Tumor Cell Penetration Research Articles

A human xenograft model for testing early events of epithelial neoplastic invasion

We report on a model of human prostate tumor cell invasion using the SCID (severe combined immunodeficient) mouse diaphragm. Tumor cells were injected into SCID mice intraperitoneally and the diaphragms harvested three to five weeks later. Electron microscopy showed tumor cell penetration of the mesothelial cell layer and adhesion to the underlying basement membrane on the inferior surface of the mouse diaphragm, where colonies developed. Immunohistochemistry showed invasion by tumor cells through the basement membrane into the muscle of the diaphragm, presence of human tumor cells among the muscle cells and the presence of selected proteins on the invasion front of the tumor cells. Digital image analysis enabled quantitative comparison of events in the metastatic cascade by variants of the tumor cell line and evaluation of the effectiveness of a putative tumor inhibitor. Results suggest that the SCID mouse diaphragm model is a convenient, effective, easily oriented and reproducible in vivo model of the early events associated with human prostate tumor cell invasion.

Identification of arachidonic acid pathways required for the invasive and metastatic activity of malignant tumor cells

Metastasis is a complex process, almost a cascade, involving multiple steps and activities. However, an important factor is that malignant cells are able to penetrate through the multiple basement membrane barriers surrounding tissues, blood vessels, nerves and muscle that would otherwise block their dissemination. Penetration of malignant tumor cells through basement membrane is an active process requiring proteolysis. We report here that inhibitors of both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism convert mouse melanoma and human fibrosarcoma cells to a non invasive state by reducing the production of MMP-2, an enzyme required for the degradation of basement membranes. Specific metabolites of each pathway, i.e. PGF 2α and 5-HPETE, are able to transcend the block and restore collagenase production, invasiveness in vitro and metastatic activity in vivo. These studies indicate a key role for arachidonic acid metabolites in metastasis and suggest novel therapeutic approaches for inhibiting the spread of cancer.

The conjugation of RGDS peptide with CM-chitin augments the peptide-mediated inhibition of tumor metastasis

A water soluble 6- O-carboxymethyl chitin (CM-chitin) containing cell adhesive Arg-Gly-Asp-Ser (RGDS) sequence, i.e. CM-chitin-RGDS conjugate was synthesized, and the inhibitory effects of this compound on lung or liver metastasis of lung-metastatic B16-BL6 melanoma or liver-metastatic L5178Y-ML25 lymphoma cells in mice was examined. CM-chitin-RGDS showed the inhibitory effects on lung metastasis of melanoma cells in a dose-dependent manner (ranging from 100 to 1000 μg) and on liver metastasis of lymphoma cells. A mixture of CM-chitin and RGDS peptide or CM-chitin alone did not show any inhibitory effect on experimental lung metastasis as compared with the conjugate CM-chitin-RGDS on a molar basis. GRGDS peptide, however, required a higher dose (3000 μg) to obtain a sufficiently antimetastatic effect. The in-vitro tumor invasion study showed that CM-chitin-RGDS was apparently more effective for the inhibition of tumor cell penetration into reconstituted basement membrane Matrigel than RGDS or the mixture of RGDS and CM-chitin on a molar basis. Intermittent i.v. administration of CM-chitin-RGDS after the inoculation of B16-BL6 cells caused significant inhibition of spontaneous lung metastasis produced by intrafootpad injection of tumor cells as compared with the multiple administration of RGDS, CM-chitin or untreated control. These results demonstrate the importance of the conjugation of RGDS peptide with CM-chitin as a polymeric carrier for the increased therapeutic potential to cancer metastasis, thus implying a possibility that RGDS-polymer conjugation may lead to the prolongation of antimetastatic action of RGDS peptide in vivo.

Pathology of brain parenchyma in meningeal carcinomatosis; immunohistochemical study with astroprotein (GFAP) and tubulin.

Effects of leptomeningeal tumor on the brain parenchyma was studied by the immunohistochemical method with astroprotein (GFAP) and tubulin in a rat model of meningeal carcinomatosis. Thickening of subpial glial lining (external glial layer) and hypertrophy of subpial astrocytes, detected by the antiserum to GFAP, was the early sign of parenchymal involvement. The glial lining was continuous as far as the tumor cells were confined to the subarachnoid space, however, penetration of tumor cells into subpial brain was associated with disruption of the glial lining. Speculative role of this lining in preventing the tumor cell to infiltrate into brain tissue was discussed. In contrast to the prominent immunohistochemical changes in astrocytes, neuronal tubulin immunoreactivity was not altered even in the late stage of the disease. The present study demonstrated that the leptomeningeal dissemination of tumor cells did cause pathologic change in brain parenchyma as was evidenced by the reactive change of astrocytes. However, the preserved immunoreaction for tubulin suggested that the nerve cell damage was not severe even at the advanced stage of the disease.

A high-capacity semi-micro method for the assay of tumor cell penetration: [formula omitted][formula omitted], [formula omitted], [formula omitted] Div. of Oncology, Dept. of Internal Medicine of the University and Dept. of Biochemistry, Biocenter of the University ∗, CH-4031 Basel, Switzerland

A high-capacity semi-micro method for the assay of tumor cell penetration: [formula omitted][formula omitted], [formula omitted], [formula omitted] Div. of Oncology, Dept. of Internal Medicine of the University and Dept. of Biochemistry, Biocenter of the University ∗, CH-4031 Basel, Switzerland

Tumor invasion and its local regulation

Introduction The ability to invade normal tissue which ~ur­ rounds a primary tumor and to subsequently metastasize to distant sites are the distinguishing features of malignant neoplasms. I In order to invade and metastasize, tumor cells must overcome several structural barriers of host tissues. These barriers include parenchymal cells (i.e., epithelium, endothelium), basement membranes (continuous, lamrnellar extracellular matrices to which parenchymal cells are attached), and connective tissue matrices. Invasive tumors arising in epithelia (i.e., urinary bladder epithelium) first penetrate the epithelial basement membrane. Once carcinoma cells have entered and invaded the underlying connective tissue, they gain access to vascular and lymphatic channels (intravasation), Tumor cell entry into the circulation requires the penetration of vascular basement membrane and endothelium. If tumor cells survive passage within vessels, they localize at fertile sites, make their way out of the vessel (extravasation), penetrate the perivascular connective tissue, an~ proliferate to form a metastatic focus (Figure. 1).1:Tumor cell penetration of each of these barriers IS accomplished by altering the physical properties of constituent host tissues in such a way that they allow the migration of cancer cells to deeper zones and that they provide space to house continuously multiplying tumor cell populations. In contrast, the noninvasive cell populations of benign neoplasms are unable to induce such alterations, and some may even isolate themselves from host tissues by a solid connective tissue capsule. The effect of non-invasive cells on the host results only in compression and displacement of surrounding tissue, but these tumor

Interactions of tumor cells with intact capillaries: a model for intravasation.

The interactions of metastatic variants of the B16 and K-1735 melanoma cell lines with intact capillaries isolated from rabbit brain were studied in vitro. The abilities of various cells to attach to and flatten out on the surface of blood vessels were monitored with phase contrast and scanning electron microscopy. In general, cells highly metastatic in vivo were capable of attaching to and flattening out on the surface of capillaries at a faster rate than cells of low metastatic potential or normal cells. In addition, K-1735 melanoma cells and normal fibroblasts were labeled with 125I-iododeoxyuridine, incubated with capillaries, and subsequently passed through filters. This procedure separated unattached cells from cells attached to capillaries. This assay provided quantitative information on the adhesion of metastatic tumor cells and normal cells to capillaries. Tumor cells attached to capillaries in significantly greater numbers than normal cells. Cells with high metastatic potential attached to the capillaries in greater numbers than cells with low metastatic potential, but not significantly. This model may be useful for elucidating some of the mechanisms involved in tumor cell attachment and penetration of the capillary wall during intravasation in vivo.

The role of cancer cell motility in invasion.

Before discussing the role of cell motility during the process of tumor cell penetration into adjacent host tissues, it seems appropriate to briefly present what is known about cellular motility in general. In its broadest sense, motility is a distinct cellular activity resulting in morphologically recognizable changes which, in a scale of increasing participation, involve (a) the cell surface alone, (b) the cell surface plus cell shape, (c) the cell surface plus cell shape plus cell position. An example of cell motility restricted to the surface are the protrusion and retraction of cytoplasmic extensions of various configurations and sizes: villi, blebs, spikes, ridges. Surface motility is the most frequent mode of cell movement. During their lifespan, cells are constantly engaged in this form of activity which serves such purposes as endoor exocytosis among others. Coupled with morphological alterations of the surface architecture are changes of cell shape. If a cell transforms its configuration from spherical to flattened, as occurs under in vitro conditions, pseudoor filopodia extend from the surface, make contact with, and establish attachment to the substrate. The overall shape of tissue-integrated cells remains more or less unchanged, and only during mitosis a transient alteration of configuration takes place. Finally, changes of cell position, consisting in the net translocation of the whole cell, are always accompanied by alterations of surface and shape. In the usual life of most tissue cells, shifts in cell position do not occur. Special conditions, as those existing during wound healing, can induce short-range excursions of various mesenchymal and epithelial cells that otherwise would remain stabilized. Regular translocation in the adult body is only performed by the professional migrators, the leukocytes. Within the context of this chapter, remarks on positional shifts exclusively refer to active cell movements. Translocative motility is completely different from the passive transport by body fluids or by a 'trottoir roulant-mechanism' under proliferation pressure, as occurs in mucous membrane epithelia. It is therefore advisable to use different terms for locomotion and for situations allowing cells to be carried away. 'Mobility' was suggested for designating the latter condition (1). However, there are situations in which cells express mobility and motility under the same circumstances. In the blood stream for instance, leukocytes and, possibly, cancer cells are passively transported, but they can also crawl along the inner surface of the vessel wall. The transition from positional stability to translocation can be of crucial importance for various

Inhibition of the arrest of hematogenously disseminated tumor cells.

Most metastases in patients occur as a result of hematogenous dissemination of tumor cells. This process of metastasis is complex and consists of several steps, foremost of which is the arrest of circulating emboli in capillary beds and the formation of a thrombus at that site. Thrombus formation in the metastasis of human cancer was described first by Billroth in 1878. It was reported that the organization of tumor cell emboli, and the subsequent penetration of tumor cells into the capillary wall, was the first stage of metastasis. Since then, many investigations and observations have been made clinically as well as experimentally to clarify the process (or mechanisms) of tumor cell arrest and how to inhibit it. Coagulative and fibrinolytic pathways were believed to have a main role in thrombus formation. However, other factors responsible for the relationship between tumor cells and the host must be also considered. Elegant and extensive studies by Fidler and Kripke demonstrated that development of metastasis is not a random process, but a selection process of specialized subpopulations of highly metastatic cells within the primary tumors. Biochemical constituents and ionic properties on cell surfaces, deformability or locomotive activities of tumor cells, as well as thrombo-plastic-fibrinolytic activities, are also important factors determining the arrest patterns of circulating tumor cells. On the other hand, host defense factors against tumor cells in the bloodstream have been attracting much attention recently in tumor immunology. Host defense factors relating the arrest of tumor cells to the establishment of metastatic foci seemed difficult to define, since many studies showed contradictory data concerning the influence of immune response on tumor cell arrest. Hemodynamic abnormality may also influence the arrest of tumor cells in the circulation. Hypercoagulability induced from host tissues is greatly associated with the arrest patterns. Platelet activities might affect thrombus formation. Nevertheless, exact explanations of the process or mechanisms inhibiting or enhancing the arrest of tumor cells after hematogenous dissemination have not been obtained. In any event, for cancer treatment, it is important to determine which substances inhibit the arrest of circulating tumor cells and how to prevent hematogenous metastasis. In this review, we will focus upon coagulative and fibrinolytic processes and then upon substances that inhibit the arrest of circulating tumor cells. Furthermore, some comments on the possible clinical applications of inhibitory substances for prevention of cancer metastasis are added.

Extracellular matrix destruction by invasive tumor cells.

The invasion of normal tissues and penetration of basement membranes by malignant cells is likely to require the active participation of hydrolytic enzymes. The four major groups of connective tissue proteins, glycoproteins, proteoglycans, collagen and elastin, vary in their quantitative distributions between different tissues. With the exception of elastin, they also vary qualitatively within each class, so that there are no 'typical' connective tissue barriers to tumor cell penetration. The matrix constituents are stabilized and organized by a variety of covalent and noncovalent interactions between the connective tissue proteins. These interactions play important roles in matrix integrity and may alter the susceptibilities of the constituents to degradative enzymes. It is likely that the complete degradation of the matrix will require the action of more than one enzyme because of differing susceptibilities to tissue proteinases. Primary and transplantable tumors produce well-characterized enzymes which may participate in invasion. These enzymes may also be involved in connective tissue turnover in other normal and pathological situations. The use of long-term tumor cell cultures has verified that tumor cells themselves are capable of producing these enzymes. However, there are many potential modulating influences operative in vivo which are absent in culture so that details of actual mechanisms and control of digestion of complex substrates are not well understood. Recent work on the degradation by tumor cells of extracellular matrices previously produced by cultured cells is likely to shed more light on pathways of tissue destruction in vivo. Experiments with tumor cell variants of defined metastatic potentials will also be useful, but invasive and metastatic abilities are not necessarily correlated. It is unlikely that simple correlations can be drawn between the production of one particular degradative enzyme by all tumor cells and the complex biological mechanisms operative during tumor invasion.

Effect of Natural Protease Inhibitors and a Chemoattractant on Tumor Cell Invasion In Vitro

The effect of natural protease inhibitors and a chemoattractant on tumor cell invasion were studied with the use of a new in vitro quantitative assay of tumor cell penetration of native connective tissue. Human amnion membrane denuded of its epithelium is composed of a continuous basement membrane (BM) attached to a dense avascular collagenous stroma. M5076 reticulum sarcoma cells, known to be highly invasive in vivo, were placed on the BM side of the amnion connective tissue. Tumor cells penetrating the full thickness of the connective tissue barrier were collected on the stromal side with a Millipore filter. N-Formylmethionyl-leucyl-phenylalanine (FMLP) at an optimal concentration of 10(-7) M stimulated the penetration of up to 600% more tumor cells into the connective tissue after 20 hours in comparison to the number of tumor cells spontaneously penetrating in serum-free media. Natural protease inhibitors blocked both FMLP-stimulated and spontaneous invasion. A bovine cartilage extract containing inhibitors of both serine proteinases and metalloproteinases caused a 500% decrease in invasion. Furthermore, a 500% inhibition of invasion was produced by a purified collagenase (metalloproteinase) inhibitor. In contrast, soybean trypsin inhibitor and bovine serum albumin did not significantly alter the invasion rate. The protease inhibitors were nontoxic and did not reduce tumor cell proliferation, attachment to the amnion, and the rate of tumor cell migration through Nuclepore filters. These data support the hypothesis that collagenolytic metalloproteinases play a necessary role in tumor cell invasion of native connective tissue.

Role of platelets in tumor cell metastases.

Platelets may have a role in the development of animal tumor metastases. Ultrastructural studies in vivo have shown arrested tumor emboli surrounded by platelets. Several tumor cell lines induce thrombocytopenia in vivo. Certain tumor cells aggregate platelets in vitro. Correlations exist between the ability of some tumor cells to aggregate platelets in vitro and their metastatic potential in vivo. Antiplatelet agents have impaired or altered the spread of certain tumor metastases. It is suggested that platelets have a role in the sequestration, adherence, and penetration of tumor cells through the blood vessel endothelial cell barrier, thus preventing their rapid clearance from the circulation and allowing extravascular formation of nests of cells. Antiplatelet agents, particularly prostaglandins, may prove useful in preventing experimental animal metastases when administered before the inoculation of tumor cells. Their potential in human malignancy, where the patient presents with an established tumor, remains to be established.

The Action of Certain Dyestuffs on the Growth of Transplantable Tumors

It was not until 28 years after the discovery of the first coaltar dye in 1856 that the selective staining action of certain dyestuffs towards various living cells and pathogenic organisms was carefully studied. Such studies included the mechanism of the penetration of the living tumor cells by dyes which were introduced into the blood stream of the hosts. The extent of injurious action on the tumor tissue varies with different dyes, insomuch as their chemical nature and the power of membrane diffusibility are not alike. In 1911, v. Wassermann, Keysser and Wassermann (1), working with cancerous mice, found by injecting a solution of selenium-eosin into the blood stream of hosts, that the eosin would carry colloidal selenium into tumors. They reported the treatment was successful. Pentimalli (2) made a careful study of the effects of aromatic selenium compounds soon after the report of v. Wassermann and found they have no curative action. But with eosin-selenium he obtained a cure in from one to two per cent of the treated animals. On the other hand, Walker (3) has shown that the colloidal selenium, with or without eosin, possessed no destructive action upon the tumors in either rats or mice. His preparation was non-toxic, in contrast to the highly toxic preparation of v. Wassermann. Similarly Uhlenhuth, Dold and Bindseil (4), Contamin, Detoeuf and Thomas (5) and Delbert (6) reported that eosin-selenium has no curative action on experimental and human cancer.