Figure 4. EPO promotes mammosphere formation, TIC self renewal, and expansion of TICs in vivo.

(A) Mammosphere formation of FACS-sorted SUM149 cells. 20,000 cells from each subpopulation were plated in triplicate in 6-well plates. PBS or EPO (1 IU/ml) were added every other day, and colonies (≥ 50 μm) were counted on day 5. (B) Representative photos of mammospheres from PBS- or EPO-treated SUM149 cells. Scale bars: 200 mm. (C) Primary mammospheres from SUM149 TICs (CD44⁺CD24^–EpCAM⁺) were dissociated and serial passaged by replating 20,000 cells for each passage. TICs cultured in PBS showed a significant exhaustion of self-renewal capacity after passage 3. (D) Mammosphere formation of Lin^–Thy1⁺CD24⁺ MMTV-Wnt1 cells cultured with PBS or EPO under 21% O₂. Non-Lin^–Thy⁺CD24⁺ cells did not grow spheres. (E) Representative photos of spheres from Lin-Thy1⁺CD24⁺ MMTV-Wnt1 cells treated with PBS or EPO. Original magnification, ×1. (F) Schematic of orthotopically implanted MMTV-Wnt1 tumors treated with PBS or EPO (500 IU/kg i.p. twice a week) for 4 weeks then assessed for TIC frequency by flow cytometry and LDA. (G) MMTV-Wnt1 orthotopic tumors were harvested and stained with antibodies specific to Thy1, CD24, a lineage cocktail, and DAPI. There were a significantly higher percentage of Lin^–Thy⁺CD24⁺ cells in tumors of mice treated with EPO. **P ≤ 0.01; ***P ≤ 0.001.