Mesenchymal Migration on Adhesive–Nonadhesive Alternate Surfaces in Macrophages

Abstract Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein‐repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive–nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive–nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive–nonadhesive alternate substrates in macrophages.

. Mesenchymal migration on the adhesive-nonadhesive alternate surface needs pre-adhesive on the FN region.
A) Cells keep a suspending round morphology on the pure PEG region. B) Macrophage adheres and spreads when it reaches a FN region. C) The cell extends out to PEG region and then retracts. D) Macrophage extends out to the PEG region and then detaches from the FN region followed by turning round. Scale bars, 10 μm from A) to D).

Figure S3. Macrophages adhere and form podosomes on non-adhesive regions.
A) Scanning electron microscopy images of macrophages on FN-PEG boundary of the alternate non-adhesive surface. Scale bar, 2 μm. B) Gentle mechanical stimulation could not move the cell extending on the PEG region. Scale bar, 5μm. C) Cells form podosomes on the PEG region in various patterns, including meshwork patterns, circle arrays, circular patterns, and stripe patterns. Scale bars, 5μm. D) Macrophages could extend to the F127 (another anti-adhesion regent) region and form podosomes. Scale bars, 10 μm. E,F,G) Macrophages could extend to the PEG region and form podosomes on alternate PEG/HMDS E), PEG/glass F), and PEG/collagen G) substrates. Scale bars, 10 μm.  membrane (violet curves) was measured every 10 s in 200 s duration prior and after the podosome appearance. E) The change of the area prior and after the podosome appearance. The area at each time point was normalized by the area at t=0. Data was obtained from 6 cells in 3 independent experiments and fitted by logistic function (red curve). The time lag from t=0 to the point when the membrane protrudes fastest was Δt ≈20 s according to the fitting. a.u., arbitrary unit.

Figure S6. Inhibition of myosin IIA facilitate cell motility on the adhesive-nonadhesive alternate surfaces while activation of myosin pathway inhibited podosome formation and migration on the adhesive-nonadhesive alternate surfaces.
A,B) Blebb promotes cell motility on the alternate non-adhesive PEG surface. Scale bar, 20 μm. C,D) Macrophages could not adhere to pure PEG surfaces treatment with Blebb C) and Y27632 D) treatment. Scale bars, 10 μm. E,F,G,H) PGE 2 E) and CalyA G) decrease cell motility on the alternate non-adhesive PEG surface. Scale bars, 10 μm. 25 μM PGE 2 F) and 5 nM CalyA H) could block the formation of podosomes. Scale bars, 5 μm.

Figure S7. Narrower FN stripes promote cell migration across non-adhesive gaps.
A) Simulation presentation of cell migration on the alternate non-adhesive surface with 10 μm FN stripe. B) Number of crossings in 40 repeats of simulation in 20 μm FN stripe and 10 μm FN stripe groups. C) Migration diagram of 10 μm FN stripe group from experimental results of movie S10. Cells could extend to the PEG region and complete crossings on the 10 μm FN stripe more easily than cells on the 20 μm FN stripe. D) Fraction of cells extending to the PEG region over time in the 20 μm and 10 μm FN stripe groups.

Movie M1. Mesenchymal migration can happen on the adhesive-nonadhesive alternate surfaces in macrophages.
Part I shows the time-lapse of a macrophage migrating from one FN region to another nonadhesive PEG region, related to Fig. 1A and B. Part II shows the migration of macrophages on stripe patterns with PEG gaps of 40 μm and 80 μm.

Movie M2. Motility of other cells on the adhesive-nonadhesive alternate surfaces
Part I shows that selected cancer cells can not extend to the PEG region. Part II shows that selected cell lines cannot extend to the PEG region. Part III shows that selected primary cells cannot migrate across the PEG region (human neutrophils can extend to the PEG region but cannot elongate and reach another FN region). Part IV shows that rat macrophages and raw264.7 cells can migrate across the PEG region, and that mouse macrophages can migrate across the F127-FN and PEG-collagen region.

Movie M3. Mesenchymal migration on adhesive-nonadhesive alternate surfaces needs preadhesion on the FN region.
Part I shows that cells cannot adhere on pure PEG until they reach an FN region. Part II shows that cells extending to the PEG region will retract or detach when failing to reach another FN region.

Movie M4. Gentle mechanical stimulation cannot move the cell extending on the PEG region.
This Movie M shows that macrophage adheres on the PEG surface.

Movie M5. Podosome dynamic during migration on the adhesive-nonadhesive alternate surfaces.
Time-lapse of macrophage labeled by SiR-actin using TIRFM. Part I shows that podosomes appear when extending to the PEG region. Part II shows that the podosomes disappear when this part reaches the FN region. Part III shows that the podosomes disappear before retraction.
Movie M6. Cells retract to the FN region upon CytoD and CK666 treatment. This Movie M shows that podosomes are essential for cell migration on the alternate non-adhesive substrates.
Movie M7. Macrophages motility was reduced by WASP siRNA. Less cells extended to PEG region on the alternate non-adhesive substrates upon WASP siRNA transfection.

Movie M8. Podosome turnover in DIC images.
The left panel shows that membrane protuberances keep turnover on the PEG region. The right panel shows that the membrane protrudes out after an adjacent podosome appears.
Movie M9. Mesenchymal migration on the adhesive-nonadhesive alternate surfaces is enhanced upon Blebb and Y27632 treatment and inhibited upon PGE 2 and CalyA. Inhibition of myosin enhanced cell motility (part I) while activation of myosin IIA decreased it (part III). Inhibition of myosin IIA promotes this motility on adhesive-nonadhesive alternate surface, but does not lead to cell adhesion on pure PEG surface (part II).

Movie M10. Simulation of mesenchymal migration on the adhesive-nonadhesive alternate surfaces.
Part I shows a cell crossing the PEG gap. Part II shows the cell detaches and re-adheres on the FN region. Part III shows the cell motility is enhanced when the generation of podosomes is upregulated. Part IV simulates that the cell migrates on triangular lattice circular patterns. MCSs, Monte Carlo steps.

Movie M11. Cell migration on narrow stripe patterns.
Part I shows the simulation result of cell migrating on stripe pattern with 10 μm FN stripe and 40 μm PEG gap. Part II shows the experimental result of cells migrating on the same stripe pattern. MCSs, Monte Carlo steps.