Methods, compositions, and systems for controlling cell migration

Abstract

A method of inducing migration of cells in a tissue comprises delivering a protein construct, or a nucleic acid encoding the protein construct, to cells of the tissue, and exposing a region of the tissue to light. Illumination of a region of the tissue can activate clustering of the light-inducible oligomerization domain of the protein construct within the cells that are exposed to light. Clustering can subsequently activate a signaling pathway that induces cell movement, e.g., cell migration. The directionality of cell migration, e.g., outward expansion of an edge of a tissue or inward migration resulting in tissue densification, is controlled by the position of the illuminated region with respect to the tissue.

Description

Methods, Compositions, and Systems for Controlling Cell MigrationRELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 653,625, filed on May 30, 2024. The entire teachings of the above application are incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number GM 144362 awarded by the National Institutes of Health (NIH) and grant numbers MCB1750663 and CBET2134935 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.BACKGROUND

[0003] Collective cell migration is a fundamental process governing multicellular phenomena such as morphogenesis, wound healing, and cancer invasion. However, programmable control over gradient shape is challenging and requires complex microfabricated devices. Furthermore, cell migration cues typically cannot be dynamically altered once patterned, and precisely manipulating electric fields requires sophisticated device design. Accordingly, methods and compositions are needed to control collective cell migration at a larger scale - sculpting tissues with high precision using patterned stimuli - and serving as a useful substrate for applications ranging from accelerated wound healing to patterning biologically relevant tissue organization.SUMMARY

[0004] The present disclosure describes inter alia methods that utilize a light-controlled Epithelial growth factor (EGF) receptor (OptoEGFR) in epithelial cell lines for, e.g., precise, programmable control of long-range tissue movements. In OptoEGFR-expressing tissues, it was shown that light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements were driven primarily by PI 3 -kinase signaling, rather than diffusible signals, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Thus, synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications including wound healing and tissue morphogenesis.

[0005] In one aspect, the disclosure provides a method of inducing migration of cells of a tissue, the method comprising: delivering a protein construct or a nucleic acid encoding the protein construct to at least a subset of the cells, wherein the protein construct comprises a light-inducible oligomerization domain and an intracellular domain of a receptor tyrosine kinase (RTK); and exposing a region of the tissue to light, thereby producing an illuminated region of the tissue and activating a signaling pathway in the cells that induces cell migration.

[0006] In some embodiments, illuminating the region of the tissue induces clustering of the protein construct in the cells in the illuminated region of the tissue by means of the lightinducible oligomerization domain. In some embodiments, clustering of the protein construct activates the signaling pathway. In some embodiments, clustering of the protein constructs comprises oligomerization of two or more constructs.

[0007] In some embodiments, the illuminated region comprises wholly illuminated cells. In some embodiments, the wholly illuminated cells are able to migrate in any direction.

[0008] In some embodiments, exposing the region of the tissue to light increases tissue fluidity.

[0009] In some embodiments, the migration comprises tissue outgrowth. In some embodiments, tissue outgrowth comprises outgrowth of an edge of the tissue within the illuminated region.

[0010] In some embodiments, migration increases speed of tissue outgrowth at least about 10%, 20%, 30%, or 40% compared to a tissue lacking the protein construct or which was not exposed to light. In some embodiments, migration increases speed of tissue outgrowth about 40% compared to a tissue lacking the protein construct or which was not exposed to light.

[0011] In some embodiments, migration comprises expansion of the tissue in at least one direction.

[0012] In some embodiments, exposing the region of the tissue to light induces collective migration of cells of the tissue toward or away from the illuminated region. In some embodiments, exposing the region of the tissue to light induces collective migration of cells of the tissue toward the illuminated region.

[0013] In some embodiments, collective migration of cells of the tissue toward the illuminated region thickens the illuminated region, increases cell density in the illuminatedregion, or both. In some embodiments, collective migration of cells of the tissue toward the illuminated region thickens the illuminated region at least two-fold. In some embodiments, collective migration of cells of the tissue toward the illuminated region thickens the illuminated region about three-fold. In some embodiments, collective migration of cells of the tissue toward the illuminated region thickens, densifies, or both thickens and densifies an area of the illuminated region located at least about 50 gm interior to an illumination boundary.

[0014] In some embodiments, the illuminated region comprises partially illuminated cells. In some embodiments, the partially illuminated cells migrate toward or away from the illuminated region. In some embodiments, the partially illuminated cells migrate toward the illuminated region. In some embodiments, migration of the partially illuminated cells toward the illuminated region stimulates migration of unilluminated cells toward the illuminated region. In some embodiments, migration of the partially illuminated cells toward the illuminated region stimulates migration of the unilluminated cells toward the illuminated region by means of cell-cell contacts. In some embodiments, the partially illuminated cells exert force on the cell-cell contacts, thereby stimulating migration of the unilluminated cells toward the illuminated region. In some embodiments, migration of the partially illuminated cells toward the illuminated region produces an area of lower cell density within the tissue, thereby stimulating migration of the unilluminated cells toward the illuminated region. In some embodiments, the area of lower cell density within the tissue reduces contact inhibition of locomotion of the unilluminated cells.

[0015] In some embodiments, the cells migrate at a velocity of at least about 1 pm, 5 pm, 10 pm, 15 pm, or 20 pm per hour during exposure to the light. In some embodiments, the cells migrate at a velocity of about 20 pm per hour during exposure to the light. In some embodiments, the migration comprises movement of a plurality of the cells of the tissue across a distance of at least about 100 pm, 200 pm, 500 pm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. In some embodiments, the migration comprises movement of a plurality of the cells of the tissue for a duration of exposing the region of the tissue to light.

[0016] In some embodiments, the tissue has a diameter of at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm. In some embodiments, the tissue has a diameter of at least about 2 mm, 3 mm, or 6 mm. In some embodiments, the illuminated region has a diameter of at least about 100 pm, 200 pm, 500 pm, 1 mm, or 2 mm. In some embodiments, the illuminated region has a diameter of at least about 200 pm, 1 mm, or 2mm. In some embodiments, the illuminated region has a diameter of at least about one thirtieth, one sixth, one third, or an entire length of a diameter of the tissue. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, or 1 x IO10cells.

[0017] In some embodiments, the tissue comprises cells from a cell line, primary cells, induced pluripotent stem cells, or a combination thereof, or wherein the tissue comprises an ex vivo tissue, tissue in situ, or a tissue graft. In some embodiments, the tissue comprises keratinocytes, epidermal epithelial cells, fibroblasts, retinal pigment epithelial cells, neurons, or a combination thereof.

[0018] In some embodiments, the protein construct or the nucleic acid encoding the protein construct is delivered at least once, twice, or three, four, five, six, seven, or eight times, to at least the subset of the cells of the tissue. In some embodiments, the protein construct or the nucleic acid encoding the protein construct is delivered to at least the subset of the cells of the tissue by means of immersion in a composition, exposure to a topically applied composition, intradermal microneedle array administration of a composition, or local injection of a composition, wherein the composition comprises the protein construct or the nucleic acid encoding the protein construct. In some embodiments, the protein construct or the nucleic acid encoding the protein construct is delivered to at least the subset of the cells of the tissue in a nanoparticle. In some embodiments, the nanoparticle comprises a lipid nanoparticle. In some embodiments, the nucleic acid encoding the protein construct is delivered to at least the subset of the cells of the tissue in a viral vector, non-viral vector, or virus-like particle.

[0019] In some embodiments, the protein construct is transiently expressed in at least the subset of the cells of the tissue. In some embodiments, the protein construct is stably expressed in at least the subset of the cells of the tissue. In some embodiments, the protein construct is expressed for at least about 1, 2, 3, 4, 5, 6, or 7 days after delivering the protein construct or the nucleic acid encoding the protein construct. In some embodiments, the protein construct is present in at least the subset of the cells for at least about 1, 2, 3, 4, 5, or 6 days or at least about 1, 2, 3, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the region of the tissue is exposed to light over a period of at least about 1, 2, 3, 4, 5, or 6 days or at least about 1, 2, 3, 3, 4, 5, 6, 7, or 8 weeks.

[0020] In some embodiments, the intracellular domain of the RTK comprises a cytoplasmic domain of Epidermal growth factor receptor (EGFR) or Fibroblast growth factorreceptor 1 (FGFR1). In some embodiments, the intracellular domain of the RTK comprises the cytoplasmic domain of EGFR. In some embodiments, the light-inducible oligomerization domain comprises a photolyase homology region of Arabidopsis thaliana Cryptochrome-2 (Cry2). In some embodiments, the protein construct further comprises and intrinsically disordered protein region (IDR). In some embodiments, the IDR comprises an N-terminal sequence of RNA-binding protein FUS (FUS), Probable ATP-dependent RNA helicase DDX4 (DDX4), or Heterogeneous nuclear ribonucleoprotein Al (HNRNPA1). In some embodiments, the IDR comprises the N-terminal sequence of FUS (FUSN).

[0021] In some embodiments, the protein construct further comprises a fluorescent domain. In some embodiments, the fluorescent domain is mCherry or FusionRed. In some embodiments, the fluorescent domain is FusionRed.

[0022] In some embodiments, the protein construct further comprises an N-terminal membrane localization tag. In some embodiments, the N-terminal membrane localization tag comprises a myristoylation tag.

[0023] In some embodiments, exposing the region of the tissue to light comprises use of a photomask to selectively expose the region of the tissue to light.

[0024] In some embodiments, the light comprises at least one wavelength of light. In some embodiments, the at least one wavelength comprises a visible wavelength between and including 400 and 800 nm. In some embodiments, the visible wavelength is about 450 nm.

[0025] In some embodiments, the light comprises a predetermined intensity. In some embodiments, the predetermined intensity comprises about 10 to about 70 mW / cm2. In some embodiments, the predetermined intensity comprises about 65 mW / cm2. In some embodiments, the predetermined intensity comprises about 12 mW / cm2.

[0026] In some embodiments, exposing the region of the tissue to light occurs for at least one predetermined period of time. In some embodiments, the predetermined period of time is at least about 3 hours. In some embodiments, the predetermined period of time is up to about 32 hours. In some embodiments, exposing the region of the tissue to light occurs at a predetermined interval for a predetermined duration over the predetermined period of time. In some embodiments, the predetermined interval is about 20 seconds to about 3 minutes. In some embodiments, the predetermined duration is about 4-5 seconds. In some embodiments, exposing the region of the tissue to light occurs for 5 seconds every 1 minute for up to about 32 hours.

[0027] In another aspect, the disclosure provides a kit for inducing migration of cells of a tissue, the kit comprising the protein construct or the nucleic acid encoding the protein construct, optionally wherein the kit comprises at least one light emitting device configured to induce self-association of the light-inducible oligomerization domain.

[0028] In yet another aspect, the disclosure provides a method of promoting wound healing in a subject in need thereof, the method comprising inducing migration of cells of a tissue. In some embodiments, the subject has at least one diabetes-related ulcer, decubitus ulcer, venous ulcer, ischemic ulcer, or chronic wound. In some embodiments, the subject has one or more of diabetes, high blood pressure, poor blood circulation, high risk for infection, or advanced age. In some embodiments, the subject is or was previously treated with or exposed to one or more agents selected from an anticoagulant, an anti-inflammatory, an antibiotic, an antimicrobial, a chemotherapeutic, an immunosuppressant, an immunomodulator, a tumor necrosis factor (TNF) inhibitor, and nicotine.

[0029] In yet another aspect, the disclosure provides a method of regenerating tissue in a subject in need thereof, the method comprising inducing migration of cells of a tissue. In some embodiments, the subject has an injury to or defect of the central nervous system. In some embodiments, the injury is a spinal cord injury. In some embodiments, the tissue is nerve tissue. In some embodiments, the method drives axon growth.

[0030] In yet another aspect, the disclosure provides a method of restoring tissue organization in a subject in need thereof, the method comprising inducing migration cells of a tissue. In some embodiments, restoring tissue organization comprises nerve regeneration. In some embodiments, the nerve regeneration comprises axon growth. In some embodiments, restoring tissue organization comprises healing a diabetic ulcer. In some embodiments, the method drives cell movement, cell proliferation, cell production of extracellular matrix, or a combination of the foregoing.

[0031] In yet another aspect, the disclosure provides a method of preparing a tissue graft, the method comprising inducing migration of cells of a tissue.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0033] FIGs. 1A-1D. Optogenetic EGFR stimulation directs tissue movement. (FIG. 1A) Schematic of Optogenetic Epidermal Growth Factor Receptor (OptoEGFR) construct for blue light-inducible EGFR clustering and signaling pathway activation. The system fuses an N-terminal membrane localization tag to the OptoDroplet system for light-inducible clustering (FUSN-FusionRed-Cry2) and the cytosolic domains of EGFR. (FIG. IB) Experiments were designed to assay for phenotypic consequences to OptoEGFR stimulation. (FIGs. 1C-1D) Light stimulation of OptoEGFR RPE-1 cells produced dramatic tissue movements, including tissue densification in illuminated interior regions (in FIG. 1C) and tissue outgrowth from illuminated tissue edges (in FIG. ID). Images show FusionRed channel; stimulation time shown in hours:minutes (hh:mm).

[0034] FIGs. 2A-2C. Light-induced cell motility is a specific feature of OptoEGFR activation and generalizes to multiple cellular contexts. (FIG. 2A) Representative fields of view showing OptoEGFR-induced tissue movement toward regions of illumination and OptoFGFR exclusion from illumination regions. (FIG. 2B) Western blot of independently treated OptoEGFR and OptoFGFR cell lysates in light and dark, showing phospho-ERK, total ERK and actin as a loading control. Both OptoEGFR and OptoFGFR showed similar levels of ERK phosphorylation upon illumination, indicating similar degrees of light-induced activation. (FIG. 2C) OptoEGFR stimulation of cell migration in MCF10A cells also expressing the Erk kinase translocation reporter (KTR) biosensor for Erk mitogen-activated protein kinase (MAPK) activity (ErkKTR-iRFP), which indicated ERK activity by a shift from nuclear to cytosolic localization. Light induced local ERK activation within the illuminated region as well as tissue movement toward the light input on a similar timescale to what was observed in RPE-1 OptoEGFR cells. Related to FIGs. 1 A-1D.

[0035] FIGs. 3A-3G. Quantifying large-scale tissue densification induced by OptoEGFR stimulation. (FIG. 3A) Schematic of large-scale tissue stimulation assay. Circular light stimuli of varying sizes were applied to the center of a confined 6-mm RPE-1 tissue and cell movements are imaged using the membrane FusionRed tag. (FIG. 3B) Quantification of tissue velocities as a function of time and distance from the tissue center. The illumination boundary is shown (dashed line). (FIG. 3C) Quantification of tissue velocity as a function of position at the fastest-moving time point for 200 pm, 1 mm, and 2 mm-diameter illumination patterns. (FIG. 3D) Maximum radial velocity and migration zone width corresponding to the curves in FIG. 3C. N = 9, 3, 3, and 3 for control and each illumination pattern, respectively. (FIG. 3E) Confocal stacks of nuclei staining for 1 mmillumination pattern, colored by tissue height at the indicated times after illumination. Scale bar: 300 pm. (FIG. 3F) Sum -projection along the radial coordinate for tissues in FIG. 3E. Scale bar: 300 pm. (FIG. 3G) Quantification of data in FIG. 3F showing relative cell density and tissue height as a function of radial position. N = 3 and 5 for control and illuminated tissues, respectively.

[0036] FIGs. 4A-4I. Large-scale optical illumination drives tissue flows at a millimeter scale. (FIGs. 4A-4B) Experimental setup for illumination light shield and DMD. The laser-cut photomask (shown in FIG. 4A) was placed on top of the LED-based transmitted light turret, which was used to deliver 450 nm blue light. (FIG. 4C) Velocity vector field for tissue flows near the 1 mm diameter illumination pattern. (Scale bar: 300 pm). (FIGs. 4D- 4E) Maximum radial velocity (in FIG. 4D) and migration zone width (in FIG. 4E) plotted over time for 200 pm, 1 mm, and 2 mm illumination patterns. For larger patterns, tissue flow was sustained over the time course with a peak at about 15 h; for the 200 pm pattern, tissue movement was primarily observed during approximately the first 10 h with a peak at about 3 h. (FIGs. 4F-4G) Peak radial velocity and migration zone width shown as a function of illumination dose. Light inputs of identical intensity were delivered for 5 sec every 20 sec, for 5 sec every 1 min, or for 4 sec every 2.5 min. The OptoDroplet light-responsive module in OptoEGFR has a dark-state reversion time of about 2-3 min, so each stimulus type was expected to drive different intermediate activity levels that were sustained over time. (FIGs. 4H-4I) Persistent tissue deformation was observed even 43 h after removal of the optogenetic stimulus. Brightfield images show results immediately after applying a 36 h stimulus or after waiting an additional 43 h after stimulation. Related to FIGs. 2A-2F.

[0037] FIGs. 5A-5H. Global illumination drives tissue fluidization and enhanced outgrowth. (FIG. 5A) Initial and final (24 h) images of OptoEGFR RPE-1 tissues in darkness or under global 450 nm illumination. Scale bar: 500 pm. (FIGs. 5B-5C) Quantification of tissue radius over time (in FIG. 5B) and radial tissue velocity computed as the rate of change of tissue radius growth (in FIG. 5C). (FIG. 5D) Mean edge speed compared between control and globally illuminated tissue. N = 15 and 18 for illuminated and control tissues, respectively. (FIG. 5E) Kymograph of radial velocity as a function of position and time, measured by particle image velocimetry on expanding tissues. (FIG. 5F) Quantification of radial velocity as a function of position at 15 h after barrier removal. N=11 or 15 for illuminated and control tissues, respectively. (FIG. 5G) Kymograph of cell density as a function of position and time for illuminated and unilluminated expanding tissues. (FIG.5H) Quantification of cell density as a function of position at 23 h after barrier removal. N = 11 and 15 for illuminated and control tissues, respectively.

[0038] FIGs. 6A-6G. Interrogating the molecular basis for light-induced collective cell migration. (FIG. 6A) Schematic of experiment to test for the role of diffusible signaling and an illumination boundary on cell movement. Two tissues were plated with a 300 pm gap and allowed to freely expand while one tissue was illuminated. (FIG. 6B) Outgrowth rates as a function of distance from tissue boundary at the near and far edges of the unilluminated tissue in A. N=8 tissues across 2 experiments. (FIG. 6C) Images of tissues stimulated as in FIGs. 3A-3G with a 200 pm illumination circle in the presence of the indicated chemical inhibitors. Scale bar: 500 pm. (FIG. 6D) Quantification of peak radial velocity (left) and migration zone width (right) for illuminated tissues treated with each compound. N = 6, 6, and 5 tissues across 2 experiments for control, N-blebbistatin, and the matrix metalloproteinase inhibitor TAPI-1 respectively. (FIG. 6E) Images of tissues stimulated as in FIG. 6C in the presence of the indicated chemical inhibitors.

[0039] FIGs. 7A-7B. Effect of chemical inhibitors on light-induced migration in MCF10A cells. (FIG. 7 A) Representative images of MCF10A OptoEGFR and ErkKTR- iRFP expressing cell 18 h after treatment with no input, a local pattern of 450 nm light (“light only”), the matrix metalloproteinase inhibitor TAPI-1, the Rho kinase inhibitor Y27632 which is expected to inhibit myosin activation, the EGFR inhibitor gefinitib, the MEK inhibitor cobimetinib, or the PI 3 -kinase inhibitor PI- 103. ErkKTR-iRFP fluorescence imaging shown. (FIG. 7B) Quantification of mean radial tissue velocity over the 18 h time course in each stimulus condition shown in FIG. 7A. Related to FIGs. 6A-6G.

[0040] FIGs. 8A-8D. Local and global cues drive OptoEGFR-induced light-induced collective cell migration. (FIG. 8A) Conceptual model for how different illumination geometries affect collective cell movement. Left: Partially illuminated cells experienced a directional cue mediated by PI 3-kinase, driving movement into the illuminated region. This movement can exert force on neighboring unilluminated cells or leave a lower-density gap to drive these neighboring cells’ movement into the light, repeating the cycle. Right: whole-cell illumination produced by global light stimuli drove distinct effects, including increased tissue fluidity and more rapid outgrowth velocities. (FIGs. 8B-8D) Driving complex tissue patterning with a combination of interior and edge illumination patterns. Left: illumination pattern applied to a circular tissue; Middle: FusionRed fluorescence imaging after 48 h of illumination and expansion; Right: a mathematical model implementing tissue flux atillumination boundaries and diffusion captures qualitative features of the tissue pattern. Scale bar: 1 mm.

[0041] FIG. 9. Model simulation of tissue convergence from FIG. 3G. 1 mm diameter (500 pm radius) circular pattern of illumination was simulated in the model and the density of tissue was determined after 20 and 32 h simulation time. The depletion zone and peak of cell density at the illumination boundary were evident from the simulation, which only incorporated boundary-driven tissue flux into the illuminated region and diffusion-based tissue spreading. Related to FIGs. 8A-8D.DETAILED DESCRIPTION

[0042] A description of example embodiments follows.

[0043] Before the present invention is described in further detail, it is to be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0044] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, a limited number of the exemplary methods and materials are described herein.

[0046] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0047] The phrase “and / or”, as used herein, should be understood to mean “either or both” of the elements so conjoined, z.e., elements that are conjunctively present in some cases and disjunctively present in other cases.

[0048] It should also be understood that, unless clearly indicated to the contrary, in any methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.Methods of inducing migration of cells

[0049] The disclosure provides inter alia a method of inducing migration of cells of a tissue comprising delivering a protein construct or a nucleic acid encoding the protein construct to at least a subset of the cells of the tissue, and exposing a region of the tissue to light. Illumination of a region of the tissue can activate clustering of the light-inducible oligomerization domain of the protein construct within the cells that are exposed to light. Clustering can subsequently activate a signaling pathway that induces cell movement, e.g., cell migration. The directionality of cell migration, e.g., outward expansion of an edge of a tissue or inward migration resulting in tissue thickening, is controlled by the relative position of an illuminated region within the entire tissue. Additional examples of methods for inducing migration of cells in a tissue can be found, for example, in Suh K, Thornton R, Farahani PE, Cohen D, Toettcher J. Large-scale control over collective cell migration using light-controlled epidermal growth factor receptors. bioRxiv [Preprint], 2024 May 31 :2024.05.30.596676. doi: 10.1101 / 2024.05.30.596676. Update in: Cell Syst. 2025 Mar 19;16(3): 101203. doi: 10.1016 / j.cels.2025.101203. PMID: 38853934; PMCID:PMC 11160748, the entire contents of which are incorporated herein by reference70, and in Suh K, Thornton RH, Nguyen L, Farahani PE, Cohen DJ, Toettcher JE. Large-scale control over collective cell migration using light-activated epidermal growth factor receptors. Cell Syst. 2025 Mar 19;16(3): 101203. doi: 10.1016 / j.cels.2025.101203. Epub 2025 Mar 3. PMID: 40037348, the entire contents of which are incorporated herein by reference71.

[0050] In some embodiments, migration of cells of a tissue comprises large-scale, e.g., tissue-scale, migration. In some embodiments, large-scale or tissue-scale migration comprises movement of cells of a tissue, e.g., a plurality of cells of a tissue. In some embodiments, large-scale or tissue-scale migration comprises movement of a majority of target cells of a tissue, e.g., a majority of cells targeted for induction of migration. In some embodiments,large-scale or tissue-scale migration comprises movement of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%, at least 98%, or at least 99% of target cells of a tissue. In some embodiments, large-scale or tissue-scale migration comprises movement of a sufficient number of target cells of a tissue to be therapeutically effective, e.g., in a treatment for wound healing. In some embodiments, a therapeutically effective number of target cells comprises hundreds, thousands, tens of thousands, hundreds of thousands, millions, tens of millions, hundreds of millions, billions, or tens of billions of cells or more. In some embodiments, large-scale or tissue-scale migration comprises movement of cells of a tissue, e.g., a plurality of cells of a tissue, across a distance on the order of millimeters to centimeters. In some embodiments, migration of cells of a tissue comprises small-scale migration. In some embodiments, small- scale migration comprises movement of up to 10%, up to 20%, up to 30%, up to 40%, or up to 50% of target cells of a tissue. In some embodiments, small-scale migration comprises movement of cells of a tissue, e.g., a plurality of cells of a tissue, across a distance on the order of micrometers.

[0051] In some embodiments, migration of cells of a tissue comprises collective movement of a plurality of cells in similarly oriented directions. In some embodiments, migration of cells of a tissue comprises collective movement of a plurality of cells toward a particular area or away from a particular area. The term “collective movement” indicates overall movement of a plurality of cells. In some embodiments, collective movement comprises advancement of a tissue edge, wherein a plurality of cells participate in the collective movement, and wherein all cells of the tissue need not participate or participate to the same extent in the collective movement.

[0052] In some embodiments, migration of cells of a tissue comprises coordinated movement of a plurality of cells in similarly oriented directions. In some embodiments, migration of cells of a tissue comprises coordinated movement of a plurality of cells toward a particular area or away from a particular area. In some embodiments, coordinated movement comprises interactions between cells that stimulate cell movement.Methods of controlling direction of migration

[0053] As further described in the Examples herein, activating clustering of the protein construct and a downstream migration signaling pathway leads to cell movement, e.g., movement of individual cells. In some embodiments, movement among cells in a tissue,whether directional e.g., migration) or localized (e.g., back-and-forth) depends on the degree and direction of illumination. Cell movement and migration can increase tissue fluidity.

[0054] Exposing the region of the tissue to light can induce collective migration of cells of the tissue toward or away from the illuminated region. For example, a protein construct comprising the cytoplasmic domain of Epidermal growth factor receptor (EGFR) can induce migration of cells toward an illuminated region, while a protein construct comprising the cytoplasmic domain of Fibroblast growth factor receptor 1 (FGFR1) can induce migration of cells away from an illuminated region.

[0055] The direction of migration can be influenced by the relative location of partially illuminated cells within the tissue, e.g, cells that are on the boundary of the illuminated region. Partially illuminated cells can migrate toward or away from the illuminated region according to the activity of the receptor tyrosine kinase of the protein construct. Partially illuminated cells containing a protein construct comprising EGFR can migrate toward the illuminated region. Directional migration of the partially illuminated cells, e.g, toward the illuminated region, can stimulate migration of unilluminated cells toward the illuminated region. A migration-stimulating signal among unilluminated cells can be propagated by means of cell-cell contacts with the partially illuminated cells that are migrating as a result of the light-induced clustering of the protein construct. Without being bound by theory, the partially illuminated cells can exert directional force on the cell-cell contacts, whereby unilluminated cells are pulled along cell-cell contacts in the direction of the partially illuminated cells’ migration. Alternatively, and without being bound by theory, migration of the partially illuminated cells toward the illuminated region may result in a lower-density region, e.g., a space no longer occupied by the migrating cells, thereby reducing the contact inhibition of locomotion affecting neighboring unilluminated cells, allowing the unilluminated cells to move into that space. As partially illuminated cells move toward the illuminated region, they may become wholly illuminated cells. Similarly, as unilluminated cells move into spaces previously occupied by partially illuminated cells, the unilluminated cells may become partially illuminated.

[0056] The direction of migration can also be influenced by wholly illuminating a tissue, e.g., cells containing the protein construct. In such examples, wholly illuminated cells contribute to outgrowth of a tissue, e.g., radial expansion away from a center of a tissue.

[0057] In some embodiments, exposing the region of the tissue to light comprises use of a photomask to selectively expose the region of the tissue to light. One or more photomasksmay be implemented in limiting light exposure to a particular area of a tissue, e.g., an internal area to induce thickening or an edge to induce directional outgrowth.Methods of increasing speed of tissue outgrowth

[0058] A cell comprising the protein construct and which is wholly illuminated is able to migrate in any direction. In a tissue that is entirely illuminated, the increased localized movement of the cells comprising the protein construct results in a higher rate of outgrowth, e.g., outward migration of cells from the edge of the tissue. Illumination of the protein construct can increases speed of tissue outgrowth at least about 10%, 20%, 30%, or 40% compared to a control tissue, e.g., a tissue lacking the protein construct or which was not exposed to light. Illumination of an entire tissue does not confer a directional preference for outgrowth, and therefore can lead to expansion of the tissue in any direction, e.g., in at least one direction.Methods of tissue thickening and densification

[0059] In some embodiments, methods of inducing migration of cells thickens, densifies, or both thickens and densifies a tissue. Tissue thickening, densification, or both can be accomplished by inducing collective migration of cells toward an illuminated region that is already occupied by cells. For example, a region within a tissue may be illuminated and result in movement of cells from an unilluminated region into the illuminated region, and may thicken the illuminated region at least two-fold, at least three-fold, or at least four-fold. In some embodiments, cells in a three-dimensional environment, e.g., in vivo or cultured on a scaffold or extracellular matrix, can migrate and thicken an illuminated region at least twofold, at least three-fold, at least four-fold, or more. As cells migrate into the illuminated region, thickening can affect an area within the illuminated region, e.g., an area located at least about 50 pm interior to an illumination boundary. For example, if an illuminated region is circular and has a diameter of about 250 pm, the concentric thickened area may have a diameter of about 150 pm.Methods of controlling speed and distance of migration

[0060] As used herein, the terms “speed” and “rate,” when used with respect to migration of cells in a tissue, are used interchangeably. “Velocity” indicates a direction of migration inaddition to a rate; in instances where a direction is not noted, “velocity” may be considered to have a similar or same meaning as “speed” or “rate.”

[0061] The direction of migration can be controlled by the relative position of an illuminated region within a tissue; the distance and rate of migration can be controlled by the interval and duration of exposure to light.

[0062] For example, migration can comprise movement of at least a subset of the cells of the tissue across a distance of at least about 100 pm, 200 pm, 500 pm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, or more, depending on the duration and configuration of light exposure. Without being bound by theory, appropriate apparatus and conditions could facilitate light exposure capable of inducing cell migration over a period of hours, days, weeks, or longer, and could therefore induce long-distance cell migration. In some embodiments, an area of the tissue may be exposed to light for a predetermined period of time, for example, at least about 3 hours or up to about 32 hours.

[0063] For example, to induce migration across a millimeter to centimeter (mm-to-cm) scale distance in a particular direction over time, a light source may be adjusted to move the illuminated region in the desired direction at a rate commensurate with the velocity of migration, e.g., at a rate no greater than a maximum radial velocity of the cells. In some embodiments, a light source or light exposure therapy might be configured to move at a rate of less than or about 1 pm, 5 pm, 10 pm, 15 pm, or 20 pm per hour, thereby facilitating a migration velocity of at least about 1 pm, 5 pm, 10 pm, 15 pm, or 20 pm per hour during exposure to the light.

[0064] In addition to an overall period of time during which the region of the tissue is exposed to light, the exposure to light may occur at intervals and for variable durations at said intervals. In other words, exposing the region of the tissue to light may occur at a predetermined interval for a predetermined duration over the predetermined period of time. For example, a predetermined interval may be about 20 seconds to about 3 minutes and a predetermined duration may be about 4-5 seconds. In some embodiments, exposing the region of the tissue to light occurs for a duration of 5 seconds at 1 -minute intervals, that is, every 1 minute. In some embodiments, the exposure at intervals may last for a predetermined period of time, e.g., for up to about 32 hours.Cells and tissues

[0065] The disclosed methods of inducing migration of cells in a tissue may be implemented with a variety of tissues comprising a variety of cell types. For example, the tissue may comprise cells from a cell line, primary cells, induced pluripotent stem cells, or a combination thereof, or an ex vivo tissue, tissue in situ, or a tissue graft. Example cell types include keratinocytes, epidermal epithelial cells, fibroblasts, retinal pigment epithelial cells, human embryonic kidney cells, skin cells, neurons, or a combination thereof. In some embodiments, skin cells comprise keratinocytes, epidermal epithelial cells, fibroblasts, or any combination of the foregoing. In some embodiments, a tissue comprises keratinocytes, epidermal epithelial cells, fibroblasts, or any combination of the foregoing. In some embodiments, migration of cells in a tissue comprises migration of keratinocytes, epidermal epithelial cells, fibroblasts, or any combination of the foregoing. In some embodiments, migration of cells in a tissue comprises migration of keratinocytes. In some embodiments, migration of cells in a tissue comprises migration of epidermal epithelial cells. In some embodiments, migration of cells in a tissue comprises migration of fibroblasts. In some embodiments, migration of cells in a tissue comprises migration of keratinocytes, epidermal epithelial cells, and fibroblasts.

[0066] In some embodiments, a tissue is a cultured tissue, e.g., grown as a monolayer or three-dimensional colonies. A tissue, e.g., a cultured tissue, may have a diameter of at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm. In some embodiments, an illuminated region of a tissue is smaller than the entire tissue, e.g., the illuminated region may have a diameter of at least about 100 pm, 200 pm, 500 pm, 1 mm, or 2 mm. In some embodiments, the illuminated region has a diameter of at least about one thirtieth, one sixth, one third, or an entire length of a diameter of the tissue. In some embodiments, a tissue comprises at least about 4 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, or 1 x 1010cells.

[0067] In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, or 1 x 1010cells. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 102cells. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 103cells. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 104cells. In some embodiments, migration ofcells of the tissue comprises migration of at least about 1 x 105cells. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 106cells. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 107cells. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 108cells. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x 109cells. In some embodiments, migration of cells of the tissue comprises migration of at least about 1 x IO10cells.

[0068] In some embodiments, migration of cells of the tissue comprises migration of cells on the order of hundreds of cells. In some embodiments, migration of cells of the tissue comprises migration of cells on the order of thousands of cells. In some embodiments, migration of cells of the tissue comprises migration of cells on the order of tens of thousands of cells. In some embodiments, migration of cells of the tissue comprises migration of cells on the order of hundreds of thousands of cells. In some embodiments, migration of cells of the tissue comprises migration of cells on the order of millions of cells. In some embodiments, migration of cells of the tissue comprises migration of cells on the order of tens of millions of cells. In some embodiments, migration of cells of the tissue comprises migration of cells on the order of hundreds of millions of cells. In some embodiments, migration of cells of the tissue comprises migration of cells on the order of billions of cells. In some embodiments, migration of cells of the tissue comprises migration of cells on the order of tens of billions of cells.

[0069] In some embodiments, a tissue may comprise primary cells, induced pluripotent stem cells, or both. Such embodiments may be useful, for example, in personalized medicine applications wherein cells are collected from a patient or donor, cultured and, optionally, differentiated, prior to the induction of migration.

[0070] In some embodiments, a tissue may comprise an ex vivo tissue, tissue in situ, or a tissue graft. Non-limiting examples include, for example, an ex vivo tissue that may comprise a tissue collected from a patient or donor; a tissue in situ that may comprise an injured, diseased, or developmentally disordered tissue in a patient; and a tissue graft may comprise a graft that is being prepared for implantation or that has been implanted into a patient.

[0071] In some embodiments, a tissue comprises a mixture of cells that comprise the protein construct and cells that do not comprise the protein construct. For example, the protein construct or a nucleic acid encoding the protein construct may be delivered to a tissue, yet uptake of the protein construct or nucleic acid might be successful in only a subset of allthe cells of the tissue. The evidence disclosed herein supports a mechanism by which illuminated cells containing the activated construct can stimulate unilluminated cells or cells that do not contain the construct to migrate.Protein constructs

[0072] The present disclosure also provides protein constructs comprising a lightinducible oligomerization domain and an intracellular domain of a receptor tyrosine kinase (RTK). The light-inducible oligomerization domain facilitates clustering of the protein constructs in response to light exposure; clustering may involve oligomerization of two or more constructs within a cell, e.g., oligomerization of at least two, at least three, or at least four constructs. In some embodiments, the protein constructs further comprise an intrinsically disordered protein region (TDR).

[0073] Examples of a light-inducible oligomerization domain include, without limitation, Arabidopsis thaliana Cryptochrome-2 (Cry2) photolyase homology region (PHR) (Cry2PHR), Cry2PHRcomprising an E490G point mutation (Cry2olig), phytochrome B (PhyB), phytochrome-interacting factors (PIF), light-oxygen-voltage sensing (LOV) domains, or the light-switchable fluorescent protein Dronpa.

[0074] In some embodiments, a protein construct further comprises one or more functional regions, intrinsically disordered protein regions (IDRs), cleavage tags, target proteins, or a combination thereof, in addition to the light-inducible oligomerization domain, RTK intracellular domain, and, optionally, a fluorescent domain, a localization tag, or both. Additional examples of protein constructs are found in U.S. Patent No. 10,533,167, the entire contents of which are incorporated herein by reference. In some embodiments, a protein construct comprises one or more modifications (e.g., chemical modifications, post- translational modifications).

[0075] The light to which the protein construct is exposed can comprise at least one wavelength of light that is selected to elicit a clustering response from the light-inducible oligomerization domain of the protein construct and may, for example, comprise a visible wavelength between and including 400 and 800 nm. In some embodiments, the visible wavelength is about 450 nm, e.g., a wavelength that induces clustering among protein constructs comprising a Cry2PHRdomain. The light may also comprise a predetermined intensity, e.g., about 10 to about 70 mW / cm2. In some embodiments, light has an intensity ofabout 65 mW / cm2near the source of emission and an intensity of about 12 mW / cm2as it reaches the tissue.Methods of delivering a protein construct or encoding nucleic acid

[0076] The protein construct or nucleic acid encoding the protein construct may be delivered to the tissue once or multiple times (e.g., twice, or three, four, five, six, seven, or eight times, etc.). In some embodiments, the protein construct is delivered multiple times. The number of times the protein construct is delivered to the tissue may depend on one or more factors, including, for example, the transient or stable nature of the delivery, the immunogenicity of the delivery composition, and the intended use or need of a subject. In some embodiments, the protein construct or the nucleic acid encoding the protein construct is delivered at least once, twice, or three, four, five, six, seven, or eight times, to at least the subset of the cells of the tissue. In some embodiments, the protein construct or the nucleic acid encoding the protein construct is delivered at least nine times or at least ten or more times to at least the subset of the cells of the tissue.

[0077] The protein construct or the nucleic acid encoding the protein construct can be delivered to at least the subset of the cells of the tissue by means of immersion in a composition, exposure to a topically applied composition, intradermal microneedle array administration of a composition, or local injection of a composition, wherein the composition comprises the protein construct or the nucleic acid encoding the protein construct. Immersion in the composition generally refers to cells in a culture comprising liquid medium. Exposure to a topically applied composition may refer to, in some examples, a liquid, gel, or cream comprising the protein construct or nucleic acid being applied, e.g., to skin or an open wound. The composition may be administered, for example, by a microneedle array, e.g., a microneedle patch, to the dermal layer, e.g., skin around a wound. A local injection may be used to administer the composition to a site of injury, e.g., nerve or spine injury.

[0078] A variety of compositions may be used in the deliver of the protein construct or the nucleic acid encoding the protein construct to the tissue. For example, the protein construct or nucleic acid may form part of a nanoparticle, e.g., a nanoparticle that, aside from the protein construct or nucleic acid, comprises biologically inert material. In some embodiments, a nanoparticle encapsulates the protein construct or nucleic acid in a non- immunogenic material. In some embodiments, the nanoparticle comprises a lipid nanoparticle.

[0079] The protein construct may be transiently expressed in at least a subset of the cells of the tissue. The protein construct need not be expressed or present in all cells of a tissue in order to induce migration of cells in the tissue. The protein construct may also be transiently expressed throughout the tissue, e.g, in substantially all of the cells of a tissue. “Substantially all” cells of the tissue can refer to cells that survive a selection step after delivering the protein construct or nucleic acid encoding the protein construct to a population of cells. In some embodiments, “throughout the tissue” can refer to successful uptake if of the protein construct or nucleic acid in a target cell type or population within a tissue, e.g, and may not necessarily include non-target cells, such as stromal cells, vascular cells, and resident immune cells.

[0080] The protein construct may be expressed, e.g., transiently expressed, for at least about 1, 2, 3, 4, 5, 6, or 7 days after delivering the protein construct or the nucleic acid encoding the protein construct. The protein construct itself may be present in at least the subset of the cells for at least about 1, 2, 3, 4, 5, or 6 days or at least about 1, 2, 3, 3, 4, 5, 6, 7, or 8 weeks. The duration of the expression or presence of the protein construct may depend on several factors, including, for example, the method of delivery, the intended use or therapeutic application, and the presence of stabilizing features within the protein construct.

[0081] In some embodiments, a nucleic acid encoding the protein construct may be delivered to at least the subset of the cells of the tissue in a plasmid, a viral vector, a non-viral vector, or a virus-like particle. For example, a lentivirus-derived vector may be used to achieve stable expression of the construct in vitro. The nucleic acid may be DNA or RNA (e.g., mRNA, circular RNA), and may be unmodified or modified (e.g., include on or more chemically modified nucleotide bases).Kits

[0082] Also provided herein are kits for inducing migration of cells, comprising a protein construct of the disclosure or nucleic acid encoding the protein construct. In some embodiments, the kit further comprises at least one light emitting device configured to induce self-association, z.e., clustering, of the light-inducible oligomerization domain of the protein construct. In some embodiments, the kit further comprises a means for delivering (e.g., means for injecting, means for applying) the protein construct or encoding nucleic acid to cells or a tissue. In some embodiments, the kit further comprises instructions for use.Uses

[0083] The methods of inducing migration of cells disclosed herein may be used for a variety of purposes, including, for example, in the promotion of wound healing, in the regeneration of tissue, in the restoration of tissue organization, and / or in the preparation of a tissue graft. In some embodiments, the methods of the disclosure are useful for promoting wound healing in a subject in need thereof. In some embodiments, the subject has at least one diabetes-related ulcer, decubitus ulcer (e.g., bed sore), venous ulcer (e.g., venous leg ulcer), ischemic ulcer (e.g., arterial ulcer), or chronic wound. In some embodiments, a chronic wound comprises a wound that fails to heal within an expected time frame. A chronic wound may be characterized by a non-advancing margins, although other signs of the healing process may be present, e.g., coagulation, inflammation, tissue remodeling, and cell proliferation. Inducing migration of cells in a tissue of a chronic wound may ameliorate a deficiency in cell and / or tissue migration during the healing process, which may be of particular use to a subject that has one or more of diabetes, high blood pressure, poor blood circulation, high risk for infection, advanced age, or other condition that impairs wound healing. In some embodiments, the subject is or was previously treated with or exposed to one or more agents selected from an anticoagulant, an anti-inflammatory, an antibiotic, an antimicrobial, a chemotherapeutic, an immunosuppressant, an immunomodulator, a tumor necrosis factor (TNF) inhibitor, and nicotine.

[0084] In some embodiments, the methods of the disclosure are useful for the regeneration of tissue. For example, a method of regenerating tissue in a subject in need thereof may comprise inducing migration of cells of a tissue according to the present disclosure. In some embodiments, the subject has an injury to or defect of the central nervous system, e.g., a spinal cord injury.

[0085] In some embodiments, the methods of the disclosure are useful in the restoration of tissue organization in a subject in need thereof. For example, the protein construct or nucleic acid encoding the protein construct may be delivered to cells of a tissue of a subject or to donor cells, which are then delivered to the tissue of the subject. Light exposure can be used to direct the migration of cells toward or away from an illuminated area, depending on the cytoplasmic domain of an RTK that is in the protein construct. Such a method may beuseful, for example, when an injured or diseased tissue can be healed, e.g., remodeled, to restore healthy tissue organization and function. In some embodiments, restoration of tissue organization comprises nerve regeneration, wherein target cells (e.g., cells to which the protein construct or nucleic acid encoding the protein construct is delivered) comprise neurons, and wherein the protein construct is used to drive axon growth. In some embodiments, restoration of tissue organization comprises healing of diabetic ulcers, wherein the target cells are epidermal cells or fibroblasts, and wherein the protein construct is used to promote cell movement, proliferation, and / or extracellular matrix production to aid in wound closure.

[0086] In some embodiments, the methods of the disclosure are useful in the preparation of a tissue graft. For example, a method of preparing a tissue graft may comprise inducing migration of cells of a tissue according to the disclosure. Methods of preparing a tissue graft may be useful, for example, in promoting cell migration through a nerve guide conduit or other scaffold.Compositions

[0087] Compositions comprising the protein construct or nucleic acid encoding the protein construct described herein can be formulated for administration to a subject (e.g., a human). Accordingly, in various embodiments, the compositions described herein further comprise one or more pharmaceutically acceptable carriers or excipients. Suitable pharmaceutical carriers typically will contain inert ingredients that do not interact with the agent or nucleic acid. Examples of pharmaceutical carriers include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate-buffered saline, Hank’s solution, Ringer’s lactate, solutions appropriate for supporting the health of immune cells (e.g., solutions containing glucose, amino acids, growth factors, and / or other nutrients or immune stimulators), and the like. Formulations can also include small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifying agents, solubilizing agents, pH buffering agents, wetting agents). For inhalation, the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer or nebulizer or pressurized aerosol dispenser).

[0088] Standard pharmaceutical formulation techniques can be employed, such as those described in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Suitable pharmaceutical carriers for parenteral administration include, for example, sterilewater, physiological saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate-buffered saline, Hank’s solution, Ringer’s lactate and the like. Formulations can also include small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifying, solubilizing, pH buffering, wetting agents). Methods of encapsulation compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art.

[0089] A nucleic acid in the compositions described herein can be administered to a subject as a neutral compound or as a salt or ester. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic or tartaric acids, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Salts of compounds containing an amine or other basic group can be obtained, for example, by reacting with a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like. Compounds with a quaternary ammonium group also contain a counteranion such as chloride, bromide, iodide, acetate, perchlorate and the like. Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base, for example, a hydroxide base. Salts of acidic functional groups contain a countercation such as sodium or potassium.

[0090] In some embodiments, the pharmaceutically acceptable carrier is selected from a liposome, a nanoparticle, an exosome, a micelle, a polymeric matrix or a gel matrix, wherein the DNA oligonucleotide molecule is contained in, or is in a complex with, the liposome, nanoparticle, exosome, micelle, polymeric matrix or gel matrix.

[0091] In some embodiments, the compositions described herein include one or more additional therapeutic agents.Subjects and Treatments

[0092] As used herein, “subject” refers to a mammal (e.g., human, non-human primate, cow, sheep, goat, horse, dog, cat, rabbis, guinea pig, rat, mouse). In some embodiments, the subject is a human. A “subject in need thereof’ refers to a subject (e.g., patient) who has, or is at risk for developing, a disease or condition (e.g., a chronic wound) that can be treated (e.g., improved, ameliorated, prevented) by administration of a composition described herein.

[0093] As used herein, the terms “treat,” “treating,” or “treatment,” mean to counteract a medical condition (e.g., a chronic wound) to the extent that the medical condition is improved according to a clinically-acceptable standard (e.g., advancement of wound margins).

[0094] As used herein, an “effective amount” refers to an amount of a composition or therapeutic agent as described herein that, when administered to a subject, is sufficient to achieve a desired therapeutic effect in the subject under the conditions of administration, such as an amount sufficient to promote (e.g., initiate, maintain and / or enhance) wound healing. The therapeutic effectiveness of a composition described herein can be determined by any suitable method known to those of skill in the art using any suitable standard.

[0095] In certain embodiments, the methods described herein are used in combination with administration of at least one other therapeutic drug / agent. When administered in a combination therapy, administration of a composition comprising the protein construct or a nucleic acid encoding the protein construct described herein can be done before, after or concurrently with the other therapeutic agent (e.g., administration of a chemotherapeutic agent, such a paclitaxel or doxorubicin). When co-administered simultaneously (e.g., concurrently), the oligonucleotide molecule or composition and other therapeutic agent can be in separate formulations or the same formulation. Alternatively, the composition and other therapy can be administered sequentially, as separate compositions, within an appropriate time frame (e.g., a treatment session / interval such as 1.5 to 5 hours) as determined by a skilled clinician (e.g., a time sufficient to allow an overlap of the pharmaceutical effects of the therapies). The chosen mode of administration can vary depending on the particular agent selected. The actual dose of the protein construct or nucleic acid, as well as the actual treatment regimen, can be determined by a skilled physician, taking into account the nature of the condition being treated and patient characteristics.Additional Non-limitins Embodiments of the Disclosure1. A method for controlling large-scale tissue movement with one or more wavelengths of light, comprising: delivering a synthetic light-activatable receptor to a tissue in need of tissue repair, the synthetic light-activatable receptor configured to be activatable by at least one wavelength of light; and controlling light conditions used to activate the synthetic light-activatable receptor to drive tissue repair over time.2. The method of Embodiment 1, wherein the synthetic light-activatable receptor comprises Opto-EGFR.3. The method of Embodiment 1, wherein the tissue is skin tissue or spinal tissue.4. The method of Embodiment 1, wherein the tissue is tissue other than skin tissue or spinal tissue.5. The method of Embodiment 1, wherein tissue movement velocity driven by the synthetic light-activatable receptor is at least 5 pm / hour.6. The method of Embodiment 5, wherein tissue movement velocity driven by the synthetic light-activatable receptor is at least 10 pm / hour.7. The method of Embodiment 6, wherein tissue movement velocity driven by the synthetic light-activatable receptor is at least 15 pm / hour.8. The method of Embodiment 1, wherein controlling light conditions includes projecting the at least one wavelength of light through a photomask and onto the tissue to illuminate the synthetic light-activatable receptor.9. The method of Embodiment 1, wherein projecting the at least one wavelength of light through a photomask and onto the tissue induces tissue flow, the tissue flow defined by a velocity vector field.10. The method of Embodiment 1, wherein controlling light conditions includes having a light source generate the at least one wavelength of light at a duty cycle of at least 0.25.11. The method of Embodiment 1, wherein controlling light conditions includes having a light source generate the at least one wavelength of light at a duty cycle of no more than 0.25.12. The method of Embodiment 11, wherein the duty cycle is at least 0.025.13. The method of Embodiment 1, wherein controlling light conditions includes controlling light conditions for a period of time of at least 1 hour.14. The method of Embodiment 1, wherein controlling light conditions includes controlling light conditions for a period of time of at least 4 hours.15. The method of Embodiment 1, wherein controlling light conditions includes controlling light conditions for a period of time of at least 12 hours.16. The method of Embodiment 1, wherein controlling light conditions includes controlling light conditions for a period of time of at least 24 hours.17. The method of Embodiment 1, wherein controlling light conditions includes controlling light conditions for a period of time of at least 36 hours.EXEMPLIFICATION

[0096] Collective cell migration is a fundamental process governing multicellular phenomena such as morphogenesis, wound healing, and cancer invasion1 3. The ability to control collective migration - sculpting tissues with high precision using patterned stimuli - could improve understanding of this fundamental tissue-scale process and serve as a useful substrate for applications ranging from accelerated wound healing to patterning biologically relevant tissue organization.

[0097] Over the past decade, various tools have been developed to achieve programmable control over collective cell migration. Tailored ligand gradients can drive chemotactic responses, but programmable control over gradient shape is challenging and requires complex microfabricated devices4 6. Micropatteming chemotactic ligands and extracellular matrices can control cellular behavior at high spatial resolution, but these cues typically cannot be dynamically altered once patterned7 9. Directing collective migration using electric fields is a promising approach, as electrical cues can drive electrotaxis in many cell types and can be rapidly adjusted in multiple spatial dimensions10 l4. However, the mechanisms by which cells sense and respond to electric fields are still poorly understood, and precisely manipulating electric fields requires sophisticated device design10,15.

[0098] Optogenetics also represents a promising approach for guiding cell and tissue motility. Light can be focused precisely in space, rapidly applied / removed, and patterned using optical approaches. Moreover, a wealth of light-controlled signaling proteins have been previously developed that could potentially interface with cell motility programs, including light-controlled GTPases and their regulators16,17, phosphoinositide 3-kinase (PI3K)18, andreceptor tyrosine kinases19. Work has demonstrated light-based guidance of individual or small groups of cells16'17'20 22and even morphogenesis in the early Drosophila embryo23,24, yet optogenetic control of mammalian tissues at macroscopic (millimeter or larger) length scales has not yet been achieved, despite its role in applications ranging from tissue regeneration to organoid production in defined geometries.

[0099] Whether light-gated receptor tyrosine kinases (RTKs) could serve as a platform for achieving optogenetic control over collective cell migration was investigated. Receptor tyrosine kinases play essential roles in cell and tissue movement in many different contexts ranging from wound healing and regeneration25,26to developmental collective migration of border cells27and neural crest cells28. RTKs also interface with many different potential modulators of cell motility, including Proto-oncogene tyrosine-protein kinase Src family kinases29, PI 3-kinase18,30, and Extracellular signal-regulated kinase / Mitogen-activated protein (Erk / MAP) kinase signaling20, enabling them to potentially orchestrate complex downstream programs. RTKs are typically activated by the association of individual receptor molecules upon ligand binding, and multiple groups have developed optogenetic RTK variants based on fusion with protein domains that undergo dimerization or oligomerization upon illumination19,31 34. Two light controlled RTKs were previously developed - OptoFGFRl and OptoEGFR - in which the intracellular domains of Fibroblast growth factor receptor 1 (FGFR1) and Epidermal growth factor receptor (EGFR) were fused to the OptoDroplet protein phase separation system35, resulting in rapid, potent, reversible, and spatially controllable activation of either receptor8,9.

[0100] As disclosed herein, the OptoEGFR system was used to drive large-scale (e.g., mm to cm), light-controlled collective migration of mammalian cells. OptoEGFR stimulation had distinct effects on collective migration depending on the geometry of the tissue and illumination pattern. Tissue densification was produced when a local light input applied to an interior region within a continuous monolayer, driven by converging cell movement into the illuminated region. Conversely, illumination of a tissue edge drove rapid tissue expansion at speeds about 40% faster than unilluminated control tissues. An overall increase in tissue motility and outward migration speed was observed when tissues were globally illuminated. Overall, these data suggest that OptoEGFR can both act as a local directional cue to guide collective migration, and as an overall amplifier of directional cell movement initiated by other non-optogenetic sources. Pharmacological perturbations and tissue patterning experiments revealed that large-scale tissue movements (e.g., migration) were primarilydriven by physical interactions between cells, not diffusible ligand gradients; that ERK signaling and myosin-driven contractility were dispensable for tissue movement; and that PI3K signaling activity was required for the effect. These data are consistent with a model where boundaries of the light pattern drive directional tissue flows, a principle that can be used to guide tissue patterning into more complex structures.

[0101] Optogenetics is a powerful tool for spatiotemporal control of cellular behavior. Previous studies focused on subcellular level control21,33,35; in contrast, the present disclosure investigated the possibility of macroscopic, tissue-level behavioral control using light illumination. Illumination of millimeter-scale OptoEGFR-expressing retinal pigment epithelial (RPE) tissue induced two profound phenotypes for tissue-scale movement: (1) tissue densification into local regions of illumination, and (2) accelerated outgrowth at tissue edges. These phenotypes might initially seem contradictory, as the same stimulus (blue light) and cellular context (OptoEGFR cells) can either trigger formation of high-density domains within a tissue or expand outward to low density, depending on the geometry of the tissue and light pattern.

[0102] To study tissue flows into local regions of illumination, circular illumination patterns were projected to the inside of the tissue resulted in tissue movement that was distributed over about 1 mm, with a peak speed at the illumination boundary. The extent of the collective migration was dependent on the size of the illumination pattern, with larger migration speeds and more sustained movement obtained with larger stimulation patterns (FIGs. 3C-3D). Without being bound by any theory, this phenomenon might be explained by the high cell densities that are reached in the small illumination patterns, which have a high perimeter but low area, thus driving tissue flow into a region of limited size. There was also an intermediate light dose for driving tissue migration (FIGs. 4F-4G), with more frequent illumination dramatically inhibiting tissue flows. It was counterintuitive that higher light doses did not trigger a more potent migratory response, which might be explained through dose-dependent effects of EGFR signaling on downstream signaling or motility programs.

[0103] To study the effects of OptoEGFR illumination on tissue outgrowth, expansion of tissues under global illumination was monitored. The outgrowth of the tissue could be accelerated by about 40% (FIGs. 5A-5D). Illumination not only affected the edge of the tissue but also enabled free edge expansion to propagate deeper into the tissue, decreasing cell density at the tissue center (FIGs. 5E-5H). These findings were reminiscent of the solidlike to fluid-like tissue phase transition termed un-jamming that has been reported in otherepithelial contexts53,54. The RPE-1 cell line’s tissue architecture exhibited spider web-like tissue structure, not a classic epithelial geometry with tightly packed configuration cell bodies filling the entire space, where morphology of each cell (shape index) is used to determine the phase of the tissue based on the energy barrier for cellular junction restructuring50,64.

[0104] The results described herein support a model that resolves the apparent contradiction of densification at interior regions and outgrowth at cell boundaries (FIG. 8A). At the illumination boundary, cells that are partially illuminated migrate directionally toward the illuminated region. Their unilluminated neighbors then enter the illumination boundary, either by being pulled along cell-cell contacts or by migration into the lower-density region produced by their neighbor’s movement. These cells are now exposed to a partial light stimulus and the process repeats. Consistent with this model, it was found that cells must be present at the illumination boundary for directional migration into the illuminated region to occur (FIG. 6A). Cells that were wholly illuminated did not experience a directional cue and were free to expand in any direction, including outward from a tissue edge.

[0105] The disclosed study also sheds light on the essential molecular mechanisms for RTK-driven tissue flows. Combining light stimulation with small-molecule inhibitor treatment revealed that PI 3 -kinase signaling was essential for light-induced collective cell migration, emphasizing that OptoEGFR acts at the level of cell direction-sensing, producing a collective migration polarity. These data suggest that OptoEGFR-driven cell migration operates via principles distinct from those suggested in recent studies of EGFR-driven cell movement in Madin Darby canine kidney (MDCK) epithelial monolayers. The experiments disclosed herein suggest that neither Disintegrin and metalloproteinase domain-containing protein 17 (ADAMI 7) activity nor Epidermal growth factor (EGF) ligands diffusion is required to coordinate tissue-scale cell movements downstream of OptoEGFR stimulation. Further, the disclosed experiments found that Erk activity is neither necessary (using mitogen-activated protein kinase (MEK) inhibitor treatment) nor sufficient (using OptoSOS stimulation; OptoSOS is an optogenetic system to directly activate Ras GTPase (Ras)ZERK signaling downstream of RTKs) for light-induced tissue movement in either RPE-1 cells or MCF10A human epithelial fibrocystic breast cells. These data suggest that RTKs can trigger cell movement through a variety of distinct intracellular mechanisms depending on cellular context, and MDCK collective cell migration may represent a distinct mode from the cell lines studied here.

[0106] Overall, the disclosed study demonstrated that light-controlled tissue movement represents a powerful and controllable means to drive tissue rearrangements, which could find utility in applications where tissue organization is disrupted such as wound healing, tissue regeneration, and restoring proper tissue organization in cases of developmental disorders.Example 1 : Materials and MethodsCell culture

[0107] RPE cells (hTERT RPE-1 human hTERT-immortalized retinal pigment epithelial cells, American Type Culture Collection (ATCC), CRL-4000) were cultured in full media comprising DMEM / F12 (Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12) (Gibco, Waltham, MA, USA; Catalog No. 11320033) supplemented with 10% fetal bovine serum (R&D Systems, Minneapolis, MN, USA; Catalog No. 26140079), 1% L-glutamine (Gibco, Catalog No. 25030081), and 1% penicillin / streptomycin (Gibco, Catalog No.15140122). MCF10A-5E cells65(human epithelial fibrocystic breast cells) were cultured in full media comprising DMEM / F12 supplemented with 5% horse serum (Invitrogen, Waltham, MA, USA; Catalog No. 16050122), 20 ng / mL EGF (PEPROTECH®, Gibco, Catalog No. AF-100-15-1MG), 0.5 pg / mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA; Catalog No. H0888), 100 ng / mL cholera toxin (Sigma-Aldrich, Catalog No. C8052), 10 pg / mL insulin (Sigma-Aldrich), and 1% penicillin / streptomycin. All cells were maintained at 37°C and 5% CO2. Cells were tested to confirm the absence of mycoplasma contamination.Plasmid construction

[0108] All constructs were cloned into the pHR lentiviral expression plasmid66using inFusion cloning (Takara Bio, San Jose, CA, USA; Catalog No. 638943). Linear DNA fragments were produced by PCR using high-fidelity (HiFi) polymerase (Takara Bio, Catalog No. 639298), followed by treatment with methylation-sensitive restriction enzyme Dpnl to remove template DNA. PCR products were then isolated through gel electrophoresis and purified using the NUCLEOSPIN® gel purification kit (Takara Bio, Catalog No.740609.250). Linear DNA fragments were then ligated using inFusion assembly and amplified in Stellar competent Escherichia coli (Takara Bio, Catalog No. 636766). Plasmidswere purified by miniprep (QIAGEN, Germantown, MD, USA; Catalog No. 27104) and verified by whole-plasmid sequencing (Plasmidsaurus, South San Francisco, CA, USA).Cell line generation

[0109] Constructs were stably expressed in cells using lentiviral transduction. First, lentivirus was produced by co-transfecting HEK293T LX (Lenti-X human embryonic kidney 293 simian virus 40 large T antigen-expressing) cells (Takara Bio, Catalog No. 632180) with pCMV-dR8.91, pMD2.G66, and the expression plasmid of interest. 48 hours later, viral supernatants were collected and passed through a 0.45 pm filter. Cells were seeded at about 40% confluency and transduced with lentivirus 24 hours later. 24 hours post-seeding, culture medium was replaced with medium containing 10 pg / mL polybrene and 150-300 pL viral supernatant was added to cells. Cells were then cultured in virus-containing medium for 48 hours. Populations of cells co-expressing each construct were isolated using fluorescence- activated cell sorting on a Sony SH800S cell sorter (San Jose, CA, USA). Sequentially bulk- sorted populations were collected for all experiments. Cell lines used in this study (RPE, MCF10A) were validated using short tandem repeat (STR) profiling (ATCC Catalog No. 135-XV).Tissue patterning

[0110] 35 mm glass bottom dish (Cellvis, Mountain View, CA, USA; Catalog No. D35-20-1.5-N) was coated with lOpg / ml human fibronectin (EMD Millipore, Burlington, MA, USA; Catalog No. FC010) for 30 minutes at 37°C then washed three times with deionized water (DI). Surface of the dish was completely dried by nitrogen blowing. For the tissue seeding stencil, a 250pm thick polydimethylsiloxane (PDMS) membrane (Stockwell Elastom erics, Philadelphia, PA, USA; Catalog No. BISCO® HT-6240) was cut by the SILHOUETTE CAMEO® vinyl cutter (Silhouette America, Inc., Lindon, UT, USA). The stencil was treated with 2% PLURONIC® F-127 surfactant polyol (Invitrogen, Catalog No. P6866) solution diluted in phosphate buffered saline (PBS) for 30 minutes at 37°C followed by three times wash with DI and drying with nitrogen. The dried stencil was attached to the fibronectin coated glass bottom dish.

[0111] Cells were washed once with PBS and treated TrypLE (Gibco, 12604-013) for 7 minutes at 37°C to be detached from the cell culture dish. The TRYPLE® dissociation enzyme treated cell solution was diluted with the culture medium and centrifuged for 3 minutes under 1500 revolutions per minute (RPM). After the centrifugation, the supernatant was aspirated and the cell pellet was dissolved into the culture medium. The resuspended cell solution was carefully seeded into the stencil using a micropipette. The concentration of the cell solution was aimed to be between 1.25E6 to 1.5E6 cells / ml. Seeding volume was determined by the empirical equation: seeding volume (pl) = stencil area(mm2) x conversion constant (0.44ul / mm2). To facilitate cell adherence, the cell seeded dish was incubated for 1 hour at 37°C before being flooded with the culture medium. 15 hours after flooding, the culture medium was exchanged to serum-free starvation medium consisting of DMEM / F12 (Gibco, Catalog No. 11320033), 1% L-glutamine (Gibco, Catalog No. 25030081), and 1% penicillin / streptomycin (Gibco, Catalog No. 25030081). Imaging was performed 3 hours after the media exchange.

[0112] For OptoEGFR activity validation experiment (FIGs. 2A-2C), cells were imaged on glass-bottom, black-walled 96-well plates (Cellvis, Catalog No. P96-1.5H-N) coated with fibronectin. Wells of 96-well plates were first incubated with 10 pg / mL fibronectin dissolved in PBS at 37°C for a minimum of 30 minutes. Cells were then seeded on glass-bottom 96- well plates at about 40,000 cells / well 1 day prior to imaging. To increase adhesion, cell suspension was plated into 100 pL of media and then spun down in a tabletop centrifuge for 30 seconds. After confirming the adhesion of cells, an additional 100 pL of full media was added. The growth medium of cells was replaced with serum-free starvation medium 3 hours prior to imaging.Light guard generation for illumination pattern projection

[0113] The measurement of the scaling factor (length of physical pattern on light guard / length of projected illumination pattern on the dish) was measured with circular pattern light guard. A black plastic weighing boat (Heathrow Scientific, Vernon Hills, IL, USA; Catalog No. HS1423CC) was cut with a laser cutter to generate the light guard. The light guard was attached to the empty slot of the polarizer. The transmitted light source was turned on and the illumination pattern was focused by adjusting the height of the condenser turret. The image of the illumination pattern was captured and the diameter of the illuminated circle was measured with the Imaged software (Abramoff et al., “Image Processing with Imaged”. BiophotonicsInternational, 2004). The scaling factor was calculated by dividing the diameter of the circular hole in the test light guard by the diameter of the circular illumination. The measured scaling factor for the Nikon (Tokyo, Japan) Ti-2 system was 2.61. Based on the value, light guards with desired illumination patterns were designed and manufactured in the same way as the test light guard (FIGs. 4A-4I).Live-cell imaging

[0114] For small-scale patterning experiments (e.g., FIGs. 1A-1D), imaging was performed on a Nikon Ti microscope with an iXon EM-CCD camera (Oxford Instruments, Abingdon, United Kingdom) using a 20x objective. Patterned optogenetic stimuli were applied using a Mightex (Pleasanton, CA, USA) Polygon 4000 digital micromirror device (DMD) and an X-Cite XLED 450 nm light source (Excelitas, Pittsburgh, PA, USA). To prevent evaporation of media while imaging, 50 pL of mineral oil (VWR, Radnor, PA, USA) was pipetted onto wells prior to mounting samples on the microscope. Optogenetic stimulation was achieved with the DMD set to a value of 75% and 200 pm diameter region of interest (ROI) which resulted in a measured intensity at the objective lens of 65 mW / cm2.

[0115] For large-scale patterning experiments e.g., FIGs. 3A-3H), imaging was performed on a Nikon Eclipse Ti-2 microscope with a Qi-2 camera, red fluorescent protein (RFP) channel for RPE and MCF10A, using a lOx objective. Live-cell imaging was performed within the custom-made incubator box which maintained 37°C and supplied humidified 5% CO2 air flow. Images were captured every 10 minutes. To project illumination patterns, light guards were attached to the empty slot of the polarizer. The transmitted light source’s blue light emitting diode (LED) was used to apply 450 nm illumination to the tissue, with a measured intensity at the sample plane of 12 mW / cm2. The default illumination frequency for all figures was 5 seconds / minute except where otherwise indicated.Illumination frequency was adjusted to 4 seconds / 2.5 minutes for the high throughput assay in FIGs. 6C-6G. For live nuclear imaging, tissues were incubated in serum-free media with 10 pM Janelia Hoechst 646 DNA probe for 1 hour before imaging in the Cyanine 5 (Cy5) channel.Immunostaining

[0116] To quantify the three-dimensional (3D) structure of the tissue (FIGs. 3E-3G), the tissues were fixed and stained with NUCBLUE® formulation of the DAPI (common name,4’,6-diamidino-2-phenylindole; IUPAC name, 2-(4-carbamimidoylphenyl)-U / -indole-6- carboximidamide) dye (Invitrogen, Catalog No. R37605) after the experiment. The tissues were treated with 4% paraformaldehyde (PF A) diluted in PBS for 45 minutes. 2 drops of NUCBLUE® were added to each dish. Nuclei stained tissues were imaged by the W1 confocal unit, using the 20x objective.Inhibition Assays

[0117] Cells were plated according to the protocols outlined above for the small and tissue scale conditions. After the 3-hour starvation period, media containing the desired inhibitor concentration was added to each experimental well immediately before imaging. The following small molecule inhibitors were used (Table 1).Table 1. Small molecule inhibitors used in inhibition assays.

[0118] The following are additional identifiers, e.g., International Union of Pure and Applied Chemistry (IUPAC) names and American Chemical Society Chemical Abstract Service (CAS) numbers, for the small molecules named in Table 1. Gefitinib: IUPAC name, A-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine; CAS No. 184475-35-2. Cobimetinib: IUPAC name, [3,4-difhioro-2-(2-fhioro-4- iodoanilino)phenyl]-[3-hydroxy-3-[(25)-piperidin-2-yl]azetidin-l-yl]methanone; CAS No. 934660-93-2. MK2206: IUPAC name, 8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl-2JT- [l,2,4]triazolo[3,4-f][l,6]naphthyridin-3-one;dihydrochloride; CAS No. 1032350-13-2. PI-103: IUPAC name, 3-(6-morpholin-4-yl-8-oxa-3,5,10-triazatricyclo[7.4.0.02’7]trideca- l(9),2(7),3,5,10,12-hexaen-4-yl)phenol; CAS No. 371935-74-9. G66976: IUPAC name, 3- (23-methyl-14-oxo-3,13,23-triazahexacyclo[14.7.0.02’1°.04’9.011’15.017’22]tricosa- l,4,6,8,10,15,17,19,21-nonaen-3-yl)propanenitrile; CAS No. 136194-77-9. TAPI-1 : IUPAC name, (27?)-A-[(25)-l-[[(25)-l-(2-aminoethylamino)-l-oxopropan-2-yl]amino]-3-naphthalen- 2-yl-l-oxopropan-2-yl]-7V-hydroxy-2-(2-methylpropyl)butanediamide; CAS No. 163847-77- 6. / ?ara-Nitro-Blebbistatin: chemical name, (S)-4'-nitro-Blebbistatin; IUPAC name, (3aS)-3a- hydroxy-6-methyl-l-(4-nitrophenyl)-2,3-dihydropyrrolo[2,3-b]quinolin-4-one; CAS No.1621326-32-6. Y27632: chemical name, trans- - [(1 ?)- 1- Aminoethyl] -7V-4- pyridinylcyclohexanecarboxamide dihydrochloride; IUPAC name, 4-[(17?)-l-aminoethyl]-A- pyridin-4-ylcyclohexane- 1 -carboxamide.

[0119] The following are names related to targets listed in Table 1 that are not elsewhere described. AKT1 : RAC-alpha serine / threonine-protein kinase. AKT2: RAC -beta serine / threonine-protein kinase. AKT3: RAC-gamma serine / threonine-protein kinase. MMP: metalloproteinase. MTOR: Serine / threonine-protein kinase mTOR. PKC: protein kinase C.

[0120] The following is information related to suppliers listed in Table 1 that is not elsewhere described. MCE: MedChemExpress, Monmouth Junction, NJ, USA. Selleck Chem: Selleck Chemicals LLC, Houston, TX, USA. Cayman Chem: Cayman Chemical Company, Ann Arbor, MI, USA. Tocris: Tocris Bioscience, Bio-Techne Corporation, Minneapolis, MN, USA.Immunoblotting analysis

[0121] To collect cell lysate, cells were cultured in 10 mm tissue culture dishes, washed with PBS and lysed in RIPA buffer (ThermoFisher, Waltham, MA, USA; Catalog No.89900). Cell scrapers were used to separate adherent cells from the culture dish, and immediately placed on ice inside 1.5 mL EPPENDORF® tubes. Lysates were centrifuged at 13,000 RPM at 4°C for 10 minutes, the supernatant was collected and the pellet was discarded. NUPAGE® polyacrylamide gel electrophoresis (PAGE) lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Catalog No. NP0007) was added to each sample before being heated at 95°C for 10 minutes and then placed on ice. Sample proteins were then separated via sodium dodecyl sulfate (SDS)-PAGE and transferred to nitrocellulose membranes using the iBlot 2 Gel Transfer (Invitrogen, Catalog No. IB21001). Membranes were blocked with Odyssey Blocking Buffer (LLCOR, Lincoln, NE, USA; Catalog No. 927-60001) for 1 hour at room temperature preceding primary antibody incubation using a 1 : 1 mixture of Odyssey Blocking Buffer and tris-buffered saline with Tween (TBST) (ThermoFisher, Catalog No. J77500.K8) diluted to lx concentration at 4°C overnight. The following primary antibodies were used: Phospho-p44 / 42 MAPK (Erkl / 2) XP® ( Cell Signaling Technology (CST), Danvers, MA, USA; Catalog No. 4370), p44 / 42 Erkl / 2 (CST Catalog No. 4696), P-Actin (CST Catalog No. 3700), and Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) (CST Catalog No. D4C6R). The following secondary antibodies were used: IRDYE® 800CW Goat anti-Rabbit IgG (LI-COR, Catalog No. 926-32211) and IRDYE® 680RD Goat anti-Mouse IgG (LI-COR, Catalog No. 926-68070). Immunofluorescence imaging was conducted using the LI-COR Odyssey Clx system.Tissue migration analysis

[0122] Local velocity vector field of the tissue was generated by particle image velocimetry (PIV). PIVLab MATLAB® (The MathWorks Inc., Natick, MA, USA) plugin was used for this analysis67. Pass 1 and 2 window sizes were assigned as 200 pixels and 100 pixels for the timelapse image sequences captured with the lOx objective. Overlap between box was 50%. Further analysis was conducted after deducing radial velocity from the velocity vector field. Radial velocity was calculated by multiplying the speed to the cosine of angle difference between velocity vector and vector pointing toward the center of the illumination (FIGs. 3A-3G; FIGs. 7A-7B) or the tissue (FIGs. 5A-5H). Peak radial velocity was defined as the peak value of radial velocity of the entire tissue. Migration zone width was measured by the distance from the illumination boundary to the point where the radial velocity value drops to the threshold value (1 pm / hour), a sufficiently high value that was not attained by unilluminated tissue.Pseudo density analysis

[0123] Confocal stack of nuclei stained tissue was processed by the sum Z-proj ection function in ImageJ. Radial average intensity from the illumination center was calculated with the ImageJ plugin Radial Profile Angle. The radial intensity data was binned with a 50 pm binning window. Pseudo density was deduced by normalizing the fluorescence intensity by the average intensity of the control tissue.Tissue height analysis

[0124] Confocal stack of nuclei stained tissue was radially resliced via reslice function in ImageJ. The radial reslice stack was further processed with the sum Z-proj ection function in Image! The obtained merged XZ slice image was segmented with the threshold function in ImageJ. Local tissue height was measured with this binary image and was binned with a 50 pm binning window.Radius and edge expansion speed analysis

[0125] RPE tissue was segmented using the Bernsen method, a locally adaptive binarization method, in auto local threshold function in ImageJ. Area of the segmented tissue image was measured by the regionprop function in MATLAB®. Radius of the tissue was calculated by fitting the tissue area to the circle. Edge expansion speed was deduced as the speed of radius increment.Cell density analysis

[0126] Cy5 nuclei channel images were segmented by StarDist ImageJ plugin68,69.Centroid of each nucleus was calculated with the regionprop function in MATLAB®. Local cell density was measured by (Number of centroids in the ROI) / (Area of ROI). Dimension of ROI was 100 pixel x 100 pixel square and 50% overlap between the adjacent ROIs.Single cell analysis

[0127] The Cy5 channel nucleus image stack was tracked via the TrackMate ImageJ plugin. Persistence and speed were calculated using a custom MATLAB® script.Statistical test

[0128] The Mann Whitney U-test was applied for statistical tests to compare differences between two groups. For the group size below 50, t-test analysis in Prism (Dotmatics, Boston, MA) was used. For the group size above 50, 50 observations were random-sampled with replacement for each data group. The P-value was calculated from these subsets. The mean P- value of 50 repeats of this process was used to decide statistical significance of the difference between two groups. A custom MATLAB® script was used for this process.Mathematical modeling

[0129] A mathematical model was constructed to obtain qualitative insights for how tissues might flow under patterned light inputs. The model comprises a continuous variablec(x,y) representing the density of tissue at each position in two-dimensional (2D) space. It also incorporates an arbitrarily-drawn light input u(x,y) that takes binary values (1 for illumination at that position; 0 for darkness). The model incorporated two cellular processes: a diffusion term (with diffusion constant / J) to represent tissue spreading over time and a flux term at boundaries of the binary illumination input in the direction of the light with rate k. This partial differential equation system was simulated by discretizing the x and y coordinates into 101 bins and simulating the resulting 10,201 element ordinary differential equation system, where each element was defined as:where was related to the gradient of the light stimulus, and was defined as 1 for elements where the input u changed from 0 to 1 and -1 for elements where the input changed from 1 to 0 for pairs of elements along the x or y direction and Axyis the length scale associated with each discretized spatial element (e.g., the total length scale of simulation divided by 101). MATLAB® code implementing the model is available at http s : / / github . com / toettchl ab / Suh-Thornton2024.

[0130] To obtain approximate values for the parameters D and k, the 1 mm diameter tissue densification pattern of FIG. 3G (see FIG. 9) was simulated to qualitatively match the length and time scale of tissue movement into the illuminated region, which led to values £>=50 pm2 / min and k=0.3 min-1. The complex pattern of FIGs. 8A-8D was then simulated using the same parameters.Example 2: OptoEGFR stimulation triggers both local tissue convergence and enhanced outgrowth

[0131] Initially, the cell motility effects of OptoEGFR and OptoFGFR light-controlled receptor tyrosine kinases32,33were characterized (FIGs. 1A-1B). In each case, the intracellular domains of the receptor tyrosine kinases was fused to the FusionRed fluorescent protein as well as the membrane OptoDroplet system35, which is composed of a myristoylation tag to drive membrane localization, the FUS disordered N-terminal sequence, and the Arabidopsis thaliana Cryptochrome-2 (Cry2) photolyase homology region (PHR) (Cry2PHR) domain which undergoes oligomerization upon illumination with 450 nm light36,37.

[0132] OptoEGFR or OptoFGFR was introduced using lentiviral transduction into RPE-1 cells, an immortalized human retinal pigmental epithelial cell line38. Confluent monolayers of each cell line were plated and locally stimulated with pulses of 450 nm blue light delivered every 2-3 minutes using a digital micromirror device on a microscope at an intensity of 65 mW / cm2. Cells were imaged in the FusionRed channel, which marked OptoEGFR expression and localization. Illumination elicited profound changes in tissue organization, with OptoEGFR cells undergoing rapid and sustained movement into the illuminated region (FIG.IC). Local illumination at the edge of an OptoEGFR-expressing tissue produced a distinct effect, with cells rapidly moving outward from the edge to fill the illuminated region (FIG.ID)

[0133] Light-induced migration phenotypes were not a general feature of optogenetic receptor tyrosine kinase activation. Rather than rapid light-induced migration into illuminated regions, OptoFGFR-expressing cells were gradually excluded from the illumination region (FIG. 2A), consistent with prior observations of retraction away from illuminated regions in individual OptoFGFR-expressing NIH3T3 mouse fibroblasts32. Similar degrees of ERK phosphorylation with both OptoEGFR and OptoFGFR-expressing cells were observed (FIG. 2B), suggesting that this difference in cellular responses was not driven by an absolute difference in receptor activity but rather different intracellular signaling pathways engaged by the two receptors.

[0134] Similar OptoEGFR-driven tissue movement was also observed in a second human cell line, MCF10A breast epithelial cells expressing the Erk kinase translocation reporter (KTR) biosensor for Erk mitogen-activated protein kinase (MAPK) activity, ErkKTR-iRFP39. Illumination drove rapid export of the ErkKTR from cells only within the illuminated region as well as tissue convergence in an analogous manner to what was observed in RPE-1 cells (FIG. 2C). These data confirm that illumination drove localized OptoEGFR activation, and that the light-induced tissue movement triggered by OptoEGFR generalized across multiple cellular contexts.

[0135] The migration data present an apparent paradox because the same optogenetic tool and light stimulus drove opposing effects: either convergent motion and an increase in cell density when illumination was applied at interior positions, or divergent outgrowth and expansion from a tissue edge. In subsequent experiments, both types of motion were quantified and the basis for light-induced tissue movement was dissected to resolve this paradox.Example 3: OptoEGFR tissue densification is driven by collective migration at the illumination boundary

[0136] To better understand and quantify how OptoEGFR stimulation drives convergent motion in a confluent monolayer, stimulus and imaging conditions were scaled up, e.g., to the mm-cm scale, in living tissues. Local stimuli can often elicit global responses in collective systems40,41. Collective cell behaviors depend heavily on tissue size and shape42, so precise arrays of replicates of 6-mm diameter circular tissues were engineered using a tissue stenciling approach43to increase throughput, improve statistical power, and ensure directly comparable tissues. Typically, localized optogenetic stimuli are applied to cells and tissues using digital micromirror devices through the imaging light path, which restricts patterned stimuli to a single field of view. To expand optical stimulation to a larger length scale, various illumination patterns were instead projected through the transmitted light path using laser cut photomasks (see Example 1: Methods) placed directly in the light path of the condenser of an inverted microscope, which allowed illumination of OptoEGFR-expressing RPE-1 cells over centimeter length scales (FIGs. 4A-4B). An automated microscope was then programmed to uniquely align each of the large tissues with specific patterns on the photomask to allow use of one photomask to stimulate multiple tissues (see Example 1: Methods).

[0137] This approach was used to apply circular illumination patterns with 200 pm, 1 mm, and 2 mm diameters at the center of confined, 6 mm diameter RPE-1 tissues and cells were imaged in the FusionRed channel (FIG. 3A). Light stimulation drove rapid tissue movement into the boundary of the illuminated region that gradually filled in toward the center. Outside the illumination boundary, a broad region of decreased cell density was also observed, suggesting that cells were displaced from hundreds of micrometers away from the surrounding tissue into the illuminated region, indicating a large correlation length. The converging migratory behavior of the illuminated tissue was quantified with particle image velocimetry (PIV) analysis on the time-lapse images (FIG. 3B) to produce spatial maps of migration dynamics. Confirming the qualitative observations, a local velocity vector map showed strong converging motion generated at the illumination boundary and nearby unilluminated tissue, oriented toward the illumination center (FIG. 4C). A kymograph of theradial component of tissue velocity revealed that the convergent motion was relatively stable over time, extending about 500 pm from the illumination boundary (FIG. 3B).

[0138] Next, the spatial profile of tissue velocities was quantified for each illumination pattern (FIG. 3C), focusing on the time point at which the maximum velocity was achieved for each illumination pattern (3 hours for 200 pm illumination pattern tissue; 15 hours for 1- and 2 -mm illumination pattern tissues) (FIGs. 4D-4E). A sharp, relatively symmetric peak in tissue velocity was observed near the border of the illumination area, with slower movement farther into the illuminated region or in the unilluminated exterior region (FIG. 3C). Tissue movement was fastest and most sustained for millimeter-scale illumination patterns, which produced a about 3 -fold higher peak velocity compared to the 200 pm illumination pattern (FIG. 3D). These data suggest that localized optogenetic stimulation is well suited for driving tissue reorganization even at macroscopic length scales.

[0139] Notably, the zone in which directional migration was observed was confined to a region near the illumination boundary of similar width for both the 1 mm and 2 mm illumination pattern (FIG. 3D). For the 2 mm diameter pattern, cells within the illuminated region more than 1 mm from the light boundary did not undergo substantial movement, despite consistent illumination. These data suggest that light-induced collective migration was confined near the interface between illuminated and unilluminated tissues.

[0140] Illumination dose was systematically varied for a fixed geometry to test how the strength of OptoEGFR activation alters migratory responses (FIG. 4F). Compared to the base case (5 seconds per minute of 450 nm light exposure), increasing the illumination frequency by three-fold (5 seconds every 20 seconds) dramatically decreased overall tissue migration, whereas a lower illumination dose (4 seconds every 2.5 minutes) modestly decreased movement speed. These data suggest that the extent of tissue migration varied with both the illumination pattern geometry and the illumination schedule of OptoEGFR activation, and might reflect an intermediate level of RTK activity for driving cell migration or competing timescales for light-induced changes in cell / tissue mechanics.

[0141] If cells migrate over long distances to enter regions of light-induced OptoEGFR activity, a dramatic increase in cell density or a transition from 2-dimensional to 3- dimensional tissue organization might be expected over time. Indeed, light-induced collective migration also led to pronounced tissue thickening that was evident in confocal z-stacks of the illuminated tissue (FIG. 3E) as well as radial profiles obtained by summing across radial slices of the nuclear intensity image (FIG. 3F). Both tissue height and cell density rosesharply to a peak about 50 pm interior to the illumination boundary to values approximately 3-fold higher than unilluminated control tissues (FIG. 3G). Depletion of cells outside the illumination area was also observed, consistent with the elongated morphology of cells just outside the illumination area (FIG. 1C). Light-induced tissue densification persisted for at least 40 hours after a shift back to darkness (FIGs. 4G-4H), consistent with a model where localized light stimuli drive irreversible cell rearrangements and permanent changes to tissue structure. Taken together, these data demonstrate that optogenetic EGFR stimulation drove rapid collective migration toward sites of illumination, leading to millimeter-scale changes in tissue organization.Example 4: Global tissue illumination drives tissue fluidization and rapid outgrowth

[0142] Next, the effect of OptoEGFR stimulation at tissue boundaries was measured. Local illumination at a tissue edge combined two effects: light-induced edge outgrowth and light-induced convergence at interior boundaries. To simplify the geometry, light was globally applied to unconfined tissues and did not produce a light boundary within the tissue, and thus avoided regions of local tissue convergence. 2-mm diameter circular tissues were grown in a confining stencil, allowed to reach confluency, and then the stencil was removed and tissue outgrowth was monitored for 24 hours in the presence or absence of global 450 nm illumination (FIG. 5A). Illuminated tissues indeed exhibited more rapid outgrowth compared to unilluminated controls, with movement that extended deeper within the interior of the tissue (not shown). Edge expansion was quantified by fitting the expanding tissue to a circle at each time point (FIG. 5B) and the speed of outgrowth was estimated from the rate of radial growth (FIG. 5C). Illuminated tissues grew consistently faster than their unilluminated counterparts, with about 40% increased speed on average over the time course (FIG. 5D). These data indicate that OptoEGFR stimulation exerted global effects on tissue movement, increasing the rate of expansion of unconfined tissue.

[0143] Globally illuminated OptoEGFR tissues exhibited increased collective motion not only at tissue edges but also at interior regions (not shown). To quantify this effect, the local velocity field of the entire tissue was mapped using PIV analysis (FIG. 5E). In the control case, outward tissue flow was confined to the outermost about 500 pm, with minimal movement at interior positions (FIG. 5E, shaded region). This observation is consistent with prior studies of expanding tissue monolayers42,44,45as well as the prior observation of decreased movement at high cell densities termed contact inhibition of locomotion (CIL)46 48.Tissue-scale CIL has been interpreted as a jamming transition49’50that coincides with high cell density46,51,52. In contrast, interior regions of illuminated OptoEGFR-RPE-1 tissues gradually began to flow outward (FIG. 5E). Strikingly, illuminated tissues maintained outgrowth speeds at interior positions that were even higher than the peak speeds observed at the periphery of control tissues (FIG. 5F). This may indicate a general fluidization of the bulk as the previously ‘solid-like’ interior gave way to increased motility.

[0144] To better characterize the interplay between migration and cell density, Hoechst Janelia Fluor 646 live-cell dye was used to stain cell nuclei and monitor cell density throughout the tissue (FIGs. 5G-5H). Despite initially similar density profiles, illuminated tissues gradually decreased in cell density in coordination with increased outward tissue speed, whereas control tissues retained the high-density interior that is usually observed epithelial monolayer expansion42’43. In summary, global illumination of dense tissues with free edges enhanced the tissue’s outgrowth and promoted fluidization of interior regions. The increase in cell movement and decrease in cell density of illuminated tissues was reminiscent of epithelial tissue unjamming, the transition of tissue phase from static solid-like phase to motile fluid-like phase53,54.Example 5: Light-induced tissue movement depends on cell-cell contact and PI 3-kinase signaling

[0145] What processes downstream of OptoEGFR stimulation drive collective migration? Many cellular processes have been implicated in RTK-directed cell migration. Recent studies in Madin Darby canine kidney (MDCK) cells provide evidence of EGFR-related collective cell migration being driven by a feedback loop between intracellular Erk kinase activity, cell contractility, and ADAM17-triggered shedding of epidermal growth factor (EGF) to stimulate Erk activity neighboring cells20,21, and prior studies also implicate myosin contractility initiated by Rho kinase (ROCK)55and PI 3-kinase activity18,22,56as RTK- dependent drivers of cell motility. Cellular processes responsible for the profound light- induced tissue reorganization were explored.

[0146] The basic principles governing large-scale cell movement in the disclosed system were identified. Prior experiments revealed oriented cell movement towards the light input that appeared to be restricted to 1 mm region centered on the illumination boundary (FIG. 3C). How did the illumination boundary drive tissue movement, and how did a localized light stimulus produce effects hundreds of microns away? Two broad classes of tissue levelcoordination were considered: diffusion of ligands from illuminated to unilluminated regions, and mechanical coupling, where movement of illuminated cells was sensed through cell-cell contacts or changes in cell density57,58.

[0147] To discriminate between these broad classes of models, an experiment was designed to determine if differences in illumination could be transmitted across a physical discontinuity between two tissues59(FIG. 6A). Two identical tissues were seeded, separated by a 300 pm gap, a distance which was shorter than the light-induced migration zone produced in the unilluminated region of a continuous tissue (FIG. 3D). Then, the right-hand tissue was illuminated and the outgrowth speed of the unilluminated left-hand tissue was monitored, comparing outgrowth between the tissue edges that were proximal and distal to the illuminated tissue (FIG. 6A). Quantification of tissue outgrowth revealed no difference in outgrowth speeds at the near and far edges (FIG. 6B). No large-scale migration was observed toward the illuminated region, suggesting that directed migration required the projection of a light-dark boundary on cells, not in the gap between cells.

[0148] Independent experiments were also performed to specifically test for roles of EGF ligand release and cell contractility in tissue-scale motility, as suggested in recent work20. Confluent 3 mm-diameter tissues were prepared and a 100 pm-diameter central region was illuminated to induce local tissue densification in the presence of the ADAMI 7 inhibitor TAPI-1 or the contractility inhibitor N-blebbistatin (a non-photosensitive variant of the classic myosin inhibitor blebbistatin). Neither N-blebbistatin nor TAPI-1 treatment blocked light-induced tissue densification or long-range cellular movements (FIG. 6C), and tissue movements reached similar peak velocities in all three cases (FIG. 6D). However, N- blebbistatin treatment appeared to broaden the migration zone deeper into the unilluminated tissue, consistent with prior observations in MDCK cells that blebbistatin can reduce cell-cell friction and lead to larger regions of coordinated migration60(FIG. 6D, right). Overall, these results suggest that diffusible ligand stimulation was dispensable for large-scale tissue movements downstream of OptoEGFR, and that cell-cell contact was required for transmission of information between regions of local OptoEGFR activation and neighboring unilluminated tissues.

[0149] To gain further insight into the signaling pathways involved in coordinating OptoEGFR-induced cell movement, the light-induced migration assay of FIG. 6C was performed in the presence of kinase inhibitors directed at nodes in the EGFR pathway: EGFR, PI3K, AKT, MEK, and PKC (FIG. 6E). As expected, the EGFR inhibitor gefitinibcompletely prevented light-induced tissue movement (FIGs. 6E-6F). In contrast, cells retained strong light-induced movement in the presence of the MEK inhibitor cobimetinib, the PKC inhibitor G66976, and the Akt inhibitor MK-2206. Substantial cell death was observed throughout the tissue during a 24-hour incubation with the MEK inhibitor cobimetinib, consistent with mitogen-activated protein kinase (MAPK) signaling for longterm cell survival. Consistent with the dispensability of MEKZErk signaling for tissue movement in this system, MCF10A cells expressing OptoSOS, an optogenetic system to directly activate RasZERK signaling downstream of RTKs61,62, had no effect on tissue movement in MCF10A cells also expressing the ErkKTR biosensor, despite similar Erk activation within the illuminated region in both cases (not shown). In contrast, the PI 3-kinase inhibitor PI- 103 was the only downstream inhibitor tested to completely block light-induced migration of OptoEGFR RPE-1 cells, phenocopying receptor inhibition by gefitinib. This was also consistent with the role of PI 3-kinase in directed cell migration and emphasizes that OptoEGFR activation is likely to act directly at the level of front-rear cell polarity63, rather than purely ‘pulling’ cells along by contraction. Similar inhibitor results were also obtained in OptoEGFR MCF10A cells (FIGs. 7A-7B), suggesting that the mechanisms underlying OptoEGFR-induced cell movements were general across cellular contexts.Example 6: Illumination boundaries provide directional information to sculpt tissue organization

[0150] Taken together, the disclosed results suggest a model for how OptoEGFR stimulation drive tissue movements in both illuminated and unilluminated regions (FIG. 8A). At the illumination boundary, partial illumination of individual cells triggered localized activation of EGFR and its downstream effector PI 3-kinase, leading to cell movement into the illumination region. Consistent with this picture, optogenetic PI 3-kinase stimulation has been observed to act as a directional cue to guide motility of individual cells18,22. Nearby unilluminated cells would then move toward the illumination boundary, either through forces applied to cell-cell contacts or to fill the gap left by their neighbor at the illumination boundary, leading them to be partially illuminated and repeating the process. The disclosed data also indicates that a second set of phenomena modulate cell movement within illuminated regions, where OptoEGFR stimulation increases both tissue fluidity and edge outgrowth speed (FIG. 8A; FIG. 5A).

[0151] To gain confidence in this conceptual model, an experiment was conducted to test whether it would be sufficient to recapitulate arbitrary, complex patterns of light-controlled cell movement. A mathematical model was implemented, which simulated light-controlled tissue flows on the same geometry as the experiments. The model assumed a continuous tissue with two sources of tissue movement (1) outward diffusion into un-occupied space (using an effective diffusion parameter Z>, and (2) cell flux at illumination boundaries into the illuminated region (using a boundary flux parameter k). The two model parameters D=50 pm2 / min and k=l min'1were qualitatively estimated from the observed rates of tissue outgrowth and light-induced movement throughout the disclosed experiments (see Example 1: Methods; FIG. 9). The continuum model was a qualitative, simplified implementation to investigate the consequences of a minimal set of biological assumptions (cell flux at illumination boundaries) and did not capture more complex tissue features such as density- driven jamming or light-induced tissue fluidization.

[0152] A complex illumination pattern that incorporated multiple domains of tissue densification and outgrowth was generated (FIG. 8B) and applied to both an OptoEGFR RPE-1 monolayer and the mathematical model. The evolution of a complex 3 -dimensional tissue structure was observed, with regions of light-induced tissue densification at regions of interior illumination, as well as enhanced outgrowth from illumination at tissue edges (FIG. 8C). This pattern was qualitatively matched by model simulations, where tissue densification was driven by flux of cells into the illuminated region, and enhanced outgrowth at the tissue boundary resulted from the higher cell density produced by this cell influx. Tissue flux at illumination boundaries was a principle that was sufficient to explain many light-induced tissue movements and is likely to be useful as a starting point for sculpting complex tissue architectures with more sophisticated illumination protocols.REFERENCESE Ridley, A. 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[0153] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0154] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of inducing migration of cells of a tissue, the method comprising: a) delivering a protein construct or a nucleic acid encoding the protein construct to at least a subset of the cells, wherein the protein construct comprises: i) a light-inducible oligomerization domain, and ii) an intracellular domain of a receptor tyrosine kinase (RTK); and b) exposing a region of the tissue to light, thereby producing an illuminated region of the tissue and activating a signaling pathway in the cells that induces cell migration.

2. The method of claim 1, wherein illuminating the region of the tissue induces clustering of the protein construct in the cells in the illuminated region of the tissue by means of the light-inducible oligomerization domain.

3. The method of claim 2, wherein clustering of the protein construct activates the signaling pathway.

4. The method of claim 2 or 3, wherein clustering of the protein constructs comprises oligomerization of two or more constructs.

5. The method of any one of claims 1-4, wherein the illuminated region comprises wholly illuminated cells.

6. The method of claim 5, wherein the wholly illuminated cells are able to migrate in any direction.

7. The method of any one of claims 1-6, wherein exposing the region of the tissue to light increases tissue fluidity.

8. The method of any one of claims 1-7, wherein the migration comprises tissue outgrowth.

9. The method of claim 8, wherein tissue outgrowth comprises outgrowth of an edge of the tissue within the illuminated region.

10. The method of claim 8 or 9, wherein migration increases speed of tissue outgrowth at least about 10%, 20%, 30%, or 40% compared to a tissue lacking the protein construct or which was not exposed to light.

11. The method of any one of claims 8-10, wherein migration increases speed of tissue outgrowth about 40% compared to a tissue lacking the protein construct or which was not exposed to light.

12. The method of any one of claims 1-11, wherein migration comprises expansion of the tissue in at least one direction.

13. The method of any one of claims 1-12, wherein exposing the region of the tissue to light induces collective migration of cells of the tissue toward or away from the illuminated region.

14. The method of any one of claims 1-13, wherein exposing the region of the tissue to light induces collective migration of cells of the tissue toward the illuminated region.

15. The method of claim 13 or 14, wherein collective migration of cells of the tissue toward the illuminated region thickens the illuminated region, increases cell density in the illuminated region, or both.

16. The method of any one of claims 13-15, wherein collective migration of cells of the tissue toward the illuminated region thickens the illuminated region at least two-fold.

17. The method of any one of claims 13-16, wherein collective migration of cells of the tissue toward the illuminated region thickens the illuminated region about three-fold.

18. The method of any one of claims 13-17, wherein collective migration of cells of the tissue toward the illuminated region thickens, densifies, or both thickens and densifies an area of the illuminated region located at least about 50 pm interior to an illumination boundary.

19. The method of any one of claims 1-18, wherein the illuminated region comprises partially illuminated cells.

20. The method of claim 19, wherein the partially illuminated cells migrate toward or away from the illuminated region.

21. The method of claim 19 or 20, wherein the partially illuminated cells migrate toward the illuminated region.

22. The method of claim 20 or 21, wherein migration of the partially illuminated cells toward the illuminated region stimulates migration of unilluminated cells toward the illuminated region.

23. The method of any one of claims 20-22, wherein migration of the partially illuminated cells toward the illuminated region stimulates migration of the unilluminated cells toward the illuminated region by means of cell-cell contacts.

24. The method of claim 23, wherein the partially illuminated cells exert force on the cellcell contacts, thereby stimulating migration of the unilluminated cells toward the illuminated region.

25. The method of any one of claims 20-24, wherein migration of the partially illuminated cells toward the illuminated region produces an area of lower cell density within the tissue, thereby stimulating migration of the unilluminated cells toward the illuminated region.

26. The method of claim 25, wherein the area of lower cell density within the tissue reduces contact inhibition of locomotion of the unilluminated cells.

27. The method of any one of claims 1-26, wherein the cells migrate at a velocity of at least about 1 pm, 5 pm, 10 pm, 15 pm, or 20 pm per hour during exposure to the light.

28. The method of any one of claims 1-27, wherein the cells migrate at a velocity of about 20 pm per hour during exposure to the light.

29. The method of any one of claims 1-28, wherein the migration comprises movement of a plurality of the cells of the tissue across a distance of at least about 100 pm, 200 pm, 500 pm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm.

30. The method of any one of claims 1-29, wherein the migration comprises movement of a plurality of the cells of the tissue for a duration of exposing the region of the tissue to light.

31. The method of any one of claims 1-30, wherein the tissue has a diameter of at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm.

32. The method of any one of claims 1-31, wherein the tissue has a diameter of at least about 2 mm, 3 mm, or 6 mm.

33. The method of any one of claims 1-32, wherein the illuminated region has a diameter of at least about 100 pm, 200 pm, 500 pm, 1 mm, or 2 mm.

34. The method of any one of claims 1-33, wherein the illuminated region has a diameter of at least about 200 pm, 1 mm, or 2 mm.

35. The method of any one of claims 1-34, wherein the illuminated region has a diameter of at least about one thirtieth, one sixth, one third, or an entire length of a diameter of the tissue.

36. The method of any one of claims 1-35, wherein migration of cells of the tissue comprises migration of at least about 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, or 1 x 1010cells.

37. The method of any one of claims 1-36, wherein the tissue comprises cells from a cell line, primary cells, induced pluripotent stem cells, or a combination thereof, or wherein the tissue comprises an ex vivo tissue, tissue in situ, or a tissue graft.

38. The method of any one of claims 1-37, wherein the tissue comprises keratinocytes, epidermal epithelial cells, fibroblasts, retinal pigment epithelial cells, neurons, or a combination thereof.

39. The method of any one of claims 1-38, wherein the protein construct or the nucleic acid encoding the protein construct is delivered at least once, twice, or three, four, five, six, seven, or eight times, to at least the subset of the cells of the tissue.

40. The method of any one of claims 1-39, wherein the protein construct or the nucleic acid encoding the protein construct is delivered to at least the subset of the cells of the tissue by means of immersion in a composition, exposure to a topically applied composition, intradermal microneedle array administration of a composition, or local injection of a composition, wherein the composition comprises the protein construct or the nucleic acid encoding the protein construct.

41. The method of any one of claims 1-40, wherein the protein construct or the nucleic acid encoding the protein construct is delivered to at least the subset of the cells of the tissue in a nanoparticle.

42. The method of claim 41, wherein the nanoparticle comprises a lipid nanoparticle.

43. The method of any one of claims 1-42, wherein the nucleic acid encoding the protein construct is delivered to at least the subset of the cells of the tissue in a viral vector, non-viral vector, or virus-like particle.

44. The method of any one of claims 1-43, wherein the protein construct is transiently expressed in at least the subset of the cells of the tissue.

45. The method of any one of claims 1-43, wherein the protein construct is stably expressed in at least the subset of the cells of the tissue.

46. The method of claim 44 or 45, wherein the protein construct is expressed for at least about 1, 2, 3, 4, 5, 6, or 7 days after delivering the protein construct or the nucleic acid encoding the protein construct.

47. The method of any one of claims 1-46, wherein the protein construct is present in at least the subset of the cells for at least about 1, 2, 3, 4, 5, or 6 days or at least about 1, 2, 3, 3, 4, 5, 6, 7, or 8 weeks.

48. The method of claim 46 or 47, wherein the region of the tissue is exposed to light over a period of at least about 1, 2, 3, 4, 5, or 6 days or at least about 1, 2, 3, 3, 4, 5, 6, 7, or 8 weeks.

49. The method of any one of claims 1-48, wherein the intracellular domain of the RTK comprises a cytoplasmic domain of Epidermal growth factor receptor (EGFR) or Fibroblast growth factor receptor 1 (FGFR1).

50. The method of claim 49, wherein the intracellular domain of the RTK comprises the cytoplasmic domain of EGFR.

51. The method of any one of claims 1-50, wherein the light-inducible oligomerization domain comprises a photolyase homology region of Arabidopsis thaliana Cryptochrome-2 (Cry2).

52. The method of any one of claims 1-51, wherein the protein construct further comprises an intrinsically disordered protein region (IDR).

53. The method of any one of claim 52, wherein the IDR comprises an N-terminal sequence of RNA-binding protein FUS (FUS), Probable ATP-dependent RNA helicase DDX4 (DDX4), or Heterogeneous nuclear ribonucleoprotein Al (HNRNPA1).

54. The method of claim 53, wherein the IDR comprises the N-terminal sequence of FUS (FUSN).

55. The method of any one of claims 1-54, wherein the protein construct further comprises a fluorescent domain.

56. The method of claim 55, wherein the fluorescent domain is mCherry or FusionRed.

57. The method of claim 56, wherein the fluorescent domain is FusionRed.

58. The method of any one of claims 1-57, wherein the protein construct further comprises an N-terminal membrane localization tag.

59. The method of claim 58, wherein the N-terminal membrane localization tag comprises a myristoylation tag.

60. The method of any one of claims 1-59, wherein exposing the region of the tissue to light comprises use of a photomask to selectively expose the region of the tissue to light.

61. The method of any one of claims 1-60, wherein the light comprises at least one wavelength of light.

62. The method of claim 61, wherein the at least one wavelength comprises a visible wavelength between and including 400 and 800 nm.

63. The method of claim 62, wherein the visible wavelength is about 450 nm.

64. The method of any one of claims 1-63, wherein the light comprises a predetermined intensity.

65. The method of claim 64, wherein the predetermined intensity comprises about 10 to about 70 mW / cm2.

66. The method of claim 65, wherein the predetermined intensity comprises about 65 mW / cm2.

67. The method of claim 66, wherein the predetermined intensity comprises about 12 mW / cm2.

68. The method of any one of claims 1-67, wherein exposing the region of the tissue to light occurs for at least one predetermined period of time.

69. The method of claim 68, wherein the predetermined period of time is at least about 3 hours.

70. The method of claim 68 or 69, wherein the predetermined period of time is up to about 32 hours.

71. The method of any one of claims 68-70, wherein exposing the region of the tissue to light occurs at a predetermined interval for a predetermined duration over the predetermined period of time.

72. The method of claim 71, wherein the predetermined interval is about 20 seconds to about 3 minutes.

73. The method of claim 71 or 72, wherein the predetermined duration is about 4-5 seconds.

74. The method of any one of claims 1-73, wherein exposing the region of the tissue to light occurs for 5 seconds every 1 minute for up to about 32 hours.

75. A kit for inducing migration of cells of a tissue, the kit comprising the protein construct or the nucleic acid encoding the protein construct of any one of claims 1-74, optionally wherein the kit comprises at least one light emitting device configured to induce self-association of the light-inducible oligomerization domain.

76. A method of promoting wound healing in a subject in need thereof, the method comprising inducing migration of cells of a tissue according to any one of claims 1- 74.

77. The method of claim 76, wherein the subject has at least one diabetes-related ulcer, decubitus ulcer, venous ulcer, ischemic ulcer, or chronic wound.

78. The method of claim 76 or 77, wherein the subject has one or more of diabetes, high blood pressure, poor blood circulation, high risk for infection, or advanced age.

79. The method of any one of claims 76-78, wherein the subject is or was previously treated with or exposed to one or more agents selected from an anticoagulant, an antiinflammatory, an antibiotic, an antimicrobial, a chemotherapeutic, an immunosuppressant, an immunomodulator, a tumor necrosis factor (TNF) inhibitor, and nicotine.

80. A method of regenerating tissue in a subject in need thereof, the method comprising inducing migration of cells of a tissue according to any one of claims 1-74.

81. The method of claim 80, wherein the subject has an injury to or defect of the central nervous system.

82. The method of claim 81, wherein the injury is a spinal cord injury.

83. The method of any one of claims 80-82, wherein the tissue is nerve tissue.

84. The method of any one of claims 80-83, wherein the method drives axon growth.

85. A method of restoring tissue organization in a subject in need thereof, the method comprising inducing migration cells of a tissue according to any one of claims 1-74.

86. The method of claim 85, wherein restoring tissue organization comprises nerve regeneration.

87. The method of claim 86, wherein the nerve regeneration comprises axon growth.

88. The method of claim 85, wherein restoring tissue organization comprises healing a diabetic ulcer.

89. The method of claim 85 or 88, wherein the method drives cell movement, cell proliferation, cell production of extracellular matrix, or a combination of the foregoing.

90. A method of preparing a tissue graft, the method comprising inducing migration of cells of a tissue according to any one of claims 1-74.

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