Colon cancer metastasis in mouse liver is not affected by hypercoagulability due to Factor V Leiden mutation

Clinical trials have shown life-prolonging effects of antithrombotics in cancer patients, but the molecular mechanisms remain unknown due to the multitude of their effects. We investigated in a mouse model whether one of the targets of antithrombotic therapy, fibrin deposition, stimulates tumour development. Fibrin may provide either protection of cancer cells in the circulation against mechanical stress and the immune system, or form a matrix for tumours and/or angiogenesis in tumours to develop. Mice homozygous for Factor V Leiden (FVL), a mutation in one of the coagulation factors that facilitates fibrin formation, were used to investigate whether hypercoagulability affects tumour development in an experimental metastasis model. Liver metastases of colon cancer were induced in mice with the FVL mutation and wild-type littermates. At day 21, number and size of tumours at the liver surface, fibrin/fibrinogen distribution, vessel density and the presence of newly formed vessels in tumours were analysed. Number and size of tumours did not differ between mice with and without the FVL mutation. Fibrin/fibrinogen was found in the cytoplasm of hepatocytes and cancer cells, in blood vessels in liver and tumour tissue and diffusely distributed outside vessels in tumours, indicating leaky vessels. Vessel density and angiogenesis varied widely between tumours, but a pre-dominance for vessel-rich or vessel-poor tumours or vessel formation could not be found in either genotype. In conclusion, the FVL mutation has no effect on the development of secondary tumours of colon cancer in livers of mice. Fibrin deposition and thus inhibition of fibrin formation by anticoagulants do not seem to affect tumour development in this model.     

Experimental chemotherapy and concepts related to the cell cycle

Scheduling of chemotherapy is limited by damage to normal tissues, and tolerated schedules are dependent on normal tissue recovery. Most anticancer drugs are more toxic to proliferating cells and the fall and recovery of granulocyte counts after chemotherapy may be explained by the effect of drugs on rapidly proliferating precursor cells in the bone marrow. It is argued that serious toxicity due to myelosuppression most often occurs because of damage to proliferating precursors that may be recognized in bone marrow rather than to stem cells. In contrast, therapy that is aimed at producing cure or long-term remission of tumours must be directed at killing tumour stem cells. The evidence that tumours contain a limited population of cells which can repopulate the tumour after treatment (and are therefore tumour stem cells) is reviewed critically. While there is quite strong evidence for a limited population of target cells, evidence from studies on metastases suggests that the tumour cells which may express this stem cell property may change with time. The stem cell concept has major implications for predictive assays. Although colony-forming assays appear to have a sound biological background for predicting tumour response, technical problems prevent them from being used routinely in patient management. Cells in tumours are known to be heterogeneous and at least three types of heterogeneity may influence tumour response to drug treatment: the development of subclones with differing properties including drug resistance; variation in cellular properties due to differentiation during clonal expansion; and variation in properties due to nutritional status and micro-anatomy. Heterogeneity in drug distribution within solid tumours may occur because of limited drug penetration from blood vessels, and nutrient-deprived cells in solid tumours may be expected to escape the toxicity of some anticancer drugs as well as being resistant to radiation because of hypoxia. This may occur both because nutrient-deprived cells have a low rate of cell proliferation, and also because of poor drug penetration to them. There is a need for improved understanding of the mechanisms that lead to cell death in tumours. If these mechanisms were understood, it might be possible to simulate them by therapeutic manoeuvres. Recent research from our laboratory suggests that the combination of low extracellular pH and hypoxia may be very toxic to cells in nutrient-deprived regions. Drugs which limit the cell's ability to survive in regions of acid pH may provide strategy for therapy of nutrient-deprived cells.     

Proliferation kinetics of endothelial and tumour cells in three mouse mammary carcinomas

Abstract. An autoradiographic study of three corded mouse tumours is reported. The proliferation characteristics of both tumour cells and endothelial cells were studied. The doubling time of these three tumours differed by a factor of 2.6 but there was only a small difference in the intermitotic time. All three tumours showed a very high cell loss factor (˜0.80) and the differences in growth rate resulted mainly from differences in the growth fraction .
The endothelial cell proliferation rates differed markedly in the three tumours, with labelling indices ranging from 18% in the faster tumours to 4.5% in the slowest. The potential doubling times for endothelium, calculated from these values, were much slower than the tumour cell cycle time or the tumour potential doubling time, but were two to four times faster than the volume doubling time of the tumour.
It appears likely that the endothelial proliferation rate influences the growth fraction, but similar high cell loss factors can occur in tumours with a four-fold difference in endothelial cell production rates. Inadequate branching of blood vessels seems likely to be at least as important as inadequate production of endothelial cells. It is not possible to determine whether slow tumour cell production evokes a slower endothelial growth or vice versa.     

Brain tumour stem cells: the undercurrents of human brain cancer and their relationship to neural stem cells

Conceptual and technical advances in neural stem cell biology are being applied to the study of human brain tumours. These studies suggest that human brain tumours are organized as a hierarchy and are maintained by a small number of tumour cells that have stem cell properties. Most of the bulk population of human brain tumours comprise cells that have lost the ability to initiate and maintain tumour growth. Although the cell of origin for human brain tumours is uncertain, recent evidence points towards the brain's known proliferative zones. The identification of brain tumour stem cells has important implications for understanding brain tumour biology and these cells may be critical cellular targets for curative therapy.     

Vascularization is a general requirement for growth of plant and animal tumours

Solid-tumour growth in animals as in humans depends on angiogenesis. Tumours that fail to induce the formation of new blood vessels do not enlarge beyond a few millimetres in diameter. Plant tumours induced by Agrobacterium tumefaciens can reach diameters of more than 100 mm, thus raising the question of how they are sufficiently supplied with nutrients and water. Until recently, these rapidly growing tumours were considered unorganized or partly organized masses. However, in analogy to animal and human tumours, growth of leaf and stem tumours depends on neovascularization. Plant tumour cells induce the formation of a sophisticated vascular network consisting of water-conducting vessels and assimilate-transporting sieve elements. Similar to animal and human tumours that overexpress angiogenic growth factors, plant tumours overexpress the T-DNA-encoded vascularization-promoting growth factors auxin and cytokinin upon AGROBACTERIUM: infection. High auxin levels induce ethylene emission from the tumours, which has a strong impact on tumour and host stem, as well as on root structure and function. Ethylene apparently stimulates abscisic acid synthesis in the leaves above the tumour, which reduces transpiration and thus protects the host plant from rapid wilting. Hence, for the elucidation of phytohormone-dependent vascular development in plants, such tumours are regarded as an excellent model system. The comparison of analogous requirement of neovascularization for tumour growth in plants, as in animals and humans, is discussed in terms of interdisciplinary strategies of possible prevention and therapy.     

Roles of p38 MAPKs in invasion and metastasis

Cells from primary tumours need to go through several steps to become fully metastatic. During this process, cancer cells acquire the ability to invade, migrate across the surrounding tissue, enter into the circulation and colonize distant organs. In the present paper, we review recent progress in understanding how the p38 MAPK (mitogen-activated protein kinase) signalling pathway participates in the different steps of metastasis. Experimental evidence suggests that tumour cells need to modulate p38 MAPK activity levels to successfully metastasize.     

Serotonin-producing pancreatic endocrine tumour. Histological, ultrastructural and immunohistochemical study of a case

Serotonin-producing pancreatic endocrine tumours are rare neoplasms which in most cases exhibit malignant biological behaviour. These tumours, in the majority of the well-documented cases, are composed of argyrophil- and argentaffin-positive cells which contain large pleomorphic neurosecretory granules. In contrast, argyrophilic non-argentaffin pancreatic endocrine tumours with tumour cells containing round neurosecretory granules are exceptional. In this study we describe such a tumour not associated with clinical evidence of carcinoid syndrome in a 60-year-old woman. Histological examination revealed tumour extension in pancreatic lymphatic vessels and veins but no evidence of locoregional or distant metastases. Ten months after surgery the patient showed no recurrence of the disease. Immunohistochemistry revealed cytoplasmic serotonin production in the tumour cells which were negative for anti-gastrin, insulin, glucagon, somatostatin, pancreatic polypeptide (PP), vasoactive intestinal peptide (VIP) and ACTH. This study emphasizes the usefulness of combined ultrastructural and immunohistochemical investigations in order to identify and characterize the rare pancreatic endocrine tumours with serotonin production.     

CYTOKINETICS OF TUMOUR AND ENDOTHELIAL CELLS AND VASCULARIZATION OF LUNG METASTASES IN C3H/HE MICE

A model of lung metastases was developed using intravenous injection of tumour cell aggregates of spontaneous C3H/He mammary tumours in syngeneic mice. the growth rate of lung tumours decreased with increasing tumour volume, with mean host survival of 46 days. the cytokinetics of individual tumours ranging between 0.004 and 4.2 mm³ in volume were studied. the labelling index (LI) ranged between 12 and 17%, the DNA synthesis time (T_s) being 9–10 hr. the growth fraction (GF) ranged between 26 and 38%. the cell cycle time (T_c) was found to be 18–19 hr. the LI and the GF decreased with increasing tumour volume doubling time (T_d). No correlation was found between the tumour volume and T_c. the LI of endothelial cells within these tumours, ranging between 0.004 and 4.2 mm³ in volume was 14–15% and endothelial cell proliferation was not affected by tumour growth. Vascular parameters were also determined for these tumours as a function of tumour volume. Vascular volume increased with increasing tumour size while the percentage of capillary vessels decreased. the cellular volume to capillary volume ratio increased with increasing tumour volume. Necrosis was observed in 0.27 mm³ tumours and increased with increasing tumour size. The results from these studies suggested that the age-dependent decrease in proliferative activity of tumour cells growing in the lung is related to change in effective vascularity.     

PV1 down‐regulation via shRNA inhibits the growth of pancreatic adenocarcinoma xenografts

PV1 is an endothelial‐specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour‐bearing mice by single‐dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down‐regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC‐1 and BxPC‐3). The effect observed is because of down‐regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down‐regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis.     

Transplantability and metastatic potential of chemically induced rat brain tumours

From clinical observations it is known that brain tumours in principle do not metastasize. An explanation for this phenomenon is not available. The few described cases of distant metastases from primary brain tumours all occurred after surgery of the central nervous system. Furthermore, the brain does not contain a lymphatic system. The major question in this matter is whether the inability of CNS tumours to metastasize is based on a specific tumour bound property or on specific local factors. Since an experimental model for this situation was not available we induced brain tumours in rats. About 130 WAG/Rij and Sprague Dawley rats (males and females) were treated with the neurocarcinogen ethylnitroso-urea (ENU) within 24 hours after birth. Tumours appeared at the age of 6 to 29 months. All tumours were removed after killing the host and transplanted subcutaneously into syngeneic rats. Histologically the tumours were mostly oligodendrogliomas, schwannomas and several mixed glial tumours. Metastases from these primary tumours were not observed. The transplanted tumours showed distant metastases in 52% of the cases. Metastases occurred mainly in lungs, liver and lymph nodes. From these observations it is concluded that the absence of metastases from primary brain tumours is probably not related to a specific property of brain tumours. Further research is emphasized on specific local factors.     

PDGFRbeta+ perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival

The microvasculature consists of endothelial cells and their surrounding pericytes. Few studies on the regulatory mechanisms of tumour angiogenesis have focused on pericytes. Here we report the identification of tumour-derived PDGFRbeta (+) (platelet-derived growth factor receptor beta) progenitor perivascular cells (PPCs) that have the ability to differentiate into pericytes and regulate vessel stability and vascular survival in tumours. A subset of PDGFRbeta (+) PPCs is recruited from bone marrow to perivascular sites in tumours. Specific inhibition of PDGFRbeta signalling eliminates PDGFRbeta (+) PPCs and mature pericytes around tumour vessels, leading to vascular hyperdilation and endothelial cell apoptosis in pancreatic islet tumours of transgenic Rip1Tag2 mice.     

Endothelial cell differentiation of human breast tumour stem/progenitor cells

Breast tumour stem cells have been reported to differentiate in the epithelial lineage but a cross-lineage potential has not been investigated. We aimed to evaluate whether breast tumour stem cells were able to differentiate also into the endothelial lineage. We isolated and cloned a population of breast tumour stem cells, cultured as mammospheres that expressed the stem markers nestin and Oct-4 and not epithelial and endothelial differentiation markers, and formed serially transplantable tumours in SCID mice. When cultured in the presence of serum, mammosphere-derived clones differentiated in the epithelial lineage. When cultured in the presence of VEGF, the same clones were also able to differentiate in the endothelial lineage acquiring endothelial markers and properties, such as the ability to organize in Matrigel into capillary-like structures. In the transplanted tumours, originated from mammospheres, we demonstrate that some of the intratumour vessels were of human origin, suggesting an in vivo endothelial differentiation of mammosphere-derived cells. Finally, endothelial cell clones originated from mammospheres were able, when implanted in Matrigel in SCID mice, to form after 7 days a human vessel network and, after 3–4 weeks, an epithelial tumour suggesting that in the endothelial-differentiated cells a tumourigenic stem cell population is maintained. In conclusion, the results of the present study demonstrate that stem cells of breast cancer have the ability to differentiate not only in epithelial but also in endothelial lineage, further supporting the hypothesis that the tumour-initiating population possesses stem cell characteristics relevant for tumour growth and vascularization.     

Investigations on the usefulness of CEACAMs as potential imaging targets for molecular imaging purposes

Members of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) family are the prototype of tumour markers. Classically they are used as serum markers, however, CEACAMs could serve as targets for molecular imaging as well.In order to test the anti CEACAM monoclonal antibody T84.1 for imaging purposes, CEACAM expression was analysed using this antibody. Twelve human cancer cell lines from different entities were screened for their CEACAM expression using qPCR, Western Blot and FACS analysis. In addition, CEACAM expression was analyzed in primary tumour xenografts of these cells. Nine of 12 tumour cell lines expressed CEACAM mRNA and protein when grown in vitro. Pancreatic and colon cancer cell lines showed the highest expression levels with good correlation of mRNA and protein level. However, when grown in vivo, the CEACAM expression was generally downregulated except for the melanoma cell lines. As the CEACAM expression showed pronounced expression in FemX-1 primary tumours, this model system was used for further experiments. As the accessibility of the antibody after i.v. application is critical for its use in molecular imaging, the binding of the T84.1 monoclonal antibody was assessed after i.v. injection into SCID mice harbouring a FemX-1 primary tumour. When applied i.v., the CEACAM specific T84.1 antibody bound to tumour cells in the vicinity of blood vessels. This binding pattern was particularly pronounced in the periphery of the tumour xenograft, however, some antibody binding was also observed in the central areas of the tumour around blood vessels. Still, a general penetration of the tumour by i.v. application of the anti CEACAM antibody could not be achieved despite homogenous CEACAM expression of all melanoma cells when analysed in tissue sections. This lack of penetration is probably due to the increased interstitial fluid pressure in tumours caused by the absence of functional lymphatic vessels.     

Growth and vascular structure of human melanoma xenografts

The growth and the vascular structure of five human melanomas grown in athymic nude mice were studied. Four growth parameters (tumour volume doubling time, fraction of cells in S-phase, growth fraction, cell-loss factor) were analysed against each of four vascular parameters (length of vessels with diameters in the range 5-15 micron, total vessel length, total vessel surface, total vessel volume--all per unit of histologically intact tumour volume). Statistically significant linear correlations between the parameters were found for any of the combinations. However, there was a consistent trend in the data: the tumour volume doubling time and the cell-loss factor tended to decrease while the fraction of cells in S-phase and the growth fraction tended to increase with increasing vascular density, whichever vascular parameter was considered. This finding indicates that the vascular density is among the factors which are decisive for the growth rate of tumours. However, the present work does not exclude the possibility that intrinsic properties of the tumour cells may also be important.     

Growth and Vascular Structure of Human Melanoma Xenografts

Abstract The growth and the vascular structure of five human melanomas grown in athymic nude mice were studied. Four growth parameters (tumour volume doubling time, fraction of cells in S-phase, growth fraction, cell-loss factor) were analysed against each of four vascular parameters (length of vessels with diameters in the range 5–15 μm, total vessel length, total vessel surface, total vessel volume-all per unit of histologically intact tumour volume). Statistically significant linear correlations between the parameters were found for any of the combinations. However, there was a consistent trend in the data: the tumour volume doubling time and the cell-loss factor tended to decrease while the fraction of cells in S-phase and the growth fraction tended to increase with increasing vascular density, whichever vascular parameter was considered. This finding indicates that the vascular density is among the factors which are decisive for the growth rate of tumours. However, the present work does not exclude the possibility that intrinsic properties of the tumour cells may also be important.     

Evidence for high activity of xylem parenchyma and ray cells in the interface of host stem and Agrobacterium tumefaciens-induced tumours of Ricinus communis

Rapidly developing tumours at hypocotyls of Ricinus communis, induced by Agrobacterium tumefaciens strain C58, were characterized by strong differentiation of vascular bundles and their functional connection to the host bundles. The stem/tumour interface showed increased xylem, with numerous vessels accompanied by multiseriate unlignified rays. To know how nutrients efficiently accumulate in the tumour sink tissue, cell electropotentials (E(m)) in cross-sections were mapped. The measured cells were identified by injected Lucifer Yellow. Xylem and phloem parenchyma cells and stem/tumour-located rays hyperpolarized to E(m) values of about -170 mV, which suggest high plasma membrane proton pump activities. Rapidly dividing cells of cambia or small tumour parenchyma cells had low E(m). The tumour aerenchyma and the stem cortex cells displayed values close to the energy-independent diffusion potential. The lowest values were recorded in stem pith cells. Cell K(+) concentrations largely matched the respective E(m). The pattern of individual cell electropotentials was supplemented by whole organ voltage measurements. The voltage differences between the tumour surface and the xylem perfusion solution in stems attached to the tumours, the trans-tumour electropotentials (TTP), confirm the findings of respiration-dependent and phytohormone-stimulated high plasma membrane proton pump activity in intact tumours, mainly in the xylem and phloem parenchyma and ray cells. TTPs were inhibited by addition of NaN(3), CN(-) plus SHAM or N(2) gas in the xylem perfusion solution and by external N(2) flushing. The data provide functional evidence for the structural basis of priority over the host shoot in nutrient flow from the stem to the tumour.     

Metastases and the Normalization of Tumour Blood Vessels by ICRF 159: A New Type of Drug Action

Profound modification of the structure and arrangement of the blood vessels has been shown in tumours after treatment with ICRF 159. X-ray angiography, carbon black (Pelikan ink) labelling, and intravital staining with lissamine green were used to demonstrate the changes. Alteration of the morphology of the blood vessels at the edge of a tumour may affect the escape of malignant cells and the rate of blood flow (and thus the concentration of anticancer drugs) through the tumour.     

In vivo labelling with halogenated pyrimidines of squamous cell carcinomas and adjacent non-involved mucosa of head and neck region

The frequency and distribution of labelled cells were studied immunohistochemically in 37 squamous cell carcinomas (SCC) of head and neck after in vivo infusion of IdUrd and BrdUrd. Tumours were classified according to their labelling patterns. Low and moderate grade SCC consisted of tumour islands separated by interstitial tissue. In some tumours labelled cells only appeared near the basal layer while in others proliferative cells were evenly distributed within the neoplastic island. In anaplastic carcinomas labelled cells were distributed either randomly or around blood vessels (cord structures). While the basal layer in adjacent normal epithelium contained very few labelled cells (LI = 1.6 ± 0.2%), the LI of basal cells in tumour islands were much higher than the average LI of the tumour (47.2 ± 2.8% and 23.8 ± 1.6%, respectively). In patients who had received cytotoxic therapy up to two months before the biopsy, the LI in the basal layer of normal epithelium was 19.0 ± 3.5%. In sequential biopsies obtained 1–2 weeks after the infusion of IdUrd and BrdUrd some labelled tumour cells were found in necrotic foci and in pearl structures. Additionally, in six tumours, we found areas of cells labelled with IdUrd alone, even though the IdUrd infusion had been followed by a BrdUrd infusion 1 h later. This is in agreement with the phenomenon of intermittent tumour blood flow described earlier in experimental tumours.     

Fine-needle aspiration of renal angiomyolipoma: a report of four cases

OBJECTIVE: Renal angiomyolipoma is an uncommon benign tumour composed of smooth muscle cells, blood vessels and adipose tissue. The cytological findings of this tumour are described. METHODS: A retrospective analysis of four cases of angiomyolipoma diagnosed on fine-needle aspiration cytology (FNAC) during the period 1998-2004 was carried out. All the aspirations were carried out under ultrasonographic image guidance. RESULTS: Smears from three cases showed oval- to spindle-shaped tumour cells, cohesive stromal fragments embedded in adipose tissue and branching blood vessels in a haemorrhagic background. No mitotic figures were seen. Smears from one case showed adipose tissue and blood. In this case, sections from the cell block showed mature adipose tissue and small blood vessels. CONCLUSION: The diagnosis of angiomyolipoma can be made by FNAC under image guidance and a cell block may be quite helpful in making a correct diagnosis. It is important to establish a correct preoperative diagnosis as treatment of these tumours is conservative and this obviates the need for total nephrectomy.     

Expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in pituitary tumours

The present study was performed to investigate HIF-1alpha (hypoxia-inducible factor-1alpha) expression in a large number of immunohistochemically and ultrastructurally characterized surgically removed pituitary tumours. The potential relation of HIF-1alpha with outcome variables as well as the presence of HIF-1alpha expression in the tumours treated with dopamine agonists and octreotide, a long-acting somatostatin analogue was also investigated. HIF-1alpha immunoreactivity was confined to the nucleoplasm whereas the nucleoli were unconspicuous. The distribution of HIF-1alpha was evident in the tumours whereas normal adenohypophysial cells showed no HIF-1alpha staining. HIF-1alpha expression was detected not only in the tumour cells but also in endothelial cells lining the blood vessels within the tumour. ACTH producing adenomas showed the lowest level of HIF-1alpha expression whereas pituitary carcinomas and GH producing adenomas had the highest counts. The statistical study demonstrated no significant correlation between HIF-1alpha expression, patient age, gender, tumour, size, invasiveness, cell proliferation rate and vascularity. These results suggest that the behaviour of pituitary tumours does not primarily depend of HIF-1alpha expression. Our study demonstrated an increase HIF-1alpha expression in bromocriptine treated PRL producing pituitary adenomas compared with untreated tumours but no increase in octreotide treated tumours.