Figure 3.

miR100 inhibits tumor growth of SUM159 cells in vivo. A, 50,000 pTRIPZ-SUM159-miR100 cells were injected into the fourth fat pads of NOD/SCID mice. The treatment started as indicated on the top of the growth curve. miR100 was induced by adding doxycycline (DOX, 1 mg/mL) in drinking water. miR100 late, doxycycline was added to the drinking water after the average tumor size reached to about 10 mm³; miR100 early, doxycycline was added to the drinking water right after SUM159 cells were inoculated to the fat pads. miR100 induction inhibits SUM159 tumor growth in advance setting and completely blocks tumor formation in adjuvant setting. B, tumors from CTRL and miR100 (late) were collected and cells were isolated from each tumor. ALDH was accessed by the Aldefluor assay on viable dissociated cells. C, Ki67 stainings were performed by immunohistochemistry on fixed sections; both Ki67⁺ cells (brown) and Ki67⁻ tumor cells were counted in at least five random fields. D, serial dilutions of cells obtained from CTRL and miR100 (late) were implanted in the fourth fat pads of secondary mice, which received no further treatment. E, extreme limiting dilution analysis for the group CTRL or miR100 (late) was calculated on the website http://bioinf.wehi.edu.au/software/elda/. Briefly, utilizing the online calculation form, we input the number of cells injected and the number of mice used in each group and compared the CSC frequency between CTRL group and miR100 group. F-H, fresh isolated cells from human primary breast tumor xenografts (UM2, MC1, UM1) were infected with pTRIPZ-miR100 lentivirus in suspension and doxycycline was added to the medium for 1 to 2 days and RFP-positive cells were collected. A total of 100,000 cells from noninfected tumor cells or pTRIPZ-UM2 (F), MC1 (G), or UM1 (H) -miR100 cells was injected into the fourth fat pads of NOD/SCID mice. The treatment started right after injection as indicated on the top of the growth curve. miR100 was induced by adding doxycycline (1 mg/mL) in drinking water. *, P< 0.05; Error bars, mean ± SD. I, NOD/SCID mice bearing SUM159 xenografts were treated with either Tf-LipA-negative control (Nano-NC2) or Tf-LipA-miR100 (Nano-miR100; n = 8 mice per cohort) by tail vein injection at a dose of 25 µg miRNA mimics per mouse every other day (EOD). Treatment with miR100 anti-CD44 nanovector significantly inhibited tumor growth (P < 0.01) compared with the negative control group. Data, the tumor volume before initiation of treatment.