ARTICLE DOI: 10.1038/s41467-018-06641-z OPEN Matrix mechanical plasticity regulates cancer cell migration through confining microenvironments Katrina M. Wisdom 1, Kolade Adebowale 2, Julie Chang3, Joanna Y. Lee 1, Sungmin Nam1, Rajiv Desai4, Ninna Struck Rossen5, Marjan Rafat 5, Robert B. West6, Louis Hodgson 7 & Ovijit Chaudhuri 1 Studies of cancer cell migration have found two modes: one that is protease-independent, requiring micron-sized pores or channels for cells to squeeze through, and one that is pro- 1234567890():,; tease-dependent, relevant for confining nanoporous matrices such as basement membranes (BMs). However, many extracellular matrices exhibit viscoelasticity and mechanical plasti- city, irreversibly deforming in response to force, so that pore size may be malleable. Here we report the impact of matrix plasticity on migration. We develop nanoporous and BM ligand- presenting interpenetrating network (IPN) hydrogels in which plasticity could be modulated independent of stiffness. Strikingly, cells in high plasticity IPNs carry out protease- independent migration through the IPNs. Mechanistically, cells in high plasticity IPNs extend invadopodia protrusions to mechanically and plastically open up micron-sized chan- nels and then migrate through them. These findings uncover a new mode of protease- independent migration, in which cells can migrate through confining matrix if it exhibits sufficient mechanical plasticity. 1 Department of Mechanical Engineering, Stanford University, Stanford, CA 94305, USA. 2 Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA. 3 Department of Bioengineering, Stanford University, Stanford, CA 94305, USA. 4 School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA. 5 Department of Radiation Oncology, Stanford University, Stanford, CA 94305, USA. 6 Department of Clinical Pathology, Stanford University, Stanford, CA 94305, USA. 7 Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Correspondence and requests for materials should be addressed to O.C. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:4144 | DOI: 10.1038/s41467-018-06641-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06641-z arcinoma progression and metastasis require that cancer matrix viscosity, matrix architecture in the tumor micro- Ccells traverse basement membranes (BMs): first through environment may also exhibit elevated mechanical plasticity, the BM separating epithelial and stromal tissue, and then enabling cell-generated forces to induce permanent micro- across the BM lining blood vessels (Fig. 1a)1,2. Invadopodia are structural rearrangements in the matrix. This raises the possibility the actin-rich, invasive protrusions that enable cancer cells to that cells can carry out invasion into, and migration through, invade the BM, and they are thought to do so by secreting pro- confining matrices using cell-generated forces to dilate pores if teases to degrade the BM3,4. Recent studies suggest that without those matrices are sufficiently plastic. matrix degradation, nanometer-scale pores of BM would physi- Here we assess the role of matrix plasticity in mediating cally limit invasion, as cells are unable to squeeze through elastic invasion and migration of confined cancer cells. We develop IPN or rigid pores smaller than roughly 3–5 μm in diameter5–11. hydrogels, which are nanoporous, present BM ligands to cells, However, pore size may be malleable—particularly in tumor tis- and enable mechanical plasticity to be modulated independent of sue. While it has been long appreciated that tumor tissue is up to stiffness. Cells in high plasticity (HP) IPNs carry out protease- an order of magnitude stiffer than normal tissue12, noninvasive independent migration through the IPNs, while cells in low clinical imaging has also revealed breast tumor tissue to be more plasticity (LP) IPNs mostly do not migrate. Cells in HP IPNs first viscous, or liquid-like, than normal tissue13. The elevated visc- utilize invadopodia protrusions to mechanically and plastically osity of tumor tissue is thought to arise in part from abnormal open up channels, and then generate protrusive forces at the tissue cross-linking that accompanies breast cancer leading edge to migrate through them. Together, these findings progression13,14. Because matrix plasticity can be related to establish that cells can migrate through confining matrices such a b Human mammary tumor indentation c Human mammary tumor sample Invasion out of Blood vessel Basement 0 primary tumor intravasation membrane Invading 40 Hours 1 2 3 4 cancer cell Indentation 1st Invadopodium 20 2nd Mammary Force (mN) stroma Mammary Extravasation 0 duct Before indentations After indentations 0.0 0.5 1.0 Indentation depth (mm) deMammary tumor tissue fg 4 ns #### Perm. deformation Low plasticityMedium plasticity High plasticity Ionically 0.15 **** Total indentation crosslinked ** 60 alginate network 3 **** Basement 0.10 40 membrane 2 matrix Loss tangent 0.05 1 20 Molecular weight Alginate + 2+ Young's modulus (kPa) 10 12 15 10 12 15 Indentation plasticity (%) differences 0.00 0 molecular weight + Ca 0 LP MP HP Human Mouse LP MP HP h i jkPlasticity: 100 Pa creep Plasticity: 10 Pa creep l m Particle diffusion 100 40 100 Pa 0 Pa 100 #### HP 1.0 Creep Recovery LP 50 80 **** MP 1.0 HP IPN **** 30 MP 40 LP MP IPN ** HP Elastic LP IPN **** 60 **** 30 20 0.5 Strain 0.5 40 ns Total Viscoelastic 20 10 Normalized strain 20 Permanent 10 Permanent strain (%) Partition coefficient Permanent strain (%) plastic/malleable Permanent strain (%) 367 3333 0.0 0 0 0 0.0 Time 0 5000 10,000 PA LP MP HP Silly 0.05 0.10 20 30 40 Time (s) putty rBM col-1 Loss tangent Nominal AuNP HP IPN LMW Alg size (nm) Fig. 1 Mechanical plasticity of interpenetrating networks of alginate and reconstituted basement membrane matrix (IPNs) can be independently tuned. a Schematic depicting invasion of basement membranes (green) during invasion and metastasis. b Schematic depicting the indentation tests performed on human mammary tumor tissue, and the corresponding force vs. indentation depth curves (green arrow—permanently retained indentation; red arrow— drop in peak force during second indentation; dotted line—25% of initial peak force). Subplot shows indentation test profile. c Before and after images of an indented mammary tumor sample. Indentation region outlined by dotted circle, and discolored tissue regions indicated by black arrows. Scale bar is 1 mm. d Indentation plasticity measurements of human tumor (two specimens from a tumor sample) and mouse tumor specimens (one sample each from four separate mice). e Schematic of approach to tuning matrix plasticity in IPNs of alginate (blue) and reconstituted basement membrane (rBM) matrix (green). f, g Young’s moduli (f) and loss tangent (g) of the different IPN formulations. The differences in loss tangent indicated are significantly different (**P < 0.01, ****P < 0.0001, ANOVA; ns not significant), as is the increasing loss tangent across this series of IPNs (####P < 0.0001, Spearman’s rank correlation). h Schematic depicting the elastic, viscoelastic, and plastic (permanent) portions of a material response in a creep and recovery test. i Representative creep and recovery tests of IPNs. j Permanent strain of IPNs, polyacrylamide gels (PA), and silly putty from creep and recovery tests. Statistically significant differences are indicated (**P < 0.01, ****P < 0.0001, ANOVA), plasticity across the IPNs (####P < 0.0001, Spearman’s rank correlation). k Permanent strain of HP IPN, alginate hydrogel, rBM matrix, and col-1 gels from creep–recovery tests. Statistically significant differences compared to HP IPN are indicated (****P < 0.0001, ANOVA). In f–k, bars indicate means and error bars indicate 95% confidence interval of the indicated biological replicates. l Permanent strain and loss tangent are correlated in the IPNs (R2 = 0.7953). m Partition coefficients for PEGylated gold nanoparticles of the indicated size encapsulated in IPNs for 4 days 2 NATURE COMMUNICATIONS | (2018) 9:4144 | DOI: 10.1038/s41467-018-06641-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06641-z ARTICLE as BMs or dense stromal tissue independent of proteases, if the Cells migrate in HP IPNs. Using this materials system for 3D cell matrix exhibits mechanical plasticity. culture, we first investigated whether matrix plasticity affected breast cancer cell morphology in confining microenvironments. Results Highly invasive MDA-MB-231 cells were fully encapsulated in the IPNs and stimulated with 50 ng mL−1 epidermal growth Mechanical plasticity in breast tumors. First, we assessed factor (EGF) (Fig. 2a). Striking morphological differences were mechanical plasticity in breast tumors. Indentation testing of observed between MDA-MB-231 cells in different IPNs after one human breast tumor tissue revealed that these tissues exhibit day, with cells adopting circular morphologies in LP IPNs, highly substantial mechanical plasticity, as they permanently sustained protrusive morphologies in HP IPNs, and intermediate substantial deformations following indentation and exhibited a morphologies in the MP IPNs (Fig. 2b, c and Supplementary loss in the peak force sustained in successive indentations Fig. 3a). Differences in morphology due to plasticity were also (Fig. 1b–d and Supplementary Fig. 1). Similar