Establishment and characterization of five human malignant mesothelioma cell lines derived from pleural effusions.

Malignant mesothelioma (MM) is an aggressive tumour of the serosal cavities which is associated with exposure to asbestos. Studies of this tumour have been limited by a paucity of well-characterized human MM cell lines. In this study, 5 human MM cell lines were established from pleural effusions of patients with this malignancy. All 5 patients were males with known crocidolite asbestos exposure, who had received no treatment for their disease and in whom the diagnosis was confirmed by cytology, histology and electron microscopy (EM). These lines have been in culture from 11 to 25 months, and all of them for more than 18 passages. The appearance of the cells in culture was extremely varied; in 3 of the lines they were spindle-shaped with few vacuoles (JU77, LO68 and ONE58); in 1 line they had a thick, stellate shape with vacuoles (NO36) and in 1 they were very pleomorphic in both shape and size with irregular membranes and numerous vacuoles [DeH128 (M)]. Upon reaching confluence, cells in 3 of the 5 lines assumed the cobblestone-like pattern characteristic of epithelial-type cells, whereas in the other 2 (LO68 and ONE58) they remained spindle-shaped. All 5 lines demonstrated a loss of contact inhibition (i.e., piling) at confluence. Minimum doubling times varied significantly from 18 hr (JU77) to more than 30 hr [DeH128 (M)]. Cytological examination showed characteristic mesothelial/mesothelioma morphology, and epithelial membrane antigen (EMA) and cytokeratin were demonstrated in cells from all 5 lines. These cells lacked CEA and epithelial mucin. The presence of cell junctions, glycogen and numerous long, thin, branching microvilli was readily demonstrable by EM. All lines had abnormal karyotypes, with the modal chromosome number varying from 40 to 80. Variable chromosome numbers, numerous structural rearrangements and unrecognizable marker chromosomes were readily observed; however, the only consistent change seen was del 6q21 in 4 of the 5 lines. The establishment of these 5 cultured human MM cell lines now provides an opportunity for comparative study of several aspects of the biology of MM in vitro as well as screening new treatment modalities.     

Differential characteristics of two newly established human breast carcinoma cell lines

Two human breast carcinoma cell lines, EP and MW, were established in culture from malignant pleural effusions. In addition to producing tumors in antithymocyte serum-immunosuppressed mice, both cell lines showed epithelial characteristics and anchorage-independent growth in soft agar. EP and MW differed in morphology (spindle-shaped versus round), chromosomal mode (hyperdiploid versus near triploid), estrogen receptor content (43.8 versus 5.1 fmol/mg protein), cloning efficiency (0.24 versus 15%), and activities (milliunits/10(6) cells) of creatine phosphokinase (25.7 versus 62.6) and lactate dehydrogenase (346.7 versus 778.5). Electron microscopy revealed that MW cells had more perinuclear filamentous material and more frequent intracytoplasmic vacuole formation than did EP cells. While having no effect on MW cells at the concentrations studied (10(-5) to 10(-11) M), beta-estradiol (10(-7) M) stimulated the growth of EP cells by 106% over the hormone-depleted control. In a variety of systems, EP was consistently the more drug sensitive of the two lines. In vitro, EP was significantly (p less than 0.001) more sensitive to methotrexate, vincristine, and 5-fluorouracil, respectively. In antithymocyte serum-mouse xenografts, EP displayed a greater response to three different dosages of a combination of cyclophosphamide, methotrexate, and 5-fluorouracil. One such dosage (cyclophosphamide, 32.0 mg/kg/day; methotrexate, 13.0 mg/kg/day; 5-fluorouracil, 190.0 mg/kg/day; for 1 day) reduced EP and MW tumor weights to 5.9 and 41% of controls, respectively. These results correlated well with the clinical responses.     

Characteristics of cell lines established from human gastric carcinoma

We report the establishment and characterization of four continuous cell lines derived from human primary and metastatic gastric carcinomas, and we compare their properties with a panel of colorectal carcinoma cell lines previously established and reported by us. Our success rate in culturing gastric carcinomas was relatively low, especially from primary tumors, compared to colorectal carcinoma. These observations may reflect the relatively modest number of gastric carcinoma cell lines established (mainly from Japan), compared to the abundance of colorectal carcinoma lines established worldwide. All four gastric lines expressed the surface glycoproteins carcinoembryonic antigen and TAG-72 and three lines expressed CA 19-9. Two of the lines expressed aromatic amino acid decarboxylase but lacked other markers for neuroendocrine differentiation. All four lines were positive for vasoactive intestinal peptide receptors but lacked gastrin receptors. In addition, two lines expressed receptors for muscarinic/cholinergic receptors but not beta-adrenergic receptors. Cytogenetic evidence for gene amplification was present in the cell lines. All four lines contained varying numbers of double-minute chromosomes. One line, SNU-16, was amplified for the c-myc proto-oncogene and contained four homogeneously staining regions. While c-myc and c-erb-B-2 RNA were expressed by all lines, there was no evidence of amplification or overexpression of several other proto-oncogenes and growth factors. The multiple properties we have described in our gastric carcinoma cell lines are remarkably similar to those found in the panel of colorectal carcinoma cell lines. These properties include morphology, growth characteristics, expression of surface glycoproteins, partial expression of neuroendocrine cell markers, frequent chromosomal evidence of gene amplification, and occasional amplification of the c-myc proto-oncogene. Our four well characterized cell lines should provide useful additions to the modest number currently available for in vitro studies of gastric carcinoma.     

Immunological characterization of cell lines established from malignant and normal human urothelium

Cell lines from normal or malignant human urothelium, described elsewhere by morphological, cell kinetic and genotypical criteria, have been characterized further by their response to antibody or lymphocyte mediated cytotoxicity in vitro. Four groups of cell lines were included: (1) two fast proliferating cell lines from normal urothelium, (2) three fast proliferating cell lines from transitional cell carcinoma (TCC) together with two transformed sublines of normal derived cell lines, (3) six slowly proliferating TCC lines, and (4) a line from squamous cell carcinoma. HLA antigen expression was demonstrated in the cell lines of groups 1 and 3, but not in lines from group 2 or 4. The sensitivity to spontaneous lymphocyte mediated cytotoxicity (SLMC) of cells in group 1 and 2 exceeded that of cells from group 3 by a factor of 8. The TCC line, HU 456, was more susceptible to SLMC than T 24. Specifically increased cytotoxicity of lymphocytes from patients suffering from interstitial cystitis (IC) against HU 609 from normal urothelium indicated that this line expresses tissue specific antigens, and at the same time that an immune reaction may be part of the pathogenesis of IC. By a sensitive test for antibody-dependent, cell-mediated cytotoxicity (ADCC) antibody activity against HU 456 was found in only one of nine TCC patients and none of five clinical controls. A pronounced non-disease-related blocking serum activity, frequently found, may account for some of the negative findings. The SLMC against HU 456 could be inhibited but not abolished by Fab fragments of rabbit anti-human immunoglobulin, indicating an ADCC mechanism as part of the SLMC.     

Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice

There are few reports describing experimental models of the growth and metastasis of human breast carcinomas. This article discusses the tumorigenic and metastatic properties of two estrogen receptor-negative breast carcinomas injected into nude mice. Tumor growth in the mammary fatpad (m.f.p.) and the subcutis was compared in female nude mice. The injection of 10(5) viable cells of two human breast carcinoma cell lines (MDA-MB-231 and MDA-MB-435) gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c. injections produced tumors and these occurred several weeks after the appearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is a favorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant metastases in 80% to 100% of recipients. The most common sites for metastasis were the lymph nodes and lungs, with a lower incidence of metastases in muscle (chest wall and thigh), heart, and brain. New variant cell lines were isolated from metastases in the lungs, brain, and heart. All the cell lines were tumorigenic in the m.f.p., and the lung- and heart-derived metastasis lines produced slightly more lung metastases than the original cell line. However, the brain metastasis variant produced significantly fewer lung metastases. Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimental lung metastases showed enhanced lung colonization potential when reinjected i.v. Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable tool for analysis of the cellular and molecular basis of the metastasis of advanced breast cancer.     

Heterogeneity of cloned cell lines established from a transplantable rat malignant fibrous histiocytoma.

Four cloned cell lines, MT-7, MT-8, MT-9 and MT-10, were established from a transplantable malignant fibrous histiocytoma (MFH) of F344 rats to investigate the histogenesis of the tumor. Cells of MT-7, MT-9 and MT-10 had fine structures characteristic of histiocytes, such as numerous cell processes, many lysosomes and well-developed cytoplasmic organelles. They stained positively for histiocytic lysosomal and antigenic markers. In addition, MT-9 cells contained microfilaments and well-developed RER in their cytoplasm, suggesting that they may be facultative fibroblasts. MT-8 cells stained weakly for histiocytic markers and had scant cytoplasmic organelles. They were identified as undifferentiated mesenchymal cells. The tumors induced in syngeneic rats by inoculating MT-7 or MT-10 consisted of a mixture of the pleomorphic, myxoid and storiform types of MFH, and those by MT-9 were of the storiform type. Cells forming these tumors stained positively for histiocytic markers. Tumors induced by MT-8 consisted of undifferentiated cells negative to these stainings. The histogenesis of MFH is surmised to be related to various differentiation stages shifting from undifferentiated cells to histiocytic cells capable of acting as facultative fibroblasts.     

Novel cell lines established from pediatric brain tumors

The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat “fingerprints” matching that of the patient’s tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors.     

Establishment of high and low metastasis cell lines derived from a human tongue squamous cell carcinoma

Malignant tumors are composed of cells with different phenotypic properties and only certain cell subpopulations present metastatic potential. The establishment of cell lines with high or low metastatic potential is necessary to investigate the molecular mechanisms of the metastatic process. However, human oral squamous cell carcinoma (SCC) cell lines that are suitable for the above investigation are scarce. High and low metastatic cells were obtained from a primary lesion of a patient with tongue carcinoma who had not received any therapy. Two distinct cell lines were selected, UM1 with a scattered growth pattern and loose cell-cell adhesion, and UM2 with a colony-formed growth pattern and firm cell-cell adhesion. The expression of E-cadherin in UM1 was clearly lower than that in UM2. UM1 exhibited a higher motility, invasive and metastatic activity than UM2 in vivo and in vitro. A low invasive and a metastatic oral SCC cell line, useful to investigate invasion and metastasis mechanisms, have been established.     

Collagenases in human breast carcinoma cell lines

The intense stromal response to some human tumors is termed the desmoplastic reaction. It is found with most human breast carcinomas. Dissolution of this response, containing predominantly fibrous proteins such as collagen and elastin, can occur with treatment. We have undertaken a study of the collagenases of the breast tumor desmoplastic reaction using a tissue culture model composed of human breast tumor cell lines and various human fibroblasts. The breast tumor cells had the higher collagenase activity, particularly the ZR75-31A cell line. Activity was 10-fold higher than that of the stromal cells. The enzyme was secreted into the media and required trypsin pretreatment for activity to be manifest. Partial purification was achieved of the major collagenase species. The protein was a metalloprotease and, like other mammalian collagenases, had a relative molecular weight of 60,000. Classical 3/4 and 1/4 cleavage products of the triple helical collagen substrate were demonstrated, typical of most mammalian collagenases. Only types I and III collagens were suitable substrates for this enzyme, with no apparent preference between the two. The breast tumor collagenases were not responsive to hormones; however, stimulation of activity was apparent in the absence of proteolytic pretreatment. This may represent conversion of the procollagenases of the breast tumor cells to the active form by an estrogen-sensitive plasminogen activator secreted by the same tumor cells.     

Intrapleural administration of CDDP against malignant pleural effusions in breast cancer

Malignant pleural effusion in the patients with breast cancer commonly occurs, and is a life-threatening factor. The present paper shows the usefulness of intrapleural administration of CDDP in six cases. A decrease of pleural effusions was observed in all cases. Treatment was effective in two cases of CR and four cases of PR. A median survival from initiation of intrapleural therapy is 17 months (range 2-47 months). This procedure produced distinctly fewer side effects than intravenous administration. The results of this trials suggest that CDDP should be considered as an active agent in the treatment of malignant pleural effusion in the patients with breast cancer.     

Elastases in human breast carcinoma cell lines

Elastosis, the deposition of large amounts of elastin, is characteristic of the desmoplastic reaction to human breast carcinoma. Dissolution of the elastin often occurs following treatment regimens that involve steroid hormones or their antagonists. Elastinolytic activities must be invoked to account for the loss of this elastin-cotaining stroma. We have utilized a tissue culture model to explore the molecular aspects of this phenomenon. The elastases of several human fibroblast and breast carcinoma cell lines were examined. The tumor cells had 10- to 30-fold higher elastase activity than did the fibroblasts. Three separate elastinolytic activities were observed in the tumor cell lines, and partial purification was achieved. The effect of steroid hormones on these elastases was examined. No stimulation of activity was found with any of the hormones, in any combination. However, there was marked inhibition of elastase with estradiol, progesterone, and dexamethasone of the ZR75-1 cell line. This is the estrogen receptor positive line that is estrogen responsive. The corticosteroids also inhibited the elastases of the estrogen receptor positive, non-responder cell line ZR75-30. No effect was seen on the elastases of receptor negative cells ZR75-31A with any of the steroid hormones. Stimulation of elastinolytic activities in these tumor cells must occur by some as yet unidentified pathway.     

Two cell lines with epithelial cell-like characteristics established from malignant fibrous histiocytomas.

Two malignant fibrous histiocytoma (MFH) cell lines were established: one from a storiform-pleomorph subtype and the other from a myxoid one (codes, MFH-3 and MFH-4). Light microscopic examination revealed large rounded cells, growing mostly separately, in both cell lines. Their ultrastructure was different in various aspects. The MFH-3 cells showed abundant lysosomal activity, a well-developed Golgi apparatus, and a few desmosome-like cell contacts. The MFH-4 cells had a well-developed rough endoplasmic reticulum, delicate bundles of tonofilaments, the formation of pseudoacini, and the presence of small completely developed desmosomes. Based on immunostaining and immunoblotting assays of cultured cells, both cell lines expressed immunoreactivity for vimentin; cytokeratins 7, 8, and 18; desmin; and laminin, but they lacked reactivity for cytokeratins 10 and 19, neurofilament, alpha-smooth muscle actin, S-100 protein, collagen type IV, carcinoembryonic antigen, and antigens specific for macrophages. Fibronectin and, to a variable extent, glial fibrillary acid protein and epithelial membrane antigen (EMA) were detectable in MFH-3 cells only. Furthermore, a 60-kilodalton band was present in both cell lines which was reactive for cytokeratins 8 and 18. The MFH-3 cells had the capacity to grow as xenografts with a carcinoma-like pattern. The cells retained their immunoreactivity for vimentin and cytokeratin 8 and showed the presence of desmosomes. Several of these immunophenotypic features also were noticed in established sarcoma cell lines and in short-term cultures of fibroblasts, smooth muscle cells, and endothelial cells. However, experimental data on the two MFH cell lines show that the MFH cell line may express some immunophenotypic and ultrastructural features considered to be specific for epithelial cells. The MFH cells may originate from multipotential mesenchymal cells with a capacity to differentiate to fibroblast-like cells, and less frequently, to epithelial cells, smooth muscle cells, and Schwannian cells. Such a differentiation became evident when these cells were adapted to culture conditions or grew in nude mice.     

Biologic properties of three newly established human esophageal carcinoma cell lines

Three epithelial cell lines, CE-48T/VGH, CE-69T/-VGH, and CE-81T/VGH, were established from human squamous cell carcinoma of the esophagus. The cells were polygonal with a high nucleus-to-cytoplasm ratio. Many cells were multinucleate. Electron microscopy revealed the presence of tonofilaments and desmosomes. Chromosome analysis showed that these 3 cell lines were heteroploids of human origin. When transplanted into BALB/c (nu/nu) mice, CE-69T/VGH and CE-81T/VGH produced tumors, the histology of which proved to be carcinomas. All 3 cell lines secreted carcinoembryonic antigen. However, the secretion patterns were different. These 3 cell lines may provide useful models for the study of human esophageal cancer.