Tumorigenesis model

Tumorigenesis experiments were approved by the University of Iowa Institutional Animal Care and Use Committee (IACUC) under Protocol 8121508, in compliance with Public Health Service Policy, Animal Welfare Regulations and the Guide for the Care and Use of Laboratory Animals (Jackson Labs). All efforts were taken to minimize animal suffering. Female NOD.CB17-PRKDCSCID/J mice (Cat.# 001303; Jackson Laboratories) were injected in a posterior mammary fat pad with 1x10⁶ MB-231 cells. To test the effect of the anti-CD44 mAb on tumor formation, affinity-purified anti-CD44 mAb P3D2 was mixed at 5 mg/kg of mouse with the suspension of MB-231 cells, in a final volume of 200 μl, in PBS, pH7.2. As a control, affinity purified IgG1 was mixed with MB-231 cells. Mice were tested in three separate experiments. All mice were euthanized when a palpable tumor was detected in the immediate region of the injection sites of control mice. Mice were dissected, and the mammary fat pads excised. The region of the fat pad with a tumor in control mice and a corresponding region in mAb treated mice were fixed in 4% formaldehyde then transferred to 70% ethanol, paraffin-embedded at the Central Microscopy Research Facility at the University of Iowa, sectioned and stained with hematoxylin and eosin-Y [50].