Immunophenotyping and metastatic localization of breast cancer.

Monoclonal antibody SM 92 is involved in the immunophenotype of gastrointestinal and liver cells, SM 43 in ovarian cells, and SM 13 in lung cells. Based on a study of 61 breast adenocarcinoma patients, we found that tumors reacting with SM 92 appear associated with liver metastases, SM 43 with ovarian metastases, and SM 13 with lung metastases. These associations are highly significant. They lend some support to the concept that tumor cells that metastasize tend to go to sites where cells normally have the same surface antigens.

Establishment, characterization, chemosensitivity, and radiosensitivity of two different cell lines derived from a human breast cancer biopsy.

In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro.

Characterization of a new surface epitope specific for human epithelial cells defined by a monoclonal antibody and application to tumor diagnosis.

In an attempt to characterize the antigens attached to cells of a line established from a human squamous cell carcinoma of the tongue (CAL 27), BALB/c mice were immunized with whole CAL 27 cells; hybridomas were then produced using spleen cells of the animals and cells of an NS1 syngeneic myeloma. A hybridoma secreting a monoclonal antibody was obtained (CALAM 27); CALAM 27 was directed against an epitope attached to the CAL 27 cells. CALAM 27, IgG2a, reacted with a membrane antigen specific to all epithelial cells. After immunoprecipitation, this antigen corresponded to two bands (Mr 22,000 and 54,000). Reactivity disappeared when the tissue was embedded in paraffin but was conserved after fixation with acetone or methanol. This antigen was conserved for both benign and malignant epithelial cell pathologies. The action of CALAM 27 was tested on 80 samples of pleural effusions, ascites, and cerebrospinal fluid samples; after conventional cytological examinations, CALAM 27 failed to recognize either reactive mesothelial cells or meningothelial cells. In addition, the cell structure recognized by CALAM 27 is not found on certain lymphoid tissue cells. CALAM 27 also failed to react with small cell carcinoma of the lung. Its strictly epithelial specificity therefore permits its use for the diagnosis of micrometastases of carcinoma in ascites and cerebrospinal fluid, in pleural effusions, and in bone marrow. CALAM 27 may also prove useful in confirming diagnosis of pathologies suspected to be of epithelial origin.

Characterization of cal39, a new human cell-line derived from a vulvar squamous-cell carcinoma.

We have obtained a permanent cell line from a squamous cell carcinoma of the vulva. These cells, named CAL39, exhibited morphological, ultrastructural, and immunochemical characteristics of epithelial cells. They were tumorigenic in nude mice. Our observations indicate that the behavior of the cell line in the nude mouse and in culture is similar to that of the original cancer from which it was derived. We have compared these cells with A431 which are currently the best studied model cells of the same tumor origin. Like A431, CAL39 had an elevated number of high affinity EGF receptors, and showed an amplification of DNA sequences at 11q13. We have compared the cytogenetic features of the amplification units in the two cell lines. Unexpectedly, an HSR was present, in both cases, on a marker chromosome which was a derivative of chromosome 11. This result addresses the issue of the in situ amplification of chromosomal DNA.

A new epithelial membrane antigen (Calam 27) as a marker of carcinoma in serous effusions.

A new monoclonal antibody (Calam 27) that reacts with a membrane antigen present on cells of epithelial origin, but not on cells of mesothelial origin, was investigated as a means of distinguishing between mesothelial cells and malignant cells in cytologic smears of serous effusions from patients with carcinoma. Immunofluorescence staining of cells in 151 effusions from 109 patients with different diseases showed a good correlation between the cytologic diagnosis on routine preparations and the staining with Calam 27. Calam 27 was also used to study the ploidy and cell cycle kinetics of carcinoma cells versus reactive mesothelial cells and normal cells by flow cytometry; these experiments confirmed that Calam 27 is not reactive with mesothelial cells. In conclusion, Calam 27 staining can help to confirm the cytodiagnoses in cases with carcinomatous effusions.

In vitro clonogenicity in relation to kinetic and clinicopathological features of breast cancer.

The clonogenicity in soft agar and the labeling index (LI) which represents the percentage of cells in the DNA synthesis phase, were studied in 59 breast cancer patients and these parameters were related to other known clinicopathological features, namely age, histological grading, estrogen and progesterone receptors and the status of axillary lymph nodes. Out of 59 tumors, 49 could be successfully cloned in soft agar and the mean plating efficiency (PE) was 0.1%. Low grade tumors were more frequently encountered in tumors which did not form colonies (P = 0.025). Cloned tumors had a higher mean LI (P = 0.05). A high PE was associated with low estrogen receptors (ER) (P = 0.03). Clonogenicity was not related to patient age, progesterone receptors (PR) or the status of axillary lymph nodes. These results suggest that a successful in vitro cloning and a high PE are associated with unfavorable prognostic factors in breast cancer.

[CAL 54, a new cell line derived from a human renal carcinoma: characterization and radiosensitivity]. / CAL 54, une nouvelle lignée cellulaire dérivée d'un carcinome rénal humain: caractérisation et radiosensibilité.

A new cell line (CAL 54) was isolated from a malignant pleural effusion of a patient with renal carcinoma. CAL 54 is a continuous and stable cell line. Immunochemical staining showed simultaneous expression of cytokeratin, epithelial membrane antigen and vimentin. Cytometric flow analysis of DNA content reveals one major hyperdiploid population. Histological aspect of the tumor induced in the nude mouse showed well differentiated adenocarcinoma with papillary structure. Radiation response of these cells was evaluated by the colony-forming method and the data were fitted with the linear-quadratic model. Survival at 2 Gy (SF2) was 0.28 and the mean inactivation dose (D) = 1.50 Gy, ranking this cell line among the radiation sensitive cells. CAL 54 may be an informative cell line to investigate radiation effects in the management of renal tumours.

[Characterization of a human cell line from an anaplastic carcinoma of the thyroid gland]. / Caractérisation d'une lignée cellulaire humaine provenant d'un carcinome anaplasique de la thyroïde.

A new cell line derived from a thyroid anaplastic carcinoma, CAL 62, has been established in culture. This line is constituted by highly tumorigenic cells. Their epithelial phenotype is stable in culture. Immunochemical staining for human thyroglobulin is negative. Cytogenetic analysis showed a gain of chromosome 20, the translocation i (14q), and breakpoints of centrometric chromatine. These results are similar to those previously reported by other investigators. CAL 62 radiosensibility has been studied. The survival curve of the in vitro irradiated cells has been adjusted with a linear-quadratic model. This cell line is thus showed to be radioresistant. Cell line CAL 62 constitutes an appropriate model for in vitro studies of thyroid anaplastic carcinoma.

Establishment and characterisation of a new tumorigenic cell line with a normal karyotype derived from a human breast adenocarcinoma.

A new cell line (CAL51) was isolated from a malignant pleural effusion of a woman with metastatic breast cancer. These cells grow in continuous culture and exhibit the morphological, ultrastructural and immunohistochemical features of epithelial cells of mammary origin. They are tumorigenic in nude mice and clone in soft agar. Oestrogen receptors are not detected. CAL51 consists of a homogeneous population of cells with normal chromosomes even after the use of high resolution banding. Cytogenetic analysis of the cells from the tumour induced by CAL51 in the nude mouse confirmed the normality and the stability of the karyotype. All breast cancer cell lines established to date present abnormal karyotypes; CAL51 cell line may be more informative than cell lines with aberrant karyotypes for investigating essential genetic differences between normal and malignant mammary gland cells.

Two new human tumor cell lines derived from squamous cell carcinomas of the tongue: establishment, characterization and response to cytotoxic treatment.

Two new permanent cell lines derived from squamous cell carcinomas of the tongue, CAL 27 and CAL 33, have been established in culture. Both cell lines were isolated in standard culture media without epidermal growth factor or fibroblast feeder layer to avoid obtaining clones of more differentiated cells. Analysis of the morphology, ultrastructure, karyotype and immunohistochemical properties of these two cell lines demonstrated that they are both well characterized, uncontaminated by HeLa cells, and do in fact correspond to transformed epithelial cells that have conserved certain characteristics of the original Malpighian epithelium. CAL 27 and CAL 33 have relatively long doubling times (35 and 43 h respectively). Their response to 14 drugs used for cancer chemotherapy was evaluated by a short term assay based on tritiated thymidine incorporation after exposure to the drugs. CAL 27 was more resistant than CAL 33 in all cases but one. Although cytogenetic examination revealed both lines to be malignant, neither CAL 27 nor CAL 33 produced colonies in soft agar; both lines were tumorigenic after inoculation into nude mice. This study clearly demonstrates the diversity of cancers of a given histologic form, in agreement with the diversity noted previously in vivo. Isolated without the use of any selection criteria, these cell lines constitute appropriate models for the study of human tumors.

Establishment of two new human bladder carcinoma cell lines, CAL 29 and CAL 185. Comparative study of cell scattering and epithelial to mesenchyme transition induced by growth factors.

We describe here two new human urothelial carcinoma cell lines, CAL 29 and CAL 185, established from two patients with high-grade tumours and which display very different properties in vitro. We have shown that CAL 29 cells were tumorigenic in mice and expressed characteristic features of both cell scattering and transition from epithelial to mesenchymal phenotype (EMT) after triggering by the EGF receptor ligands, TGFalpha and EGF. At the opposite, the CAL 185 cells were not tumorigenic in mice and neither scattered nor expressed vimentin intermediary filaments in the presence of growth factors. We further demonstrated that CAL 29 cell scattering was reversible after growth factor removal and that both scattering and EMT were markedly impaired after treatment with MEK, Src and PI3-kinase inhibitors suggesting that these kinases might be important components of the cellular responses to EGF and TGF-alpha leading to scattering and EMT. These agents could help to understand the intracellular pathways involved in invasiveness and to find new targets for limiting metastasis. In conclusion, these two new cell lines could be good models to dissect the molecular mechanisms involved in invasion and metastasis development in human bladder cancer.

Establishment, characterisation and partial cytokine expression profile of a new human osteosarcoma cell line (CAL 72).

Permanent human osteosarcoma cell lines are important tools for the study of bone cancer. As representative of an osteoblastic phenotype, they partly reflect their normal osteoblastic counterparts and, thus, may represent appropriate models to investigate the mechanisms involved in bone remodelling and in haematopoietic differentiation. In the present work, we describe a new human cell line, CAL 72, obtained from an osteosarcoma of the knee of a 10-year-old boy. These cells grow in continuous culture, and karyotypic analysis has revealed clonal abnormalities in number and structure, especially loss of chromosome Y. These cells exhibit morphological, immuno-histochemical and molecular characteristics of the osteoblastic lineage. Using RT-PCR, we have shown that the CAL 72 cell line expresses high levels of mRNA coding for several cytokines, such as G-CSF, GM-CSF, IL-1beta and IL-6. In view of this expression profile, the CAL 72 phenotype appears to be closer to normal primary osteoblasts than other reported osteosarcomas. Moreover, these cells express mRNA for both HGF and its receptor c-MET, suggesting that this autocrine loop might contribute to the invasiveness of the tumour from which CAL 72 originated.