Fig 2: Analysis of CTCs of the EpCAM-depleted fraction from the CellSearch system.a Cells from MDA-MB-231 cell line were spiked into healthy donors’ blood and first enriched EpCAM-based. The EpCAM-depleted fraction (EDF) was collected and the remaining tumour cells were subsequently enriched based on Trop-2/CD-49f. b EpCAM-depleted fractions from seven patients were collected and divided into two parts. From one part, CTCs were enriched EpCAM-based and from the other part, CTCs were enriched based on Trop-2/CD-49f. The P value was determined by paired two-sided t test (* indicates a P value <0.05). c CTC positivity rates of EpCAM-based enrichment from blood and subsequent Trop-2/CD-49f-based enrichment from the EpCAM-depleted fraction of 15 metastatic breast cancer patients. d Mutations detected by targeted next-generation sequencing on DNA from CTCs from the EpCAM-depleted fraction and primary tumour tissue. Values indicate the variant allele frequency. Cutoffs of 12.5% or 5% were applied for CTCs or tissue samples, respectively. The grey box indicates that the respective region was not covered in that sample.

Fig 3: Comparative analysis of EpCAM high- and low-expressing CTCs.a Cells from MCF7 and MDA-MB-231 cell lines were spiked into healthy donors’ blood samples, enriched based on Trop-2 and CD-49f and stained on glass slides. Anti-EpCAM mean fluorescence intensity (MFI) was determined as reference. b CTCs were enriched Trop-2/CD-49f based, stained on glass slides, and the anti-EpCAM MFI was determined. Kaplan–Meier plots of progression-free survival (PFS) (c) and overall survival (OS) (d) of patients positive or negative for CTCs with an anti-EpCAM MFI below 12.5. Kaplan–Meier plots of progression-free (e) and overall survival (f) of patients positive or negative for CTCs with an anti-EpCAM MFI above 75. The P values were determined by the log-rank test. g CTCs from three independent patients were stained in suspension and isolated in order to analyse chromosomal aberrations and mutations. Upon whole-genome amplification (WGA) cells with high EpCAM expression (red) and low EpCAM expression (green) were selected for low-pass sequencing based on the WGA products’ quality. Further cells were analysed by mutation analysis (blue circled). h Chromosomal aberrations of intra-patient EpCAM high- and low-expressing CTCs from three patients were detected by low-pass sequencing. Dendrograms are based on hierarchical clustering. i Mutations in intra-patient EpCAM high- and low-expressing CTCs were detected by targeted next-generation sequencing. Values indicate variant allele frequency. A cut-off of 12.5% was applied. Grey boxes indicate that the respective region was not covered. No mutations were found for patient 3. A.U. artificial unit.

Fig 4: Expression of targets for CTC enrichment.Gene expression data from 135 CTCs and 195 CTCs, respectively, were obtained from gene expression omnibus (national centre for biotechnology information; GSE144494 and GSE144495). a Correlation coefficients for the correlation between the expression of EpCAM and the respective CTC enrichment marker in CTCs were calculated by non-parametric Spearman correlation. The P values were adjusted to multiple testing by Benjamini–Hochberg correction (* indicates a P value <0.05, ** indicates a P value <0.01, *** indicates a P value <0.001). b Comparison of the expression of the respective CTC enrichment marker in EpCAM-positive and -negative CTCs. Significance levels were determined by the two-sided Mann–Whitney test and were adjusted to multiple testing by Benjamini–Hochberg correction.