) viruses such as hepatitis viruses do not infect the rodent liver. A mouse bearing a liver in which the hepatocytes have been largely repopulated with h-hepatocytes would overcome some of these disadvantages. We have established a practical, efficient, and large-scale production system for such mice. Accumulated evidence has demonstrated that these hepatocyte-humanized mice are a useful and reliable animal model, exhibiting h-type responses in a series of in vivo drug processing (adsorption, distribution, metabolism, excretion) experiments and in the infection and propagation of hepatic viruses. In this review, we present the current status of studies on chimeric mice and describe their usefulness in the study of peroxisome proliferator-activated receptors

An experimental study of human melanoma cells cutured in vitro

This thesis records the results of a series of experiments that were designed to examine the biology of human malignant melanoma cells cultured in vitro. The studies were so planned as to document phenotypic differences that exist between melanomas, to define respects in which melanoma cell differentiation could be modulated and to correlate biochemical variability with in vivo behaviour as measured in the nude mouse. Melanoma cell lines were established from biopsy material obtained from 7 patients at Groote Schuur Hospital. Two of these lines synthesized tyrosinase and melanin at a rate that was directly related to cell density. The five remaining lines did not pigment. ii All of the lines showed aneuploidy; 5 of the 7 showed anchorageindependent growth; and 6 of the 7 grew as lethal tumours in nude mice. As has been found with all other melanomas studied, these cells released a plasminogen activator that was chemically and immunologically identical to tissue activator. One of the lines proved to be an exception to this general rule in that it synthesized urokinase-type enzyme. Unlike most other human cells cultured in vitro, melanoma cells proved to be relatively refractory to hormonal stimuli. Addition of estrogen, progesterone, testosterone, dexamethasone or melanocyte-stimulating hormone to the culture medium had very little effect on cellular release of plasminogen activator, upon cell growth, or upon cellular morphology. Although remarkably resistant to hormonal influences, cellular release of plasminogen activator did appear to be inhibited to a striking degree by cocultivation with normal skin fibroblasts. This observation led to the discovery of a phenomenon in which fibroblasts of many types bound tissue-type plasminogen activator and so removed it from the medium. This was accompanied by an apparent change in molecular weight of the melanoma cell enzyme from 72K daltons to approximately 115K daltons, suggesting the presence of a 40-SOK binding molecule. iii In an attempt to influence in vitro differentiation, the tumour promoter tetradecanoylphorbol acetate, and the differentiation-inducing retinoid, retinoic acid, were added to the two pigmenting cell lines. The effects of these compounds on induction of tyrosinase activity, morphological change or plasminogen activator release differed. In the one cell line, tetradecanoylphorbol acetate caused morphological maturation with a decrease in the rate of plasminogen activator release and no obvious effect upon pigmentation. This line was relatively resistant to the action of retinoids. The other pigmenting line responded hardly at all to the tumour promoter. Retinoic acid, on the other hand, inhibited the induction of tyrosinase activity, yet caused an inhibition of growth and plasminogen activator release. A number of interesting observations could be made in experiments in which melanoma cells were inoculated into nude mice. Firstly, the growth rate of the tumours ~n vivo correlated poorly with the doubling times of the corresponding cells cultured in vitro. Secondly, despite a marked inhibitory effect of fibroblasts on plasminogen activator in vitro, coinjection of fibroblasts and melanoma cells in vivo greatly enhanced tumour growth when small tumour cell inocula were used and shortened the latent period for tumour appearance with larger inocula. Thirdly, melanomas growing in nude mice differed strikingly in their ability to elicit a desmoplastic response. Tumours in which large amounts of host connective tissue were deposited tended to be heavily contaminated with murine fibroblasts when re-established in vitro. This contamination was not seen with tumours that contained very little connective tissue. These results point to the existence of a melanoma-associated fibrogenic factor. Finally, by excision of the primary tumour, it was possible to avoid death of the animal from local complications and so allow time for metastases to develop. In three mice, metastatic melanoma deposits could be detected by this device, so establishing a protocol for the use of nude mice as valid models for the experimental study of metastatic spread of human tumours

Neutrophil Leucocyte Locomotion in 3-D Gel Matrices

The invasive and locomotory behaviour of neutrophil leucocytes was studied using 3-D collagen gel matrices made from reconstituted rat tail tendon. Other techniques used included time-lapse filming of neutrophils moving over protein-coated glass, and chemiluminescence