Fig. 1
Single-cell clones derived from parental 4T1 cells exhibit varying characteristics. A One thousand 4T1, LM.4T1, or HM.4T1 cells were seeded into a 8-well Lab-Tek II chamber slide and cultured for 4 days at 37 °C and then stained with Diff-Quik. The photographs show distinct morphologies of LM.4T1 and HM.4T1 cells. Arrows indicate spheroids. The scale bar indicates 100 μm. B One thousand 4T1, LM.4T1, or HM.4T1 cells were seeded into 96-well plates and allowed to grow for four days. The cell numbers were determined by the standard MTT assay. The results are presented as the mean ± SEM. Statistical significance was analyzed by one-way ANOVA with multiple comparisons. ****p < 0.0001, n = 9. C LM.4T1 and HM.4T1 cells (1 × 105 cells in 100 μl PBS) were transplanted into the 3rd mammary pad of BALB/c mice, and the sizes of tumors were measured, and tumor volumes were calculated. The results are presented as the mean ± SEM. n = 5. D, E Mice were euthanized 4 weeks after injection, and the weight of tumors and the number of lung metastases were evaluated. The results are presented as the mean ± SEM. Statistical significance was analyzed by unpaired Student’s t test. *p < 0.05, n = 5. F LM.4T1 and HM.4T1 cells were cultured in 6-well plates. When cells became 80% confluent, total protein and total RNA were isolated. The levels of E-cadherin, \(\upbeta\)-catenin, and Snail protein were examined by western blotting (n = 3). The levels of E-cadherin (n = 3) and Snail mRNA (n = 4) were examined by RT-qPCR. The results are presented as the mean ± SEM. Statistical significance was analyzed by two-way ANOVA with multiple comparisons. **p < 0.05. G Sphere formation assay using LM.4T1 and HM.4T1 cells. For sphere generation, one thousand cells were seeded into 96-well ultra-low attachment plate and grow for 4 days. The diameter of each sphere measured by using the Image J software and the volume of each sphere were calculated. The scale bar indicates 50 μm. The results are presented as the mean ± SEM. Statistical significance was analyzed by unpaired Student’s t test. ****p < 0.0001, n = 6. H One thousand 4T1, LM.4T1, or HM.4T1 cells were cultured in 96-well ultra-low attachment plates, and the growth of each cell type was compared by MTT assay. The results are presented as the mean ± SEM. Statistical significance was analyzed by one-way ANOVA with multiple comparisons. ****p < 0.0001, n = 9. I The ability of cell migration and invasion in 3D was examined by sphere invasion assay. After four-day culture to form spheroids (day 0), medium was replaced with Matrigel and the invasion of cells was observed on day 5 and 10. The invasion area was measured by using the Image J software. The results are presented as the mean ± SEM. Statistical significance was analyzed by unpaired Student’s t test. **p < 0.001, n = 5. The scale bar indicates 100 μm