Rules of contact inhibition of locomotion for cells on suspended nanofibers Jugroop Singha, Aldwin Pagulayanb, Brian A. Camleyc,d,1, and Amrinder S. Naina,b,1 aDepartment of Biomedical Engineering and Mechanics, Virginia Tech, Blacksburg, VA 24061; bDepartment of Mechanical Engineering, Virginia Tech, Blacksburg, VA 24061; cDepartment of Physics and Astronomy, Johns Hopkins University, Baltimore, MD 21218; and dDepartment of Biophysics, Johns Hopkins University, Baltimore, MD 21218 Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved February 7, 2021 (received for review June 9, 2020) Contact inhibition of locomotion (CIL), in which cells repolarize and (11–13). Recently, micropatterned substrates have been used to move away from contact, is now established as a fundamental understand restricted motility, developing one-dimensional (1D) driving force in development, repair, and disease biology. Much of collision assays where cell migration is constrained to straight what we know of CIL stems from studies on two-dimensional (2D) lines, allowing for a greater occurrence of cell–cell collisions to substrates that do not provide an essential biophysical cue—the quantify rates and outcomes of different types of cell–cell in- curvature of extracellular matrix fibers. We discover rules control- teractions (11, 13–15). These interactions do not necessarily re- ling outcomes of cell–cell collisions on suspended nanofibers and semble the stereotyped CIL behavior. Broadly, experiments and show them to be profoundly different from the stereotyped CIL simulations (16–18) have observed the following: 1) the classical behavior on 2D substrates. Two approaching cells attached to a stereotype of CIL with two cells contacting head-on, with both single fiber do not repolarize upon contact but rather usually mi- cells repolarizing (referred to as “reversal” or “mutual CIL”); 2) grate past one another. Fiber geometry modulates this behavior; after a head-on collision, only one cell reverses (“training” or when cells attach to two fibers, reducing their freedom to reorient, “nonmutual CIL”); and 3) cells manage to crawl past or over one only one cell repolarizes on contact, leading to the cell pair migrat- another, exchanging positions (“walk past” or “sliding”). Within ing as a single unit. CIL outcomes also change when one cell has the well-studied neural-crest cell explants, walk past is extremely recently divided and moves with high speed—cells more fre- rare (11), but it can occur in epithelial cells, especially in those quently walk past each other. Our computational model of CIL in that have been metastatically transformed or that have decreased fiber geometries reproduces the core qualitative results of the ex- BIOPHYSICS AND COMPUTATIONAL BIOLOGY periments robustly to model parameters. Our model shows that E-cadherin expression (15). the increased speed of postdivision cells may be sufficient to ex- Both 2D substrates and micropatterned stripes provide con- plain their increased walk-past rate. We also identify cell–cell ad- trollable and reproducible environments but neither fully models hesion as a key mediator of collision outcomes. Our results suggest the details of in vivo native cellular environments, which consist that characterizing cell–cell interactions on flat substrates, chan- of extracellular matrices (ECM) of fibrous proteins, with these nels, or micropatterns is not sufficient to predict interactions in a fibers having different radii. Our earlier in vitro recapitulation of matrix—the geometry of the fiber can generate entirely new the effects of fiber curvature showed that both protrusive and behaviors. migratory behavior is sensitive to fiber diameter (19–21). Fur- CELL BIOLOGY thermore, we have shown that suspended, flat 2D ribbons do not contact inhibition of locomotion | cell motility | collective migration | cell capture the protrusive behavior observed on suspended round biology fibers (19); thus, we wanted to inquire if the CIL rules developed on 1D collision and 2D assays extend to contextually relevant ell migration is an essential component of various physio- Clogical processes such as morphogenesis, wound healing, and Significance metastasis (1). Cell–cell interactions in which cell–cell contact reorients cell polarity are necessary for the correct function of When cells heal a wound or invade a new area, they coordinate many developmental events (2). One of the earliest such inter- their motion. Coordination is often studied by looking at what actions known was termed “contact inhibition of locomotion” happens after pairs of cells collide. Postcollision, cells often (CIL) by Abercombie and Heaysman over five decades ago in exhibit contact inhibition of locomotion—they turn around and chick fibroblasts cultured on flat two-dimensional (2D) sub- crawl away from the point where they touched. Past knowl- strates (2–4). In CIL, two approaching cells isolated from the rest edge of repolarization on contact comes from studies on flat of the cell population first make contact, followed by protrusion surfaces, unlike cells in the body, which crawl along fibers. We inhibition at the site of contact, which leads to cell repolarization discover that cells on single fibers walk past one another, but through formation of new protrusions away from the site of cells in contact with multiple fibers stick to one another and contact. Subsequently, cells migrate away from each other in the move as pairs. This outcome changes to walk past after cell direction of newly formed protrusions (1). This sequence can, division. Our experiments and models reveal how the envi- – however, be altered in specific conditions such as metastasis in ronment regulates cell cell coordination after contact. which a loss of CIL allows malignant cells to invade fibroblast — Author contributions: A.S.N. conceived the study; B.A.C. and A.S.N. designed research; B. cultures this is a loss of CIL between different cell types (het- A.C. developed and implemented the theoretical model; J.S., A.P., B.A.C., and A.S.N. erotypic CIL) (4, 5). Recent work has also begun to identify the performed research; J.S., A.P., B.A.C., and A.S.N. analyzed data; J.S., B.A.C., and A.S.N. molecular players that initiate and regulate CIL, including Rac wrote the paper. activity, microtubules, Eph/Ephrin binding, and E- and The authors declare no competing interest. N-cadherin expression (6–10). This article is a PNAS Direct Submission. CIL is most commonly studied and analyzed on flat 2D sub- Published under the PNAS license. strates using several invasion and collision assays (2, 3, 11). By 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. contrast, cells traveling in matrix in vivo are constrained to move This article contains supporting information online at https://www.pnas.org/lookup/suppl/ along narrow fibers. A common shortcoming in featureless 2D doi:10.1073/pnas.2011815118/-/DCSupplemental. assays is thus the inability to study CIL under natural constraints Published March 18, 2021. PNAS 2021 Vol. 118 No. 12 e2011815118 https://doi.org/10.1073/pnas.2011815118 | 1of9 Downloaded by guest on October 2, 2021 fibrous environments. To understand CIL in fibrous environ- fibers but at times difficult to optically discern (Movie S2). Thus, ments that mimic native ECM, we use suspended and aligned we selected our ∼500 nm diameter fibers as the central model nanofiber networks to study CIL behavior in NIH/3T3 fibroblast system. We also immediately observed that cells underwent mi- cell–cell pairs exhibiting two distinct elongated morphologies: totic division, and daughter cells subsequently migrated with a spindle, attached to a single fiber and parallel cuboidal, attached large increase in speed in both spindle and parallel-cuboidal to two fibers (22). We further investigate the effect of cell divi- configurations (Fig. 1D). sion on CIL by studying the encounters of cells that have recently divided (daughter cells) with other cells; these recently divided CIL Collision Outcomes in Two Approaching Spindle Cells. Next, we cells are much faster, consistent with earlier work (23). Our work wanted to observe and quantify the outcomes of spindle-cell CIL allows us to determine the types and rates of cell–cell contact interactions. We looked at 47 randomly selected approaching outcomes—the “rules of CIL”—in a biologically relevant system spindle-cell–cell collisions without cell division from 40 experi- with a controlled geometry. These rules are radically different ments. We found that spindle cells approaching each other from the known stereotypical behavior in 2D assays, but the mostly walked past each other without repolarizing (66%, essential features of these rules emerge robustly from a minimal Fig. 2A and Movies S3 and S4). Since spindle cells on fibers form computational model of CIL in confined geometries. focal adhesions at the poles (22), they can shift position around the fiber, thus allowing them to walk past one another. We also Results observed nonmutual CIL (30%) where upon contact only one Fiber Spacing and Diameter Control Cell Shape; Cell Division Controls spindle repolarized, and both cells continued to migrate as a Speed. To develop the rules of CIL in fibrous environments cohesive unit (“train”) in the new migration direction (Movie mimicking native ECM, as shown schematically in Fig. 1A (24), S5). Very rarely, 4% of the time, we see a mutual CIL response we must first specify a controllable geometry. We