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Fig. 2 | Breast Cancer Research

Fig. 2

From: Exosomal Wnt7a from a low metastatic subclone promotes lung metastasis of a highly metastatic subclone in the murine 4t1 breast cancer

Fig. 2

Co-injection of LM.4T1 augmented the lung metastasis of HM.4T1 cells. A One hundred and thousand LM.4T1, HM.4T1 cells, or 1:1 cell mixture were injected into the 3rd mammary pad of BALB/c mice. Tumor sizes were monitored for 28 days, and the volume of tumors was calculated. The results are presented as the mean ± SEM. n = 5. B After 4 weeks of tumor growth, mice were euthanized, and tumor weights were measured. The results are presented as the mean ± SEM. n = 5. C The number of metastatic tumor nodules in the lung was quantified and compared among three different groups. The results are presented as the mean ± SEM. Statistical significance was analyzed by one-way ANOVA with multiple comparisons. *p < 0.05, ***p < 0.001, n = 4. D One hundred and thousand 1:1 cell mixture of RFP-labeled LM.4T1 cells and GFP-labeled HM.4T1 cells was injected into the 3rd mammary pad of BALB/c nude mice. Four weeks after the injection, metastasized cells in lung tissues were examined under a fluorescent microscope (Olympus stereoscopic microscope SZX12). Arrows indicate three tumors detected on lung surface. Photographs of Bouin’s solution-fixed lungs are also presented. E Two thousand cells were allowed to grow for 7 days in 96-well ultra-low attachment plates. The diameter of each spheroid was measured by Image J software, and the volume of each spheroid was calculated. The scale bar indicates 100 μm. The results are presented as the mean ± SEM. Statistical significance was analyzed by unpaired Student’s t test. ****p < 0.0001. n = 5. F One thousand 1:1 cell mixture of LM.4T1-RFP cells and HM.4T1-GFP cells was allowed to grow for 4 days in 96-well ultra-low attachment plates. The localization of each cell type was examined under a fluorescence microscope (KEYENCE, All-in-one fluorescence microscope BZ-X700). Scale bar indicates 100 μm. G Medium was replaced with Matrigel, and the cells were cultured for an additional 10 days. Cells in the invading front (box 1) and in the center of spheroids (box 2) were examined using a confocal microscope (Carl Zeiss Confocal Laser Scanning Microscope LSM780). Scale bar indicates 200 μm

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