Characterization of two newly established cell lines derived from squamous cell carcinomas of the oesophagus.

This study describes characteristics of two newly established cell lines (OSC-1 and OSC-2), derived from two oesophageal squamous cell carcinomas. Morphologically, OSC-1 cells and OSC-2 cells grew in epithelial cobblestone patterns with cells piling up to 4 cells. Ultrastructurally, both cell lines showed formation of desmosomes; however, tonofilaments were only formed by OSC-2 cells. Immunohistochemical investigations revealed coexpression of vimentin and cytokeratin in OSC-1 cells and OSC-2 cells. A cytokeratin subtype typical for mature squamous epithelia (cytokeratin 13) was expressed only in OSC-2 cells. OSC-1 cells showed tumour formation in nude mice, whereas OSC-2 cells did not. Cytogenetic analysis revealed that OSC-1 cells had a hyperdiploid karyotype and OSC-2 cells had a near-triploid karyotype. In both cell lines, the formation of multicellular spheroids could be induced. In conclusion, in comparison with OSC-2 cells the OSC-1 cells were characterized by a poorer degree of differentiation and by a more aggressive growth behaviour in vivo.

Establishment and characterization of four cell lines derived from human head and neck squamous cell carcinomas for an autologous tumor-fibroblast in vitro model.

To study interactions between tumor cells and stromal elements, we established carcinoma cell lines as well as tumor-derived and skin fibroblast cultures from four patients with squamous cell carcinoma of the head and neck. For the characterization of the tumor cell lines we a) determined population doubling times, b) assessed morphological features by light and electron microscopy, c) investigated the expression of typical markers by immunohistochemistry, including various intermediate filaments and surface antigens, d) compared these findings with expression patterns in the respective original tumor specimens, e) evaluated p53 mutations in tumor specimens and cell lines, f) performed chromosome analysis, g) investigated the tumorigenicity in athymic mice, and h) tested the formation of both tumor and mixed tumor-fibroblast multicellular spheroids. Tumor cell cultures were considered established cell lines when maintained and passaged over a period of two years after primary explantation. The in vitro morphology of the cell lines showed well preserved characteristics of squamous cell carcinoma, and electron microscopy as well as immunohistochemistry revealed their squamous type of differentiation. All cell lines presented the same p53 genotype as the respective original tumors. Furthermore, they were successfully xenotransplanted into nude mice and formed both pure and mixed three dimensional spheroids. This experimental model allows the in vitro and in vivo investigation of various tumor-fibroblast interactions.

A novel human malignant fibrous histiocytoma cell line of the heart (MFH-H) with secretion of hematopoietic growth factors.

Primary malignant fibrous histiocytoma (MFH) exhibits an extremely adverse prognosis. Investigations into the principles determining the biological aggressiveness of this cardiac tumor would be facilitated by an appropriate in vitro model. Therefore, we report on the first permanent cell line (MFH-H), derived from a human cardiac MFH. The original tumor had shown coexpression of cytoskeletal filaments typical of mesenchymal (vimentin), epithelial (cytokeratins) and neurogenic (neurofilaments) differentiation. This potential for multidirectional differentiation was observed in the MFH-H cell line as well and indicated marked plasticity of gene activation acquired during the process of neoplastic transformation. Pronounced genetic alterations also became evident from cytogenetic analysis, which revealed a highly variant karyotype with multiple numeric and structural chromosomal aberrations. Secretion of G-CSF, GM-CSF and M-CSF was shown to be another feature of deregulated gene expression in MFH-H cells. Direct autocrine effects of their hematopoietic growth factors, however, were precluded by the lack of the corresponding receptors. In conclusion, the cell line MFH-H will provide an appropriate in vitro model to analyze the biological properties of this cardiac malignancy in more detail, especially with regard to a possible immunomodulating capacity of MFH-derived hematopoietic growth factors.

In vitro and in vivo organ cultures containing respiratory epithelium.

A combination of two methods, organ culture and transplantation under the kidney capsule of nude mice, can be a suitable tool to study the mechanism of carcinogenesis of the human respiratory epithelium and to detect risks in environmental pollution. We used two tissues containing the respiratory epithelium--rat lung and human vocal cord. Gelita, a porous material used in surgery for haemostasis, produces a good support for the growing organ cultures of both tissues. These tissues continue to grow and to differentiate after transplantation under the kidney capsule. No pronounced immunological reaction of the nude mouse was detected. These conditions not only allow us to test various carcinogenic substances and their combinations, but as well enable us to detect an eventual malignant growth.

Dual role of fibroblasts in tumor cell growth.

Vast experience with cultivating biopsies from human tumors indicates that in most cases the admixture of fibroblasts has a negative effect on growth of tumor cells. Only rarely is observed help provided by the fibroblasts. It has also long been known that fibroblasts can inhibit by contact themselves and also produce growth factor(s) promoting cell proliferation. We have used three permanent squamous cell carcinoma lines and fibroblasts derived from biopsies of trachea to study this paradox. The inhibitory activity of fibroblasts is contact-dependent in a cell membrane-bound factor, which is trypsin sensitive. We prepared microsomal fractions (MF) from aged (more than 40 passages) fibroblasts and followed their effect on proliferation of the tumor cell lines in MTT assay. MF from all three fibroblast lines inhibited the tumor cells. Most regularly was this phenomenon observed with line UM-SCC 22B. Not all tumor cells are immortal. On the contrary, a large portion of them undergoes terminal differentiation. With the line UM-SCC 22B we tested the possibility that MF from fibroblasts can increase the portion of tumor cells which will senescence after few mitoses. The immortal cells were defined as cells capable in 8 or 13 days of forming colonies of more than 50 or 200 cells. The senescent cells were defined as cells not capable of producing within the same period of time colonies of more than 12 or 30 cells. The MF was able to increase the number of small colonies, i.e. the number of senescent tumor cells. The fibroblasts of the same passage level were releasing soluble growth factor(s) promoting growth of cells. Fibroblasts can apparently simultaneously inhibit growth by contact and release factor(s) promoting growth of cells. The final outcome is a result of the balance between these two forces. This balance is regulated by many intrinsic and extrinsic forces.

Effect of lymphokine-activated killer cells on head and neck tumours in nude mouse model.

The present study was designed to investigate the in vivo effect of local application of lymphokine-activated killer (LAK) cells on the growth of tumours implanted under the renal capsule in nude mice, and especially to test whether large granular lymphocytes (LGL), regarded as natural killer (NK) cells, are the main precursor of LAK cells in vivo. Our results showed that the local application of LAK cells inhibited the growth of tumours in the head and neck region. The growth of tumours implanted under the renal capsule was inhibited by local application of 1 x 10(7) recombinant interleukin-2 (rIL-2) activated non-adherent lymphocytes, but the inhibitory effect was almost the same as produced by 3 x 10(6) rIL-2-activated LGL application. The findings indicate that the rIL-2-activated LGL are the main effectors in inhibiting tumour growth. In addition, rIL-2-activated non-adherent lymphocytes as well as LGL significantly prolonged the number of days of 50% survival and mean survival time of nude mice, in which HLaC78 cells, from a laryngeal tumour cell line, were injected into the subrenal capsule space with effector cells at various effector: target (E:T) ratios. The results indicate that the application of LAK cells may be useful in the treatment of patients with head and neck tumours.

[Establishing three dimensional tumors in vitro and the reaction of the lymphokine-activated killer cells (LAK) to the three dimensional tumors]. / Etablierung dreidimensionaler Tumoren in vitro und Wirkung von lymphokin-aktivierten Killer (LAK)-Zellen auf die dreidimensionalen Tumoren.

In this study, we attempted to develop a technique, by which three-dimensional tumors were produced from two cultured head and neck tumor cell lines (Hep2, KB) and a colon adenocarcinoma cell line (HT29) using fibrinogen, thrombin, and double layered agar system. The three-dimensional tumor was large enough to perform the histologic study, which showed no significant histologic difference in comparison with the histologic findings of the xenografted tumor on nude mice. Furthermore, we applied this assay model to evaluate the antitumor effect of lymphokine activated killer (LAK) cells on the three-dimensional tumor produced by the technique. When tumor cells were cocultured with LAK cells, the damage of the three-dimensional structure due to the degeneration of tumor cells was observed. These findings suggest that the three-dimensional tumor may be useful to evaluate the antitumor effect of LAK cells in term of head and neck solid tumors.

[Expression of cellular antigen of hypopharyngeal carcinoma in different culture conditions]. / Expression von zellulären Antigenen eines Hypopharynxkarzinoms unter verschiedenen Kulturbedingungen.

BACKGROUND: The need to improve therapy regimes, determine prognosis, and study biological properties of tumors extracorporally led to development of different experimental systems. In order to approach the in vivo situation, specific properties of the tumors of origin should be retained by the cells in culture over relatively long periods. However, culture conditions may change expression of cellular antigens. METHODS: Cryosections of a hypopharyngeal carcinoma were compared in this respect with different cultivation systems (2-dimensional monolayers [ML], 3-dimensional multicellular tumor spheroids [MTS] and substrate cultures on Gelita) in regard to expression of cytokeratins (CK) 1, 7, 10, 14, 18 and 19, vimentin, neurofilament (NF) kD200 and 68, ganglioside GD2, oncogene products (P53 mutant and wild), and membrane-associated antigens (HLA-ABC and -DR, epidermal growth factor receptor EGFR). RESULTS: Semiquantitative immunohistochemical methods revealed differences in expression of CK1, 14 and 19, GD2, and P53 mutant between these systems. CONCLUSION: Pronounced expression of markers in MTS compared to original biopsy and monolayer emphasizes the importance of cell-cell contact and 3-dimensionality or metabolic stress. However, weak marker expression within substrate cultures may reflect loose cell-cell contact observed. In these experiments, the antigenic configuration of MTS resembled the one of the original tumor more than the other culture systems: monolayer and growth on substrate. As vimentin and NF are not expressed by healthy epithelial cells of adults, occurrence of intratumoral vimentin and NF could point to derepression of early differentiation antigens.

Induction of transformation of human respiratory epithelium in vitro. Preliminary investigation.

Malignancy is the result of multistep transformational changes of normal somatic cells. In the case of respiratory epithelial malignancies this process lasts for several years. Many methods have been explored to mimic this process in an extracorporal model. In the present investigation we combined several of these methods. Organ cultures were prepared from tracheal specimens and were then consecutively treated with human papilloma virus, benzo(a)pyrene, methylnitronitrosoguanine and tetradecanoyl phorbol acetate. Identical numbers of organ cultures from the same specimen were maintained without exposure to carcinogens. After 6 weeks these cultures were further cultivated either in mixed cultures (MC) with autologous isotopic fibroblasts or under the kidney capsule of the nude mouse (SRC). These two methods were combined after a few months: MC cells were transplanted under the SRC or SRC transplants were explanted in cell culture. This long-term selection procedure revealed striking differences between control and treated organ cultures. Three-dimensional structures containing epithelial cells were isolated from both organ cultures but survived more than 3 months only from treated cultures. Only MC from treated organ cultures produced nodules under SRC. The incidence and morphology of the nodules in the SRC were directly related to carcinogen treatment, with more nodules with pronounced epithelial cell atypia obtained from treated organ cultures. MC and SRC showed the importance of a time factor for selecting cells with changed growth behavior--increased time increased the incidence of such cells.

[Morphologic and immunohistochemical studies of organ cultures of human tracheal biopsies]. / Morphologische und immunhistochemische Untersuchungen an Organkulturen humaner Trachealbiopsien.

Most likely the transformation of epithelial cells to carcinoma cells takes place during the process of differentiation. In order to study in vitro carcinogenesis, an experimental system for organ cultures was developed in which human respiratory epithelial from tracheal biopsies differentiate within six weeks. The mucosal and submucosal layer of small tracheal biopsies was cut into pieces measuring 3 x 3 and 5 x 5 mm2, respectively, and placed on Gelita cubes (Braun-Melsungen, Germany) measuring 10 x 10 x 10 mm3 with the epithelium facing up. The culture medium (RPMI 1640 supplemented with 10% fetal calf serum, 200 mM L-glutamine, 100 IU/ml penicillin, and 100 micrograms/ml streptomycin) was added in a way that the tissue lay between the medium and air. Gelita deteriorates in about two to three weeks. However, the cultures are easily transferred to fresh Gelita cubes using the rest of the old Gelita. These organ cultures were fixed at regular intervals, and histological sections were stained with hematoxylin eosin or monoclonal antibodies against cytoskeletal intermediate filament proteins. These morphological and histological studies revealed that the epithelial cells differentiated under these conditions in at least 39 days. The mesenchymal elements remained viable without showing strong proliferation. The implication of these techniques for studying carcinogenesis in vitro is discussed.