Fig. 5: Illustration of different cell migration patterns under intensive force application with CD-PNIPAM-RGD and CD-PNIPAM-RTD.

a Schematic of integrin αvβ3 activated with CD-PNIPAM-RGD and time-lapse brightfield images showing cell migration over a 4-day period. Scale bar: 100 μm. b Fluorescent images at day 4 from a. Scale bar: 100 μm. c Representative images displaying enhanced SNAI1 expression in amoeboid cells compared with mesenchymal cells. Scale bar: 10 μm. d Representative images displaying enhanced E-cadherin expression in mesenchymal cells compared with amoeboid cells. The immunostaining images of actin are highlighted in green and the nuclei (DAPI, 4,6-diamidino-2-phenylindole) in blue, SNAI was presented in yellow and E-cadherin was presented in red. e Schematic of integrin αvβ6 activated with CD-PNIPAM-RTD and time-lapse brightfield photograph showing cell migration over a 4-day period. Scale bar: 100 μm. f–h Quantitative analysis of cell phenotype distributions from cells in hydrogels functionalized with CD-PNIPAM-RGD or CD-PNIPAM-RTD after 4 days of force application, used to evaluate the effects of (f) pulsed frequency, (g) NIR power densities, and (h) actuator molecular weight (Mw). f–h Each data point represents an independent spheroid (biological replicate, n = 9 spheroids/group, 3 experiments). For (f) *P = 0.01565, *P = 0.01766, ns = 0.62788, ns = 0.75417, (g) *P = 0.03366 *P = 0.01495, ns P = 0.97658, ns P = 0.58146 (h) **P = 0.00126, *P = 0.02328, ns P = 0.84325, ns P = 0.34575. i–k Quantitative analysis of cell maximum migration length after 4 days of force application, used to evaluate the effects of i pulsed frequency, j NIR power densities, and k actuator molecular weight (Mw). (i-k) The sample sizes for each condition are as follows: (i) n = 54, 54, 30, 65, 65, 60 cells (j) n = 54, 65, 30, 24, 25, 60 cells (k) n = 53, 48, 30, 51, 50, 60 cells from left to right taken from 9 spheroids respectively. For (i) *P = 0.04095, ***P < 0.001, *P = 0.04568, **P = 0.0928, (g) *P = 0.04611 ***P < 0.001, *P = 0.04282, ***P < 0.001 (h) ***P < 0.001, ***P < 0.001, **P = 0.0764, ***P < 0.001. The data in f-k are presented as mean values  ±  S.D. Data from different conditions were compared using a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. For CD-PNIPAM-RGD actuators, increased illumination frequency, power density, and actuator molecular weight enhanced both cell MAT and migration. In contrast, for CD-PNIPAM-RTD actuators, these increases improved cell migration but did not significantly affect the MAT ratio.