Spatial organization of three-dimensional cocultures of adriamycin-sensitive and -resistant human breast cancer MCF-7 cells.

Genetic and cellular heterogeneity is one of mechanisms involved in increasing tumour aggressiveness during neoplastic progression. Development of drug-resistant tumour cell subpopulations is a major problem in clinical oncology. Multi-drug resistant tumour cells survive when exposed to cytotoxic agents.

A method for three-dimensional coculture of cancer cells combined to any other type of cells maintained organotypically.

A three-dimensional cell coculture method is presented where cancer cells can be maintained alone or combined with other cell types in longterm culture in order to reconstitute some of the interactions between the different cell elements in tumors in vivo. The cells are accumulated by centrifugation to form 'nodules' which are cultivated on a semisolid agar medium at medium/air interface. The nodules are not mere cell aggregates, they are able to develop morphological and functional differentiation as well as tissue-like membrane junctions.

Interactions of lymphokine-activated killer cells and A549 lung carcinoma nodules maintained in three-dimensional culture.

Lymphokine-activated killer (LAK) cells were cocultured in the presence of interleukin-2 (IL-2), with pulmonary surfactant secreting A549 lung carcinoma nodules and maintained in continuous three-dimensional culture for 2-6 days in an attempt to test the response of tumor cells which produce LAK cell inhibitory substances. The A549 nodules secrete mucus which envelops them. This mucus is also secreted inside pseudoalveolar structures characteristic of these nodules.

Effects of lymphokine-activated killer-cells on melanoma nodules maintained in 3-dimensional culture.

Lymphokine activated killer (LAK) cells were cocultivated from 2 to 6 days with WM266 metastatic melanoma cells maintained as nodules in organotypic culture. The LAK cells in suspension were allowed to deposit freely on the nodule surface from where they could infiltrate spontaneously into the nodules. Immunohistochemical studies were done to localize the LAK cells as well as electron microscopical observations for effector/target membrane contacts.

Fibroblast-mediated differentiation in human breast carcinoma cells (MCF-7) grown as nodules in vitro.

The growth of cells in 3-dimensional form as nodules in vitro facilitates studies of in vivo cellular interactions. Taking advantage of this technique, human breast carcinoma cells (MCF-7) were co-cultured with stromal fibroblasts isolated from either normal or tumorous breast tissue to study the influence of such fibroblasts on tumor-cell growth and differentiation. Ten days after co-culture of carcinoma cells with fibroblasts from normal tissue at a 1:10 ratio, the size of nodules began to increase and stabilize by day 30 while the fibroblast number decreased and finally disappeared.

Lymphokine-activated killer cells induce differentiation in MCF-7 breast carcinoma nodules but not in mastosis nodules maintained in three-dimensional culture.

In order to better understand the interaction between activated lymphocytes and breast carcinoma cells, we studied the degree of infiltration, the membrane contacts established and their cytostatic and cytolytic effects in MCF-7 nodules maintained in three-dimensional culture. A comparison was made with nodules of a nonmalignant, immortalized mastosis cell line. Histological, immunohistochemical and electron microscopical observations were performed as well as DNA synthesis measurements in the two components of the coculture.

[Effects of LAK cells activated by IL-2 on MCF-7 human breast cancer cell line maintained in organotypic culture].

Lymphokine Activated Killer (LAK) cells, stimulated by interleukin 2 (IL-2) have a pronounced antitumor effect in the therapy of melanoma and renal cancers. LAK cells were cultivated in presence of the nodules of the human breast adenocarcinoma cell line MCF-7 maintained in organotypic culture to study the interactions between lymphocytes and breast tumor cells. After two days of co-culture, the proliferation of MCF-7 nodules and that of LAK cells was diminished about five folds.

Differential effects of butyrate derivatives on human breast cancer cells grown as organotypic nodules in vitro and as xenografts in vivo.

The antiproliferative and cytodifferentiating effects of a new stable butyric derivative, monobut-3, were compared using human MDA-MB-231 breast cancer cells grown in three dimension as either in vitro tumor nodules or in vivo xenograft tumors. In in vitro tumor nodules, monobut-3 exhibited marked growth inhibitory effects consistent with the results obtained in monolayer cell cultures. Some functional cell differentiation was also detected in treated nodules.

Molecular heterogeneity of somatostatin analogue BIM-23014C receptors in human breast carcinoma cells using the chemical cross-linking assay.

Distinct proteins complexed with somatostatin and the somatostatin analogue BIM-23014C were revealed in human breast cancer cells using the cross-linking assay. One BIM-23014C-specific complex (Mr 57,000) was observed in MCF-7 (monolayer, nodule, and tumor) and T47D. Growth inhibition of MCF-7 tumor xenografts by BIM-23014C was dose related in the 6-day subrenal capsule assay.

Mapping the cellular distribution of labelled molecules by SIMS microscopy.

We took advantage of one of the main possibilities of ion microscopy, ie isotopic analysis, to study the cellular distribution of molecules labelled either with carbon 14 or with stable isotopes of low natural abundance such as nitrogen 15 and deuterium. The surface of the sample is bombarded with an ion beam (O2+, Cs+ etc). Secondary ions emitted from the sample are filtered by a mass spectrometer and the distribution of the labelling isotope is recorded.

Effects of hypergravity on lung carcinoma cells maintained in continuous organotypic culture.

The effects of hypergravity levels ranging from 1 to 15 g were studied on A549 lung adenocarcinoma cell line, cultivated as nodules. This organotypic culture model preserves as closely as possible the cellular structures and differentiation functions of the in vivo situation. Nodules submitted to hypergravity conditions for 27 d did not show any change of cell growth, protein and DNA contents, compared with controls.

Short- and long-term synergistic effects of human tumor necrosis factor and interferon-gamma on A549 human lung cancer cells maintained in three-dimensional culture.

We have studied the short- and long-term effects of human recombinant tumor necrosis factor (TNF) and TNF/recombinant human interferon-gamma (IFN-gamma) mixtures on A549 human lung carcinoma cells maintained in organotypic culture. Continuous treatments with 2 x 10; 2 x 10(2); 2 x 10(3) and 2 x 10(4) U/ml TNF or with mixtures of TNF/IFN-gamma at 2 x 10(2) and 10(3) U/ml, respectively, were administered. Nodule growth, cell proliferation and cell survival were studied.

Increase of epidermal growth factor receptor expression associated with a lack of antiproliferative effect of IFN-beta in human lung cancer nodules in organotypic culture.

The possible relationship between the effects of alpha 2-, beta- and gamma-interferons (IFNs) on the growth of alveolar II pulmonary tumor cells (A549) maintained in tridimensional organotypic culture (nodules) and the modulation of epidermal growth factor receptor (EGF-R) expression was investigated. Treatment with rHu IFN-alpha 2 or IFN-gamma which results in the inhibition of the growth of A549 nodules had no effect on the binding of 125I-EGF to these cells. In contrast, treatment with rHu IFN-beta which exhibits no antiproliferative activity on A549 nodules resulted in a reproducible increase of the binding of 125I-EGF.

Human lung cancer nodules in organotypic culture: no evidence of correlation between the antiproliferative effects of interferons and the induction of 2',5'-oligoadenylate synthetase.

Alveolar II pulmonary tumor cells (A549), maintained in continuous tridimensional organotypic culture, were used in an attempt to investigate whether there could be a relationship of the 2',5'-oligoadenylate (2,5A) synthetase pathway to the antiproliferative activity of interferons (IFNs) in this particular tumor cell model. IFN-alpha 2, -beta and -gamma were used separately and in combinations. IFN-alpha 2 and -gamma demonstrated an inhibitory effect on the nodule growth, whereas IFN-beta did not.

Potentiation of antiproliferative activity by mixtures of human recombinant IFN-alpha 2 and -gamma on growth of human cancer nodules maintained in continuous organotypic culture.

Alveolar II pulmonary tumor cells (A549 cells) maintained in continuous tridimensional organotypic culture were used to evaluate the eventual potentiation effect of mixtures of recombinant human interferon-alpha 2 and -gamma on growth inhibition of the tumor nodules. A continuous 45 day treatment (interferon renewed three times a week) with 10, 10(2) or 10(3) U/ml of IFN-alpha 2 or -gamma combined with a fixed high dose (10(3) U/ml) of either IFN-alpha 2 or -gamma resulted in an additive or synergistic growth inhibition according to the doses used. There was a close dose-effect relation, the percentage of inhibition increasing proportionally to the variable IFN doses added to the fixed high dose; moreover, the growth inhibition effect occurred earlier with the mixtures than with IFNs used separately.

Effects of human recombinant interferons-alpha 2, -beta and -gamma on growth and survival of human cancer nodules maintained in continuous organotypic culture.

Alveolar II pulmonary tumor cells (A549 cells) maintained in continuous tridimensional organotypic culture were used to test the effects of recombinant human interferons -alpha 2, -beta and -gamma on growth inhibition and survival of the tumor nodules. The organotypic culture method has several advantages: the three-dimensional structures of the cells as well as some cell differentiation are maintained and the extremely low traumatizing culture conditions offer injured cells the maximum chance of survival. A continuous treatment lasting 65 days (three weekly interferon changes) with 10, 10(2), 10(3) and 10(4) U/ml doses of the three interferons led to growth inhibition and necrosis only in the presence of the two highest doses (10(3) and 10(4) U/ml) of IFN-alpha 2 and -gamma.

Effects of combined treatments of cis-diamminedichloroplatinum(II), 5-fluorouracil, and X-rays on growth of human cancer nodules maintained in continuous organotypic culture.

The effectiveness of combined treatments of cis-diamminedichloroplatinum(II) (cis-DDP), 5-fluorouracil (5-FUra), and X-rays on growth inhibition of human pulmonary cancer nodules maintained in continuous organotypic culture was tested. To obtain the most effective growth inhibition, a cis-DDP treatment had to be preceded by an X-irradiation, whereas a 5-FUra treatment had to be postirradiated. This indicates the importance of the order in which the different combinations must be done.

Growth stimulation by misonidazole of lung carcinoma cells maintained in continuous organotypic and monolayer cultures.

Inasmuch as misonidazole is a drug used in clinical trials for sensitizing radioresistant hypoxic cells in solid tumors, it seemed of interest to study its effects in human tumor cells maintained in tridimensional organotypic cultures. This type of culture involves: spatial organisation of the cells with fairly undisturbed differentiation patterns, minimal traumatizing culture conditions, and offers the possibility to follow post-treatment growth patterns over several months without disturbing the cultures. Misonidazole exhibited a radiosensitizing effect on irradiated nodules derived from a lung adenocarcinoma, and on cells of this tumor growing in monolayers.

Long term regeneration of cis-platinum and X ray treated human tumor nodules maintained in continuous organotypic culture.

A simple method for maintaining tumor cells in continuous three-dimensional culture, derived from Wolff's organotypic technique, has been used to study the effects of cis-platinum and X rays on growth inhibition, regrowth and long term regeneration of cultures maintained in low traumatizing conditions (absence of enzymatic dissociation of the cells and the possibility of avoiding subculturing, if necessary). The tumor nodules were derived from cells of the A 549 lung carcinoma cell line. The nodules developed an alveolar structure.

Spontaneous structural changes in DNA during fibroblast aging and the establishment process in vitro.

The molecular weight of single-stranded DNA isolated from human fibroblasts decreased in phase III by comparison with phase II. Mouse fibroblast DNA isolated during the growth crisis had a decreased molecular weight compared to the initial DNA. Established mouse cells recovered this high molecular weight DNA.

Changes in DNA alkali-sensitive sites during senescence and establishment of fibroblasts in vitro.

DNA molecular weight was studied in human embryonic and mouse newborn lung fibroblasts in vitro at different passages of the culture using alkaline and neutral sucrose gradient techniques. Reduction of molecular weight of single-stranded DNA due to alkaline-sensitive sites appeared spontaneously during the growth decline of the mouse cells. These changes disappeared when the mouse fibroblasts became a permanent cell line.