Augmentation of anti-tumor effects of methotrexate by distilled water on Dunn osteosarcoma in mouse air pouch

The anti-tumor effects of hypoosmotic solution of MTX in distilled water (DW) on Dunn osteosarcoma were evaluated in mouse air pouches. Dunn osteosarcoma cell suspension (1×105 cells in 0.1 ml of medium) was inoculated into the mouse subcutaneous air pouch that had formed 7 days after the initial injection of air. Two hours after the inoculation of tumor cells, 5 ml of various concentrations of MTX (from 0 to 1×10−3 M) dissolved in DW or PBS were injected into the air pouch. Five minutes later, the entire solution in the air pouch was aspirated. The mice were sacrificed 3 weeks after the inoculation of tumor cells and the air-pouch tissue was transected in the coronal plane with the largest area of tumor mass. The sections were stained with H&E and the area was measured with the NIH Image program. The largest area of tumor mass in the air pouch treated with 1×10−3 M of MTX in DW was 11.8±3.4 mm2 (N=5), which was significantly (P<0.005) smaller than that in PBS (51.7±8.3 mm2). These findings suggested that hypoosmotic solution in DW might augment the anti-tumor effect of MTX on sarcoma cells.