Antonella Mannini

Tumoral and macrophage uPAR and MMP-9 contribute to the invasiveness of B16 murine melanoma cells

The aim of this study was to investigate whether tumor cells as well as tumor-associated macrophages (TAMs) contribute to the generation of protease activities essential to tumor cell invasiveness, such as matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9), and the urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR). We found that the enhanced invasiveness through Matrigel-coated filters of B16 murine melanoma cells stimulated with IFNγ was associated with an higher expression of uPAR and MMP-9 in these cells. Moreover, treatment with anti-MMP-9 or anti-uPAR monoclonal antibodies abrogated the increase of invasiveness in IFNγ-stimulated melanoma cells, suggesting a cooperation of uPA system and MMP-9 in cytokine-stimulated invasiveness. Invasiveness through Matrigel was also enhanced in B16 melanoma cells exposed to a medium conditioned by TAMs, represented in our experimental model by thioglycollate-elicited macrophages co-cultivated with melanoma cells. Macrophages isolated from these co-cultures were found to express higher levels of uPAR and MMP-9 compared to macrophage cultures alone, and the pro-invasive activity of the co-culture-conditioned medium was abrogated by anti-MMP-9 monoclonal antibodies, but not anti-uPAR monoclonal antibodies. Furthermore, the enhanced uPAR and MMP-9 expression in macrophages co-cultivated with tumor cells seems a rather specific phenomenon, generated through a cell-to-cell contact mechanism. On the whole, our data point to a cooperation between tumor cells and macrophages elicited by tumor cells themselves in generating key enzymes essential in the promotion of tumor invasiveness, such as uPAR and MMP-9.

Enhancement of lung-colonizing potential of murine tumor cell lines co-cultivated with activated macrophages

In order to explore the influence of activated macrophages on tumor cells, we cultured a series of weakly metastatic clones isolated from the murine T3 fibrosarcoma line (T3 clones) and the B16-F10 melanoma cells on feeder layers of C. parvum- or thioglycollate-elicited macrophages, or ÔresidentÕ (unstimulated) macrophages. Co-cultivation with C. parvum-elicited macrophages, but not with resident macrophages, induced an increase of the lung-colonizing potential of T3 clones and B16-F10 cells. An enhancement of lung-colonizing potential was also found in tumor cells grown in media conditioned by C. parvum-elicited macrophages. Thioglycollate-elicited macrophages also generated a pro-clonogenic activity which was however effective only on T3 clones but not on B16-F10 cells. The increase in the lung-colonizing potential of tumor cells stimulated by activated macrophages was retained to some degree after subcultivation in tissue culture medium or transplantation into syngeneic animals. In conclusion, our data support the notion that macrophages at different states of activation may enhance lung colonization of tumor cells by inducing a partially stable change of phenotype.

An enhanced apoptosis and a reduced angiogenesis are associated with the inhibition of lung colonisation in animals fed an n -3 polyunsaturated fatty acid-rich diet injected with a highly metastatic murine melanoma line

Both epidemiological and experimental studies indicate that dietary n-3 PUFA inhibit carcinogenesis and tumour growth. Metastatic diffusion has also been found to be affected in animals fed diets containing purified n-3 PUFA or fish oil. In the present study, we investigated whether the metastatic diffusion of a highly metastatic variant (F10-SR cells) isolated from the B16 melanoma F10 line was affected by feeding host animals a diet containing 5 % fish oil. In these animals, compared with those fed a diet containing 5 % maize oil, there was a reduced number of metastatic pulmonary colonies. The immunohistochemical analysis of appropriate markers revealed that the antimetastatic effect of dietary n-3 PUFA was not related to a reduction of proliferation, but rather to an enhanced apoptotic activity. The reduction of von Willebrand factor immunoreactivity found in pulmonary colonies of F10-SR cells grown in fish oil-fed animals indicates that a decrease of angiogenesis contributes to the antimetastatic effect of dietary n-3 PUFA. This conclusion stands in spite of the higher expression of vascular endothelial growth factor observed in pulmonary colonies grown in fish oil-fed animals.

Effect of Phosphatidylcholine Structure on the Adenylate Cyclase Activity of a Murine Fibroblast Cell Line

To determine which structural characteristics of membrane phospholipids influence adenylate cyclase activity, we measured basal and sodium fluoride- or forskolin-stimulated activity in a murine fibroblast cell line,i.e., Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid-depleted FCS (LD-FCS) or LD-FCS complexed with different phosphatidylcholine (PC) molecular species. Cells grown in the presence of LD-FCS showed a substantial decrease in their basal and NaF-stimulated adenylate cyclase activities; however, their forskolin-stimulated activity was not altered, suggesting that the enzymes catalytic site is not affected by changes in membrane lipids. Media supplemented with different LD-FCS/PC complexes were shown to prevent the LD-FCS-mediated reduction of basal and NaF-stimulated adenylate cyclase activity to different extents. Addition ofcis-9-16∶1/cis-9-16∶1,cis-9-18∶1/cis-9-18∶1 orcis-9-18∶1/cis-9,12-18∶2sn-glycerophosphocholine (GPC) completely restored adenylate cyclase activity, whilecis-11-18∶1/cis-11-18∶1 GPC was not effective and only a partial recovery was observed with 16∶0/16∶0, 16∶0/cis-9-18∶1 andtrans-9-18∶1 GPC. Considering the structural features of these seven PC molecular species, the findings suggest that an optimal lipid environment is conferred to the enzyme by the presence of thecis double bonds, each located in Δ9 position of the PC acyl chains. The limited effect ofcis-9-16∶1/cis-9-18∶1 GPC andcis-9-18∶1/cis-9-16∶1 GPC suggests that an equal length of the terminal hydrocarbon chains extending bevond the Δ9 double bonds is also important. Moreover, complete restoration of adenylate cyclase activity in cells exposed to 16∶0/cis-9,12-18∶2 GPC suggests that twocis-9,12 double bonds located on the same chain are as effective as twocis-9 double bonds each located on two different chains of PC. As the four double bonds of 16∶0/cis-5,8,11,14-20∶4 GPC had no effect, a mere increase in the number of double bonds seems insufficient to build an optimal lipid microenvironment for the enzyme.

Dietary n-3 polyunsaturated fatty acids enhance metastatic dissemination of murine T lymphoma cells

Epidemiological investigation and animal studies have shown that dietary n-3 PUFA prevent the development and progression of certain types of cancer. However, conflicting results have been reported by the few studies that focused on the effect of dietary n-3 PUFA on the development of metastases. In the present study, we investigated the metastatic dissemination of murine T lymphoma lines with different metastatic potential transplanted into mice fed a fish oil diet, compared with mice fed a maize oil diet. Transplantation of highly metastatic S11 cells into animals fed a fish oil diet induced a large lymphomatoid infiltration in the spleen, associated with an eight-fold increase in spleen weight, compared with normal animals on the same diet. In contrast, only a limited increase in spleen weight was found in animals transplanted with S11 cells while fed a maize oil diet. No significant increase in spleen weight was found in animals transplanted with low-metastatic 164T2 cells regardless of whether they were fed a fish oil or a maize oil diet. At the end of experiment, an overt cachexia was shown by animals fed a fish oil diet transplanted with S11 cells, but not by those transplanted with 164T2 cells. The particularly high pro-metastatic effect of dietary n-3 PUFA on S11 cells rules out the generalisation that dietary n-3 PUFA inhibit tumour growth and progression.

Enhancement of Nitric Oxide Release in Mouse Inflammatory Macrophages Co-cultivated with Tumor Cells of a Different Origin

In the present study we investigated whether synthesis of nitric oxide (NO) by macrophages is affected by contact with tumor cells. Although it is well known that NO generated by macrophages influences different activities related to tumor progression, there is limited information on the modulatory role of tumor cells on NO release by macrophages. The experimental protocol used in our study consisted in the determination of NO secreted by macrophages, either resident or inflammatory, co-cultivated with tumor cells (B16 melanoma and L929 fibrosarcoma cells) at different cell densities and macrophage:tumor cell ratios. This experimental in vitro protocol simulates the different interactions between macrophages and tumor cells that occur during the development of a tumor mass. We found that the co-cultivation with tumor cells induced an increased secretion of NO in macrophages provided that they express an inflammatory phenotype, and they were challenged with LPS or IFNγ/LPS. Two more variables were found to be critical in the increase of NO generation in inflammatory macrophages cultivated with tumor cells: a high cell density and a prevalence of tumor cells over macrophages. The enhancement of NO secreted in inflammatory macrophages stimulated by tumor cells was not observed in normal murine fibroblasts co-cultivated with tumor cells.

Biological properties associated with the enhanced lung-colonizing potential in a B16 murine melanoma line grown in a medium conditioned by syngeneic Corynebacterium parvum-elicited macrophages.

A previous study by our laboratory showed that the peritoneal murine Corynebacterium parvum-elicited macrophages released into their growth medium an activity which enhanced the ability of B16-F10 melanoma cells to form experimental metastases in the lung of syngeneic mice. In the present study, we used a clone of B16-F10 line (F10-M3 cells) to investigate whether the increase in lung-colonizing potential due to the pro-clonogenic activity released by C. parvum-elicited macrophages was associated with biological properties characteristic of a metastatic phenotype. We have found that the pulmonary retention, growth rate in lung parenchyma, invasiveness through Matrigel, adhesiveness to IL-1-activated endothelium and MHC class I expression were increased in F10-M3 cells stimulated by the macrophage pro-clonogenic activity. By using an in vitro experimental protocol, the enhancement of lung-colonizing potential in the stimulated melanoma cells turned out to be a transient phenomenon as was the increase of invasiveness through Matrigel and the higher expression of MHC class I antigens. In conclusion, the melanoma cells stimulated by the pro-clonogenic activity released by C. parvum-elicited macrophages showed changes in biological parameters which are relevant to metastatic diffusion. These changes appeared as a temporary phenomenon which sustains the view that the metastatic phenotype represents a transient biological character influenced by host factors.

Diminution of the development of experimental metastases produced by murine metastatic lines in essential fatty acid-deficient host mice

In a previous study we found that the capacity for spontaneous metastases of tumors developed after subcu-taneous transplantation of RSV-transformed Balb/c 3T3 cells was reduced in essential fatty acids (EFA)-deficient host animals. In the present study, we have extended our investigation by considering the requirement of EFA for the formation of lung colonies obtained by i.v. injection of two metastatic murine cell lines of different origin: (1) T3 cells, a highly metastatic cell line isolated from a fibrosarcoma, and (2) the F10 variant of B16 melanoma (B16-F10 cells). We found that EFA deficiency reduces the lung colonization of both T3 cells and B16-F10 cells without affecting the retention of tumor cells in the lung. NK cells did not seem to be involved in the diminution of lung colonization in EFA-deficient animals. Furthermore, by examining histologically the lung parenchyma at successive intervals after tumor cell injection, we found that, in comparison with control mice, EFA-deficient animals had fewer lung colonies and a prevalence of smaller microcolonies during the entire period of observation. This led us to conclude that the diminution in development of tumor colonies in the lungs of EFA-deficient host animals was related to a reduced growth rate of tumor cells at this site.

The complex metabolic network gearing the G 1 /S transition in leukemic stem cells: Hints to a rational use of antineoplastic agents

We defined the stem cell profile of K562 line, demonstrating the expression of the Embryonic Transcription Factors Oct3/4, Sox2, Klf4 and Nanog. This profile was associated with a high vulnerability to the physiological oxidizable substrate pyruvate. remarkably, this substrate was shown to be innocuous, even at the highest doses, to normal differentiated cells. This vulnerability is based on a complex metabolic trim centered on the cellular redox state expressed by the NADP/NADPH ratio geared by the mitochondrial respiratory chain. Flow cytometry revealed that the inhibition of this chain by antimycin A produced cell accumulation in the S phase of cell cycle and apoptosis. This block negatively interferes with the aerobic synthesis of purines, without affecting the anaerobic synthesis of pyrimidines. This imbalance was reproduced by using two antifolate agents, LY309887 and raltitrexed (TDX), inhibitors of purine or pyrimidine synthesis, respectively. All this revealed the apparent paradox that low doses of TDX stimulated, instead of inhibiting, leukemia cell growth. This paradox might have significant impact on therapy with regard to the effects of TDX during the intervals of administration, when the drug concentrations become so low as to promote maintenance of dormant cancer cells in hypoxic tissue niches.

The inhibition of lung colonization of B16-F10 melanoma cells in EFA-deficient animals is related to enhanced apoptosis and reduced angiogenesis

Previous studies conducted in our laboratory showed that the reproduction of spontaneous and experimental metastases was reduced in host animals deprived of essential fatty acids (EFA). In the present study, we have explored the possibility whether apoptosis, proliferation, and angiogenesis might be involved in the antimetastatic effect of EFA deficiency. To this aim, in pulmonary colonies developed from B16-F10 cells in EFA-deficient animals or in animals fed a 5% corn oil diet, we performed an immunohistochemical analysis of bcl-2/bax proteins, PCNA, and VEGF and von Willebrand Factor (vWF), typical markers of apoptosis, proliferation, and angiogenesis, respectively. Apoptosis was also evaluated by detecting DNA fragments in metastatic cells. We found that the reduction of pulmonary colonies grown in EFA-deficient animals was associated with a high expression of apoptotic activity as revealed by the presence of apoptotic nuclei and a high immunoreactivity for bax. Cell proliferation seemed not to be influenced by EFA deficiency in view of the observation that PCNA was highly expressed in pulmonary colonies of control as well as EFA-deficient animals. Pulmonary colonies developed in EFA- deficient animals showed a lower expression of VEGF and a decreased microvessel density, indicating that a reduced angiogenesis contributes to the antimetastatic effects of EFA deficiency. Our analysis of the results invokes the possibility that a relationship between angiogenesis and apoptosis may account for the diminution of the development of experimental metastases in the lungs of EFA-deficient animals.