Figure 3. L-DOXR cells promote cancer growth and tumor progression in TNBC.
(A) Cell proliferation assay of WT and L-DOXR cells by cell counting. (B) Ki67 immunofluorescence staining and intensity measurement in eight randomly selected fields to evaluate proliferative ability. Scale bars: 20 μm. (C) Wound healing assay to measure cell migration. The gap between cells was measured and shown as a bar graph (bottom). Scale bars: 50 μm. (D, E) Timeline showing subcutaneous injection of 1×107 WT cells and L-DOXR cells followed by DOX injection (2 mg/kg) once a week when tumor volume reached 150 mm3 (n=6 per group). A timeline demonstrating the subcutaneous injection of 1×107 WT cells and L-DOXR cells, followed by injection of DOX (2 mg/kg) into the tail vein (n=6 per group) once a week when the tumor volume reached 150 mm3. Representative tumors shown in photographs. (F) Tumor size measured with calipers every three days for up to 36 days. (G) Representative images of hematoxylin and eosin (H&E) staining (upper) and immunohistochemical staining for PCNA on paraffin sections of tumor tissues (bottom). Scale bars: 50 μm. (H, I) H&E staining of a TNBC tissue microarray with different tumor grades (grades 1, 2, 3, and negative) to detect L-DOXR cells. The number of L-DOXR cells was counted and analyzed from five randomly selected fields on each slide. The black boxes are magnified, and the orange arrows indicate L-DOXR cells. Scale bars: 500 μm. Data presented as mean ± SEM; ***p<0.001; Student’s two-tailed, unpaired t-test (A, B); one-way ANOVA with Bonferroni’s post-test (C, F, I).