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

Abstract

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 IFNgamma/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.

Arginine metabolism in tumor-associated macrophages in cutaneous malignant melanoma: evidence from human and experimental tumors

“…The production of NO by thioglycollate-elicited macrophages was rather limited (12.3 F 3.8 nmol/10 6 macrophages) and was not enhanced in macrophages cocultivated with tumor cells (9.2 F 4.2 nmol/10 6 macrophages) (Fig. 5), a finding also reported in a previous article from our laboratory [23]. In contrast, when macrophages were cocultivated with tumor cells in the presence of IFN-c/LPS, they generated a greater quantity of NO than IFN-c/LPS-stimulated macrophage monolayers.…”

“…Cocultures were prepared by seeding tumor cell suspensions on macrophage or fibroblast monolayers. Cell density and ratio between macrophages and tumor cells or fibroblasts were chosen based on previous experiments [23]. …”

Dual role of macrophages in the response of C26 colon carcinoma cells to 5-fluorouracil administration

“…As other studies previously suggested, the increased production of NO could be a phenomenon strictly related to the tumor cell-macrophage interaction (12). Previous studies demonstrated that nanomolar concentrations of NO produced by inducible/endothelial nitric oxide synthases in human colon and ovarian carcinoma cells as a result of administration of cytotoxic drugs other than 5-FU, such as doxorubicin or cisplatin, ensure protection against ROS-induced apoptosis in these cancer cells (39,45,46).…”

“…32652/01.07.2014). Mice were previously injected intraperitoneally with 1 ml of 3% thioglycollate (Fluka) (12). After 3 days, chemically elicited macrophages (with inflammatory and antitumor action) were collected, and co-cultures were prepared by seeding C26 tumor cell suspensions on macrophage monolayers.…”

“…After 3 days, chemically elicited macrophages (with inflammatory and antitumor action) were collected, and co-cultures were prepared by seeding C26 tumor cell suspensions on macrophage monolayers. The complex interaction of these macrophages with tumor cells in a co-culture system has been shown to enable tumor cell-macrophage crosstalk and to promote macrophage polarization into TAMs which favor tumor progression (12,13). In our experiments, co-cultures of macrophages and tumor cells were created at a cell density ratio of 1:4, as it has previously been demonstrated that this cell density ratio ensures the optimal cytokine interplay between tumor cells and macrophages, which provides an approximation of the physiological conditions of colon carcinoma development in vivo (13).…”