Figure 3
From: Molecular characterization of breast cancer CTCs associated with brain metastasis
The assessment of proliferation-related biomarkers in breast cancer patient-derived CTCs. a Ki67 staining in PanCK+ and CD44+/CD24− CTC subtypes isolated from breast cancer patient blood. Scale bar = 10 μm. b uPAR and int-β1 expression in single CTCs designated as Ki67Low (n = 60) or Ki67High (n = 48). p-value was calculated by two-way matched ANOVA. Despite a statistically significant overall difference, 20–30% individual CTCs in both groups had similar uPAR and int-β1 expression. c Ki67 expression in single CTCs designated as uPAR−/int-β1− (n = 17) or uPAR+/int-β1+ (n = 11). p-value calculated by unpaired t-test. Of note, 7 out of 17 uPAR/int-β1 CTCs had higher Ki67 expression than the rest of the group. d Demonstration of Ki67, uPAR, and int-β1 staining of single CTCs by DEPArrayTM. Note that the CTC in the third row, is designated as uPAR−/int-β1, but the Ki67 expression is higher than the Ki67Low CTC shown in the first row. e Flowchart depicting PBMC processing for concomitant cell surface and nuclear staining for single-step analysis by flow cytometry. PCNA was replaced by cleaved PARP (Asp214) in the last eight samples to accommodate an apoptosis marker in the nine-color panel. f MTN index of Ki67Low vs. Ki67High CTCs assessed by the PermiflowTM method. g Difference in Ki67 status of BCBM vs. no BCBM patient-derived CTCs (n = 8). p-value calculated by two-way matched ANOVA test. h Difference in uPAR/int-β1 expression status in BCBM vs. no BCBM patient-derived CTCs (n = 8). p-value calculated by two-way matched ANOVA test. i uPAR and int-β1 expression in CTC groups designated as Ki67Low or Ki67High (n = 32). p-value calculated by two-way matched ANOVA. j Ki67 expression in CTC groups designated as uPAR−/int-β1− or uPAR+/int-β1+ (n = 16). p-value calculated by unpaired t-test. k Circos plot depicting cancer-related gene exon insertions/deletion (in/del) mutations in uPAR+/int-β1+ and uPAR−/int-β1− CTC subsets obtained from BCBM and no BCBM patients. From outer to inner circle: uPAR+/int-β1+ and uPAR−/int β1− CTCs derived from BCBM patient; uPAR+/int β1+ and uPAR−/int-β1− CTCs derived from no BCBM patient. 881/884 in/del mutations found in BCBM subsets, whereas 434/211 in/del in no BCBM subsets. Error bars represent s.e.m
