Type I collagen inhibits differentiation and promotes a stem cell-like phenotype in human colorectal carcinoma cells.

BACKGROUND: Human colorectal cancer is caused by mutations and is thought to be maintained by a population of cancer stem cells. Further phenotypic changes occurring at the invasive edge suggest that colon cancer cells are also regulated by their microenvironment. Type I collagen, a promoter of the malignant phenotype in pancreatic carcinoma cells, is highly expressed at the invasive front of human colorectal cancer. METHODS: This study investigates the role of type I collagen in specifying the colorectal cancer cell phenotype. The effect of type I collagen on morphology, localisation of cell-cell adhesion proteins, differentiation and stem cell-like characteristics was examined in a panel of human colorectal carcinoma cell lines. RESULTS: Human colorectal carcinoma cells grown on type I collagen in serum-free medium show an epithelial-mesenchymal-like transition (EMT-like), assuming a more flattened less cohesive morphology. Type I collagen downregulates E-cadherin and beta-catenin at cell-cell junctions. Furthermore, type I collagen inhibits differentiation, increases clonogenicity and promotes expression of stem cell markers CD133 and Bmi1. Type I collagen effects were partially abrogated by a function-blocking antibody to alpha2 integrin. CONCLUSION: Together, these results indicate that type I collagen promotes expression of a stem cell-like phenotype in human colorectal cancer cells likely through alpha2beta1 integrin.

Collagen IV synthesis is restricted to the enteroendocrine pathway during multilineage differentiation of human colorectal epithelial stem cells.

The human large intestine is lined by a rapidly renewing epithelial monolayer where cell loss is precisely balanced with cell production. The continuous supply of new cells is produced by undifferentiated multipotent stem cells via a coordinated program of proliferation and differentiation yielding three epithelial lineages: absorptive, goblet and enteroendocrine. Cell-matrix interactions have been suggested to be regulators of the multilineage differentiation program of the colorectal crypt but the expression of matrix proteins or their receptors does not appear to have the subtlety expected for this task. We have developed an in vitro model system of intestinal epithelial stem cells to facilitate the direct analysis of stem cells undergoing lineage commitment and differentiation. Using this culture system, we can now directly investigate the role of cell-matrix signalling in stem-cell decisions. In this study, collagen-IV synthesis has been followed in monolayers of multipotent cells that have been induced to differentiate into absorptive, goblet and enteroendocrine cells. Our experiments demonstrate that commitment to the enteroendocrine lineage is specifically accompanied by the expression of type-IV collagen that remains enteroendocrine-cell associated. Undifferentiated cells, absorptive cells and goblet cells do not express collagen IV. To confirm that the differential lineage-specific expression of collagen IV observed in the model system was representative of the in vivo situation, collagen-IV synthesis was analysed in isolated human colorectal crypts and tissue sections using immunocytochemistry and in situ hybridisation. These studies confirmed the in vitro findings, in that implementation of the enteroendocrine differentiation program involves synthesis and accumulation of a collagen-IV matrix. Thus, human colorectal enteroendocrine cells are unique in the colorectal crypt in that they assemble a cell-associated collagen-IV-rich matrix not observed on other colorectal epithelial cells. This study provides the first evidence for differential matrix synthesis between colorectal epithelial lineages in human colorectal epithelium. The specialised pericellular environment of the enteroendocrine cells might explain some of the unique phenotypic characteristics of this cell lineage. Furthermore, these findings suggest a potential mechanism whereby individual epithelial cells could modulate their cell-matrix signalling even while rapidly migrating in heterogeneous sheets over a shared basement membrane.

Prostaglandin J2 and 15-deoxy-delta12,14-prostaglandin J2 induce proliferation of cyclooxygenase-depleted colorectal cancer cells.

Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Nonsteroidal anti-inflammatory agents (NSAIDS) inhibit growth of various CRC cell lines by both COX-dependent and COX-independent pathways. To specifically examine the effect of COX and PGs on proliferation in CRC cells, we introduced an antisense COX-2 cDNA construct under the control of a tetracycline (Tc)-inducible promoter into a CRC cell line, HCA-7, Colony 29 (HCA-7) that expresses COX and produces PGs. In the presence of Tc, PG production in COX-depleted cells was reduced 99.8% compared with either uninduced transfectants or parental HCA-7 cells. This decrease in PG production was associated with a concomitant 60% reduction in DNA replication. Subsequently, we examined the effects of various PGs to modulate cell growth in COX-depleted HCA-7 or COX-null HCT-15 cells by quantifying [3H]thymidine incorporation and/or growth in collagen gels. We report that J-series cyclopentenone PGs, particularly PGJ2 and 15-deoxy-delta12,14-PGJ2, induce proliferation of these cells at nanomolar concentrations. Lipids extracted from parental HCA-7 cell conditioned medium stimulated mitogenesis in COX-depleted HCA-7 cells and COX-null HCT-15 cells. Using chromatographic and mass spectrometric approaches, we were able to detect PGJ2 in conditioned medium from parental HCA-7 cells. Taken together, these findings implicate a role for cyclopentenone PGs in CRC cell proliferation.

Antioxidants reduce cyclooxygenase-2 expression, prostaglandin production, and proliferation in colorectal cancer cells.

Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Recent observations suggest that reactive oxygen intermediates play a role in tumor cell growth regulation and expression of the inducible COX, COX-2. We therefore evaluated the effects of various antioxidants on COX expression and cellular growth in the human CRC cell line HCA-7. The antioxidants pyrrolidinedithiocarbamate (PDTC), N-acetylcysteine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and U74006 decreased PG production, intracellular redox status, and cellular growth in a concentration-dependent manner. The decrease in cellular growth was associated with the induction of apoptosis. Unlike the selective COX inhibitors 1-[(4-methylsulfonyl)phenyl]-3-trifluoromethyl-5-[(4-fluoro)phenyl]pyraz ole (SC 58125) and (2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS 398) that inhibit COX-2 catalytic activity, these antioxidants decreased COX-2 expression at the transcriptional level. Combined treatment of HCA-7 cells with PDTC and SC 58125 resulted in an additive decrease in PG levels and anchorage-dependent and -independent growth. Furthermore, whereas antioxidants or SC 58125 reduced tumor growth in vivo, coadministration of PDTC and SC 58125 resulted in actual tumor regression. These results suggest that combined therapy with NSAIDs and antioxidants might be useful in the prevention and/or treatment of CRC.

Inhibition of growth and enhancement of differentiation of colorectal carcinoma cell lines by MAb MR6 and IL-4.

We have previously shown that expression of gp200-MR6, a molecule that is functionally associated with the interleukin-4 receptor (IL-4R), is lost from breast carcinoma cells as malignancy increases. Here we have analysed a series of colorectal carcinoma cell lines and show a similar decrease with increasing malignancy. Moreover, analysis of the HRA-19 cell line, which can exhibit a poorly or a well-differentiated phenotype according to culture conditions, shows that gp200-MR6 is weakly expressed on the former but strongly expressed on the latter. Functional analysis using either IL-4 or monoclonal antibody (MAb) MR6 and the well-differentiated cell line SW1222 revealed that MAb MR6 acts as an agonist for IL-4, with both reagents causing a dose-dependent inhibition of cell division, but greatly enhancing the glandular differentiation of SW1222 in three-dimensional collagen gels. These observations suggest that the gp200-MR6 molecule may act as the product of a tumour suppressor gene and that its loss may be a primary event in tumourigenesis.

Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2.

A considerable amount of evidence collected from several different experimental systems indicates that cyclooxygenase-2 (COX-2) may play a role in colorectal tumorigenesis. Large epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in persons taking aspirin or other nonsteroidal antiinflammatory drugs on a regular basis. One property shared by all of these drugs is their ability to inhibit COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. In this study, we selected two transformed human colon cancer cell lines for studies on the role of COX-2 in intestinal tumorigenesis. We evaluated HCA-7 cells which express high levels of COX-2 protein constitutively and HCT-116 cells which lack COX-2 protein. Treatment of nude mice implanted with HCA-7 cells with a selective COX-2 inhibitor (SC-58125), reduced tumor formation by 85-90%. SC-58125 also inhibited colony formation of cultured HCA-7 cells. Conversely, SC-58125 had no effect on HCT-116 implants in nude mice or colony formation in culture. Here we provide evidence that there may be a direct link between inhibition of intestinal cancer growth and selective inhibition of the COX-2 pathway.

Epidermal growth factor receptor activation induces nuclear targeting of cyclooxygenase-2, basolateral release of prostaglandins, and mitogenesis in polarizing colon cancer cells.

Nonsteroidal antiinflammatory drugs reduce the risk of colon cancer, possibly via cyclooxygenase (COX) inhibition. The growth factor-inducible COX-2, which is overexpressed in neoplastic colonic tissue, is an attractive target to mediate this effect. Herein we have exploited the ability of a human colon cancer cell line, HCA-7 Colony 29, to polarize when cultured on Transwell (Costar) filters to study COX-2 production and the vectorial release of prostaglandins (PGs). Administration of type alpha transforming growth factor to the basolateral compartment, in which the epidermal growth factor receptor (EGFR) resides, results in a marked induction of COX-2 immunoreactivity at the base of the cells and the unexpected appearance of COX-2 in the nucleus. The increase in COX-2 protein is associated with a dose- and time-dependent increase in PG levels in the basolateral, but not apical, medium. Amphiregulin is the most abundantly expressed EGFR ligand in these cells, and the protein is present at the basolateral surface. EGFR blockade reduces baseline COX-2 immunoreactivity, PG levels, and mitogenesis in a concentration-dependent manner. Two specific COX-2 inhibitors, SC-58125 and NS 398, also, in a dose-dependent manner, attenuate baseline and type alpha transforming growth factor-stimulated mitogenesis, although PG levels are decreased > 90% at all concentrations of inhibitor tested. These findings show that activation of the EGFR stimulates COX-2 production and its translocation to the nucleus, vectorial release of PGs, and mitogenesis in polarized HCA-7 Colony 29 cells.

Multilineage differentiation of cloned HRA-19 cells in serum-free medium: a model of human colorectal epithelial differentiation.

Colorectal epithelium is composed of polarised absorptive enterocytes, mucus-producing goblet cells and enteroendocrine cells. All these cell lineages are thought to arise from multipotential stem cells located near the base of the crypt, but the mechanisms which control differentiation and commitment of cells to a particular lineage are poorly understood. We have used the human rectal adenocarcinoma cell line, HRA-19, to investigate the regulation of expression of lineage-specific markers. HRA-19 cells have multipotential characteristics, forming absorptive, mucous and endocrine cells when grown as xenografts. However, HRA-19 cells grown in vitro in culture medium containing 10% foetal calf serum show negligible expression of the differentiated phenotypes observed in vivo. These findings initially suggested that the absence of positive stimuli from extracellular matrix, stromal cells and/or soluble factors present in vivo resulted in the lack of differentiation in vitro. The subsequent demonstration of a marked inhibitory effect of foetal calf serum on differentiation provided an alternative explanation for the differences between in vivo and in vitro differentiation. In addition, the inhibition of differentiation differed widely between batches of foetal calf serum and limited the usefulness of the system for studying the regulation of differentiation. This manuscript describes the development of chemically defined culture conditions (Dulbecco's Eagles medium supplemented with insulin, transferrin and ascorbic acid) which reproducibly induced the multilineage differentiation of HRA-19 cells into absorptive, mucous and endocrine cells. Morphological characteristics and the expression of lineage-specific markers, as determined by immunocytochemistry, identified absorptive, goblet and endocrine cells in HRA-19 monolayers grown in this serum-free medium. Differentiation of cloned HRA-19 cells in to the three cell lineages proceeds in the absence of stromal cells and without exogenous extracellular matrix, although these factors may subsequently be shown to modulate the rate of cell differentiation. These chemically defined culture conditions will facilitate the study of differentiation in the HRA-19 cell line in the absence of the complex mixture of growth factors, hormones and differentiation inhibitory factor(s) present in foetal calf serum.

Organisation and gel contraction by human colonic carcinoma (HCA-7) sublines grown in 3-dimensional collagen gel.

The role of cell-matrix interactions in controlling phenotypic heterogeneity in human colonic carcinoma sublines has been investigated. Four cell lines (colony 1, colony 3, colony 6 and colony 30) previously isolated from a single human colonic carcinoma cell line, HCA-7, were grown in 3-dimensional collagen gels. In collagen, the growth of the 4 sublines ranged from well-organised glandular structures (colony 30) to elongated branching structures (colony 3). The capability of cells to organise into glandular structures in collagen correlated with the degree of differentiation observed in their xenografts. Certain sublines, most notably colony 3, were able to contract the collagen gel. Gel contraction could be partially inhibited by a function-blocking antibody directed to the alpha 2 integrin chain but not by an antibody directed to the alpha 3 integrin chain demonstrating a role for alpha 2 integrin in the contraction process. In addition, colony 3 cultures treated with the function-blocking alpha 2 antibody formed more compact structures with limited outgrowth, suggesting a role for alpha 2 integrin in cell migration. Gel contraction and cell migration in collagen gel was largely restricted to 1 subline, colony 3. The subsequent demonstration that alpha 2 integrin is involved in both of these processes suggests that integrin expression and function has a role in generating the phenotypic heterogeneity exhibited by these cell lines.

Endocrine and mucous differentiation by a cloned human rectal adenocarcinoma cell line (HRA-19) in vitro: inhibition by TGF-beta 1.

Colorectal epithelium is composed of absorptive, mucous and endocrine cells, all of which are considered to arise from a common stem cell located in the crypt base. However, the factors controlling the commitment to differentiate are poorly understood. This is partly due to the lack of in vitro model systems for the study of differentiation in colorectal epithelium. The HRA-19 cell line, established from a human rectal adenocarcinoma, has been shown to have multipotential characteristics with cloned HRA-19 cells able to differentiate into absorptive, mucous and endocrine cells when grown as xenografts. The lack of such differentiated cells in HRA-19 monolayers in vitro suggests that differentiation is controlled by extracellular matrix, stromal cells and/or soluble factors. Such observations show that differentiation in HRA-19 cells can be controlled by extrinsic factors and therefore provide a model system for studying control of differentiation in colorectal epithelium. Unfortunately, the restriction of differentiation to xenografts of the cell line limits the degree to which this differentiation can be manipulated. In this study, the possibility that HRA-19 cells could be induced to differentiate in vitro under appropriate conditions has been investigated. Endocrine and mucous cells were identified by immunocytochemistry with differentiation-related antibodies and histology of monolayers. Preconfluent HRA-19 cells grown in 10% foetal calf serum formed a well polarised monolayer with apical tight junctions and sparse microvilli, but cells with mucous or endocrine phenotypes were only very occasionally observed. However, endocrine and mucous cells could reproducibly be demonstrated in postconfluent monolayers grown in 1% foetal calf serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of multiple cell types from a single human colonic carcinoma: tumourigenicity of these cell types in a xenograft system.

The HCA-7 cell line was established from a moderately well differentiated mucinous adenocarcinoma of the colon, which showed histological heterogeneity. This was reflected in the morphological heterogeneity in early passages of the HCA-7 cell line but diminished as the cells were passaged in vitro. Nine subpopulations were isolated from early passage cultures of the HCA-7 line and maintained as cell lines. Each subpopulation demonstrated a unique set of stable biological characteristics in vitro. When established in vivo, there was a wide variation in xenograft generation time. The parent cell line gave rise to six distinct xenograft patterns, two of which had been observed in the primary carcinoma. Individual subpopulations yielded characteristic tumours which were composed of between one and four organizational patterns. Xenografts differed in both the organization of cells and the cell differentiation within the different patterns. Mucous cells were absent from some tumours while abundant in others. The subpopulations isolated from the HCA-7 cell line provide a new and extensive model system for studying the generation and maintenance of phenotypic heterogeneity in colorectal carcinoma.

Control of fluid transport in human rectal adenocarcinoma cells (HRA-19) in monolayer and collagen gel cultures.

HRA-19a1.1. cells, derived from a primary human rectal adenocarcinoma, form polarised monolayers when grown on tissue-culture plastic. HRA-19 monolayers have a heterogeneous morphology even after 150 passages in vitro or single cell cloning. The morphological changes observed in HRA-19 monolayers were postulated to be the result of vectorial fluid transport leading to accumulation of fluid between cells. To test this hypothesis, a variety of agents that control ion transport in colorectal epithelium were tested for their effect on HRA-19 morphology. Forskolin, cholera toxin and prostaglandin E2 all markedly changed HRA-19 monolayer morphology, with the rapid disappearance of intercellular spaces. These agents all stimulate Cl- secretion in colorectal epithelium, i.e. transport from basolateral to apical surface, and therefore would be expected to reduce fluid accumulation at the basolateral side of the cell. Conversely, vasopressin, which stimulates absorption of Na+ and water across colorectal epithelium, leads to a small increase in intercellular spaces in the monolayer. In collagen gel cultures, addition of cholera toxin, forskolin or prostaglandin E2 resulted in a large increase in colony size. In such treated cultures, the colonies were 'bubble-like', often composed of a single rim of flattened cells, which appeared to encompass a fluid-filled space. Similar morphological changes were observed when HRA-19 cells were co-cultured with 3T3 cells. This effect was probably due, at least in part, to prostaglandin production by the 3T3 cells, as the effect could be markedly reduced by the addition of indomethacin to these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of polarity and differentiation on antibody localization in multicellular tumour spheroid and xenograft models and its potential importance for in vivo immunotargeting.

Two monoclonal antibodies (MAbs) AUAI and HMFGI recognize antigens located on different membrane domains of polarized epithelial cells. We have assessed the accessibility of these antigens in multicellular tumour spheroids produced in culture using a well-polarized (HRA-19) and a non-polarized cell line (LoVo) of human large-bowel carcinoma origin. Multicellular spheroids of HRA-19 cells develop polarity, so that the membrane which is in contact with the culture medium (apical) becomes antigenically distinct from the membrane facing the centre of the spheroids (basolateral). This was confirmed by immunostaining sections of spheroids with 2 MAbs, AUAI and HMFGI. AUAI recognizes an antigen located exclusively on the basolateral membranes of polarized epithelial cells, and stained only internal membranes in spheroid sections. Conversely HMFGI, which recognizes an antigen located on the apical membranes, stained only the periphery of the spheroids. These 2MAbs were then radiolabelled with 125I and incubated with live spheroids for 4 hr at 37 degrees C. Autoradiographs of spheroid sections showed a marked difference between the 2 MAbs. 125I-HMFGI-radioantibody localized exclusively on the spheroid surface in a pattern identical to the in vitro immunostaining pattern, while 125I-AUAI radioantibody showed no binding in spite of the uniform presence of antigen on all tumour cells basolaterally. This appeared to be the result of the inaccessibility of basolateral antigenic sites in well-polarized epithelial cells because of the tight junctions connecting these cells at their apical surfaces. In contrast to the HRA-19 cell line LoVo, spheroids do not develop polarity; as a result, when stained with AUAI, variable antigenic expression all over the cell surface was seen. Autoradiographs of these spheroids showed 125I-AUAI binding with a penetration to a depth of about 1-3 cells, while HMFGI which shows no reactivity with this cell line in vitro, did not bind. This phenomenon was further investigated in xenografts of the HRA-19 cell line. It was shown that in a well-differentiated adenocarcinoma where the tumour cells forming acini are arranged in a polarized fashion, the luminal antigenic sites may be inaccessible to the injected MAb. The striking differences in binding of MAbs on polarized and unpolarized tumours indicate the importance of cell polarization and exact location of antigenic sites for in vivo immunotargeting.

Polarity and differentiation of human rectal adenocarcinoma cells in suspension and collagen gel cultures.

HRA-19a1.1. cells, derived from a primary human rectal adenocarcinoma, form polarized monolayers when grown on tissue-culture plastic. The apical membrane of the cells is in contact with the culture medium while the basolateral surface is attached to the plastic substratum. Cells cultured on non-tissue-culture plastic form floating colonies. Cells within these colonies are orientated so that their apical membrane is in contact with the culture medium while the basolateral membrane faces the centre of the colony. When these colonies are embedded in collagen gel the cells organize to form glandular structures similar to those observed in xenografts of this cell line. In addition a reversal in the orientation of cell polarity is observed with the basolateral membrane now in contact with the collagen gel, while the apical membrane faces lumina within the colony. This interaction is specific to collagen gel, as in a control experiment where colonies are embedded in agarose gel, neither a reversal in polarity nor the formation of glandular structures is observed. These results demonstrate an induction of glandular organization in human rectal adenocarcinoma cells by collagen gel. In addition an increased absorptive cell differentiation and reversal in cell polarity is observed in response to collagen gel.

Clonal origin of columnar, mucous, and endocrine cell lineages in human colorectal epithelium.

Human colorectal epithelium is composed mainly of columnar, mucous and endocrine cells; origin of these cell lineages from a multi-potential stem cell at the base of the crypt (the Unitarian hypothesis) has been proposed but not yet demonstrated. Gut endocrine cells have variously been considered of neural crest or endodermal origin, but conclusive evidence, particularly in humans, is lacking. It has been shown that in mouse gastrointestinal tract, a single progenitor cell gives rise to both columnar and mucous cells, but it has yet to be demonstrated that such a progenitor cell can also give rise to endocrine cells. Here, a single human rectal adenocarcinoma cell has been shown to differentiate into columnar, mucous and endocrine cells; therefore all epithelial lineages are of clonal origin. Additionally, these results show that human colorectal enteroendocrine cells, at least in neoplastic epithelium, have an endodermal origin.

Calcium- and cyclic AMP-dependent chloride secretion in human colonic epithelia.

Three stable epithelial cell lines (HCA-7, HCA-7-Col 1 and HCA-7-Col 3) all derived from the same human adenocarcinoma have been cultured on collagen-coated Millipore filters. These epithelial monolayers have been used to record short circuit current (SCC) in response to of secretagogues. Similar monolayers, but grown on plastic dishes, were used for measurements of tissue cyclic AMP. Lysylbradykinin, applied to either side of the monolayers, increased SCC in HCA-7 cells but had little effect on the other two lines. The responses showed rapid desensitization, which could be prevented by cooling to 4 degrees C. Responses to kinin were not significantly attenuated by piroxicam, an inhibitor of cyclo-oxygenase. Other secretagogues, vasoactive intestinal polypeptide (VIP) and carbachol also increased SCC in monolayers. The responses to VIP were greatest in HCA-7-Col 1 monolayers while responses were virtually absent in HCA-7-Col 3. A similar profile was seen with carbachol except that responses of HCA-7 and HCA-7-Col 1 monolayers were more equal. With one exception the responses to VIP and carbachol showed sidedness, acting only from the basolateral side. The effects of the secretagogues were inhibited by piretanide, a loop diuretic, applied basolaterally. It is presumed that SCC responses represent electrogenic chloride secretion. Treatment with forskolin increased SCC in HCA-7 and HCA-7-Col 1 monolayers with little effect in HCA-7-Col 3. Nevertheless cyclic AMP levels were elevated most in HCA-7-Col 3 and least in HCA-7-Col 1 monolayers, in reciprocal relationship to the functional response. A23187 increased SCC when applied to HCA-7 and HCA-7-Col 3 monolayers with little effect on HCA-7-Col 1. The differential responses of the three human cell lines provide unique opportunities to discover the functional responsibilities of entities involved in the chloride secretory process. HCA-7-Col 3 cells which generate high levels of cyclic AMP in response to forskolin but which fail to show a substantial chloride secretory response may be a useful model of some disease conditions.

Accessibility of antigenic sites recognized by AUA1, HMFG1 and HMFG2 monoclonal antibodies: its influence on antibody binding of live cells.

We assessed the immunoreactivity of live and alcohol-fixed monolayers of HRA-19, a rectal adenocarcinoma cell line, to the monoclonal antibodies AUA1, HMFG1 and HMFG2. Differences in staining patterns between live and alcohol-fixed colonies were found. The well-polarized cells forming the centers of the monolayer colonies showed strong membrane staining when the cells were alcohol-fixed prior to AUA1 incubation, but showed no staining when the cells were alive during the incubation. When AUA1 incubation was done both before and after alcohol fixation, membrane staining was again seen, ruling out the possibility of antigenic modulation. Incubation of live cells with AUA1 together with EDTA showed strong staining of dissociating cells. It is concluded that AUA1 antigenic sites, which on polarized cells are basolateral in location, are inaccessible to the antibody-containing culture fluid, which bathes the apical aspects of the cells, but they become accessible after alcohol fixation, or treatment with EDTA. HMFG1 antigenic sites are located on the apical cell membrane, and accordingly, no differences were seen between incubation of live and alcohol-fixed cells when incubated with HMFG1. The antigenic sites of HMFG2 are partly intracellular, and in our monolayer model, the staining of live cells was weaker and more scarce than on alcohol-fixed cells. It is concluded that immunostaining of cytological and histological material of tumours may not adequately predict antibody binding on live cells, and thus, these findings are of importance in the context of selection of monoclonal antibodies for clinical radio-immunotargeting.

Establishment and characterisation of six human colorectal adenocarcinoma cell lines.

The establishment and characterisation (morphology, ultrastructure, tumourigenicity) of six cell lines from primary human colorectal adenocarcinomas is described. These lines were established from surgical specimens, from 49 unselected patients, without the use of 'feeder' cells, 'conditioned' medium or passage of cells in nude mice. The six cell lines exhibit considerable variation in morphology, CEA secretion and tumourigenicity in nude mice. At least two of the lines retain some of the differentiated characteristics of colorectal epithelium.