Figure 6. Effects of CD133 Depletion and Enrichment on Orthotopic TN Breast Cancer Model.

8- to 10-week-old female NSG Mice were injected with either 10⁶ standard MDA-MB-231 cells (13.9% ± 1.1 CD133⁺), Enriched CD133⁺ cells (22.1% ± 1.3 CD133⁺), or depleted CD133⁻ cells (1.1% ± 0.4 CD133⁺), in the mammary fat pad through the 4^th nipple. CD133 Cells were either enriched or depleted by magnetic bead isolation as described in Supplemental Figure S7. Injection was performed in 50 μL 50:50 Matrigel/PBS while the mouse was under general anesthesia. (A) Tumor growth was monitored every two days by caliper and recorded as mm³. *P<0.05 with respect to readings statistically significant from the Unaltered control group, by 2-tailed Student’s t test. (B) Animals were euthanized, and their tumors were isolated once reaching 1000mm³. Tumor tissue was homogenized, and cells were stained with αCD133-PE, αHuman-BV421-IgG, αEpCAM-AF647 and ghost red viable dye before analysis with an LSRFortessa H0081. Cells positive for ghost red viability and negative for αHuman dyes were removed from analysis. All remaining αHuman positive cells were characterized for expression levels of CD133 and EpCAM. Data was collected for each sample until a total of ~10,000 ± 150 alive and human cells were obtained for analysis (i.e., alive tumor cells). A representative Flow Cytometry Diagram can be seen in supplemental Figure S7. The data is presented as the mean ± standard deviation of three independent FACs trials.