Figure 1.
Establishment of breast cancer stem cells (FTH-BCSCs) that overexpress the myc-tagged heavy chain subunit of ferritin (myc-FTH) and EGFP.
(A) Flow cytometric analysis of the surface markers, CD44 and CD24 in BCSCs isolated from human breast cancer specimens. (B) Flow cytometric analysis of the surface markers, CD44 and CD24 in FTH-BCSCs expressing myc-FTH and EGFP. (C) Immunocytochemistry of BCSCs and FTH-BCSCs (bar, 50 µm). (D) Western blot analysis for myc-FTH and EGFP in BCSCs and FTH-BCSCs. (E) Measurement of cellular iron in BCSCs and FTH-BCSCs treated with increasing concentrations of ferric ammonium citrate (FAC). The results were obtained from 4 independent experiments and are presented as means ± SD. *P<0.05.
Table 1.
Evaluation of the incidence of tumors in NOD/SCID mice implanted with different numbers of BCSCs and FTH-BCSCs.
Figure 2.
MRI images of phantom cells and xenograft tumors.
(A) In vitro MRI images (left) and color-coded maps (right) of agarose phantoms of BCSCs and FTH-BCSCs treated with or without 25 µM FAC. (B) R2* values measured from MRI images of BCSC and FTH-BCSC phantoms. *P<0.05. (C) In vivo MRI images (left) and color-coded maps (right) of BCSC and FTH-BCSC xenograft tumors in the mammary fat pads of NOD/SCID mice. (D) The distributions of the R2* values obtained for the BCSC and FTH-BCSC tumors at 3 weeks post-transplantation.
Figure 3.
Volumetric MRI images and distribution of R2* values in control (BCSC and FTH-BCSC) and docetaxel-treated (BCSC Doc and FTH-BCSC Doc) xenograft tumors.
(A) Follow-up volumetric MRI images of BCSC, BCSC Doc, FTH-BCSC and FTH-BCSC Doc tumors. (B-D) Distribution of R2* values in BCSC, FTH-BCSC, BCSC Doc and FTH-BCSC Doc tumors at day 0, 5 and 14. The orange box indicates the pixels over the threshold (mean +3 SDs) in the FTH-BCSC Doc tumors. E, analysis of the percentage of pixels over the threshold (mean +3SDs) in each tumor group. *P<0.05.
Table 2.
Mean R2* values of control (BCSC and FTH-BCSC) and docetaxel-treated (BCSC Doc and FTH-BCSC Doc) xenograft tumors.
Figure 4.
Histological analysis of the peripheral and central portions of control (BCSC and FTH-BCSC) and docetaxel-treated (BCSC Doc and FTH-BCSC Doc) xenograft tumors.
(A–D) MRI images and H&E staining of BCSC, BCSC Doc, FTH-BCSC and FTH-BCSC Doc tumors obtained 4 weeks after the implantation. The red and the black boxes on the MRI images indicate the corresponding areas to H&E staining images of the periphery and the center of each tumor. The white dotted lines on the MRI images indicate the boundary where the R2* values were differed in the BCSC Doc and FTH-BCSC DOC tumors. The black dotted lines on H&E staining indicate the demarcation between viable and nonviable cells area in the central portions of the BCSC Doc and FTH-BCSC Doc tumors (x100; bar in H&E, 100 µm, bar in MRI, 500 µm). MRI images before magnification and cropping are in the Supplement (Figure S5). (E) Immunostaining of EGFP and myc-FTH in FTH-BCSC and FTH-BCSC Doc tumors. The white dotted line indicates the demarcation between the areas of viable and nonviable cells in the central portion of FTH-BCSC Doc tumors (x400; bar, 50 µm).
Figure 5.
Immunohistochemical analysis of the peripheral and central portions of control (FTH-BCSC) and docetaxel-treated (FTH-BCSC Doc) xenograft tumors.
(A) Double staining for CD44 and CD24 expression performed in FTH-BCSC and FTH-BCSC Doc tumors (x400; bar, 100 µm). (B) The percentage of CD44+/CD24−, CD44+/CD24+, CD44−/CD24+, CD44−/CD24− and EGFP-positive cells in the periphery and center of the FTH-BCSC and FTH-BCSC Doc tumors. *P<0.05. (C) Cell proliferation marker (phospho-histone 3, PH3) expression in the FTH-BCSC and FTH-BCSC Doc tumors (x200; bar, 100 µm). (D) The percentage of PH3-positive/EGFP-positive cells in the periphery and center of the FTH-BCSC and FTH-BCSC Doc tumors. **P<0.01.