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. Here, we studied in a three-dimensional (3D) coculture system, called "ex vivo nodules", how drug-resistant and sensitive tumour cells settle down in a 3D space. For this, we cocultured adriamycin-sensitive (MCF-7S) and -resistant (MCF-7R) human breast cancer cells in long term nodules. We showed that both types of cells are able to grow separately or in coculture until five weeks in spheroidal forms. MCF-7R cells did not loose their multi-drug resistance when cultured in nodules as measured by RT-PCR. Curiously, the exterior aspects of mixed (MCF-7S/ MCF-7R) nodules and MCF-7R nodules were similar whereas MCF-7S nodules were completely different. Nevertheless, morphologically these three nodule types were distinct, in particular in their density. Immunostaining showed that in mixed nodules, MCF-7R cells were arranged at the periphery, whereas the MCF-7S cells are in the central part of the nodules. Even if the mechanism of this arrangement remained unclear, this work shows that three-dimensional cell culture is well adapted to the study of the relationships between adhesion mechanisms and drug-resistance.

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. Studies on short-term and long-term effects of anticancer treatments are possible and their long-term regrowth can be obtained. Especially, in nodules containing cell mixtures, the localization of the different cell types can be determined and their specific differentiation. An example showing stroma-like formations and collagen production in breast cancer cell and breast fibroblast containing nodules is presented.

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. The mucus contains the pulmonary surfactant and sialomucins, both LAK cell inhibitory substances. The spontaneous infiltration into the neoplastic tissue and the membrane contacts established between the two cell types were studied by means of histological, immunohistochemical and electron-microscopic methods. Free-floating LAK cells were allowed to sediment and adhere freely to the nodule surface. The cytostatic and cytolytic effects of LAK cells were tested using thymidine incorporation into DNA and flow cytometry. Despite the presence of a mucus envelope, LAK cells adhered to the A549 nodule surface and penetrated spontaneously into them in the presence of IL-2; they settled mainly in the pseudoalveolar structures where they became apoptotic. According to electron-microscopic observations performed on the second day of coculture, the LAK cells, which remained between the cancer cells, established mostly pinpoint contacts with the carcinoma cells, forming cytoplasmic fusions. These fusions indicate the induction of pores in both the cancer cell and the LAK cell membranes. Electron-microscopic observation also displayed LAK-cell-associated apoptotic and necrotic carcinoma cells. However, at this stage of the coculture (day 2), the DNA synthesis rate of the A549 nodules still remained unchanged; it diminished by approximately 3 times on day 4 and almost stopped on day 6: nodule disintegration was then complete. In the free-floating LAK cell component of the cocultures, DNA synthesis was already strongly inhibited (26x) by the second day. Nevertheless, their cytolytic effect remained unaltered, as was tested on A549 monolayer cells. The presence of tumor necrosis factor (TNF) in the coculture supernatant has been demonstrated, and when coculturing took place in the presence of monoclonal TNF antibody, nodule proliferation was significantly enhanced (up to 145%). Our results indicate that, despite the presence of pulmonary surfactant and sialomucins containing mucus, LAK cells were capable of killing lung carcinoma cells in three-dimensional culture at an early stage of coculture (day 2) by direct cell-to-cell contact. Total nodule disintegration, however, was complete much later (on day 6), and taking into account the low amount of LAK cells in the cancer tissue, this seemed to be the result of an indirect effect implying, in particular, the presence of soluble TNF.

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. Proliferation of the nodules was tested and also that of the LAK cells after coculturing using tritiated thymidine incorporation into DNA. Cell death was determined by arrest of thymidine incorporation and total nodule disintegration. Infiltration rate of LAK cells into the nodules was low: after coculturing 5% of the nodule cells were LAK cells. Although close membrane contacts and cytoplasmic fusions between effector and target cells leading to tumor cell apoptosis were observed, this direct cytolytic process seemed to be too infrequent for the induction of total nodule disintegration at day 6. Therefore, the indirect pathway to cytolysis might be predominant implying, among other cytokines, soluble TNF. On the other hand, LAK cell proliferation diminished strongly after coculturing (down to 11%) but the cytotoxicity was significantly enhanced (18% higher) suggesting an enhancement of differentiation. This might account for the peculiar efficacy of LAK cells on melanomas in vivo and it would be of interest to study this phenomenon further.

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. Concurrently, the carcinoma cells underwent a progressive redifferentiation process which histologically resulted in the appearance of highly developed papillar and tubular structures after 2 months in culture. The production of mucins was further evidence that these cells had undergone differentiation. By contrast, when MCF-7 cells were grown alone or with fibroblasts isolated from a breast carcinoma, the nodules continued to exhibit their characteristic histodedifferentiation properties and did not grow. The re-establishment of a normal epithelial state of differentiation in MCF-7 carcinoma nodules indicates that the phenotypic characteristics of tumor cells are reversible and are influenced or controlled by the stromal environment by which these tumor cells are surrounded or in contact with. Overall, our results open the possibility of exploiting the effects that connective tissue cells have on tumor-cell differentiation for use in prevention and treatment of cancer.

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. The lymphokine-activated killer (LAK) cells adhered more frequently to the carcinoma nodules than to the mastosis nodules. They actively penetrated into both of them. The penetration remained peripheral, and only a few cells migrated more deeply. The LAK cells established close cell-to-cell contacts with the two types of nodules, and intercellular gaps were formed: damaged cells could be seen near the activated killer cells. In MCF-7 nodules, a 5-fold inhibition of proliferation occurred, and extensive necrotic zones developed; this was accompanied by a general tendency for glandular redifferentiation. In mastosis nodules, necrosis also developed but no cell differentiation occurred and proliferation was less inhibited (2 times). Interleukin-2 alone enhanced DNA synthesis in mastosis nodules but had no effect on MCF-7 nodules, and no extending necrosis could be seen in both types of nodules. The cytolytic effects of LAK cells combined with their redifferentiating effect in MCF-7 breast carcinoma nodules may be a useful indication for further breast cancer therapy research.

[Effects of LAK cells activated by IL-2 on MCF-7 human breast cancer cell line maintained in organotypic culture]. / Effets des cellules LAK en présence d'IL-2 sur la lignée carcinomateuse mammaire humaine MCF-7 maintenue en culture organotypique.

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. The cytotoxic effect of the latter, appreciated by Chrome 51 release was unchanged after the coculture. In histological sections, the penetration of the LAK cells into the MCF-7 nodules was accompanied by an increase of tumor necrosis but also by a glandular differentiation of cancerous tissue. Polarized epithelial cell formations bording neoplasic lumens with intracytoplasmic vacuoles filled with mucus, appeared in the nodules. The immunohistochemistry underlines the presence of T lymphocytes marked by UCHL1 and CD3 antibodies and of Natural Killer (NK) cells marked by IOT10, located between the MCF-7 cancer cells. In electron microscopy, the membrane contacts were tight and were accompanied by the appearance of secondary lysosomes and nuclear alterations. The relatively low infiltration level of the nodules may lead to the supposition that an indirect mechanism will intervene in this dual action of a LAK cells: increase of necrosis, although partially, and development of glandular and functional differentiation.

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. In in vivo xenografts, monobut-3 significantly decreased MDA-MB-231 tumor take but did not affect the rate of tumor growth. No difference was noted in the histological characteristics of the xenografts between untreated and treated mice. Moreover, once monobut-3 treatment was discontinued, tumor growth rapidly resumed in tumor-free animals. The decreased efficacy of monobut-3 in in vivo MDA-MB-231 xenografts as compared to in vitro tumor nodules indicates that factors related to host environment may still limit the clinical effectiveness of this compound.

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. Three complexes (Mr 27,000, 42,000, and 57,000) were detected in MDA-MB-231, and no complex was visible in HBL-100. No correlation was found between receptors for BIM-23014C and epidermal growth factor in these lines. Twenty-seven of 30 human breast tumors (90%) had at least one BIM-23014C receptor. Sixteen had three complexes (Mr 27,000, 42,000, and 57,000). Six had the two complexes (Mr 27,000 and 57,000), two had Mr 42,000 and 57,000 complexes, two had just the Mr 27,000 complex, and one had just the Mr 42,000 complex. The presence of the three BIM-23014C receptors was positively correlated (P less than 0.05) to the low amount of sex steroid receptors (less than 20 fmol/mg) [seven of eight (estrogen receptor negative, progesterone receptor negative) versus four of 14 (estrogen receptor positive, progesterone receptor positive)]. Another positive correlation was established between the absence of progesterone receptors and the presence of these three complexes [12 of 16 (progesterone receptor negative) versus four of 14 (progesterone receptor positive)]. This high percentage of BIM-23014C receptor-positive biopsies and its inhibitory activity would support its clinical potential for the treatment of breast cancer.

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. In this way, we obtained images showing the characteristic distribution of 14C-thymidine and D-arginine in human fibroblasts, and of 15N-adenine in organotypic cultures of human breast cancer cells. The spatial resolution on the acquired images was close to 0.1 micron when using the UPS-ONERA ion microprobe. The sensitivity of the method for detecting carbon 14 is far greater than that of autoradiography and the technique is both fast and quantitative. On the other hand, the capacity of ion microscopy for studying the tissular distribution of molecules labelled with stable isotopes, opens the way for biological and pharmacological tracer studies of human diseases.

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. Also, cellular differentiation, as regards intracellular phospholipid composition and more particularly phosphatidylcholine content, appeared undisturbed. The only obvious effect of hypergravity was a modification of the structural organization, with a disappearance of the large alveoli present at the surrounding of control nodules and the development of a dense cellular mass instead.

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. On the 2nd day of treatment with TNF, only the highest dose (2 x 10(4) U/ml) diminished cell proliferation significantly, as measured by tritiated thymidine uptake into DNA. On the 10th day, only the lowest TNF dose (2 x 10 U/ml) induced no significant growth inhibition. Necrosis and nodule disintegration were apparent in the 2 x 10(4) U/ml-treated nodules where DNA synthesis decreased. In this case, using agar cloning assays, no cell survival could be observed. Similar results could be obtained with TNF at low concentration (2 x 10(2) U/ml) in combination with INF-gamma (10(3) U/ml), showing a synergistic effect on inhibition of cell proliferation. In the long-term experiments, with the lower TNF doses, in situ evidence of regrowth was observed (outgrowing zones in the nodules) on about the 40th day of treatment, and nodule recovery was confirmed by the resumption of DNA synthesis measured on the 50th day of treatment. No regrowth, however, occurred when the IFN-gamma/TNF combination was used, and the nodules disintegrated completely on the 35th day of treatment without evidence of any cellular survival.

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. Scatchard analysis of the EGF binding data indicated that a 48-hour exposure period resulted in an increase in the apparent number of cell surface EGF-R but did not significantly alter receptor affinity. Results from Northern blot analysis showed that the enhanced expression of EGF-R on the A549 nodule cells treated with IFN-beta correlates with an increase in mRNA for EGF-R. No modification of the EGF-R mRNA expression was observed in nodules treated with IFN-alpha 2 or IFN-gamma. In this model, our results suggest a relationship between resistance to antiproliferative effect of IFN-beta and modulation of EGF-R.

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. Moreover, combinations of IFN-alpha 2 and -gamma resulted in a significant synergistic antiproliferative activity; IFN-beta only potentiated slightly the effect of IFN-gamma. All three IFNs induced an increase in the 2,5A synthetase activity, indicating a discrepancy with the pattern of anticellular activity. Furthermore, whereas the combination of IFN-alpha 2 and -gamma resulted in a synergistic antiproliferative effect, no synergism was observed in the induction of the enzyme. These results show that there is a lack of correlation between the sensitivity or the resistance to IFNs of A549 tumor nodules and the induction of the 2,5A synthetase activity.

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. Furthermore, the growth inhibition observed with 2000 U/ml of the mixture (1000 U/ml of each IFN) was greater than that induced by 2000 U/ml of IFN-alpha 2 or -gamma used alone. A 35-day treatment with IFN-alpha 2 1000 U/ml plus IFN-gamma 1000 U/ml led to a complete growth inhibition and necrosis of the nodules. These data demonstrate that IFN-alpha 2 and -gamma cooperate to potentiate the IFN antiproliferative activity.

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. IFN-beta had no inhibitory effect. Some nodules, especially at the lower dose levels (10(2) U/ml), showed enhanced growth in presence of the three types of interferons. After stopping the treatments, all the necrotic and disintegrating nodules resumed growth. Growth of the recovered nodules was followed in the absence of interferon for another period of 70 days. The growth rate of IFN-beta and -gamma-treated nodules was similar to that of the controls, but was slowed down for the regenerated IFN-alpha 2-treated nodules. Hence, in A549 organotypic cancer nodules and under our experimental conditions, only high doses of IFN-alpha 2 and -gamma appeared to have a partial cytolytic, but finally no tumoricidal action; IFN-beta was inactive. At the lower doses growth stimulation was found during the treatments with the three interferons.