Nitric oxide synthase inhibition by N-G-nitro-L-arginine methyl ester inhibits tumor-induced angiogenesis in mammary tumors

Using a murine breast cancer model, we earlier found a positive correlation between the expression of nitric oxide synthase (NOS) and tumor progressio n; treatment with inhibitors of NOS, N-G-methyl-L-arginine (NMMA) and N-G-n itro-L-arginine methyl ester (L-NAME), had antitumor and antimetastatic eff ects that were partly attributed to reduced tumor cell invasiveness. In the present study, we used a novel in vivo model of tumor angiogenesis using s ubcutaneous implants of tumor cells suspended in growth factor-reduced Matr igel to examine the angiogenic role of NO in a highly metastatic murine mam mary adenocarcinoma cell line. This cell line, C3L5, expresses endothelial (e) NOS in vitro and in vivo, and inducible (i) NOS in vitro on stimulation with Lipopolysaccharide and interferon-gamma. Female C3H/HeJ mice received subcutaneous implants of growth factor-reduced Matrigel inclusive of C3L5 cells on one side, and on the contralateral side, Matrigel alone; L-NAME an d D-NAME (inactive enantiomer) were subsequently administered for 14 days u sing osmotic minipumps, Immediately after sacrifice, implants were removed and processed for immunolocalization of eNOS and NOS proteins, and measurem ent of angiogenesis, Neovascularization was quantified in sections stained with Masson's trichrome or immunostained for the endothelial cell specific CD31 antigen. While most tumor cells and endothelial cells expressed immuno reactive eNOS protein, iNOS was localized in endothelial cells and some mac rophages within the tumor-inclusive implants. Measurable angiogenesis occur red only in implants containing tumor cells. Irrespective of the method of quantification used, tumor-induced neovascularization was significantly red uced in L-NAME-treated mice relative to those treated with D-NAME, The quan tity of stromal tissue was lower, but the quantity of necrotic tissue highe r in L-NAME relative to D-NAME-treated animals. The total mass of viable ti ssue (ie, stroma and tumor cells) was lower in L-NAME relative to D-NAME-tr eated animals. These data suggest that NO is a key mediator of C3L5 tumor-i nduced angiogenesis, and that the antitumor effects of L-NAME are partly me diated by reduced tumor angiogenesis.