Characterization of quantitative mucin variants from a human colon cancer cell line

Colonic mucins are high molecular weight glycoproteins produced by goblet cells of colonic epithelium. Some studies have indicated that patients with colonic cancers that produce high amounts of mucin have a poorer prognosis than patients whose tumors produce low amounts of mucin. At present, however, the role of mucin in affecting the behavior of colon cancer cells is not well understood. To further elucidate the relationship between cellular mucin content and the growth characteristics and morphology of tumor cells, we utilized a replica plating technique and immunoscreening method to identify and purify variant clones of the human colon cancer cell line LS174T that produce high and low levels of mucin. This procedure enabled us to isolate two high mucin-containing variants (HM3 and HM7) and one low mucin-containing variant (LM12). These variants exhibited different morphology. Both high mucin variants tended to form cell aggregates and suspended cells with adjoining mucoid threads. The low mucin variant formed spread monolayers on the substratum with the formation of cell processes. Metabolic labeling using [3H]glucosamine demonstrated that high mucin variants synthesized 2-fold more mucin in the cell layer and secreted 3-fold more mucin into the culture medium than the low mucin variant. The colony-forming efficiency in semisolid agar for these variants positively correlated with their mucin content. High mucin variant cells when injected into athymic nude mice formed tumors 2-fold larger than those of the parental cells while the low mucin variant formed tumors only one-half as large as those of the parental cell line. These mucin variants should provide a useful model for understanding the biological behavior of mucinous colon cancer cells in vivo and in vitro.     

Laminin expression by human pancreatic carcinoma cells in the nude mouse and in culture

The abilities of 4 established human pancreatic tumor cell lines (PANC-1, CAPAN-1, AsPc-1, and BxPc-3) to synthesize and to maintain laminin-containing basement membranes when grown in the nude mouse have been examined and compared with synthesis of the glycoprotein laminin by these tumor cells when grown in culture. Immunohistochemical visualization of basement membrane integrity within the transplanted tumors employed a monoclonal antibody specific for human laminin to clearly distinguish between human tumor-produced laminin and murine basement membranes. This technique demonstrated strikingly different degrees of basement membrane formation and laminin distribution for the 4 biologically diverse pancreatic tumors. Basement membranes were present within the differentiated, less invasive tumors, whereas structure basement membranes were absent in the poorly differentiated, invasive tumors. Regardless of their propensity to produce basement membranes in vivo, all 4 pancreatic tumor cell lines continued to synthesize laminin in culture, as was determined by immunologic assays. The most invasive cell line, AsPc-1, released large quantities of soluble laminin into conditioned culture medium. The less invasive, well-differentiated tumor cells did not release laminin into the medium, but they retained the laminin produced by them within the cell layer. The combination of in vivo and in vitro studies indicates that the extent of basement membrane loss by these human pancreatic tumors is not due to an inherent inability of the tumor cells to synthesize the structural component laminin.     

Tumorigenicity, mucin production and AM-3 epitope expression in clones selected from the HT-29 colon carcinoma cell line.

Two mucin-producing cell clones (16.2 and 12.2) and a mucin-deficient clone (15.2) were selected from the established human adenocarcinoma cell line HT-29 by limiting dilution and Alcian blue staining. The amounts of the mucin antigen detectable on the cell surface with the monoclonal antibody (MAb) AM-3 decreased in the order HT-29 greater than 16.2 greater than 12.2 greater than 15.2 = 0. The binding avidity of AM-3 antibody to cells as well as to mucin extracts from each cell line decreased in the same order, indicating that the epitope density on the cell-bound mucins was highest in HT-29 and lowest in 12.2 cells. The parental line and the mucin-producing cell clones 16.2 and 12.2 showed no contact inhibition and grew as aggregates, while the 15.2 cells were well spread and formed a regular monolayer. The mucin-producing cell lines injected into nude mice yielded solid tumors with different growth rates (HT-29 greater than 16.2 greater than 12.2), while the 15.2 cell clone was not tumorigenic at all. The relative amounts of total mucin-bound hexoses and of the mucin epitope AM-3 decreased in the xenografts in the order HT-29 greater than 16.2 greater than 12.2. The present system is suitable for investigating the role of mucins in growth of colon carcinoma cells and indicates that increased tumorigenicity in nude mice coincides with the increase in total mucin expression and the expression of the AM-3 mucin epitope in tumor tissue.     

Isolation and Characterization of an Undifferentiated Human Colon Carcinoma Cell Line (MIP-101)

An undifferentiated human colon carcinoma cell line was established from tumor tissue obtained from metastasis to the liver of colonic adenocarcinoma in a patient with fulminant Dukes D colorectal carcinoma. Histological analysis of the tumor biopsy from the liver confirmed the hospital pathology report of poorly differentiated colonic adenocarcinoma. Explants of this tumor tissue xenografted into a nude mouse were used to establish an epithelioid-like cell culture line, MIP-101. The cell line formed tumors in nude mice that hisologically appeared undifferentiated and did not stain for carcinoembryonic antigen (CEA). No CEA was present either by radio-immunoassay (RIA) of the culture supernatant or by immunoperoxidase staining of the tumors or monolayers. MIP-101 appears to be one of the most undifferentiated human colon carcinoma cells lines available. It should prove useful in the search for markers of undifferentiated colonic cancer and in studies of colonic cancer differentiation.     

Basement membrane and the SIKVAV laminin-derived peptide promote tumor growth and metastases

Summary Laminin, the major glycoprotein component of basement membrane, promotes the malignant phenotype. Cells which are adherent to laminin are more malignant than the non-adherent cells and in certain tumor cells, the number of laminin receptors is positively correlated with malignancy. Laminin also increases collagenase IV activity, an enzyme demonstrated to be critical for tumor spread. A site on laminin, containing the amino acid sequence SIKVAV, has been identified which when injected intravenously with B16F10 melanoma cells, causes an increase in the number of colonies on the surface of the lungs. This peptide does not affect tumor cell arrest in the vasculature or the immune system. It does promote angiogenesis in various in vitro and in vivo models, thereby facilitating tumor cell survival.When a complex mixture of laminin-enriched basement membrane components (Matrigel) is coinjected with tumor cells subcutaneously, tumor incidence and growth increases. Various tumor cell lines and primary isolates, which previously could not form tumors in mice, can be induced to grow rapidly in the presence of Matrigel. Slowly growing tumors or arrested tumors can also be induced to grow more quickly with additional injections of Matrigel. When an SIKVAV-containing synthetic peptide is coinjected with B16F10 tumor cells and Matrigel subcutaneously in mice, larger tumors are formed than that observed with either Matrigel or cells alone. Such studies define the role of laminin in tumor growth and spread and generate new models for studying therapeutic agents. Of particular interest is the ability to grow primary isolates which generally do not grow in mice.     

Expression of 32/67-kDa laminin receptor in laminin adhesion-selected human colon cancer cell lines.

Laminin promotes the malignant phenotype, and the expression of certain laminin receptors is increased in malignancy. Previously, we demonstrated that a laminin-adhesive subclone of a human colon cancer cell line showed increased tumorigenicity in nude mice and increased affinity of the beta1 integrin for laminin relative to the laminin-non-adhesive subclone. The total amount of either beta1 integrin protein or mRNA did not increase. As levels of the 32/67-kDa laminin receptor (67LR) correlate with malignancy, we examined 67LR expression in the laminin adhesion-selected human colon cancer cells. The laminin-adhesive subclone, which was more tumorigenic in both heterotopic and orthotopic locations than in a laminin-non-adhesive subclone, showed cell-surface membrane staining of 67LR, whereas the laminin-non-adhesive subclone showed cytoplasmic staining of 67LR. No difference in either the amount of 67LR mRNA or the amount of protein was observed in the parental cells than in the laminin-adhesive and non-adhesive subclones. When assayed on a laminin affinity column, more 67LR molecules bound to the column with cell extracts from the laminin-adhesive subclone than was observed with the non-adhesive subclone. These findings suggest that the increased tumorigenicity of laminin adhesion-selected tumour cells might be due to an alteration in the distribution and/or adhesiveness of multiple receptors including 67LR and beta1 integrin.     

Vascular endothelial growth factor and enhanced angiogenesis do not promote metastatic conversion of a newly established azoxymethane-induced colon cancer cell line

The organo-specific carcinogen, azoxymethane (AOM), produces colon tumors in mice that share many pathological features with sporadic human colorectal cancer (CRC). An important distinction between AOM-induced CRC and human CRC is lack of mucosal invasion in the murine model. To assess the role of the microenvironment in preventing the invasive phenotype, multiple benign in situ adenocarcinomas were harvested from AOM-treated mice and cultured in vitro. However, tumor cell growth was extremely limiting under standard culturing conditions. Thus, we injected tumor cells directly into nude mice and performed two serial transplants, and successfully explanted a rapidly growing epithelial tumor cell line (AJ02nm(0)). When injected subcutaneously (sc) into nude mice, AJ02nm(0) cells formed well-differentiated adenocarcinomas with minimal tumor invasive capacity. To define whether metastatic and invasive potential were related to lack of angiogenic stimuli, the AJ02nm(0) cells were transfected to overexpress murine vascular endothelial growth factor-164 (VEGF(164)). AJ02nm-VEGF cells produced rapidly growing tumors in nude mice that exhibited extensive pseudo-epithelial ductal architecture and supporting vasculature, but without increased invasive potential compared to controls. The established murine colon epithelial cell line provides a useful experimental model to further elaborate genetic and epigenetic factors that may promote or inhibit colon tumorigenesis and metastasis.     

Laminin expression by two clones isolated from the colon carcinoma cell line LoVo that differ in metastatic potential and basement-membrane organization.

In the present report we describe the characteristics of 2 clones, E2 and C5, isolated from the human colon adenocarcinoma cell line LoVo. When grafted to immunosuppressed newborn rats, these clones formed tumors that varied with regard to differentiation rate, basement-membrane organization and lung metastatic potential. Production and distribution of laminin by E2, C5 and related tumors was studied by immunohistochemistry with an anti-laminin monoclonal antibody 4C12 (MAb 4C12). In lowly metastatic E2-derived tumors, strong regular stainings were observed which were strictly peri-tumoral and corresponded to the basal lamina. Since the antibody interacted with human laminin (the graft) but not with rat laminin (the host), this result indicated that basement-membrane laminin was supplied mainly by tumor-cell synthesis. In highly metastatic C5-derived tumors, the staining obtained with MAb 4C12 was peri-cellular and unorganized. Laminin synthesis by E2 and C5 cells in sub-cultures or soon after dissociation from explanted tumors was studied by metabolic labelling with 35S-methionine under steady-state conditions followed by immunoprecipitation and SDS-PAGE. High-molecular-weight laminin comprised by disulfide-linked A and B chains, i.e., heterotrimeric laminin, was found in cell lysates and in the secretion medium of cell lines and tumor cells. In addition, B1B2 dimers and free B chains were observed in cell lysates. Quantitatively, laminin expression by E2 and C5 clones or tumor cells was not significantly different. These findings suggest that basement-membrane defects in invasive clone LoVo C5 were not due to laminin under-expression.     

Growth of murine sarcoma virus-transformed rat kidney cells in nude mice: absence of induction of host endogenous viruses.

Clonal isolates of the normal rat kidney cell line (NRK) transformed by a defective murine sarcoma virus (Kirsten strain) were injected into nude mice of BALB/c background to determine whether the growth of these cells as tumors was accompanied by the induction of host endogenous type C viruses. All the virus-transformed clones produced rapidly growing tumors in nude mice, but neither the induction of mouse endogenous viruses nor the rescue and spread of the transforming sarcoma virus were observed during the growth of tumors. The degree of expression of the tumor virus structural proteins in the transformed cells did not determine the cellular phenotype with regard to tumorigenicity in nude mice, nor did it modify the cellular growth properties in vitro. Consistent with earlier observations with simian virus 40-transformed mouse and rat cells, the ability of sarcoma virus-transformed NRK cells to initiate tumor growth in nude mice appeared to be correlated with anchorage-independent growth in vitro.     

Selection by trypsin of two sublines of rat colon cancer cells forming progressive or regressive tumors

From an established cell culture line obtained from a chemically-induced rat colon carcinoma, two sublines have been selected and isolated according to their susceptibility to trypsin-mediated detachment from plastic surfaces. Subline TR, the most resistant to the detaching effect of trypsin, gave progressive tumors in most of the syngeneic rats in which it was inoculated. Subline TS, which was easily detached by trypsin, also gave tumors in the syngeneic rats, but all these tumors disappeared within 3 or 4 weeks. Both sublines gave progressive tumors when injected into nude mice. This suggests that the parent cell line is heterogeneous and contains cell variants differing in their susceptibility to trypsin-mediated detachment from substrate and their sensitivity to host factors leading to acceptance or rejection of the inoculated tumor cells.     

In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinomas implanted into nude mice

The purpose of these studies was to select and isolate cells with increased liver-metastasizing potential from heterogeneous primary human colon carcinomas (HCCs). Cells derived from a primary HCC classified as Dukes' stage B2 were directly established in culture or were injected into the subcutis, cecum, or spleen of nude mice. Progressively growing tumors were excised, dissociated, and established in culture. Subsequent to implantation into the cecum or spleen of nude mice, cells from all four lines produced only a few liver tumor foci. HCC cells from the few liver metastases were expanded in culture and then injected into the spleen of nude mice to provide a source for further cycles of selection. With each successive in vivo selection cycle, the metastatic ability of the isolated propagated cells increased. Four cycles of selection yielded cell lines with a very high metastatic efficiency in nude mice. In parallel studies using another primary HCC classified as Dukes' stage D, we isolated cell lines that were highly metastatic in nude mice. Successive selection cycles for growth in the liver increased the metastatic properties of the HCC cells, albeit to a lesser extent than it did those of the Dukes' B2 stage HCC. The ability of the HCC cells to produce liver metastases was not due to simple trapping in the liver. In vivo distribution studies using [125I] iododeoxyuridine-labeled tumor cells revealed that, shortly after injection into the spleen, a comparable number of cells with either low or high metastatic properties arrested in the liver. The differences between the low- and high-degree metastatic cells became apparent by 24 h after injection and, by 72 h, only highly metastatic cells survived in the liver. These results demonstrate that hepatic metastasis by HCC cells is a selective process and that the nude mouse model can be useful for isolating highly metastatic HCC cells and for studying the relevant host organ factors that regulate the pathogenesis of metastasis.     

Characterization of laminin-stimulated adherence and motility in tumor cells

Laminin was shown to stimulate attachment, spreading and motility in a line of murine tumor cells. The stimulated cells began to attach and spread within 1 h and remained attached and spread throughout a 48-hour observation period. Pretreatment of the cells with laminin for 24 h beforehand did not interfere with their ability to bind laminin in a second treatment. Laminin-stimulated attachment and spreading occurred in the absence of extracellular Ca2+ or Mg2+, but not in the absence of both divalent cations. A number of cell function inhibitors blocked the adherence response, but were only partially effective even at high concentrations. These characteristics of laminin-stimulated adherence are different from those of the adherence response stimulated by peptide chemotactic factors and phorbol esters.     

Modulation of Doxorubicin sensitivity and level of p-glycoprotein expression in human colon-carcinoma cells by ectopic and orthotopic environments in nude-mice

The purpose of the study was to determine whether the organ environment can influence the response of colon cancer cells to chemotherapy. The highly metastatic human colon cancer cell line KM12L4, previously selected for production of liver metastases in nude mice, was injected into the cecal wall and into the spleen to produce liver metastases, and into the subcutis of nude mice. Doxorubicin (DOX) at 10 mg/kg or saline (control) was injected intravenously on days 7 and 16 after tumor cell injection. The in vivo response of tumors growing in the cecum, liver, and subcutaneous (s.c.) sites as well as the DOX sensitivity of cell lines established from liver and s.c. tumors were compared. Colon cancers growing s.c. were more sensitive to DOX than tumors growing in the cecal wall or liver of nude mice. The difference in response to DOX between s.c. tumors (sensitive) and liver tumors (resistant) was not due to selection of cell populations with different sensitivity to DOX, or differences in DOX distribution. PKC activity was lower in tumors of the liver and the cecum than in s.c. tumors. The expression of P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DOX. Increased levels of P-glycoprotein correlated with mdr-1, mdr-3 mRNA expression as determined by Northern analysis. Collectively, the data show that the organ environment influences the response of human colon carcinoma cells to DOX and recommend that animal models of this disease for experimental therapeutic studies employ orthotopic implantation of tumor cells.     

Laminin-dependent and laminin-independent adhesion of human melanoma cells to sulfatides

Sulfatides (galactosylceramide-I3-sulfate) but not neutral glycolipids or gangliosides adsorbed on plastic promote adhesion of the human melanoma cell line G361. Direct adhesion of G361 cells requires densities of sulfatide greater than 1 pmol/mm2. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide or seminolipid (galactosylalkylacyl-glycerol-I3-sulfate) but not to other lipids is strongly stimulated and requires only 25 fmol/mm2 of adsorbed lipid. The effects of laminin and sulfatide on adhesion are synergistic, suggesting that laminin is mediating adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed on the plastic. Although thrombospondin binds to sulfatides and G361 cells, it does not enhance, but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide. In contrast, C32 melanoma cells also adhere specifically to sulfatide, but adhesion of these cells is not enhanced by laminin or inhibited by antibodies to laminin that block laminin-dependent adhesion of G361 cells. Thrombospondin is a potent inhibitor of C32 cell adhesion to sulfatide. Fucoidan, which inhibits laminin binding to sulfatide, inhibits laminin-dependent adhesion of G361 cells by 50% at 0.2 micrograms/ml. Several other tumor cell lines also attach directly on sulfatide-coated surfaces. Laminin stimulates adhesion to sulfatide of three of the six cell lines tested. The ability of laminin to promote adhesion of tumor cells to sulfatide suggests that binding to sulfatide could participate in laminin-mediated cell-cell adhesion. Thus, many tumor cell lines can attach on sulfatide substrates using endogenous sulfatide binding proteins, and in some cells laminin but not thrombospondin can promote tumor cell adhesion to sulfatide.     

Stimulation of human prostatic carcinoma tumor growth in athymic mice and control of migration in culture by extracellular matrix.

The tumorigenicity, migration, growth and invasiveness of certain tumor cells is stimulated by basement membranes. Here we have examined the effect of Matrigel, an extract of basement membrane proteins, on the behavior of several prostate cancer cell lines, testing their growth and invasiveness in vitro and in vivo. Cells of the Tsu-prI line were more invasive than PC-3, Du-145, or LNCaP cells. Peptide inhibitors implicated laminin in the migration and invasion of these cells. When these cells were suspended in Matrigel and injected into nude mice, their growth was greatly enhanced, since large tumors formed in athymic nude mice whereas virtually no tumors were observed in the absence of Matrigel. The growth of a slowly growing line, LNCaP, was increased by exogenous basic fibroblast growth factor when injected with Matrigel. A laminin cell adhesion peptide, YIGSR, was a potent inhibitor of Matrigel-stimulated tumor growth implicating cell-laminin interactions in this process. These results suggest that tumor growth of prostate adenocarcinoma cells may be dependent both on cellular growth factors and on cell-matrix interactions mediated by laminin which facilitate the development of transplanted tumors in nude mice.     

Induction of mouse tenascin expression by a human sarcomatoid wilms' tumor cell line growing in nude mice

A new cell line from a sporadic Wilms' tumor was established and extensively characterized. In nude mice, the tumor cells rapidly formed tumors which, in histological characteristics and extracellular-matrix (ECM) composition resembled sarcomatoid Wilms' tumor. The tumor cells produced B chains of laminin, but no A chain, and laminin was deposited into the ECM in a punctate pattern typical of sarcomatoid tumors. Strong expression of tenascin was detected within the stromal ECM of the tumors. Species-specific antibodies reacting either with human or with mouse tenascin showed that tenascin was exclusively derived from mouse host cells. The human Wilms' cell line thus induced a strong stromal response with increased deposition of tenascin. The cell line may be useful for studying the behavior of sarcomatoid Wilms' tumor cells and for identifying factors that stimulate synthesis of tenascin.     

Immune response to somatic cell hybrids between ultraviolet radiation-induced regressor and spontaneous progressor C3H mouse tumor cells

We used somatic cell hybridization to determine whether the regressor phenotype exhibited by UV-induced murine tumors was dominant or recessive and whether this technique could confer immunogenic properties on nonimmunogenic syngeneic tumors. We transfected a highly antigenic UV-induced C3H mouse tumor cell line (UV-2240) with the plasmid pSV2-neo and selected G418-resistant clones. The resulting cell line was fused with a spontaneously transformed nonimmunogenic C3H progressor tumor cell line (SF-2T) that had been selected previously for resistance to 3.0 mM ouabain. These two cell lines were fused by a brief exposure to polyethylene glycol and heterokaryons isolated by growth in medium containing both G418 and ouabain. Hybrid cell lines established from individual colonies and from pools of colonies were tested for tumorigenicity in normal C3H and athymic nude mice. The results indicated that all the hybrid cell lines tested were highly antigenic in that they were completely rejected when transplanted into normal syngeneic mice but grew progressively in nude mice. Furthermore, immunization of C3H mice with the hybrid cell lines induced protective immunity against challenge with the immunizing tumor and generated cross-protective immunity against challenge with the regressor parental cell line but not against challenge with the progressor parental cell line. These results demonstrate that the regressor phenotype of the UV-2240 tumor is dominant in nature and that the immune response induced by somatic cell hybrids is uniquely directed against the dominant tumor-specific transplantation antigens expressed on the regressor tumor. This implies that introduction of tumor-specific transplantation antigens from an immunogenic tumor into a nonimmunogenic tumor, although sufficient to confer immunogenic properties to the hybrid, is insufficient to induce cross-protective transplantation immunity against the nonimmunogenic tumor.     

Overexpression of ras in mucus-secreting human colon carcinoma cells of low tumorigenicity

We have investigated the expression of the protooncogenes of the myc and ras family in HT29 cells and in three differentiated clonal cell lines derived from this colon carcinoma cell line. In contrast to the decrease in myc expression seen when leukemia cells are induced to differentiate, we have found no changes in expression of the myc gene family in differentiated colon carcinoma cells. However, a greater than 5-fold increase in expression of sequences which hybridize to Ha-ras was observed in cells which secrete mucin, with a smaller increase seen in expression of Ki-ras in the same cells. This increase was not seen in cells which exhibit vectorial transport of water and ions, and which are not mucus-secreting. All differentiated lines were less tumorigenic in nude mice than the parental HT29 cells, irrespective of the level of ras expression. These results are consistent with the reports that ras expression is highest in the most differentiated cells of the colon and is substantially decreased in metastatic human colon tumors as compared to primary colon tumors. The data also suggest that a high level of ras gene expression is a marker for a particular differentiated state in colon cells rather than being directly equated with transformation or tumorigenicity. Hence, the results may reflect on some of the discrepancies concerning ras gene expression in human colon and other tumors which appear in the literature.     

Uniqueness of each spontaneous transformant from a clone of BALB/c 3T3 cells

Seeding 2 X 10(4) cells from a clone of BALB/c 3T3 cells in agar led to the formation of about 100 small colonies (approximate diameter, 0.2 mm) and two large colonies (1-mm diameter). Seven of the former and both of the latter were isolated, and the morphology and growth properties of their cells were observed in repeated weekly passages. The seven subclones derived from the smaller agar colonies spread out on the dish and multiplied more slowly than the parental clone on plastic, but six of them produced more colonies in agar than the parental clone. The two subclones derived from the larger agar colonies had a fully transformed morphology, multiplied much faster than the parental clone on plastic, and produced a high percentage of large colonies in agar. Each of the subclones could be distinguished morphologically from the others and from the parental clone, and most of them could be distinguished on the basis of their colony-forming efficiency in agar. Most of the secondary subclones derived from an early passage on plastic of one of the two large agar clones, died out on the second passage after their isolation. Secondary subclones derived from the same subclone five weeks later had a wide range of fluctuating growth rates, but did not die out on passage. The two rapidly growing subclones derived from large agar colonies initiated fast-growing tumors in nude mice. None of the other subclones produced any visible growth in nude mice over a 3-month period. Large agar colonies of fast growing, morphologically transformed cells appeared once during further passage of the parental clone and two of the subclones. The results reveal a surprising degree of heritable diversity in morphology and growth characteristics of the progeny from a clonal line of nontransformed cells. They also indicate that, in this cell line, only those cells that have a high efficiency (greater than 20%) of large colony formation in agar have the capacity to form tumors in nude mice.