Transition from the noninvasive to the invasive phenotype and loss of alpha-catenin in human colon cancer cells.

Loss of epithelioid organization in carcinoma cell lines has been related to invasiveness and poor differentiation of tumors. We investigated the invasion in vitro of various human colon cancer cell lines. Most cell lines were noninvasive into chick heart fragments, and this correlated with an epithelioid morphotype. Only cell lines COLO320DM, SW620, and variants of HCT-8 and DLD-1 were invasive and nonepithelioid. We examined in these cell lines whether invasiveness was related to changes in the structure and function of the E-cadherin/catenin complex. E-cadherin functions as an invasion suppressor and as a cell-cell adhesion molecule when linked to the cytoskeleton via alpha-catenin plus beta- or gamma-catenin. All noninvasive cell lines showed E-cadherin linked to these catenins. The E-cadherin-dependent cell-cell adhesion function in these cell lines was demonstrated by two assays in vitro. It was interesting that all invasive cell lines showed a dysfunctional E-cadherin/catenin complex. COLO320DM, SW480, and SW620 cells were defective in E-cadherin expression, whereas the invasive variants of HCT-8 and DLD-1 lacked the alpha-catenin protein. From clonal epithelioid HCT-8 cultures with functional E-cadherin/catenin complexes, we subcloned, repeatedly, round cell variants that were again invasive and expressed no alpha-catenin protein. Our data suggest that reproducible transformations toward a more invasive phenotype in HCT-8 cells are associated with down-regulation of alpha-catenin. The mechanisms of this transformation and the level of alpha-catenin down-regulation are currently investigated.

In vivo and in vitro invasion in relation to phenotypic characteristics of human colorectal carcinoma cells.

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour.

Micro-encapsulation of MDCK-ras-e cells prevents loss of E-cadherin invasion-suppressor function in vivo.

The invasion-suppressor molecule E-cadherin mediates Ca(2+)-dependent cell aggregation and prevents invasion. E-cadherin-positive Madin-Darby canine kidney (MDCK) cells that were non-invasive in vitro formed, upon i.p. injection, tumors that were invasive. Differentiated tubular tumor areas showed an intense immuno-signal for E-cadherin at intercellular contacts, whereas undifferentiated structures did not. Cell lines derived from such tumors turned out to be invasive in vitro and showed decreased Ca(2+)-dependent cell aggregation but no change in E-cadherin immunopositivity. This combination of phenotypes indicated a loss of the E-cadherin invasion-suppressor function. Micro-encapsulation of i.p.-injected cells prevented the loss of the E-cadherin invasion-suppressor function. We concluded that this loss in vivo was dependent upon immediate contacts between tumor cells and host cells or upon host factors that could not cross the capsule membrane.

Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro.

The calcium-dependent cell-cell adhesion molecule E-cadherin has been shown to counteract invasion of epithelial neoplastic cells. Using three monoclonal antibodies, we have demonstrated the presence of E-cadherin at the surface of human MCF-7/6 mammary carcinoma cells by indirect immunofluorescence coupled to flow cytometry and by immunocytochemistry. Nevertheless, MCF-7/6 cells failed to aggregate in a medium containing 1.25 mM CaCl2, and they were invasive after confrontation with embryonic chick heart fragments in organ culture. Treatment of MCF-7/6 cells with 0.5 microgram ml-1 insulin-like growth factor I (IGF-I) led to homotypic aggregation within 5 to 10 min and inhibited invasion in vitro during at least 8 days. The effect of IGF-I on cellular aggregation was insensitive to cycloheximide. However, monoclonal antibodies that interfered with the function of either the IGF-I receptor (alpha IR3) or E-cadherin (HECD-1, MB2) blocked the effect of IGF-I on aggregation. The effects of IGF-I on aggregation and on invasion could be mimicked by 1 microgram ml-1 insulin, but not by 0.5 microgram ml-1 IGF-II. The insulin effects were presumably not mediated by the IGF-I receptor, since they could not be blocked by an antibody against this receptor (alpha IR3). Our results indicate that IGF-I activates the invasion suppressor role of E-cadherin in MCF-7/6 cells.

Metastatic T-cell hybridoma cells display target preference for invasion ('homing') in tissue culture models.

BW-O-Li1 murine T-lymphoma cells display target preference for invasion in two different in vitro models. In the precultured chick heart fragment (PHF) assay, BW-O-Li1 preferentially invaded into aggregates of MCF-7 mammary carcinoma cells rather than into PHFs. In a monolayer invasion assay (MIA), BW-O-Li1 cells preferentially invaded under 10 T1/2 mouse embryo cell monolayers rather than under MCF-7 monolayers. Thus, although BW-O-Li1 cells were perfectly able to invade into any of the targets presented, they migrated and accumulated preferentially in one of the targets when a choice was offered. We suggest that this in vitro 'homing' phenomenon can be exploited to investigate certain aspects of organ-specific metastasis.

Down-regulation of E-cadherin expression in Madin Darby canine kidney (MDCK) cells inside tumors of nude mice.

The 120-kDa cell-cell adhesion molecule E-cadherin is localized at the epithelial junctional complex and participates in the organization and maintenance of epithelia. The Madin Darby canine kidney (MDCK) cell line expresses E-cadherin in a stable way and forms polarized epitheloid structures in vitro. Harvey-murine-sarcoma-virus-transformed derivatives (MDCK-ras) produce malignant (i.e., invasive and metastatic) tumors in nude mice. We obtained evidence that E-cadherin is down-regulated in nude mouse tumors and that this down-regulation is reversible. MDCK-ras-e cell lines were cloned in vitro from MDCK-ras cell cultures. They showed an epithelioid morphotype and expressed E-cadherin at homogeneously high level. This characteristic has been conserved for at least 60 passages in vitro. MDCK-ras-e cells were not invasive in vitro. When injected into nude mice, however, they produced invasive and metastatic tumors. Primary tumors as well as large metastases were heterogeneous, showing E-cadherin-positive well differentiated epithelial structures and E-cadherin-negative undifferentiated areas. Metastasis-derived cell cultures contained both E-cadherin-positive and E-cadherin-negative MDCK-ras-e cells during early passages in vitro. During further culture, however, they regained the homogeneous E-cadherin-positive characteristic of the original MDCK-ras-e cell line. The behavior of MDCK-ras-e cells in vitro, as compared with its in vivo behavior, points to the existence of host factors which are able to down-regulate E-cadherin expression. We hypothesize that this down-regulation plays a basic role in invasion.

Influence of basement membrane molecules on directional migration of human breast cell lines in vitro.

Spheroidal cell aggregates were prepared from four tumorigenic human breast cell lines (HBL-100 and three MCF-7 variants). Cells from these aggregates were allowed to migrate towards lanes of basement membrane components coated on a glass substratum. Matrigel (reconstituted basement membrane) lanes permanently arrested the migration of one MCF-7 cell line, while migration of the others was permitted. Amongst several purified basement membrane constituents only laminin, not collagen type IV or fibronectin, was found to cause the same arrest of migration. Within the laminin molecule only the pepsin P1, not the elastase E8 fragment, efficiently arrested migration of that cell line. Although migration was inhibited by these components, time-lapse video recordings revealed that arrested cells still proliferated and actively ruffled on top of the coatings. These data suggest that, amongst several basement membrane components, laminin can function as a stop signal for cell migration. Within laminin, this activity seems to be mainly associated with the P1 fragment. We conclude that laminin is the major determinant of the barrier-function of the basement membrane, to which some cell types have become insensitive.

The flavonoid tangeretin inhibits invasion of MO4 mouse cells into embryonic chick heart in vitro.

Tangeretin, a flavonoid from citrus plants, was found to inhibit the invasion of MO4 cells (Kirsten murine sarcoma virus transformed fetal mouse cells) into embryonic chick heart fragments in vitro. The flavonoid appeared to be chemically stable in tissue culture medium, and the anti-invasive effect was reversible on omission of the molecule from the medium. Unlike (+)-catechin, another anti-invasive flavonoid, tangeretin bound poorly to extracellular matrix. It did not alter fucosylated surface glycopeptides of MO4 cells. Tangeretin seemed not to act as a microtubule inhibitor, as immunocytochemistry revealed no disturbance of the cytoplasmic microtubule complex. However, at anti-invasive concentrations of tangeretin, cell proliferation and thymidine incorporation appeared to be inhibited. When cultured on an artificial substrate, treated MO4 cells were less elongated, covered a larger surface area and exhibited a slower directional migration than untreated cells. From the decrease in ATP content in MO4 cells after tangeretin treatment, we deduce that this flavonoid inhibits a number of intracellular processes, which leads to an inhibition of cell motility and hence of invasion.

Antiinvasive activity of estramustine on malignant MO4 mouse cells and on DU-145 human prostate carcinoma cells in vitro.

Estramustine (EM) is a conjugate of estradiol and nor-nitrogen mustard (nor-HN2), which is effective in the treatment of prostate cancer. We have compared the effect of EM with that of the known microtubule inhibitor vinblastine (VLB) on the following functions of malignant MO4 mouse cells and of DU-145 human prostate cancer cells in vitro: directional migration, invasion; and the organization and the assembly/disassembly equilibrium of microtubule complexes. The circular area covered by cells migrating from an aggregate explanted on a solid substrate was taken as an index of directional migration. Invasion was studied through confrontation of MO4 or DU-145 cells with fragments of embryonic chick heart in organ culture. Microtubules were investigated immunocytochemically and through immunodetection on protein blots. VLB and EM inhibited directional migration and invasion of MO4 and DU-145 cells in a dose-dependent manner; equimolar combinations of estradiol plus nor-nitrogen mustard did not mimic these effects. At anti-invasive concentrations VLB led to partial disassembly of microtubule complexes, whereas EM resulted in an abnormal pattern of microtubule complexes without alteration of the overall assembly/disassembly equilibrium. Combined treatment with VLB and EM resulted in an enhanced VLB effect, namely complete disassembly. In all tests DU-145 cells were more sensitive to both VLB and EM than were MO4 cells, and the effects were less reversible. The present experiments showed that EM shares an anti-invasive activity with other microtubule inhibitors.

Invasiveness in vitro of mixed aggregates composed of two human mammary cell lines MCF-7 and HBL-100.

Interactions between invasive and noninvasive cells were studied via confronting cultures between mixed cell aggregates and embryonic chick heart fragments in vitro. The mixed aggregates were composed of MCF-7 and HBL-100 cells, both derived from human mammary epithelium. HBL-100 cells invade into the heart fragments when confronted as unmixed aggregates, while MCF-7 cells do not. Using mixed aggregates HBL-100 cells still invade into the heart tissue, but MCF-7 cells sort out to the periphery of the cultures and do not invade. Two mechanisms concerning the noninvasiveness of MCF-7 cells in vitro are discussed: the homotypic adhesion of the MCF-7 cell population due to the presence of numerous desmosomes, and the incapability of MCF-7 cells to migrate on extracellular laminin present in the embryonic chick heart and in the HBL-100 cell population.

Confrontation of an invasive (MO4) and a noninvasive (MDCK) cell line with embryonic chick heart fragments in serum-free culture media.

Confronting cultures of precultured embryonic chick heart fragments (PHF) with aggregates of malignant cells in vitro have been shown to be relevant for a number of aspects of tumor invasion in vivo. Preculture of the heart fragments, formation of cell aggregates, and subsequent culture of confronting pairs have so far been done only in serum-containing culture media. We describe here confronting cultures of PHF with invasive MO4 mouse cell aggregates or noninvasive MDCK dog kidney cell aggregates in serum-free media. Heart fragments precultured in the absence of serum seemed to be necrotic after confronting culture in serum-free media. However, preculturing in media supplemented with 10% fetal bovine serum allowed us to do subsequent confronting cultures in absence of serum. Cell aggregates were also prepared in serum-containing medium. MO4 cells occupied and replaced the heart tissue within 4 d, whereas MDCK cells remained at the periphery of the PHF. This indicates that serum-free confronting cultures can discriminate between invasive and noninvasive cells. The viability of individual PHF and cell aggregates cultured in the same way as in confrontations was ascertained by histology and by explantation and postculturing on a solid tissue culture substrate. Growth of the cultures was smaller in serum-free media than in media supplemented with 10% fetal bovine serum. The main advantage of serum-free culture conditions in vitro is the elimination of the influence of serum components on invasion, and the ability to examine the effect on invasion of drugs that are susceptible to inactivation by serum.

Numerical evaluation of changes in the cytoplasmic microtubule complex of C3H mouse cells by optical diffractometry and of changes in cell shape by Fourier analysis.

MO mouse cells in culture on glass were treated with taxol, or nocodazole, or incubated at 4 degrees C to alter their cytoplasmic microtubule complex (CMTC). From each treated group and from an untreated group, 30 cells stained with an antiserum against tubulin, were photographed under the photomicroscope, and negatives were analysed by optical diffractometry. Differences between groups of cells were tested by variance analysis. Phase-contrast micrographs of the same cells were used for Fourier analysis of cell shape. Both types of analyses provided numerical objective data about changes in the CMTC and in cell shape that were typical for the kind of treatment. We conclude that optical diffractometry of immunostained cells and Fourier analysis of cell shape are complementary to photomicroscopy for the study of the CMTC in cell populations cultured on an artificial substrate.

An image analysis system for the quantification of invasion in vitro.

We have tested the value of a computer-assisted image analysis program for the quantitative study of invasion in vitro using experiments that were previously described semi-quantitatively. Mouse fibrosarcoma cell (MO4) aggregates were confronted with precultured fragments of embryonic chick heart in organ culture. Confronting pairs were fixed after 1, 2, 3 and 4 days, and processed for paraffin sectioning and immunostaining with an antiserum against chick heart. The image analysis system allowed separate quantification of two aspects of invasion, namely, occupation of the heart tissue by MO4 cells and degeneration of the invaded heart tissue. Complex combination of occupation and invasion added a qualitative aspect of invasion that has not been described previously and that revealed qualitative differences in invasion under various circumstances.

Antiinvasive effect of racemic 1-O-octadecyl-2-O-methylglycero-3-phosphocholine on MO4 mouse fibrosarcoma cells in vitro.

Alkyl-lysophospholipids have been shown to possess antitumoral activity in animal and in human tumors. Among them, racemic 1-O-octadecyl-2-O-methylglycero-3-phosphocholine (ET-18-OCH3) had an antimetastatic effect in experimental tumors. We investigated the effect of ET-18-OCH3 on invasion of MO4 mouse fibrosarcoma cells and on cellular activities possibly related to invasion in vitro. Ten micrograms of ET-18-OCH3 per ml permitted growth of MO4 cells to about 75% of controls and slightly reduced trypan blue exclusion. Directional migration inferred from the area covered by MO4 cells that had migrated from an aggregate on glass was not affected. Reassembly of microtubules after treatment with 1 microgram of Nocodazole per ml occurred normally in presence of ET-18-OCH3. Invasion was completely inhibited when MO4 cell aggregates were confronted with precultured fragments of embryonic chick cardiac muscle or with fresh embryonic chick lung fragments in culture on gyratory shaker in fluid medium with 10 micrograms of ET-18-OCH3 per ml. These experiments showed that ET-18-OCH3, in contrast with microtubule inhibitors, interfered with invasion of MO4 cells in vitro at concentrations that permitted growth and directional migration of MO4 cells. Fluorescence polarization studies with the lipophylic probe diphenylhexatriene indicated that the antiinvasive effect of ET-18-OCH3 was accompanied by an overall increase of membrane fluidity. We tentatively concluded that alterations of the MO4 cellular membranes are responsible for the antiinvasive effect of ET-18-OCH3.

Effect of temperature on invasion of MO4 mouse fibrosarcoma cells in organ culture.

Invasion by MO4 mouse fibrosarcoma cells into fragments of embryonic chick heart or lung in organ culture was studied histologically and ultrastructurally at various temperatures between 12 and 40 degrees C. Invasion was absent for at least 7 days at or below temperatures of 29 degrees C. Invasion was invariably observed at or above 30.5 degrees C. Differences in invasion between 29 and 30.5 degrees C could not be ascribed to differences in growth, migration, or microtubule assembly/disassembly of MO4 cells. Neither could they be explained through differences in the attachment of MO4 cells to the heart fragments. Possible explanations for the absence of invasion at lower temperature are: altered resistance of the extracellular matrix in heart or lung fragments, and deficient expression of fucosylated glycoproteins at the surface of MO4 cells. A population of MO4 cells plated from the parent line and adapted to grow at 28 degrees C (MO(4)28 cell line) did not differ in invasiveness from the parent MO4 cells. We conclude that the temperature dependence of invasion in organ culture might indicate as yet unexplored aspects of the mechanisms of tumour invasion.

Cytoplasmic microtubules of normal and tumor cells of the leopard frog. Temperature effects.

The cytoplasmic microtubule complex (CMTC) was examined in monolayer cultures of normal tadpole mesonephros, primary renal adenocarcinoma, and an established cell line derived from a pronephric renal adenocarcinoma (PNKT-4B) of the leopard frog, Rana pipiens. Immunocytochemistry revealed typical arrays of microtubules extending from the cytocentrum to the cell periphery in all three cell types when cultured at 28 degrees C; similar results were obtained at 20 degrees C. However, the CMTC was disorganized in both tumor types, in contrast to the retention of a typical CMTC in normal tissue cultured at 7 degrees C. The response of PNKT-4B cells differed from that of normal tadpole mesonephros when treated with the microtubule inhibitor drug nocodazole. At 28 degrees C, PNKT-4B and tadpole mesonephros cells lost their CMTC with nocodazole treatment, and both were able to reconstitute CMTC when nocodazole was removed. Similarly, both lost CMTC organization with nocodazole and culture at 70 degrees C. However, while normal cells could effect a recovery at 7 degrees C after the removal of nocodazole, PONKT-4B cells were unable to restructure CMTC under the same conditions. Metastasis in the frog renal adenocarcinoma is temperature-dependent, with an elevated prevalence of metastasis in tumor-bearing frogs maintained at 28 degrees C. Few metastatic colonies are detected in tumor-bearing frogs maintained at a low temperature (7 degrees C). Other studies have indicated that microtubules, which are essential for cell motility, play an important role in the invasion by tumor cells of normal tissue fragments in vitro. The effects of temperature on metastasis of the Lucke renal adenocarcinoma are consistent with temperature-mediated changes in tumor-cell CMTC.

Glycosylated podophyllotoxin congeners: loss of microtubule inhibition and permission of invasion in vitro.

Podophyllotoxin (PPT) and its glycosylated congeners, 4'-demethylepipodophyllotoxin 9-[4,6-O-(R)-ethylidene-beta-D-glucopyranoside] (VP-16-213) and 4'-demethylepipodophyllotoxin 9-[4,6-O-(R)-thenylidene-beta-D-glucopyranoside] (VM-26), were studied for their effects on the following activities of MO4 mouse fibrosarcoma cells in vitro: growth as an aggregate in culture on a gyrotory shaker, directional migration from an aggregate explanted on glass, organization of the cytoplasmic microtubule complex as inferred from immunostaining with an antiserum against tubulin, and invasion into fragments of 9-day-old embryonic chick cardiac muscle. At concentrations that inhibited growth (greater than or equal to 0.03 microgram/ml), PPT arrested directional migration, abolished the cytoplasmic microtubule complex, and interfered with invasion. VM-26 (0.1-1.0 microgram/ml) and VP-16-213 (1-30 micrograms/ml) interfered with growth but permitted directional migration, organization of the cytoplasmic microtubule complex, and invasion. These observations imply that microtubule inhibitors are anti-invasive because they interfere with the assembly of the cytoplasmic microtubule complex and not because they inhibit growth.

Vinblastine, Vincristine and Vindesine: anti-invasive effect on MO4 mouse fibrosarcoma cells in vitro.

Inhibition of the invasiveness of MO4 mouse fibrosarcoma cells by the vinca alkaloid, vinblastine (VLB), vincristine (VCR) and vindesine (VDS), has been examined in vitro. At doses between 0.006 microgram/ml (minimal effect) and 0.1 microgram/ml (complete inhibition) these drugs interfered with the invasion of MO4 cells from an aggregate confronting a fragment of embryonic chick heart in three-dimensional culture. We have also examined the effect of these drugs on the following activities of MO4 cells: growth, directional migration and assembly of the cytoplasmic microtubule complex. Growth and directional migration were affected by the same doses of vinca alkaloids as invasion. In contrast with the vinca alkaloids, 5-fluorouracil at 1 microgram/ml inhibited growth but allowed directional migration and invasion. At a dose of 0.3 microgram/ml VLB, VCR and VDS interfered with the assembly of cytoplasmic microtubules, as visible after immunocytochemical staining with tubulin antiserum. Ultrastructural analysis demonstrated that inhibition of invasion in three-dimensional culture corresponds with abolishment of the cytoplasmic microtubule complex. Anti-invasive concentrations of VLB, VCR and VDS represent clinically achievable plasma concentrations. We concluded that the anti-invasive effect of the vinca alkaloids may contribute to their antitumor activity.

Immunohistochemical study of embryonic chick heart invaded by malignant cells in three-dimensional culture.

MO4 mouse fibrosarcoma cells, B16 mouse melanoma cells, HeLa human cervical carcinoma cells, and LICR (LOND)-HN-4 human laryngeal carcinoma cells were shown to invade into precultured fragments of 9-day-old chick cardiac muscle in three-dimensional culture. Paraffin sections from such cultures were stained with an antiserum against chick heart following the unlabeled antibody enzyme method. The immunostaining demonstrated (1) differences in the rate and the way by which the four types of malignant cells occupied and replaced the chick cardiac muscle, and (2) the phagocytic activity of the four types of invading cells in three-dimensional culture.

Phagocytic capacity of invasive malignant cells in three-dimensional culture.

To study the phagocytic capacity of invasive malignant cells, fragments of the hypoblast from chick blastoderms were confronted in three-dimensional culture with spheroidal aggregates of 1) malignant virally transformed C3H mouse cells (MO4), 2) HeLa cells and 3) embryonic chick heart cells. The hypoblast was used because it contains yolk, a marker that is absent in the confronting cells and that can be identified histologically and ultrastructurally. The confronting tissues were incubated on semi-solid agar-agar medium or in fluid medium on a gyrotory shaker. Cultures were followed for 1 to 7 days by stereomicroscopy, cinemicrophotography, light and transmission electron microscopy. Confrontation with MO4 cells of HeLa cells, known to be invasive in vitro, led to complete disappearance of the hypoblast. The fragments of hypoblast were well conserved when cultured alone or confronted with aggregates of chick heart cells. Degeneration of the hypoblast is shown at the area of contact with MO4-cell or HeLa-cell aggregates, in contrast to heart cells. Filopodia-like extensions from the MO4 or HeLa cells penetrate intercellularly, transcellularly and intracellularly into the hypoblast. Phagosomes, containing yolk and unidentified debris are observed in MO4 cells and in HeLa cells, but not in heart cells. These observations demonstrate the phagocytic capacity of invasive malignant cells.