Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites  Anam Qudrat, Abdullah Al Mosabbir, Kevin Truong  Cell Chemical Biology  Volume 24, Issue 6, Pages 703-711.e2 (June 2017) DOI: 10.1016/j.chembiol.2017.05.008 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 703-711. e2DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Detector Cells Expressing TNFR1chi and PM-Labeled RCaMP1.07 Trigger a Ca2+ Signal with Extracellular TNFα (A) Cartoon of activation mechanism of TNFR1chi. (B) Images showing TNFα detector cells stimulated with 10 ng/mL of TNFα show dim (before) and bright (after) outline of cell periphery. Images are false colored: mCherry, red. Scale bar, 40 μm. (C) Representative Ca2+ trace observed in TNFR1chi following stimulation with 10 ng/mL active TNFα. (D) Representative Ca2+ trace observed with 5× dilution of medium in a co-culture system of TNFα detector cells and TNFα source cells. (E) Type of cellular response seen in TNFα detector cells when stimulated with 10 ng/mL TNFα over an observation period of 10 min. (F) Percent cell response seen in TNFα detector cells compared with cells transfected only with the VEGFR2 tail or the Ca2+ sensor RCaMP1.07 stimulated with [ATP]f = 10 μM, [TNFα]f = 10 ng/mL, 5× media dilution, [IL-6]f = 10 ng/mL, [lipopolysaccharide (LPS)]f = 10 μg/mL, or [staurosporine (STS)]f = 100 nM. In (E) and (F), error bars denote SD. Samples were compared with one-factor ANOVA followed by a Tukey-Kramer post hoc test. Asterisks indicate significance: *p < 0.05, **p < 0.01. All experiments were repeated at least three times. See also Figure S1 and Movie S1. Cell Chemical Biology 2017 24, 703-711.e2DOI: (10.1016/j.chembiol.2017.05.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Seeking Cells Expressing TNFR1chi and CaRQ Dynamically Bleb when Stimulated with 10 ng/mL Active TNFα (A) Cartoon of activation mechanism of the TNFR1chi and CaRQ system. (B) Images showing Venus fluorescence of CaRQ expression from the TNFα seeking cells. Before stimulation of cells with 10 ng/mL active TNFα, no blebs are seen (t = 0 min). Post stimulation, blebs protrude (t = 1 min), increase in number (t = 2 min), retract (t = 3 min), and reappear (t = 4 min). Rectangular box shows zoomed region (4×). Arrows show blebs. Images are false colored: Venus, green. Scale bars, 40 μm (top left) and 10 μm (top right, applies to all subsequent panels). (C) Representative kymograph of the expansion and retraction kinetics along the normal line, L, running from the center of the cell to the periphery of a protruding bleb (left). Dotted line traces an expansion and retraction cycle; bleb expansion and retraction occur orthogonal to the cell body. (D) Percentage of TNFα seeking cells blebbing post treatment with [ATP]f = 10 μM, [TNFα]f = 10 ng/mL, or [Y-27632]f = 1 μM (i.e., ROCK I and II inhibitor). Error bars denote SD. Samples were compared with one-factor ANOVA followed by a Tukey-Kramer post hoc test. *p < 0.01. All experiments were repeated at least three times. (E) Blebbing frequency (blebs/min) of seeking cells as a function of their distance from the TNFα source. Multiple group comparisons were made with one-factor ANOVA with Tukey-Kramer post hoc test. *p < 0.01. All experiments were repeated at least three times. (F) Representative Ca2+ trace of HEK293 cells transfected with PM-labeled RCaMP1.07 and CaRQ when stimulated with [ATP]f = 10 μM. All experiments were repeated at least three times. Inset shows representative images of cells before and after stimulus. Arrow show a bleb. Images are false colored: mCherry, red; Venus, green. Scale bar, 10 μm. (G) Blebbing frequency (blebs/min) of HEK293 cells transfected with PM-labeled RCaMP1.07 and CaRQ with no, single, or oscillating Ca2+ signals. Multiple group comparisons were made with one-factor ANOVA with Tukey-Kramer post hoc test. *p < 0.01. All experiments were repeated at least three times. See also Movie S2. Cell Chemical Biology 2017 24, 703-711.e2DOI: (10.1016/j.chembiol.2017.05.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 TNFα Seeking Cells Show Directed Migration toward TNFα Sources (A) Images of TNFα seeking cells co-cultured with a cluster of TNFα source cells showing initial (t = 0 hr) and final (t = 2 hr) displacement. Dotted line bisects TNFα seeking cluster. Images are false colored: mCherry, red; Venus, green. Scale bar, 100 μm. (B) Merged images (left) and path lengths (n = 10) (right) of TNFα seeking cell displacement over 2-hr time lapse. Images are false colored: t = 0 hr, red; t = 2 hr, green. Black dots represent endpoints. Scale bars, 100 μm (left) and 1 μm (right). (C) Rose diagrams of the TNFα seeking cell cluster show that as the cells near a cluster of TNFα source cells, their spread narrows from 150° at 300 μm to 90° at 30 μm to 60° within 5 mm of the source. Arrow shows alignment axis of the two cell clusters. The initial and final samples were compared with Watson's two-sample test of homogeneity. *p < 0.001. (D) Images of TNFα seeking cells co-cultured with a cluster of TNFα source cells showing characteristic features of directed migration on days 1 and 2. Images are false colored: mCherry, red; Venus, green. Scale bar, 100 μm. (E) Transwell experiments (pictorial, left) showing cell migration in response to co-culturing with synthetic (i.e., TNFα source and null) or natural cell lines (i.e., U937 pre- and post-differentiation) as well as stimuli (i.e., [TNFα]f = 10 ng/mL, [ATP]f = 10 μM, or [Y-27632]f = 1 μM) (i.e., ROCK I and II inhibitor). “++” indicates addition of TNFα. Sample groups were compared with one-factor ANOVA followed by a Tukey-Kramer post hoc test. *p < 0.01. All experiments were repeated at least three times. See also Figures S2 and S3; Movies S3 and S4. Cell Chemical Biology 2017 24, 703-711.e2DOI: (10.1016/j.chembiol.2017.05.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 TNFα Seeking Cells Expressing VSVG and TK Fusing Cells Fuse with TNFα Sources upon Low pH Induction and Die upon Ganciclovir Treatment (A) Cartoon of the activation mechanism of TNFα fusing cells. (B) Images showing TNFα seeking cells fusing with a cluster of TNFα source cells before and after low pH (pH 5.7) induction. Arrows show syncytia. Images are false colored: mCherry, red; Venus, green; Cerulean, cyan. Scale bar, 40 μm. (C) Percentage of the number of syncytia after 7-day treatment with [ganciclovir]f = 10 μM post low pH induction (day 0). Error bars denote SEM. Sample groups were compared with one-factor ANOVA followed by a Tukey-Kramer post hoc test. *p < 0.001. (D) Percent viability of fusing cells before and after 7 days under different conditions of [ganciclovir]f = 10 μM and low pH (pH 5.7) induction. Error bars denote SEM. Sample groups were compared with one-factor ANOVA followed by a Tukey-Kramer post hoc test. *p < 0.01. All experiments were repeated at least three times. (E) Images showing TNFα fusing cells post 7-day treatment with [ganciclovir]f = 10 μM following low pH induction. Images are false colored: Venus, green; Cerulean, cyan; bf, bright field. Scale bar, 20 μm. See also Figure S4. Cell Chemical Biology 2017 24, 703-711.e2DOI: (10.1016/j.chembiol.2017.05.008) Copyright © 2017 Elsevier Ltd Terms and Conditions