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. A mixture of 5-FUra and cis-DDP was even more effective than the X-ray and drug combination but only for a short period of time; reduction of nodule size and cell loss occurred during the 2 weeks following the treatment. After this period, as happened also with nodules treated with 5-FUra alone, vigorous regrowth occurred after treatment with cis-DDP plus 5-FUra, and all the nodules regained the size of the controls sooner or later. It is to be noted that, if X-rays were applied after a 5-FUra treatment, this regrowth is inhibited at least up to 45 days after the treatment.

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.

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. At the end of phase II of the human fibroblasts, the molecular weight of single-stranded DNA also decreased, followed by the restitution of some high molecular weight DNA in the ultimate passages. When treated with 1 mM caffeine, the mouse fibroblasts during growth crisis did not survive, while cells of the established line resisted. Thus it might be possible that a DNA repair process was involved in the recovery of the mouse fibroblasts. Furthermore, results favor the hypothesis that the cells that become established are not present in the primary culture but originate in vitro.

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. Cells treated with caffeine during the growth crisis did not survive while established cells were resistant to the same conditions of caffeine treatment. A DNA repair process may play a role in establishing a permanent cell line.

Effect of low dose rate irradiation on the division potential of cells in vitro. VI. Changes in DNA and in radiosensitivity during aging of human fobroblasts.

The sensitivity to low dose rate ionizing radiation increases progressively through the lifespan of human embryonic lung fibroblasts. There is also an increase in the number of alkali-sensitive sites leading to an increase in single-strand breaks and in DNA with low molecular weight during cell lysis. These DNA changes become pronounced at the very end of the lifespan. The correlation between aging, increased radiosensitivity and accumulation of DNA damage is discussed.

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. After 1 h treatment with 15 micrograms/ml cis-platinum the growth of the nodules was slightly inhibited during the first 10 days, and then resumed normal growth. After treatment with 100 micrograms/ml cis-platinum, growth inhibition lasted longer and regrowth was observed after about 30 days. Treatment with 300 micrograms/ml of cis-platinum induced cell necrosis and loss of alveolar structures. Forty days later, regeneration occurred; two months after the drug treatment, the reconstituted nodules could be routinely subcultured. A single 15 Gy X ray dose (inducing a 0.005% survival in A 549 monolayer cells, n = 8; D0 = 1.4 Gy; Dq = 1.48) caused an early growth inhibition of about 25%. The alveolar structures disappeared. Alveolar structures reappeared in 17% of the nodules 50 days after irradiation. A slight regrowth was observed 90 days after the irradiation. A 549 nodules supported an about 20-fold higher cis-platinum concentration than monolayer cells. An almost lethal X ray dose (15 Gy, inducing a survival of 0.005%) for monolayer cells induced a prolonged lag phase in nodules followed by a slight but regular regrowth. These results support the idea that cells maintained in three dimensional culture are more resistant to radiation and drug-induced injury than monolayer cells. The organotypic culture method may be a useful tool for determining the activity of antitumoral agents.

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.

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. However, after a 4 hour contact with misonidazole at concentrations corresponding to the range of those found in the serum of treated patients, a significant stimulation of nodule growth was repeatedly observed, together with a strong increase in the frequency of sister chromatid exchanges. Similarly, after treatment of the same tumor cells in confluent monolayers, their colony forming ability was increased. These observations may account for some of the non- convincing therapeutic results obtained in clinical trials.

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.

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.

[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.

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.

Cell division and cell death during regression of the chick embryo Müllerian ducts.

In 9-day-old chick embryos, decreased DNA synthesis and enhanced necrosis were observed in a defined area of the right female Müllerian ducts, supporting the idea of the existence of a regression process in this organ. In the male ducts, decreased DNA synthesis and a low level of necrosis were present all over the studied portion of the organ.

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.

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.

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.

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.