Fig 1.
Expression of surface markers on EpCAMpos and EpCAMlow/neg cells.
Differential protein expression of EpCAM (green), Trop2, CD49f, CK8, CD146 and ADAM8 (red) in EpCAMpos (MCF7, ER+PR-HER2-, top; and SKBR3, ER-PR-HER2+, middle) and EpCAMlow/neg (MDA-MB-231, ER-PR-HER2-, bottom) cell lines is displayed by immunofluorescence staining of cytospins; blue = DAPI, 40x magnification.
Fig 2.
Adhesion of EpCAMpos/neg cells on manually spotted arrays.
2.5x104 EpCAMpos cells (pool of MCF7, SKBR3, HCC1500 and ZR-75-1; upper panel) and EpCAMlow/neg cells (MDA-MB-231; lower panel) were incubated for cell adhesion experiments on glass substrates (NEXTERION slides AL) manually coated with anti-EpCAM [Ber-EP4], anti-Trop2, anti-CD49f (0.1 mg/ml each), collagen I (Col), hyaluronic acid (HA) and laminin (Lam) (0.2 mg/ml each). Cell adhesion was visualized by Coomassie; 20x magnification.
Fig 3.
Adhesion of EpCAMpos/neg cells on multi-marker arrays.
2x105 EpCAMpos (cell pool of MCF7, SKBR3, HCC1500, ZR-75-1, TMX2-28) (A) and EpCAMlow/neg cells (MDA-MB-231) (B) were either stained with 1 μM MitoTracker Green FM (A) or MitoTracker Orange CM (B) and incubated for cell adhesion experiments on NEXTERION slides AL, coated with different antibodies and ECM molecules, alone and in combination (0.1 mg/ml each). The labeling above the single spots indicates respective capture molecules (Iso = isotype control, ms = mouse, Lam = laminin, Col = collagen, HA = hyaluronic acid, rt = rat, T = Trop2, EpC = EpCAM, 49f = CD49f, rb = rabbit). (C) Overlay image of (A) and (B); scale bars (white) = 500 μm, 20x magnification. (D) Array layout (5x5 mm) with 36 spots (spot diameter = 500 μm; pitch = 800 μm) printed on NEXTERION slides AL.
Fig 4.
Enrichment of EpCAMpos/neg cells with Dynabeads.
EpCAMpos (upper panel; MCF7, SKBR3, T47D, HCC1500 and ZR-75-1) and EpCAMlow/neg (lower panel; MDA-MB-231) cells were captured with Dynabeads coupled with antibodies for EpCAM, Trop2 and CD49f (1 μg ab/2.5x105 cells/1x107 beads). No cells bound to uncoated Dynabeads (NT = no target). Cells were imaged after a DAPI stain and are depicted as brightfield/DAPI merge; 10x magnification.
Table 1.
Patient characteristics for CTCs/primary tumors and number of CKpos/CD45neg and CKpos/CD45pos events within the EpCAM-depleted fractions.
Table 2.
Number of processed fractions and EpCAMneg CKpos/CD45neg and CKpos/CD45pos events for each marker.
Fig 5.
Number of EpCAM-enriched CTCs and potential CTCs/double positive events (CKpos/CD45pos) within the EpCAM-depleted fractions in blood of breast cancer patients.
(A) CTC count determined by EpCAM-enrichment and subsequent CK/DAPI stain in 29 blood samples of 25 patients (DIII and DIV). (B) Number of potential CTCs (top) and double positive events (bottom) within the respective EpCAM-depleted supernatants of the same blood samples; total event numbers (CKpos/CD45neg and CKpos/CD45pos) represent the sum of all events identified after 2–6 immunomagnetic enrichments for each blood/patient sample.
Fig 6.
Representative images of potential CTCs (CKpos/CD45neg) and double positive (CKpos/CD45pos) events enriched from EpCAM-depleted patient samples.
Immunofluorescence staining of (A) CTCs (top: #8, Adem-c-Met; bottom: #25, Dyna-CD49f) and (B) CKpos/CD45pos events (top: #8, Adem-c-Met; bottom: #13, Adem-Trop2) enriched after EpCAM-depletion are shown. Adem-/Dynabeads captured cells were stained for DAPI (blue), pan-CK (green) and CD45 (yellow); 40x magnification.
Fig 7.
Genomic profiling of CellCelector identified and isolated whole genome amplified EpCAMneg single cells confirms their malignant nature.
(A) One EpCAMpos CTC was selected and identified via CellSearch and re-identified with the CellCelector for a positive DAPI and CK-PE (displayed in the TRITC channel), and a negative CD45 (Cy5 channel) stain. Three EpCAMneg CTCs (I, II, III) from the CellSearch EpCAM-depleted fraction of the same patient were enriched with CD44-Adembeads and stained for DAPI/pan-CK-FITC/CD45-AF647. Single cells were isolated via the CellCelector, the whole genomic material was amplified and (B) genome wide aCGH profiles were obtained confirming that EpCAM-independent enrichment captures malignant cells. Chromosomal regions highlighted in grey show common somatic copy number alterations, light blue areas represent different chromosomal aberrations between both CTC populations.