Table 3

Tumorigenic breast cancer cells were highly enriched in the ESA+CD44+CD24−/low population

Tumors/injections
5 × 1051055 × 1042 × 1041045 × 103103500200100
Mouse passage 1
 Unsorted8/88/810/10 3/12 0/12
 CD44+CD24+ 0/10 0/10 0/14 0/10
 CD44+CD24−/low10/1010/1014/1410/10
 CD44+CD24−/lowESA+10/10*4/44/41/6
 CD44+CD24−/lowESA 0/10*0/40/40/6
Mouse passage 2
 CD44+CD24+0/9
 CD44+CD24−/low9/9
Patients' tumor cells
 CD44+CD24+0/3 0/4 0/8 1/130/2
 CD44+CD24−/low3/3 4/411/131/1
 CD44+CD24−/lowESA+2/22/2
 CD44+CD24−/lowESA 2/20/2
  • Cells were isolated from passage 1 (Mouse passage 1) T1, T2, and T3, passage 2 (Mouse passage 2) T3, and unpassaged (Patients' tumor cells) T1, T4, T5, T6, T8, and T9. CD44+CD24+Lineage populations and CD44+CD24−/lowLineage cells were isolated by flow cytometry as described in Fig. 1. The indicated number of cells of each phenotype was injected into the breast of NOD/SCID mice. The frequency of tumorigenic cells calculated by the modified maximum likelihood analysis method is ≈5/105 if single tumorigenic cells were capable of forming tumors, and every transplanted tumorigenic cell gave rise to a tumor (33). Therefore, this calculation may underestimate the frequency of the tumorigenic cells because it does not take into account cell–cell interactions and local environmental factors that may influence engraftment. In addition to the markers that are shown, all sorted cells in all experiments were Lineage, and the tumorigenic cells from T1, T2, and T3 were further selected as B38.1+. The mice were observed weekly for 4–6.5 mo, or until the mice became sick from the tumors. 

  • * Two thousand cells were injected in these experiments. 

  • Tumor formation by 5,000 T5 ESA+CD44+CD24−/lowLineage cells was detected 8–9 wk after injections, whereas tumor formation by 5,000 T5 ESACD44+CD24−/lowLineage cells was detected 10–12 wk after injections.