Mice Xenograft

Female athymic nude mice (2–3 months old) were obtained from the Frederick National Laboratory for Cancer Research animal facilities (Frederick, MD). NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NOD scid gamma, NSG) breeder mice were purchased from The Jackson Laboratory, and were maintained and bred according to the guidelines of the National Institutes of Health Animal Research Advisory Committee (NIH-ARAC). NSG mice (five females and two males) were xenografted when they were 3–4 months old. Mice were randomly assigned to two groups, each group receiving a subcutaneous injection on both sides of the flank with 3 ×10^6 H295R cells stably expressing either RARRES2 or the control vector in a 100-μl suspension (cell culture medium: Matrigel =1:1). A blinded measurement of the tumor size was taken each week. Tumor volumes were calculated using the equation x²y/2, where x is the smallest and y is the largest diameters. Mice were sacrificed when the tumor size reached 2 cm in greatest diameter. For NSG mice, there were seven mice (fourteen xenografts) in each group, but one xenograft in the RAR group never developed into measurable tumor, thus was excluded from the following analysis. The animal experiments were approved by the NIH-ARAC.