Passage Of Tumor Cells Research Articles

Oleandrin enhances radiotherapy sensitivity in lung cancer by inhibiting the ATM/ATR-mediated DNA damage response.

Despite active clinical trials on the use of Oleandrin alone or in combination with other drugs for the treatment of solid tumors, the potential synergistic effect of Oleandrin with radiotherapy remains unknown. This study reveals a new mechanism by which Oleandrin targets ATM and ATR kinase-mediated radiosensitization in lung cancer. Various assays, including clonogenic, Comet, immunofluorescence staining, apoptosis and Cell cycle assays, were conducted to evaluate the impact of oleandrin on radiation-induced double-strand break repair and cell cycle distribution. Western blot analysis was utilized to investigate alterations in signal transduction pathways related to double-strand break repair. The efficacy and toxicity of the combined therapy were assessed in a preclinical xenotransplantation model. Functionally, Oleandrin weakens the DNA damage repair ability and enhances the radiation sensitivity of lung cells. Mechanistically, Oleandrin inhibits ATM and ATR kinase activities, blocking the transmission of ATM-CHK2 and ATR-CHK1 cell cycle checkpoint signaling axes. This accelerates the passage of tumor cells through the G2 phase after radiotherapy, substantially facilitating the rapid entry of large numbers of inadequately repaired cells into mitosis and ultimately triggering mitotic catastrophe. The combined treatment of Oleandrin and radiotherapy demonstrated superior inhibition of tumor proliferation compared to either treatment alone. Our findings highlight Oleandrin as a novel and effective inhibitor of ATM and ATR kinase, offering new possibilities for the development of clinical radiosensitizing adjuvants.

Heparanase: Historical Aspects and Future Perspectives.

Heparanase is an endo-β-glucuronidase that cleaves at a limited number of internal sites the glycosaminoglycan heparan sulfate (HS). Heparanase enzymatic activity was first reported in 1975 and by 1983 evidence was beginning to emerge that the enzyme was a facilitator of tumor metastasis by cleaving HS chains present in blood vessel basement membranes and, thereby, aiding the passage of tumor cells through blood vessel walls. Due to a range of technical difficulties, it took another 16years before heparanase was cloned and characterized in 1999 and a further 14years before the crystal structure of the enzyme was solved. Despite these substantial deficiencies, there was steady progress in our understanding of heparanase long before the enzyme was fully characterized. For example, it was found as early as 1984 that activated T cells upregulate heparanase expression, like metastatic tumor cells, and the enzyme aids the entry of T cells and other leukocytes into inflammatory sites. Furthermore, it was discovered in 1989 that heparanase releases pre-existing growth factors and cytokines associated with HS in the extracellular matrix (ECM), the liberated growth factors/cytokines enhancing angiogenesis and wound healing. There were also the first hints that heparanase may have functions other than enzymatic activity, in 1995 it being reported that under certain conditions the enzyme could act as a cell adhesion molecule. Also, in the same year PI-88 (Muparfostat), the first heparanase inhibitor to reach and successfully complete a Phase III clinical trial was patented.Nevertheless, the cloning of heparanase (also known as heparanase-1) in 1999 gave the field an enormous boost and some surprises. The biggest surprise was that there is only one heparanase encoding gene in the mammalian genome, despite earlier research, based on substrate specificity, suggesting that there are at least three different heparanases. This surprising conclusion has remained unchanged for the last 20years. It also became evident that heparanase is a family 79 glycoside hydrolase that is initially produced as a pro-enzyme that needs to be processed by proteases to form an enzymatically active heterodimer. A related molecule, heparanase-2, was also discovered that is enzymatically inactive but, remarkably, recently has been shown to inhibit heparanase-1 activity as well as acting as a tumor suppressor that counteracts many of the pro-tumor properties of heparanase-1.The early claim that heparanase plays a key role in tumor metastasis, angiogenesis and inflammation has been confirmed by many studies over the last 20years. In fact, heparanase expression is enhanced in all major cancer types, namely carcinomas, sarcomas, and hematological malignancies, and correlates with increased metastasis and poor prognosis. Also, there is mounting evidence that heparanase plays a central role in the induction of inflammation-associated cancers. The enzymatic activity of heparanase has also emerged in unexpected situations, such as in the spread of HS-binding viruses and in Type-1 diabetes where the destruction of intracellular HS in pancreatic insulin-producing beta cells precipitates diabetes. But the most extraordinary recent discoveries have been with the realization that heparanase can exert a range of biological activities that are independent of its enzymatic function, most notably activation of several signaling pathways and being a transcription factor that controls methylation of histone tails. Collectively, these data indicate that heparanase is a truly multifunctional protein that has the additional property of cleaving HS chains and releasing from ECM and cell surfaces hundreds of HS-binding proteins with a plethora of functional consequences. Clearly, there are many unique features of this intriguing molecule that still remain to be explored and are highlighted in this Chapter.

Intramedullary Spinal Cord Metastasis from Renal Cell Carcinoma: A Systematic Review of the Literature

Intramedullary spinal cord metastases from renal cell carcinomas (RCCs) are rare and can cause serious diagnostic and therapeutic dilemmas. The related reports are very few. This review was aimed to perform an analysis of all reported cases with intramedullary spinal cord metastases from RCCs. In January 2018, we performed a literature search in PubMed database using a combination of the keywords “intramedullary spinal cord metastasis” and “renal cell carcinoma”. In addition, we present the clinical, neuroradiological, and histopathological findings in our patient with an intramedullary metastasis from a RCC. 17 cases were generated in our research. The mean interval from diagnosis of RCC to diagnosis of ISCM was 22 months. The median survival of surgically treated patients was 8.6 months and 8 months in patients who underwent radical surgery. Based on our review, RCCs can invade the medulla of the spinal cord several years after removal of the primary lesion. The prognosis of ISCMs from RCCs was poor. Retrograde passage of tumor cells into the spinal cord from the inferior vena cava via the epidural venous sinuses may have been the pathological mechanism for ISCM in our patient. Radical resection and radiation are effective ways of achieving recovery of neurologic function and improving quality of life. More reports are needed to enable exploration of the mechanisms of metastasis and the optimal forms of therapy.

Development and analysis of patient-derived xenograft mouse models in intravascular large B-cell lymphoma.

Intravascular large B-cell lymphoma (IVLBCL) is a distinct disease entity with the peculiar characteristic that tumor cells proliferate within vessels. Despite recent advances in understanding the disease from clinical aspects, the underlying pathogenesis remains unknown. Here we demonstrate analyses of IVLBCL biology using four xenograft mouse models established from primary IVLBCL samples. In all four models, the main characteristic of IVLBCL tumor cell proliferation within vessels was retained. Time-lapse engraftment analyses revealed that the tumor cells initially engrafted and proliferated in the sinusoids and vessels in the liver and then engrafted and proliferated in multiple organs. Intriguingly, serial passage of tumor cells from the adrenal gland of a transplanted mouse developed from primary patient bone marrow cells into a second mouse showed that the tumor cells mainly distributed into the adrenal gland in the second mouse, implying the existence of clonal selection and/or evolution at engraftment of a specific organ. Gene expression profiling analyses demonstrated that the gene set associated with cell migration was enriched for normal peripheral blood B cells, indicating that inhibition of cell migration might be involved in IVLBCL pathogenesis. In conclusion, the mouse xenograft models described here are essential tools for uncovering IVLBCL biology.

Abstract A04: Endothelial FAK activity controls vascular permeability and tumor metastasis

Abstract Tumor spread remains a primary cause of cancer mortality. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase co-activated by integrin and growth factor receptors in the control of vascular permeability. As FAK inhibitors are being tested in clinical trials, it is important to determine whether FAK signaling plays distinct roles in tumor versus stromal cell biology. Herein, we elucidate a new mechanism for endothelial cell (EC) FAK activity in the regulation of blood vessel barrier integrity and the passage of tumor cells through an EC barrier. We show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is phosphorylated by FAK in tumor-associated ECs. Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC Y658 phosphorylation. Both FAK inhibition and VEC Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. Although it is known that Src tyrosine kinase activity is important in controlling VEC tyrosine phosphorylation downstream of VEGF, we find that pharmacological Src inhibition prevents FAK activation. Moreover, FAK inhibition prevents both Src and FAK translocation to EC adherens junctions, but does not alter Src activation. Together, these results identify EC FAK as a key intermediate between Src and the regulation of EC barrier function controlling tumor metastasis. Citation Format: Christine Jean, Xiao Lei Chen, Isabelle Tancioni, Sean Uryu, Christine Lawson, Nichol Miller, Patric Turowski, Elisabetta Dejana, Sara Weis, David Cheresh, David D. Schlaepfer. Endothelial FAK activity controls vascular permeability and tumor metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A04. doi:10.1158/1538-7445.CHTME14-A04

Radical cystectomy, hysterectomy with double adnexectomy and bilateral nephroureterectomy with transvaginal extraction

ObjectivesThe onset of synchronous urothelial carcinoma in the upper or lower urinary tract is uncommon. Even more uncommon is the onset the bilateral form. The aim of this article is to describe the surgical technique of complete laparoscopic exeresis of the urinary apparatus and to add several variants of the technique that improve the patient's hemodynamics during surgery. Material and methodsWe present the technique of cystectomy with bilateral nephroureterectomy, hysterectomy with double adnexectomy and bilateral ilio-obturator lymphadenectomy by laparoscopy and transvaginal extraction of specimens from a 58-year-old patient with multiple prior vesical resections of high-grade urothelial carcinoma. The patient currently presents bladder recurrence and bilateral ureteropelvic tumor. The technique consists first of the hysterectomy and double adnexectomy along with the lymphadenectomy and cystectomy, maintaining the urethrovesical, ureterovesical and uterovaginal junctions. After changing the patient's position, both nephroureterectomies were performed. Lastly, we completed the resection of the previously mentioned segments to extract the specimens transvaginally. ResultsThe histological result was high-grade urothelial carcinoma that affected the bladder and both ureteropelvic junctions, along with endometrial carcinoma. After reviewing the literature, we found less than 10 cases in which complete exeresis of the urinary apparatus was performed and none with the technical description presented in this article. In most cases described in the literature, surgery was performed at two separate times and without preserving renal function until the end of the complete exeresis. ConclusionThis technique helps maintain diuresis for a longer time during surgery and thereby facilitates the work of the anesthesiologist and improves the patient's circulatory dynamics. Additionally, the technique prevents any type of handling of the urinary tract, thereby avoiding the passage of tumor cells to the peritoneal cavity, given that the specimens are extracted whole through the vagina.

Cistectomía radical, histerectomía con doble anexectomía y nefroureterectomía bilateral con extracción por vía transvaginal. Descripción y consideraciones específicas de la técnica quirúrgica

ObjectivesThe onset of synchronous urothelial carcinoma in the upper or lower urinary tract is uncommon. Even more uncommon is the onset the bilateral form. The aim of this article is to describe the surgical technique of complete laparoscopic exeresis of the urinary apparatus and to add several variants of the technique that improve the patient's hemodynamics during surgery. Material and methodsWe present the technique of cystectomy with bilateral nephroureterectomy, hysterectomy with double adnexectomy and bilateral ilio-obturator lymphadenectomy by laparoscopy and transvaginal extraction of specimens from a 58-year-old patient with multiple prior vesical resections of high-grade urothelial carcinoma. The patient currently presents bladder recurrence and bilateral ureteropelvic tumor. The technique consists first of the hysterectomy and double adnexectomy along with the lymphadenectomy and cystectomy, maintaining the urethrovesical, ureterovesical and uterovaginal junctions. After changing the patient's position, both nephroureterectomies were performed. Lastly, we completed the resection of the previously mentioned segments to extract the specimens transvaginally. ResultsThe histological result was high-grade urothelial carcinoma that affected the bladder and both ureteropelvic junctions, along with endometrial carcinoma. After reviewing the literature, we found less than 10 cases in which complete exeresis of the urinary apparatus was performed and none with the technical description presented in this article. In most cases described in the literature, surgery was performed at 2 separate times and without preserving renal function until the end of the complete exeresis. ConclusionThis technique helps maintain diuresis for a longer time during surgery and thereby facilitates the work of the anesthesiologist and improves the patient's circulatory dynamics. Additionally, the technique prevents any type of handling of the urinary tract, thereby avoiding the passage of tumor cells to the peritoneal cavity, given that the specimens are extracted whole through the vagina.

Absence of the Basement Membrane Component Nidogen 2, but Not of Nidogen 1, Results in Increased Lung Metastasis in Mice

Nidogen 1 and 2 are ubiquitous basement membrane (BM) components. They show a divergent expression pattern in certain adult tissues with a prominent localization of nidogen 2 in blood vessel BMs. Deletion of either nidogen 1 or 2 in mice had no effect on BM formation, suggesting complementary functions. However, studies in these mice revealed isoform-specific functions with nidogen 1-deficient mice showing neurological abnormalities and wound-healing defects not seen in the absence of nidogen 2. To investigate this further nidogen 1- or 2-deficient mice were intravenously injected with B16 murine melanoma cells, and lung metastasis was analyzed. The authors could show that loss of nidogen 2, but not of nidogen 1, significantly promotes lung metastasis of melanoma cells. Histological and ultrastructural analysis of nidogen 1- and 2-deficient lungs did not reveal differences in morphology and ultrastructure of BMs, including vessel BMs. Furthermore, deposition and distribution of the major BM components were indistinguishable between the two mouse strains. Taken together, these results suggest that absence of nidogen 2 might result in subtle changes of endothelial BMs in the lung, which would allow faster passage of tumor cells through these BMs, leading to a higher metastasis rate and more larger tumors.

Morphologic Changes in the Tumor and Liver in Mice with Transplanted RLS40 Lymphosarcoma during Increase of Its Drug Resistance

The progress of drug resistance of RLS(40) resistant lymphosarcoma and specific features of toxic lesions in the liver in polychemotherapy were studied. After intramuscular injection of tumor cells, the mice received a course of polychemotherapy. The tumor material was then collected and transplanted to intact animals, after which polychemotherapy was carried out. A total of 4 passages of tumor cells and 4 polychemotherapy courses were carried out. The expression of mdr1b, bcl-2, and p53 genes in tumor cells increased by 1.3, 2.3, and 1.6 times after 4 courses of polychemotherapy in comparison with intact tumor. Volume density of apoptoses in tumor tissue after 4 polychemotherapy courses decreased 1.7 times compared to that after single course. The increase in cytostatic load was associated with aggravation of destructive changes in the liver: the volume density of necroses in the liver increased 1.3 times after 4 passages of the tumor.

Epithelial-to-Mesenchymal Transition of Murine Liver Tumor Cells Promotes Invasion†,‡

Epithelial-to-mesenchymal transition (EMT) is predicted to play a critical role in metastatic disease in hepatocellular carcinoma. In this study, we used a novel murine model of EMT to elucidate a mechanism of tumor progression and metastasis. A total of 2 x 10(6) liver cells isolated from Pten(loxp/loxp)/Alb-Cre(+) mice, expanded from a single CD133(+)CD45(-) cell clone, passage 0 (P0), were sequentially transplanted to obtain two passages of tumor cells, P1 and P2. Cells were analyzed for gene expression using microarray and real-time polymerase chain reaction. Functional analysis included cell proliferation, migration, and invasion in vitro and orthotopic tumor metastasis assays in vivo. Although P0, P1, and P2 each formed tumors consistent with mixed liver epithelium, within the P2 cells, two distinct cell types were clearly visible: cells with epithelial morphology similar to P0 cells and cells with fibroblastoid morphology. These P2 mesenchymal cells demonstrated increased locomotion on wound healing; increased cell invasion on Matrigel basement membrane; increased EMT-associated gene expression of Snail1, Zeb1, and Zeb2; and down-regulated E-cadherin. P2 mesenchymal cells demonstrated significantly faster tumor growth in vivo compared with P2 epithelial counterparts, with invasion of intestine, pancreas, spleen, and lymph nodes. Furthermore, P2 mesenchymal cells secreted high levels of hepatocyte growth factor (HGF), which we propose acts in a paracrine fashion to drive epithelial cells to undergo EMT. In addition, a second murine liver cancer stem cell line with methionine adenosyltransferase 1a deficiency acquired EMT after sequential transplantations, indicating that EMT was not restricted to Pten-deleted tumors. EMT is associated with a high rate of liver tumor proliferation, invasion, and metastasis in vivo, which is driven by HGF secreted from mesenchymal tumor cells in a feed-forward mechanism.

Asymptomatic isolated coccygeal metastasis in low rectal carcinoma

As well as being relatively rare, osseous metastases from colorectal cancer are frequently asymptomatic and represent a late manifestation of disease. We report a case of an unidentified, asymptomatic coccygeal metastasis discovered on histological processing of the resection specimen from a patient with locally advanced rectal cancer treated with neoadjuvant chemoradiotherapy followed by radical abdomino-perineal resection with coccygectomy. The anatomical explanation for this finding may involve passage of tumour cells via the vertebral venous plexus.

TGFβ Primes Breast Tumors for Lung Metastasis Seeding through Angiopoietin-like 4

Cells released from primary tumors seed metastases to specific organs by a nonrandom process, implying the involvement of biologically selective mechanisms. Based on clinical, functional, and molecular evidence, we show that the cytokine TGFbeta in the breast tumor microenvironment primes cancer cells for metastasis to the lungs. Central to this process is the induction of angiopoietin-like 4 (ANGPTL4) by TGFbeta via the Smad signaling pathway. TGFbeta induction of Angptl4 in cancer cells that are about to enter the circulation enhances their subsequent retention in the lungs, but not in the bone. Tumor cell-derived Angptl4 disrupts vascular endothelial cell-cell junctions, increases the permeability of lung capillaries, and facilitates the trans-endothelial passage of tumor cells. These results suggest a mechanism for metastasis whereby a cytokine in the primary tumor microenvironment induces the expression of another cytokine in departing tumor cells, empowering these cells to disrupt lung capillary walls and seed pulmonary metastases.

Imaging microvascular structure with contrast enhanced MRI

Over the past 10 years there has been increasing interest in the development of novel MRI techniques that enable researchers and clinicians to study the development and structure of vasculature in pathological tissues. Although the majority of this work has been performed in studies of human tumours the techniques are potentially widely applicable in other pathological states [1–4]. All tissues depend on their vasculature for an adequate supply of nutrients and removal of waste metabolic materials. As tissues develop, an adequate and appropriately structured vascular supply must develop at the same time. This process, known as angiogenesis, is also an essential component of the behaviour of many pathological tissues including tumours and inflammatory disease [1, 5]. More importantly, the angiogenic process appears to be largely independent of the developing tissue. This has given rise to the concept of antiangiogenic therapies which could be effective in a wide range of tumours independent of the tissue type. The angiogenic process is complex and can be stimulated by any one of several mechanisms. Typically, growth of tissue which has outstripped its local blood supply results in regional hypoxia and hypoglycaemia which stimulates the release of local chemical messengers from the cells of the tissue itself. The best known of these messengers is the cytokine, vascular endothelial growth factor (VEGF) [6–9]. VEGF is a common and potent angiogenic stimulator, which is found in many pathological tissues. It is released in response to local hypoglycaemia and/or hypoxia and has several effects each of which will improve metabolic supply. In the short term VEGF will act directly on local capillaries to increase endothelial permeability resulting in an immediate increase in the supply of nutrients [10]. This increase in permeability is also believed to form an important part of the metastatic mechanism allowing passage of tumour cells into the circulation. In the medium to long term, VEGF will stimulate mitosis in endothelial cells from local blood vessels so that they divide and develop a new vascular infrastructure to supply the tumour. The angiogenic mechanism also is responsible for breakdown of local connective tissues, which allows in-growth of new blood vessels. Where the angiogenic process fails tissue development cannot occur and novel antiangiogenic therapies are currently being developed which use this mechanism for the treatment of a wide range of cancers and other pathologies [11, 12]. The increasing understanding of the role of the angiogenic process in disease progression has led to interest in methods for documenting the presence and activity of angiogenesis [12–14]. In particular there has been considerable interest in the development of reliable quantitative methods which can provide independent indicators of the status of, and changes in, microvascular structure [13, 14]. In pathological tissues the angiogenic process is often abnormal, leading to the development of distorted vascular beds characterized by an excessive proportion of blood vessels and blood vessels with abnormal morphology and flow characteristics [15]. Central areas of a rapidly growing tumour will commonly exhibit inadequate blood flow due to reduce local perfusion pressure resulting from a combination of inadequate vascularization and increased interstitial tumour pressure. Finally, the angiogenic neovasculature will exhibit increase endothelial permeability to medium and large sized molecules [16, 17]. Initial studies of microvascular structure in tumours were undertaken using histological techniques, however these are essentially unsatisfactory for a number of reasons [18, 19]. Pathological assessment relies on the acquisition of tissue samples which is clearly invasive and which can be repeated only infrequently. Furthermore, many tumours and other pathological tissues demonstrate considerable heterogeneity in microvascular structure so that isolated regional biopsies may give misleading information. These limitations have led to the development of imaging based methods for quantification of microvascular structure. These methods are commonly based on dynamic contrast enhanced imaging techniques using MRI or CT data collection combined with analytic algorithms to calculate descriptive parameters related to microvascular structure. The availability of quantitative imaging based methods to describe the microvasculature in biological tissues is potentially of considerable importance. Such methods can provide important surrogate markers of therapeutic response in trials of novel antiangiogenic therapy and the majority of applications to date have been in this area. However, these techniques can also be seen to have directly clinical relevance providing information about diagnosis, tumour grade, therapeutic response, tumour recurrence and prognosis. At the present time the majority of technical development of these techniques has been centred around the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). This article will describe the development and range of DCE-MRI techniques and the range of image analysis approaches available. The article aims to provide insight into the benefits and disadvantages of specific approaches rather than to detail the actual analysis mechanisms in detail.

Regulation of Endothelial CD44 Expression and Endothelium–Tumour Cell Interactions by Hepatocyte Growth Factor/Scatter Factor

Cancer metastasis involves the passage of tumour cells into and out of blood or lymphatic circulatory systems and requires their interaction with the endothelial cells lining these vessels. Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional protein that enhances tumour cell motility and extracellular matrix invasion and has been implicated as a mediator of metastasis. In this study, we have investigated the effect of HGF/SF on tumour cell–endothelial cell interactions. A fluorescent tumour cell–endothelial cell attachment assay demonstrated that, following endothelial monolayer stimulation with HGF/SF, tumour cell attachment to endothelium is increased. Addition of anti-CD44 antibodies in this assay inhibited the effects of HGF/SF. Western blotting studies showed that HGF/SF increased expression of the adhesion molecule CD44 in endothelial cells. These results were confirmed by both immunohistochemical staining and a cell-surface adhesion molecule ELISA. These results suggest that HGF/SF plays a key role in the initial adhesion mechanism between tumour cells and endothelial cells via up-regulation of CD44.

A basement-membrane permeability assay which correlates with the metastatic potential of tumour cells.

We describe an in vitro assay for measuring the ability of tumour cells to permeabilize basement membranes, using transwell chambers coated with the reconstituted basement membrane, matrigel. Unlike previous matrigel-based procedures which quantified passage of tumour cells across a matrigel barrier, the new assay measures the ability of tumour cells to degrade the basement membrane and increase the diffusion rate of fluorescent (FL) dextran through the barrier. The procedure has the major advantage that permeability can be rapidly and accurately quantified, either by fluorometry or by the use of radiolabelled dextran, thus avoiding tedious and subjective scoring methods. Optimal conditions for the assay are described. In addition, it is demonstrated that the assay can clearly discriminate between metastatic and non-metastatic tumour cell lines, metastatic tumours permeabilizing the basement membrane and non-metastatic counterparts failing to do so. A range of enzyme inhibitors suggested that the increase in basement-membrane permeability caused by the metastatic mammary adenocarcinoma 13762 MAT is probably dependent upon the synergistic action of several degradative enzymes, namely proteases, type-IV collagenase, and heparanase. Furthermore, the ability to permeabilize the basement membrane was dependent upon intact tumour cells; tumour cell extracts, lysates and supernatants were inactive.

Provirus insertion in Tpl-1, an Ets-1-related oncogene, is associated with tumor progression in Moloney murine leukemia virus-induced rat thymic lymphomas.

T-cell lymphomas induced in rats by Moloney murine leukemia virus acquire increasing numbers of proviruses in their genome during tumor progression in vivo and passage of tumor cells in vitro. To determine whether the proviruses progressively acquired during tumor progression play a causal role in this process, we cloned one of them from a cell line derived from the primary tumor 2772. A probe from the cellular DNA flanking the provirus was used to analyze 79 DNA samples from primary tumor tissues of 28 tumor-bearing rats and 80 DNA samples from 30 independent tumor cell lines. This analysis revealed a rearrangement in this region in the primary tumor derived from the thymus of one animal but not in a clone of the same tumor segregating in the spleen. Of the cell line DNA samples, three carried a provirus in this region. Two of these integration events had occurred independently in two clonally related sublines derived from tumor 2772, and they were followed by rapid selection in culture. On the basis of these findings this locus was named Tpl-1 (tumor progression locus 1). The Tpl-1 locus was mapped to rat chromosome 8 and to mouse chromosome 9 at a genetic distance of 1.2 +/- 0.9 centimorgans from the Ets-1 protooncogene. Although the genetic distance between Tpl-1 and Ets-1 indicates that they are different genes, analysis of Tpl-1 cDNA clones revealed that the two are closely related.

Mechanism of liver-specific metastatic tumor spread in a murine tumor model.

Malignant tumors frequently show an organ-specific metastatic spread, the causes of which are still largely unknown. Using an experimental tumor model, a methylcholanthrene-induced pleomorphic myofibrosarcoma ER 15-P of the C57Bl6J mouse, we wanted to find out whether this phenomenon is due to an adaptation or to a selection of tumor cells. After i.v. injection of tumor cells from the primary ER 15-P into the tail vein of male mice, metastases were regularly found in the lungs, mediastinal lymph nodes, and brain, as well as in the liver and kidneys, and occasionally in the adrenals. The following experimental procedures were used to isolate a tumor cell line with a possible liver preference: (1) Tumor cells from the primary ER 15-P were injected into a mesenteric vein of male mice. Tumor cells from the resulting liver colonies were again injected into the portal system of one group of mice. In a second group, part of the same cell suspension was injected into the tail vein. This procedure was performed four times. (2) Tumor cells from the primary ER 15-P were applied into the tail vein of male mice. Tumor cells from the resulting liver metastases were reinjected directly into the tail vein. This experiment was repeated three times. (3) Tumor cells from the primary ER 15-P were injected into the tail vein of male mice. Tumor cells from liver metastases were then injected, first, into the portal system of one group of male mice, and thereafter into the tail vein of another group of animals. This experiment was repeated twice. The following results were obtained: (1) By a repeated adaptation of tumor cells from the primary ER 15-P to liver tissue, no tumor cell line could be isolated that would show a preferential metastatic spread to this organ after tail-vein injection. (2) Repeated i.v. passages of tumor cells from liver metastases into the tail vein led to the selection of a tumor cell line with a tendency to liver metastasis. (3) Tumor cells selected from liver metastases induced via tail-vein injection showed, after a prolonged stay in the liver and a successive i.v. passage into the tail vein, a marked specificity for this organ. These results indicate that the liver-specific spread of tumor cells in our model is based on the selection of a tumor cell line from the primary ER 15-P influenced by the hepatic microenvironment.

Factors regulating basement membrane invasion by tumor cells.

Basement membranes serve as significant barriers to the passage of tumor cells but ones which metastatic cells can pass. This involves the production of a cascade of proteases leading to the activation of a specific collagenase that degrades the unique collagen network in basement membrane. Breast cancer cells, when estrogen dependent, show a requirement for estrogen for invasive activity. However, when these cells progress to an estrogen independent state and increased malignancy, they express an invasive phenotype constitutively. Studies with various anti-estrogens suggest that these responses are mediated via the estrogen receptor. Anti-estrogens lacking agonist activity suppress invasiveness as well as growth of the breast cancer cells.

Evidence that sulphated polysaccharides inhibit tumour metastasis by blocking tumour-cell-derived heparanases.

Recent studies in this laboratory demonstrated that several sulphated polysaccharides can inhibit metastasis of the rat mammary adenocarcinoma 13762 MAT, probably by preventing the passage of tumour cells through the walls of blood vessels. In order to directly test this possibility, 13762 MAT cells were cultured with (35S)O4(=)-labelled subendothelial extracellular matrices (ECM) and ECM degradation was monitored in either the presence or absence of different sulphated polysaccharides. Degradation products were detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent autoradiography. The 5 sulphated polysaccharides that had previously been shown to possess anti-metastatic activity were potent inhibitors of the degradation of subendothelial ECM by 13762 MAT cells. In contrast, of the 4 polysaccharides tested that failed to inhibit metastasis, 3 had no effect on ECM breakdown and one (carrageenan-kappa) was substantially less effective at inhibiting ECM degradation than the anti-metastatic preparations. It was also shown that 13762 MAT cells produce a heparan sulphate-specific glycosidase (heparanase) that degrades the heparan sulphate side-chains of the ECM, the action of this enzyme rather than that of other ECM-solubilizing enzymes being inhibited by the antimetastatic sulphated polysaccharides. Additional experiments indicated that the anti-coagulant activity of the polysaccharides probably plays a minor role in their anti-metastatic effects since heparin, almost completely depleted (98-99.5%) of heparin molecules with anti-coagulant activity by passage over an anti-thrombin III column, retained its ability to inhibit 13762 MAT heparanases and was almost as effective as unfractionated heparin at inhibiting tumour-cell metastasis. Collectively, these data suggest that sulphated polysaccharides inhibit the metastasis of 13762 MAT cells by inhibiting tumour-cell-derived heparanases involved in the penetration of the vascular endothelium and its underlying basement membrane by tumour cells.

Use of a reconstituted basement membrane to measure cell invasiveness and select for highly invasive tumor cells.

Malignant cells must traverse basement membranes during their migration to sites distant from the primary tumor. Since basement membranes are thought to be a critical barrier to the passage of tumor cells, we have constructed a model basement membrane-stromal matrix consisting of laminin and type IV collagen reconstituted onto a disk of type I collagen for use in an in vitro assay of invasiveness. Metastatic tumor cells and leukocytes are able to cross this barrier, whereas nonmetastatic tumor cells, fibroblasts, and epidermal cells cannot penetrate it. Those tumor cells that penetrate the barriers were found, when isolated and subcultured, to be more invasive and to produce more metastases than the parental population. This assay system should be useful for studying the invasiveness of tumor cells and for isolating highly invasive variants.