Journal: Nature Communications
Article Title: Molecular characterization of breast cancer CTCs associated with brain metastasis
Figure Lengend Snippet: 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 Ki67 Low ( n = 60) or Ki67 High ( 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 DEPArray TM . Note that the CTC in the third row, is designated as uPAR−/int-β1, but the Ki67 expression is higher than the Ki67 Low 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 Ki67 Low vs. Ki67 High CTCs assessed by the Permiflow TM 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 Ki67 Low or Ki67 High ( 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
Article Snippet: Anti-human FITC-CD45 (#304054, dilution 1:200), CD34 (#343504, dilution 1:200), CD105 (#323204, dilution 1:200), CD90 (#328108, dilution 1:200), CD73 (#344016, dilution 1:100), APC-Cy7-CD44 (#103028, dilution 1:100) and BV650-CD44 (#103049, dilution 1:100), BV510-CD24 (#311126, dilution 1:100), APC-int-β1 (#303008, dilution 1:100), AF647-Pan-cytokeratin (#628604, dilution 1:100), PE-PCNA (#307908, dilution 1:100), and AF594-PCNA (#307914, dilution 1:100), BV421-Ki67 (#350506, dilution 1:100), PE-uPAR (#336906, dilution 1:100) from Biolegend.
Techniques: Derivative Assay, Staining, Isolation, Expressing, Flow Cytometry, Cytometry, Marker