Establishment and characterization of human pancreatic adenocarcinoma cell line SOJ producing carcinoembryonic antigen and carbohydrate antigen 19-9

A new tumor cell line derived from a human pancreatic exocrine adenocarcinoma was established in tissue culture and was transplantable in a nude mouse. In tissue culture, the neoplastic cells grew as epithelial-like, mucin-producing cells with a population doubling time of 50-70 hrs. Chromosomes ranged from 63 to 186 with a modal number of 77. Subcutaneous injection of 1 x 10(6) cultured neoplastic cells into nude mice resulted in tumor formation histologically closely resembling the original neoplasm. Ultrastructurally, the cell line showed characteristic ductal epithelium. Immunohistochemically, carcinoembryonic antigen (CEA). Carbohydrate Antigen 19-9 (CA19-9) and DU-PAN-2 antigen were demonstrated in the original tumor, the culture cells and the transplanted tumor. The cells secreted CEA (48.7 ng/1 x 10(5) cells/24 hrs) and CA19-9 (325 U/1 x 10(5) cells/24 hrs) in spent medium as well as sera of the nude mouse. This cell line has been passaged 30 times in vitro and maintained for more than one year. These characteristics will make the cell line SOJ a valuable tool in studying various aspects of biology of human pancreatic cancer.     

Establishment of the human hepatocellular carcinoma cell line and its characteristics

c-Hc-4 has been established and maintained for more than seven years. The hepatocellular carcinoma originated in 45-year old man with liver cirrhosis. The cell grew in vitro forming a sheet of monolayered cells and firmly attaching to the inner surface of cultured flasks. Morphologically they showed epithelial-like pattern. The doubling time was about 20 hours. Their modal chromosome number was 58. Serial heterologous transplantation in nude mice was successful. The histological finding was almost the same patterns as those in the primary tumor. The cultured cells produced alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA).     

Establishment and characterization of a human chorionic gonadotropin (hCG) producing cell line (RTSG) from an ovarian epithelial cancer

A new cell line designated RTSG established in vitro from the pleural effusion of a patient with metastatic ovarian epithelial cancer has been subcultured 46 times for more than 2 years. The cells grew in a monolayered sheet, showing a tendency to pile up, with the population doubling in 48 hrs. Electron-microscopically, desmosomes were characteristically observed, suggesting the cells were of epithelial origin. Chromosomal analysis revealed aneuploidy with a tetraploid mode. The heterotransplanted tumors in nude mice were histopathologically classified as a poorly differentiated adenocarcinoma, whereas the original tumor consisted mainly of mucinous and serous cystadenocarcinoma and only partly of poorly differentiated adenocarcinoma. The cells secreted hCG (38.8 mIU/day/10(6) cells) and beta-hCG (6.1 ng/day/10(6) cells) in spent medium. Immunocytologic +-and-histochemical staining for tumor markers of the original tumor, the cultured cells and the transplanted tumors also revealed the localization of not only hCG and beta-hCG but also CA19-9 and CA-125 whose values had been elevated in the preoperative serum (hCG: 10 mIU/ml, CA19-9: 6,400 U/ml, CA-125: 225 U/ml). Results of PAS, Alcian-blue and Mucicarmine strains indicated that most of the PAS-positive substances in the cultured cells and the transplanted tumors were consistent with glycogen while the original tumor mainly contained mucin except for the lesion of poorly differentiated adenocarcinoma with glycogen. These results suggested that the cultured cells might originate from poorly differentiated adenocarcinoma cells in the original tumor.     

Establishment and characterization of strain KE-24 derived from human colonic cancer

Strain KE-24 of colonic cancer cells was established from human colonic cancer diagnosed histopathologically as poorly differentiated adenocarcinoma. Doubling time of the cancer cell line was 32.4 hrs, and the karyotype was 46, xy, t (1q:?), 6p-, 14q+. The cancer cells could be heterotransplanted in 66% of nude mice. The plating efficiency was 17% in a soft agar plate with an inoculum size of 1 x 10(3) cells/dish. The tumor cells produced and released CEA and CA19-9 in the spent medium and these cancer cells were stained with both anti-CEA and anti-CA19-9 antibodies by the immunohistological staining method. Strain KE-24 of the cancer cells could be cultured in the serum-free medium (Media-I) for more than 100 passages.     

Large-scale preparation and characterization of human colony-stimulating factor

In order to obtain human granulocytic colony-stimulating factor (G-CSF) in large quantities, a large-scale culture system of human G-CSF-producing cells has been established. The cell used for this system was T3M-1, which grew in a monolayered sheet in F-10 synthetic medium supplemented with 10% fetal bovine serum. T3M-1 cells grew in rolling bottles at the velocity of 0.5 r.p.m. with about 22 hr. of population doubling time. When the culture reached confluency, it was incubated in a serum-free medium supplemented with 1% bovine serum albumin. The conditioned medium was harvested every week, concentrated by Amicon PM-10 membrane, and loaded on a Sephadex G-75 column. The molecular weight of G-CSF was estimated at about 30,000. This G-CSF was stable over a pH range of 1.0 to 11.0 at 4°C for 21 hr. The CSF activity was destroyed by either trypsin or chymotrypsin, but resisted to RNase and DNase. A slight decrease in the activity was produced by treatment with neuramidase. G-CSF stimulated granulocytic colony formation of human and mouse marrow cells. By using the roller bottle culture system, we could obtain more than 100 liters of cultured medium in a month, which was able to form about 150,000,000 colonies of human bone marrow cells. The recovery of the human G-CSF activity from gel-filtration column was very high (91.7%), and a large increase of specific activity was obtainable (13.3-fold). This culture system is therefore expected to aid in the large-scale preparation of human G-CSF, thereby facilitating further studies on this granulopoietic factor.     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.     

Establishment and characterization of a new human colon adenocarcinoma cell line: BCS-TC2

A new cell line designated as BCS-TC2 was established in culture from a primary human colon adenocarcinoma. This cell line has been in continuous culture over a 36-month period. The cells grow as a monolayer sheet, displaying areas with a multilayered pattern as well as single cells and free-floating aggregates. The morphological, immunological, and ultrastructural features of these cells are in agreement with their epithelial origin. The characterization of this cell line indicated a 38 hr doubling time, and a colony forming efficiency of 2% in semisolid media and 22% in liquid culture, at low cell densities. These cells produce low amounts of carcinoembryonic antigen in culture (0.1 ng of CEA/10⁶ cells). Sub-cutaneous injection into athymic mice shows that these cells have a non-tumorigenic capacity. Chromosomal analysis showed a karyotype 46 XX,-15, +der (15), inv (16) (p13::q13). BCS-TC2 cell line, which maintains in culture several characteristics of the original tumor, represents a useful model system for cell biology studies of primary and non-metastatic tumors.     

Establishment and characterization of a human colonic cancer cell line expressing carbohydrate antigen 19-9 and carcinoembryonic antigen]

Colonic cancer cell strain KE43 was established from a human colonic cancer diagnosed histologically as a predominantly well differentiated adenocarcinoma with minute foci of poorly differentiated adenocarcinoma. The well differentiated adenocarcinoma cell line was identified as the major morphological picture in xenografts of KE43 and 58 in nude mice, but this changed to poorly differentiated adenocarcinoma in passage 105. Doubling time of this cancer cell line was 22.5 hours in passage 105. The modal numbers of chromosomes were 41 and 76. Cancer cells could be heterotransplanted in 100% of the nude mice. The tumor cells produced and secreted CA19-9, CEA and Laminin into the spent medium. This cell line appears to provide a useful system for studying colonic cancer in vivo and in vitro.     

Establishment and characterization of human cholaginocarcinoma, MEC, producing carbohydrate antigen 19-9

A new tumor cell line MEC was established from pleural effusion of a patient of cholaginocarcinoma. In tissue culture, the cell line grew in the sheet of variant cells and showed the epithelial-like pattern. Histologically, the cell line almost showed the same pattern as those in bile and preural effusion from the patient. Electron microscopic observation of this cell line showed the irregular microvilli on the surface of the cell and the desmosome between cells. The doubling time of the cell line was 40.8 hours. Chromosome counts ranged from 61 to 86. The cell line had 9 marker chromosomes and some variant chromosomes. The cell line was transplanted into the subcutaneous of nude mice and formed the tumor. It showed the moderately differentiated tubular adenocarcinoma the same pattern as the primary tumor. We have recognized the producing and releasing of CA19-9 in the serum from the tumor bearing nude mouse and supernate of the medium as the serum from the patient. The presentation of CA19-9 in the cytosol of the cell line and the tumor cells of nude mouse was recognized in Avidin-Biotin-Peroxidase Complex in immunoloperoxidase techniques. The cell line can grow in serum-free medium. On September, 1990, the cell line has been maintained from 70 passages during about 800 days.     

Establishment of a CSF-producing cell line from a human gastric carcinoma and characteristics of the CSF produced by that cell line

A human cell line producing colony-stimulating factor has been established in vitro from a human gastric carcinoma. The cell line was transplantable into nude mice which developed a marked neutrophilia. The cell line has been maintained for three years. The cells grew in a monolayered sheet and produced colony-stimulating factors that enhanced the formation of granulocyte and monocyte colonies in vitro with mouse bone marrow cells as the target and granulocyte colonies with human bone marrow cells as the target.     

Establishment and characterization of two squamous cell carcinoma cell lines (HYVC and HMVC) derived from vulva

Two cell lines, HYVC and HMVC, were established from cultures of the squamous cell carcinoma removed from the vulva of females of 37 and 46 years old, respectively. These cell lines were very similar in their biological characteristics, such as morphology (polygonal), growth pattern (32-43 hr of population doubling time, 50-25% of plating efficiency), heterotransplantability (1 x 10(7) cells produced squamous cell carcinomas in the nude mice), genetics (75-78 of modal chromosomal number), and producing the tumor markers (SCC and TPA). The HPV-DNA was not detected in either the HYVC or HMVC lines by using L1-PCR methods, however, it was detected in the HYVC using E6/E7-PCR.     

Establishment and characterization of a cell-line originated from human mucinous cystadenocarcinoma of the ovary

We recently established a cell line (designated 371M) derived from an ovarian mucinous cystadenocarcinoma. The tumor cells were obtained from the ascitic fluid of a 54-year-old Japanese woman while she was undergoing surgery. Adjuvant chemotherapy (combined paclitaxel and carboplatin) was administered, but was ineffective, and she died about 4 months after surgery. The 371M cells continuously propagated in vitro over a period of about 50 months and, to date, have undergone over 100 passages. They proliferated in a monolayered sheet with doubling times of 84 h and 37 h in the 10th and 34th passages, respectively. When transplanted into nude mice, the tumor histopathologically resembled the structure of the original tumor. The 371M cells secreted high levels of CA125 and CA19-9 into the culture medium. There were several abnormal chromosomes in all karyotypes selected at random. Sensitivity of 371M cells to a variety of anti-cancer drugs was examined by in vitro MTT assay, and the results suggested that CPT-11 and CDDP were more effective against 371M cells than other anti-cancer agents.     

Analysis of population cytokinetics of chick myocardial cells in tissue culture

The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.     

Effect of glutamine deprivation and glutamate or ammonium chloride addition on growth rate, metabolism and differentiation of human colon cancer cell-line HT29

Effect of glutamine deprivation (GLN- medium) and of its replacement by 4mM ammonium chloride (GLN-/NH4+ medium) or by 4mM glutamate (GLN-/Gt+ medium) was studied on growth rate, morphology and metabolism of HT29 human colon cancer cells. Growth rates were modified as follows: at the first passage, growth of GLN- cells was strongly decreased (doubling time: 192 hr vs 32 hr in control cells grown in GLN+ medium); GLN-/NH4+ cells and GLN-/Gt+ cells were found to have doubling times of 72 and 70 hr, respectively. At the 8th passage, doubling times were decreased in all cases, being: 144 hr for GLN- cells, 60 hr for GLN-/NH4+ cells and 24 hr for GLN-/Gt+ cells, which indicates a capacity of adaptation of the cell-line to new culture conditions. GLN- cells and GLN-/NH4+ cells were found to exhibit an enterocytic type of differentiation (polarization of the cell layer with apical and cystic brush border and tight junctions); GLN-/Gt+ cells remained undifferentiated and comparable to control GLN+ cells. Glycogen level varied according to the phases of the culture, with a trend to lower level in glutamine deprived cells; glucose uptake and lactate production varied as a function of the medium composition and of the phases of the culture. At the 8th passage, all the glutamine deprived cells produced less lactate than control; GLN-/Gt+ cells were found to utilize less glucose than others.     

Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines

Six new non-small-cell lung cancer (NSCLC) cell lines were established directly from human tissue or indirectly via nude mouse xenografts in serum-supplemented media with success rates of 8% and 13%, respectively. They comprised one adenocarcinoma (ADLC-5M2), two squamous cell carcinomas (EPLC-32M1, EPLC-65H), two large cell carcinomas (LCLC-97TM1, LCLC-103H), and one malignant biphasic mesothelioma (MSTO-211H). All cell lines grew adherent to culture vessels with population doubling times (PDT) of 16-40 h, formed colonies in soft agarose with efficiencies of 0.1%-5.1%, and all grew in athymic nude mice. Xenograft histologies appeared as follows: (a) undifferentiated carcinomas with feeble resemblance to the original tumors in the case of adenocarcinomas and squamous cell carcinomas; (b) large cell carcinoma with high resemblance to the original tumor; (c) an undifferentiated tumor with predominance of large epithelial cells and few fibrous cells in the case of mesothelioma. Human chorionic gonadotropin (HCG) was found by radioimmunoassay and high-affinity binding sites for epidermal growth factor (EGF) by radio-receptor assay in 4/4 cell lines. A very low activity of L-DOPA decarboxylase (DDC) was detectable only in the adenocarcinoma cell line. All cell lines overexpressed the c-myc protooncogene, and no gene rearrangement or amplification was observed. Chromosome analysis revealed modal chromosome numbers of 70-73 in ADLC-5M2, EPLC-32M1, EPLC-65H, and MSTO-211H. Cell lines derived from large cell carcinoma had modal values of 65 and 170 and a wider chromosome distribution than all other cell lines. A NSCLC specific chromosomal aberration has been undetectable until now. These cell lines may aid in elucidating the biology of NSCLC and its interrelationship to other lung tumors.     

Type-2 somatostatin receptor mRNA levels in breast and colon cancer determined by a quantitative RT-PCR assay based on dual label fluorogenic probe and the TaqMan technology

We reported previously that the expression of type 2 somatostatin receptor (sst2) was positively related to patient outcome in the childhood tumor neuroblastoma. To quantitate the expression of mRNA sst2 expression, we used a competitive RT-PCR assay. To improve the practicability of this measurement and its applicability to large groups of patients, we present here an original 'real-time' quantitative RT-PCR method, based on a dual-labeled fluorogenic probe and the TaqMan technology. By this method, we have measured sst2 mRNA expression in 24 breast cancer samples and 26 colon carcinomas as well as on the corresponding non-adjacent non-neoplastic tissue of the same patients. The proposed method has a dynamic range of 4 x 10(4) to 4 x 10(8) molecules of sst2 mRNA. The intra-assay precision of the test, evaluated as signal detection variability, was 2.4%. Accuracy, evaluated by the addition of standard RNA to unknown samples, provided a mean recovery of 98+/-2%. A significant correlation has been observed in a study performed in 24 neuroblastoma samples measured both with the proposed method and with a competitive RT-PCR assay (r=0.913, p<0.001). In our preliminary clinical study, no significant differences were observed in sst2 mRNA levels between normal and tumor specimens in both colorectal (normal tissue 5.1 x 10(7)+/-2.0 x 10(7) molecules/microg total RNA, cancer tissue 9.7 x 10(7)+/-4.2 x 10(7)) and breast tumors (normal tissue 5.5 x 10(8)+/-2.0 x 10(8), cancer tissue 4.4 x 10(8)+/-3,7 x 10(8)).However, in colorectal cancer, sst2 mRNA values of subjects with high circulating carcinoembryonic antigen (CEA) levels (>5 ng/ml) were statistically lower (2.3 x 10(7)+/-6.2 x 10(6) molecules/, microg total RNA; p<0.05) than those of subjects with low CEA concentration (1.4 x 10(8)+/-6.7 x 10(7)). Also, the sst2 mRNA ratio between normal and tumor tissue (N/T ratio) resulted significantly inversely related to CEA levels.In breast cancer, a significant difference was found between the mean N/T ratio of negative (below 10 fmol/mg protein) and positive estrogen receptor tumors (p<0.05). Analogous results were found selecting breast tumors on the basis of the progesterone receptor status (p<0.05). The proposed method is accurate, precise, sensitive and less labor-intensive than the competitive RT-PCR assay. For a correct evaluation of sst2 mRNA expression, it seems very important to measure the sst2 expression both in tumor and in the non-tumoral non-adjacent tumor specimens.     

Induction of polarized apical expression and vectorial release of carcinoembryonic antigen (CEA) during the process of differentiation of HT29-D4 cells

HT29-D4 clonal cells can be induced to differentiate by a simple alteration of the culture medium, that is, by the replacement of glucose by galactose [Fantini, J., et al. (1986) J. Cell Sci., 83:235-249] as reported for the nonclonal HT29 cells [Pinto, M., (1982) Biol. Cell, 44:193-196]. An essential property of the HT29-D4 cell line is the fact that no cell loss occurs after the medium change, so that the differentiated cells can be considered as the true counterpart of the undifferentiated one. This model is particularly suitable to study morphological and biochemical events associated with the progressive establishment of the differentiation state. We report here that carcinoembryonic antigen (CEA), a 180 kDa glycoprotein originally described as a colon tumor associated antigen, is faintly expressed at the surface of undifferentiated HT29-D4 cells. These cells release a small amount of CEA (2.5 ng/10(6) cells/24 hr) in the culture medium. Fourty-eight hours after glucose substitution by galactose, both CEA cell surface expression and release are strongly enhanced as demonstrated by immunofluorescence and immunoprecipitation studies. Ten days after the medium change, the amount of CEA released reaches a maximum value of 130 ng/10(6) cells/24 hr, which remains stable for differentiated HT29-D4 cells cultured in glucose-free, galactose-containing medium (Gal-medium) for several months. HT29-D4 cells grown in Gal-medium in porous-bottom culture dishes generate leakproof epithelial monolayers. We have successfully performed an independent radioiodination of the apical and basolateral domains of these cells, followed by immunoprecipitation. We demonstrate that CEA is expressed exclusively at the apical surface of differentiated HT29-D4 cells, since the 180 kDa polypeptide was immunoprecipitated only when the radioiodination was performed at the apical side of the monolayer. Leakproof HT29-D4 monolayers cultured in permeable chambers were also used to demonstrate that CEA was exclusively released in the medium bathing the apical side of the cells. In conclusion, this study of cell surface CEA expression and CEA release during the process of differentiation of HT29-D4 cells demonstrated that 1) CEA cell surface expression and CEA release are correlated with cell differentiation; 2) CEA is expressed in the apical brush border membrane of differentiated HT29-D4 cells; and 3) CEA release is exclusively oriented toward the apical side of the polarized monolayer.     

Signal amplification in molecular imaging by pretargeting a multivalent, bispecific antibody

Here we describe molecular imaging of cancer using signal amplification of a radiotracer in situ by pretargeting a multivalent, bispecific antibody to carcinoembryonic antigen (CEA), which subsequently also captures a radioactive hapten-peptide. Human colon cancer xenografts as small as approximately 0.15 g were disclosed in nude mice within 1 h of giving the radiotracer, with tumor/blood ratios increased by >or=40-fold (approximately 10:1 at 1 h, approximately 100:1 at 24 h), compared to a (99m)Tc-labeled CEA-specific F(ab') used clinically for colorectal cancer detection, while also increasing tumor uptake tenfold ( approximately 20% injected dose/g) under optimal conditions. This technology could be adapted to other antibodies and imaging modalities.