Fig. 4
TFF3 is an epithelial circulating tumor cell (CTC) marker involved in MDA-MB-231 migration and dissemination. A Box and whiskers plot of TFF3 expression data (log2(signal intensities)) using data from gene expression profile experiments GEP1 and GEP2. B (left) Dot plot of the secreted TFF3 concentration in conditioned media (TFF3 quantification in technical triplicates from 3 cultures per cell line), and (right) representative images of intracellular TFF3 positive cells (40x objective). C Line chart representation of the mean±standard deviation (SD) optical density measured in a time-course proliferation assay (n=3 observations in 3 lentiviral infection replicates). D (left) Dot plot of the number of migrating cells in a Boyden chamber assay, and (right) representative images of GFP expressing cells at the chamber bottom side (4x objective). E (left) Dot plot of the number of Matrigel invading cells in a Boyden chamber assay, and (right) representative images of GFP expressing cells at the chamber bottom side (4x objective). F (left) Dot plot of the number of vascular loops, and (right) representative images of GFP expressing cell loops (4x objective). G Line chart representation of the mean±SD sum of the tumor masses measured upon cell injection at the inguinal left and axillary right mammary fat pads of NOD SCID mice (n=11 shCTRL and n=11 shTFF3). H (left) Dot plot of single CTC (sCTC) counts, and (right) representative images of COX IV positive CTCs (60x oil immersion objective). I (left) Dot plot of CTC cluster (cCTC) counts, and (right) representative images of COX IV positive cCTCs (60x oil immersion objective). J (left) Dot plot of metastatic foci extent measured in lung sections, and (right) representative images of COX IV positive metastatic cells (10x objective). K (left) Dot plot of TFF3 relative expression (FC=(2-ΔΔCt); ACTB endogenous control) in different CTC subpopulations (EpCAMhi: EpCAM-enriched; HER2/EGFRhi: HER2/EGFR enriched; size-selected: isolated by filtration), and (right) representative single-fluorescence and merged channel images of a cCTC containing one EpCAM+/TFF3+ tumor cell (arrow). L (top) Bar chart of TFF3 positivity percentage according to EPCAM expression in CTC-enriched blood samples of breast cancer patients, and (bottom) 2x2 contingency table of raw values