Tumour cells were harvested from subconfluent cultures with 0.25% Trypsin-0.02% EDTA. Kilkenny, Ireland) and water. Tumour cells and culture conditions 4T1 tumour cells, a spontaneously metastasising mammary adenocarcinoma cell collection were a nice gift from Dr Fred Miller, Duke University or college. Cells were managed as monolayer cultures in Dulbecco’s Modified Eagle Medium supplemented with 10% foetal bovine serum, sodium pyruvate, non-essential amino acids, L-glutamine and vitamins (Life Technologies, Inc., GIBCOCBRL, Paisley, UK) in an atmosphere of 5% CO2 in air flow at 37C. Tumour cells were harvested from subconfluent cultures with 0.25% Trypsin-0.02% EDTA. Trypsin was neutralised with medium made up of 10% FBS, washed three times in phosphate buffered saline (PBS) and resuspended in PBS at 5105?ml?1 for injection. Only single cell suspensions of greater than 90% viability as determined by Trypan blue exclusion were used for injections. Experimental design Five104 (100?l) 4T1 cells were injected into the mammary fat pad adjacent to the left forefoot after Tnc anaesthesia was induced and maintained with inhalational halothane. Main tumours were measured on alternate days following injection of tumour cells using Vernier calipers. Tumour diameter (TD) was calculated as the square root of the product of two perpendicular diameters (Pulaski and Ostrand-Rosenberg, 1998). When imply TD was 8.40.4?mm (day 12 post injection of tumour cells), at which time micrometastases are already present in the lungs, mice were randomised into one of three groups (experiments VEGF production Five103 4T1 cells were plated at 5103/well in 96 well plates. 16?h later, SC-236 or indomethacin (5 or 10?M each) were added for 24?h. Culture supernatants were collected and VEGF measured by ELISA (R&D Systems, UK). Cells were washed twice with PBS and total protein measured using the Bicinchonic Acid method (Pierce, IL, USA). VEGF was expressed as pg VEGF g?1 cell protein. Each experiment was carried out three times in triplicate. Apoptosis Five104 4T1 cells were plated on plastic culture chamber slides (LabTek?Permanox Chamber Metformin HCl slides, Nalge Nunc International). Sixteen hours later SC-236 or indomethacin (5 or 10?M each) were added for 24?h. Cells were fixed and stained using in situ cell death detection kit (Boehringer Mannheim, East Sussex, UK). The percentage of apoptotic cells per Metformin HCl high power field (400magnification (40 objective and 10 ocular)) was recorded in each of three fields per sample. Each experiment was carried out three times in triplicate. Statistical analysis Data are expressed as meanstandard error mean (s.e.m.). Differences between and treatment groups were determined by one of Metformin HCl the ways ANOVA with Tukey Kramer test using Instat for Windows statistics bundle (Graphpad Software Inc). Data were taken as significant where control, #control. Table 1 Effect of selective COX-2 inhibition (SC-236) and non-selective COX-1+2 inhibition (indomethacin) on 4TI mammary excess fat pad tumour growth and metastasis Both SC-236 and indomethacin treatment resulted in a significant reduction in the number of spontaneous lung metastases relative to untreated controls (Table 1). Pleural effusions were present in two of the control mice whereas none of the mice in the treatment groups had evidence of pleural effusions (Table 1). The effects of COX inhibition on main tumour growth and metastasis were confirmed in a second experiment (control. Serum VEGF was measured by ELISA (Physique 4). Treatment with either SC-236 (11423.6?pg?ml?1) or indomethacin (87.218.6?pg?ml?1) significantly reduced circulating VEGF relative to controls (516.4215?pg?ml?1). Open in a separate window Physique 4 Serum VEGF levels. Blood was collected by cardiac puncture and serum VEGF measured by ELISA ( experiments. (A) VEGF production by 4T1 cells. SC-236 or Indomethacin at 5 or 10?M significantly reduced VEGF production (pg VEGF g?1 total protein) relative to controls (*(Lu directly increased tumour cell apoptosis. Microvessel density within the primary tumour has been shown to be an independent predictor of metastatic disease in breast cancer patients (De Jong (Tsujii (2000) found reduced angiogenesis in Lewis lung carcinomas produced in COX-2 knockout (COX-2?/?) mice when compared to tumours produced in wild type mice. In our study, inhibition of main tumour growth and metastasis in mice treated with COX inhibitors was associated with a significant reduction in microvessel density in the primary tumour, suggesting that these drugs exert their anti-tumour effect, at least in part, by reducing angiogenesis in the primary tumour. As the degree of tumour angiogenesis is usually predictive of metastatic disease (De Jong COX inhibition directly reduced VEGF production by the 4T1 tumour cells used in this study. These results suggest that COX inhibitors target.