Figure 3 from Breast Cancer Stem Cell–Derived Tumors Escape from γδ T-cell Immunosurveillance In Vivo by Modulating γδ T-cell Ligands
MMP14 expression increases γδ T-cell migration in basement membrane-like Matrigel and in BCSC5 tumor tissue. A, Representative dot plots (left) and histograms (right) of γδ T cells expressing mock, MMP14, or MMP14E240A on day 3 after transduction with a MOI of 5. UT cells served as control. B, Schematic illustration of the 3D migration assay in Matrigel (left). γδ T cells were seeded into ibidi μ-angiogenesis slides in 100% Matrigel. Migration toward medium supplemented with 10% FBS and 50 ng/mL CXCL12 was assessed via confocal microscopy after 48 hours. Statistical analysis (right) of the percent of γδ T cells migrating further than 10 and 100 μm (medians are indicated). Results of 8–24 wells per condition from five independent experiments are shown. Kruskal–Wallis test followed by Dunn post hoc test comparing transduced with untransduced cells. C, Migration of CMFDA-labeled UT (see also Supplementary Video S1) or MMP14 expressing (see also Supplementary Video S2) γδ T cells (green) in vibratome sections of viable BCSC5 xenograft tumors. Shown are representative Z-projections of confocal time-lapse videos from BCSC5 xenograft tumor slices stained for EpCAM (blue) and fibronectin (red) to identify tumor cell regions and stromal compartments, respectively. D, The mean speed of γδ T cells migrating in BCSC5 xenograft tumor slices is shown (left; Mann-Whitney test). The same data were analyzed with respect to the mean speed of γδ T cells in stromal ECM compartments (fibronectin+) and tumor cell regions (EpCAM+) of BCSC5 xenograft tumor slices (right; Kruskal-Wallis test followed by Dunn post hoc test comparing all groups against each other). E, The percentage of cells, which have been able to enter EpCAM+ tumor tissue (left) or which have resided over 50% of their monitored time inside EpCAM+ tumor tissue (right), was quantified in each time-lapse experiment (median, minimum to maximum). Unpaired t test, two tailed. D, Results from 15 (UT) and 11 (MMP14) time-lapse acquisitions from seven independent experiments are shown including at least 440 tracks per group. Outliers were identified and removed using the ROUT method (Q = 2%). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. ECM, extracellular matrix; EpCAM, epithelial cellular adhesion molecule; UT, untransduced.