Ivar Amund Grimstad

Direct evidence that cancer cell locomotion contributes importantly to invasion

This study was undertaken to clarify whether active locomotion of cancer cells is important for their ability to invade. The most rapidly moving cells were isolated from a cultured murine parent fibrosarcoma by successive cycles of migration through a micropore membrane. Cells were isolated by unstimulated locomotion and by haptotaxis to laminin, and the selected cells did indeed constitute rapidly locomoting subpopulations. These cells invaded biological tissues more efficiently than did the unselected parent cells. The cells selected by haptotaxis to laminin invaded most rapidly through amnion with basement membranes (containing laminin). Cancer cell haptotaxis to laminin in basement membranes thus promotes penetration of these tissue barriers. These results show in a direct manner that cancer cell locomotion is in fact important in invasion of biological tissues.

Contribution of α-d-galactopyranosyl end groups to attachment of highly and low metastatic murine fibrosarcoma cells to various substrates☆

There are much greater numbers of cell surface terminal, non-reducing alpha-D-galactorpyranosyl groups in highly malignant (metastatic) cells than are found in low malignant cells derived from the same murine fibrosarcoma. We have examined the contribution of these residues to attachment of the cells to various collagens and to plastic. Removal of these carbohydrate groups with alpha-galactosidase or blocking them with lectins from Griffonia simplicifolia seeds or with anti-blood group B antiserum all dramatically inhibited the attachment of both the highly malignant and the low malignant cells. Following removal with the enzyme, the alpha-D-galactopyranosyl end groups were rapidly resynthesized. This resynthesis was inhibited by tunicamycin, an inhibitor of de novo glycoprotein synthesis. This antibiotic also impaired cell attachment and, when used in addition to treatment with alpha-galactosidase, it inhibited cell attachment more than did treatment with the enzyme alone. The effects of all treatments on cell attachment were greater for the highly malignant than for the low malignant cells. With the latter cells, inhibition by lectin was seen only in the absence of serum, whereas the adhesion of highly malignant cells was affected in both the presence and the absence of serum. On their surface membrane the highly malignant cells express much more than do the low malignant cells of a glycoprotein that cross-reacts immunologically with laminin. The basement membrane glycoprotein laminin promotes cell attachment to collagen, and both glycoproteins contain terminal, non-reducing alpha-D-galactopyranosyl groups. Attachment of cells is a requirement for the formation of a metastasis, and thus the laminin-like molecule and the alpha-D-galactopyranosyl end groups (whether on the laminin-related moiety or on other cell surface molecules) may both be important for expression of the most malignant phenotype.

Attachment, spreading and growth in vitro of highly malignant and low malignant murine fibrosarcoma cells

Highly malignant cell lines and low-malignant cell lines isolated from three different methylcholanthrene-induced murine fibrosarcomas were examined for their ability to attach to plastic dishes and collagen-coated dishes under serumfree conditions and in the presence of serum. Most of the cells from the three highly malignant lines attached and spread under all conditions. By 72h, there was a significant increase in the number of cells indicating that at least some of the cells had undergone division (even in the absence of serum). In contrast, fewer of the cells from the three low-malignant lines attached and spread on the plastic or collagen substrates in the absence of serum or in the presence of 0.1 per cent serum. However, when 15μg laminin per dish was added along with the lowmalignant cells, they then attached and spread on the plastic and collagen-coated dishes. Previous studies have indicated that the highly malignant lines express cell surface antigens that cross-react with laminin while the low-malignant cell lines do not. We speculate that the differences between the high- and low-malignant cells in the expression of cell surface laminin-like antigens contribute to the dissimilarities in attachment and spreading capacity. These differences may also contribute to the dissimilarity between these cells in malignant potential.

A new assay for leukocyte chemotaxis using cell retrieval, electronic particle counting and flow cytometry

A new micropore membrane assay for leukocyte migration has been devised. It permits the complete retrieval in monodisperse suspension of functionally intact cells that have traversed the membrane, thus allowing the application of precise, automated techniques, including flow cytometry and electronic particle counting. Hemocytometers may also be used. Direct comparison with 2 different conventional membrane methods showed that the new method performed superiorly. It was also much more economical with regard to time and labor. This technique permitted detection of functional differences between leukocytes isolated from blood in different ways. Data on the duration of concentration gradients in chemotaxis chambers are also presented.

Growth and metastasis of hypermotile, hyperinvasive cancer cells selectedin vitro by rapid locomotion under various conditions

Cancer cells selected from a cultured murine fibrosarcoma by rapid migration through micropore membranes moved considerably faster through such membranes and invaded biological tissues much more efficiently than did the unselected parent cells [5, 8]. The present data show that populations of cells selected by unstimulated migration or by haptotaxis to laminin moved not only faster, but also in larger numbers than the parent cells. However, the selected cells were far less efficient than the parent cells in forming spontaneous lung metastases in syngeneic mice, although all cell lines were 100 per cent tumorigenic. Analysis of paired data within each group showed no relationship between the primary tumor size at any observation time and the number of lung metastases finally formed. Therefore, although the parent cell line produced primary tumors growing slightly more rapidly than did the various lines of hypermotile cells, this was probably not the main cause of the difference in spontaneous metastasis formation between the groups. Lung colonization experiments performed by intravenous injection of cells could not explain the spontaneous metastasis results.In vitro, the cells selected by rapid haptotaxis to laminin grew considerably better than the other cells in 0.1 per cent fetal bovine serum, but there were no, or only minor, differences in higher serum concentrations. Combined, these results indicate that small subpopulations of cells selected by extreme efficiency in one step of the metastasis process may be so specialized that they perform poorly in other steps. Therefore, the results do not disprove the concept that tumor cell migration plays an important part in metastasis.

Growth patterns of pulmonary metastases and primary tumours from five murine fibrosarcoma cell clones

The growth patterns, including the size, shape and regional preferences, of lung metastases from five murine fibrosarcoma cell clones were studied. Spontaneous metastases developed from tumours formed by subcutaneous inoculation of the cell clones. Lung colonies (experimental metastases) were established by i.v. injection of cells. The numbers of both spontaneously and experimentally formed subpleural lung metastases were counted through a stereomicroscope. The fraction of colonies that was located subpleurally was determined in histological sections of lungs. The growth kinetics of clonally derived primary tumours, and the number of spontaneous and experimental lung metastases, differed greatly between certain cell clones. The number of spontaneous lung metastases was correlated with the maximum size of primary tumours. No close correlation was observed between the size of the primary tumours and the size of experimental metastases. There were differences between the cell clones in the shape and regional preferences of their lung deposits. The subpleural colonies were generally larger than the intrapulmonary ones. Thus, both the regional distribution and the growth pattern of lung deposits differed between the clones.