Figure 4. Live cell, time-lapse microscopy shows reduced edge dynamics and acoustic force microscopy reveals increased binding avidity of meso^FAP in comparison to controls.

A. Representative image of sorted meso^FAP CAR T-cells (pink/salmon) interacting with AsPC-1 (cyan), CAFs (magenta), or AsPC-1 and CAFs. B. Representative images visualizing edge dynamics (percentage change of CAR T-cell area over time) of meso^FAP CAR T-cells over 20 frames (frame 1 dark blue to frame 20 white). C. Percentage change of meso^FAP CAR T-cell area over time (number of cell interactions counted: PDAC, n=28; P+C, n=25; CAF, n=42; not on target, n=25). D,F. Representative images visualizing edge dynamics of D. meso^CD19 CAR T-cells or F. UTD T-cells. E,G. Percentage change of E. meso^CD19 CAR T-cells over time (number of cell interactions counted: PDAC, n=28; P+C, n=16; CAF, n=18; not on target, n=23) or G. UTDs over time (number of cell interactions counted: PDAC n=12; P+C, n=8; CAF, n=10; not on target, n=14). H-J. Schematic showing 1:1 ratio of AsPC-1 and CAFs in the flow chamber to measure cell binding of meso^FAP (H), meso^CD19 (I), and UTD (J) avidity by acoustic force microscopy (created with BioRender.com). K. Percentage of meso^FAP, meso^CD19, or UTD bound to 1:1 AsPC-1 and CAF mixture (left) with an increasing amount of acoustic force (pN) and (right) at 1000pN as measured by z-Movi. L. Fold increase of bound meso^FAP CAR T-cells or UTD cells with the addition of FAP-TEAM on HFF fibroblasts using z-Movi. All data represent mean +/−SEM. Representative images have been chosen within 1 standard deviation from the median. Results were obtained from biological duplicates, from at least 3 different locations on the slide. Stars represent significance as determined by unpaired two-tailed t-tests. *p < 0.05, **p < 0.01 ***p < 0.001****p < 0.0001. UTD: untransduced T-cell, pN: picoNewton, P+C: PDAC+CAF.