Xenografts derived from CD44posCD24pos cells are locally invasive and contain CD44posCD24neg progeny. (a) Ca1a (top), ZR75.1 (middle), and MCF7 (bottom) breast cancer cells were FACS sorted based on their CD44, CD24 expression and injected into the abdominal mammary fat pad of nu/nu mice in 50 μl of a 25% Matrigel suspension. Latency of tumor formation following orthotopic injection of sorted CD44posCD24neg/dim (red line) or CD44posCD24pos (blue line) breast cancer cells is presented. Five mice per population were injected and palpated weekly. (b) Limiting dilution of tumor initiating cells in sorted ZR75.1 and Ca1a cells. (c) Xenografts established with either CD44posCD24neg or CD44posCD24pos cells were dissociated and evaluated for CD44/C24 expression. Host cells were excluded with mouse anti-CD45, and -H-2Kd antibodies as described in the Methods section. Representative FACS profiles of xenografts initiated with sorted Ca1a, ZR75.1, and MCF7 cells are presented. (d) Local invasion of xenografts established by injecting CD44posCD24neg or CD44posCD24pos sorted Ca1a or ZR75.1 cells into the abdominal mammary fat pad of nude mice. Representative H&E stained sections of the resulting xenografts are presented. Black arrows indicate host tissue. Light blue arrows indicate invading tumor cells.