Journal: bioRxiv

Article Title: Tumor invasion as non-equilibrium phase separation

doi: 10.1101/2020.04.28.066845

Figure Lengend Snippet: Here, the workflow is illustrated for MCF-10A macro-spheroids in collagen, but was also applied to MCF-10A microspheroids and MDA-MB-231 macro-spheroids alike. ( A ) A representative multiphoton stack of a MCF-10A macro-spheroid cultured in 2 mg/ml collagen is split into two channels, carrying separate signals: ( B ) fluorescence from DAPI-stained nuclei and ( C ) second harmonic generation (SHG) from collagen. In the case of MCF-10A micro-spheroids in Matrigel and Alginate, the two channels are given by fluorescent and bright-field images (cf. ). The two channels are processed, respectively, to identify ( D ) the 3D position of cell nuclei and ( E ) the 3D spheroid boundary using a custom algorithm. Note that the representative slices ( B-C ) displaying 2D projections of identified nuclei centers and spheroid boundary are shown overlapped onto corresponding multiphoton images. ( F ) Data on nuclei location and spheroid boundary are combined to generate a bounded Voronoi tessellation that partitions the spheroid into individual cells, from which cell volume and shape metrics are derived. Here, the 2D cross-section view of resultant Voronoi cells are shown superimposed on the corresponding multi-photon image. We note that empty spaces within the spheroid (cf. ) as well as its neighboring cells are excluded from further analysis to minimize local overestimation of cell shape ( G ) Our custom 3D nuclei detection algorithm was validated by comparing the number of cell nuclei counted by the algorithm in fixed and cleared spheroids with the number of cell nuclei estimated for spheroids that were dissociated via trypsination and manually counted using a hemacytometer. Over a wide range of spheroid sizes, using both the MCF-10A (blue squares) and MDA-MB-231 (red triangles) cell lines, manual and algorithm counts lie close to the identity line (dashed black line). Spheroids used for counting validation were formed starting from a variety of cell numbers (from ~10 3 to ~10 4 ) at the time of seeding which increased (to ~2×10 3 to ~1.5×10 4 ) over the course of the 48 hours during which spheroid formation occurred (cf. ). Both cell couting methods used spheroids from the same batch and cells at the same passage. Data are presented as mean ± SEM (n = 6-10 for manually counted spheroids and n = 3-4 for automaticaly counted spheroids of all sizes).

Article Snippet: The microstructural features of acellular collagen networks were imaged using a Bruker Ultima Investigator multiphoton microscope (MPM) and a 60x oil-immersion objective.

Techniques: Cell Culture, Fluorescence, Staining, Derivative Assay, Biomarker Discovery

Journal: bioRxiv

Article Title: Tumor invasion as non-equilibrium phase separation

doi: 10.1101/2020.04.28.066845

Figure Lengend Snippet: ( A ) Representative DIC images of macro-spheroids formed in low attachment conditions for 48 hours starting from approximately 10 3 cells in presence (+) or absence (−) of 2.5% Matrigel diluted in cell culture media. The presence of Matrigel leads to MCF-10A macro-spheroids that are larger and less regular in shape with respect to Matrigel-free controls. On the other hand, MDA-MB-231 cells form only loose aggregates in absence of Matrigel while compacting effectively in its presence. ( B ) Time-course of spheroid compaction over the course of 48 hours. The equivalent spheroid diameter d is calculated from the total cell area A thresholded from DIC time-lapse movies as . ( C) Spheroid diameter at 48 hours of compaction quantifies the differences caused by the presence of Matrigel in the different cell lines. Both cell lines form spheroids of similar sizes in the presence of Matrigel (black bars). Data are presented as mean ± SEM (n = 3 for all groups). * indicates statistical significance at p < 0.05. ( D ) 3D scanning confocal microcopy images of representative MDA-MB-231 spheroids after fixation (Control) or fixation followed by optical clearing (CUBIC), shows that DAPI-stained nuclei (red) in the spheroid core are clearly visible only after optical clearing. The depth of the confocal stacks represents the distance from the spheroid surface. ( E ) Representative equatorial cross-sections of MCF-10A and MDA-MB-231 spheroids generated in presence of Matrigel show the distribution of cell nuclei using two methods: multiphoton imaging of DAPI-stained cell nuclei after CUBIC clearing (top), and van Gieson’s stained histology slides (bottom). Note that the hollow core of MDA-MB-231 spheroids is not filled by cells (identified by the dark nuclei) but rather is occupied by matricellular proteins (shown in red).

Article Snippet: The microstructural features of acellular collagen networks were imaged using a Bruker Ultima Investigator multiphoton microscope (MPM) and a 60x oil-immersion objective.

Techniques: Cell Culture, Control, Staining, Generated, Imaging

Journal: bioRxiv

Article Title: Tumor invasion as non-equilibrium phase separation

doi: 10.1101/2020.04.28.066845

Figure Lengend Snippet: (A, B) Representative equatorial cross-sections of multiphoton images show MCF-10A macro-spheroid behavior when embedded in either 2 (low density) or 4 mg/ml (high density) collagen for 48 hours, with DAPI-stained cell nuclei shown in red and collagen fibers from SHG shown in green. In low density collagen (A) , the spheroid develops collective invasive protrusions, while in high density collagen (B) , no invasion is observed. Cell-free voids (black) are due to Matrigel used to promote spheroid formation (Methods, ); cells immediately neighboring this cell-free region are excluded from subsequent structural analysis (Methods, ). ( C ) Similar to observations from the MCF-10A micro-spheroids, Voronoi cell volumes increased from the macro-spheroid core to the periphery. In contrast to the micro-spheroids, average cell volumes from macro-spheroids cultured at both collagen densities are smaller, and suggest that cells in the macro-spheroid experience greater compressive stress (cf. ). ( D-E ) The corresponding cell shapes are shown as 2D cross-sections, color-coded according to their respective 3D Shape Index (SI). Increased and more variable SIs are localized in the region of the spheroid periphery that undergoes collective invasion ( D ). On the other hand, SIs remain narrowly distributed, in the rest of the spheroid periphery and in the core regardless of collagen density. ( F ) In fact, SIs are homogeneously distributed near the threshold for solid-fluid transition (horizontal dashed line indicates solid-fluid transition point at SI=5.4 ). SIs increased only at the invasive protrusions suggest localized unjamming and fluidization is associated with invasion. ( G-H ) Representative DIC images are shown for MCF-10A macro-spheroids cultured in 2 and 4 mg/ml collagen, with cell migratory trajectories (from optical flow, Methods) superimposed in red. Longer trajectories are observed at the collectively invading regions ( G ). The spheroid boundaries are outlined in black. The entire DIC time-lapse video capturing the dynamics of invasion over 48 hours is shown in Movie S1. ( I ) Radial distributions of average cell migratory speed quantified for the final 8-hour observation window (40-48 hours) conform to expectations from cell shapes. In both collagen densities, migratory speed is homogenously low in the spheroid core, and increased only at sites of localized invasive protrusions. Data for radial distributions are presented as mean ± STD (n = 3 for both 2 and 4 mg/ml spheroids).

Article Snippet: The microstructural features of acellular collagen networks were imaged using a Bruker Ultima Investigator multiphoton microscope (MPM) and a 60x oil-immersion objective.

Techniques: Staining, Cell Culture

Journal: bioRxiv

Article Title: Tumor invasion as non-equilibrium phase separation

doi: 10.1101/2020.04.28.066845

Figure Lengend Snippet: ( A, B ) Representative equatorial cross-sections of multiphoton images show MDA-MB-231 macro-spheroids exhibiting distinct invasion patterns when embedded in low density (2 mg/ml) versus high density (4 mg/ml) collagen for 48 hours. DAPI-stained cell nuclei are shown in red and collagen fibers from SHG are shown in green. In low density collagen (A) , these metastatic cells scatter from the spheroid core as individual, gas-like particles. Conversely, in high density collagen (B) , single-cell dominant scattering is subdued and invasion is in the form of collective, fluid-like protrusions. We note that the center of MDA-MB-231 spheroids is devoid of cells, as confirmed by staining of histological cross-sections , and thus result in a hollow shell of highly motile cells rather than a nearly solid spherical structure. Only cells that remain part of the collective are included in the structural analyses (Methods, ), hence the absence of data for the first 200 μm of the associated radial distributions. ( C ) Average Voronoi volumes suggest that MDA-MB-231 cells have larger volumes with respect to their MCF-10A counterparts (cf. ). In 2 mg/ml collagen, cell volumes remain roughly independent of radial position. In 4 mg/ml collagen, instead, cell volumes show a decreasing radial gradient. This decrease in cell volume from the spheroid core to the invasive protrusion suggests elevated stress in invading cells from confinement by the collagen matrix. ( D-E ) The corresponding cell shapes are shown as 2D cross-sections, color-coded according to their respective 3D Shape Index (SI). Regardless of collagen concentration, cells from MDA-MB-231 spheroids display higher SI with respect to MCF-10A spheroids (cf. ) ( F ) Radial distribution of average SI values is consistent with an unjammed fluid-like phase (horizontal dashed line indicates solid-fluid transition point at SI=5.4 ). In high density collagen, a radially decreasing gradient in SI suggests that cells jam while invading collectively under matrix confinement. ( G-H ) Representative DIC images are shown for MDA-MB-231 macro-spheroids cultured in 2 and 4 mg/ml collagen, with cell migratory trajectories (from optical flow, Methods) superimposed in red. The spheroid boundaries are outlined in black. The entire DIC time-lapse video capturing the dynamics of invasion over 48 hours is shown in Movie S1. Cell dynamics mirrors structural signatures of cell jamming/unjamming. ( I ) Radial distributions of RMS speed quantified for the last 8-hour observation window (40-48 hours) show that cells in MDA-MB-231 macro-spheroids have homogeneously higher speeds with respect to MCF-10A spheroids (cf. ) and are thus more fluid-like. In low density collagen, cell speed increases further as soon as cells detach from the spheroid and invade as single, gas-like particles (inset, where the radial position of the spheroid boundary is marked by a dashed vertical line). This observation supports the proposed analogy of fluid-to-gas transition. In high density collagen, RMS speed decrease radially with collective invasion, and is supportive of a fluid-to-solid transition due to confinement-induced jamming . Data for radial distributions are presented as mean ± STD (n = 3 for both 2 and 4 mg/ml spheroids).

Article Snippet: The microstructural features of acellular collagen networks were imaged using a Bruker Ultima Investigator multiphoton microscope (MPM) and a 60x oil-immersion objective.

Techniques: Staining, Single Cell, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: Tumor invasion as non-equilibrium phase separation

doi: 10.1101/2020.04.28.066845

Figure Lengend Snippet: We carried out a time-lapse experiment, including continuous DIC imaging as well as fixation and optical clearing for multiphoton microscopy, for MDA-MB-231 spheroids at distinct time points. ( A ) Representative equatorial cross-sections of multiphoton images display DAPI-stained cell nuclei (red) and collagen fibers from SHG (green) from MDA-MB-231 spheroids embedded in 2 and 4mg/ml collagen and imaged after 12, 24, and 48 hours. ( B ) Total and single cell counts over time show that cell proliferation is quite similar between spheroids embedded in various collagen densities, while there is a significantly higher single cell escape over time in spheroids embedded in 2 mg/ml collagen. ( C ) Temporal evolution of cell shape (top) and migratory speed (bottom) in spheroids embedded in 2 mg/ml (left), and 4 mg/ml (right) collagen. In 2 mg/ml collagen, MDA-MB-231 cells that remain within the primary spheroid maintained homogenous radial distributions for both cell shape and RMS speed.In contrast, in 4 mg/ml collagen, MDA-MB-231 cells within the primary spheroid have similar homogenous distributions until 12 hours of culture, but progressively develop radially decreasing trends for both cell shape and RMS speed at 24 and 48 hours.The critical cell shape (SI = 5.4) for solid-fluid transitions is indicated by a horizontal dashed line, with increasing values indicative of unjamming and decreasing values indicative of jamming. Overall, we observe that ECM confinemnt induces a jamming transition, or confinement-induced jamming, at the invasive front.

Article Snippet: The microstructural features of acellular collagen networks were imaged using a Bruker Ultima Investigator multiphoton microscope (MPM) and a 60x oil-immersion objective.

Techniques: Imaging, Microscopy, Staining, Single Cell

Journal: bioRxiv

Article Title: Tumor invasion as non-equilibrium phase separation

doi: 10.1101/2020.04.28.066845

Figure Lengend Snippet: ( A ) Representative equatorial cross-sections of multiphoton images show MDA-MB-231 spheroids after 3 days of invasion in graded collagen concentrations (1 to 4 mg/ml) along with the associated DIC minimum intensity projections (insets). Single-cell migration is observed primarily in 1 and 2 mg/ml while collective migration is observed primarily in 3 and 4 mg/ml. ( B ) Corresponding 3D rendering of cell nuclei distributions identified from automated analysis of multiphoton image stacks (Methods). Nuclei are color-coded based on whether they remain within the cell collective (blue) or are detected as single cells (red). (C) Immediately after embedding in collagen (day 0), all cells are part of the multicellular collective with no invasion at any collagen density. As the spheroid evolves over time (days 1, 2 and 3), a striking gas-like phase and corresponding single cell escape progressively emerged at lower collagen concentrations (1 and 2 mg/ml) but not higher collagen concentrations (3 and 4 mg/ml). By day 3, a switch-like biphasic reduction in the number of single invading cells emerged when collagen concentration was increased from 2 to 3 mg/ml. The temporal evolution of single cell invasion as a function of collagen concentration supports the existence of criticality between 2 and 3 mg/ml, at which point the invasive phenotype switches abruptly from single to collective invasion. Single cell counting data are shown from days 0-1-2-3 and collagen concentrations of 1-2-3-4 mg/ml, n = 3 per group, except for day 0 - 1 mg/ml (n = 2) and day 2 - 2 mg/ml (n = 9). The significance of differences due to collagen concentration and time were quantified using a one-way ANOVA and post-hoc pairwise comparisons with Bonferroni correction. Statistical significance was achieved between 1 and 2 mg/ml at day 2 (p < 0.05), and between 2 and 3 mg/ml at days 1 (p < 0.05), 2 (p < 0.01), and 3 (p < 0.01), while no significant differences were observed between 3 and 4 mg/ml. The sharp and statistically significant transition between 2 and 3 mg/ml is therefore indicated with an arrow. We examined whether this transition is due to differences in collagen structure or mechanics. ( D ) High-resolution multiphoton images show representative acellular collagen networks at 1 to 4mg/ml, with individually segmented fibers from CT-FIRE analysis as indicated by different colors. We quantified microstructural and mechanical properties of these collagen networks by combining multiphoton imaging and mechanical testing under confined compression (Methods and Supplementary Material). ( E ) Fiber density and ( F ) matrix porosity display clear trends with increasing collagen concentration between 1 and 4 mg/ml. ( G ) In contrast, compressive energy storage (under 18% compression), which quantifies nonlinear mechanical properties of the matrix, are indistinguishable between 1 and 4mg/ml. Therefore, collagen fiber density, rather than mechanics, represents a control variable for 3D cell jamming. Microstructural data are shown from 1 mg/ml (n = 12), 2 mg/ml (n = 10), 3 mg/ml (n = 12), and 4 mg/ml (n = 12) collagen gels. Mechanical data are shown from 1 mg/ml (n = 5), 2 mg/ml (n = 6), 3 mg/ml (n = 9), and 4 mg/ml (n = 5) collagen gels. * indicates statistical significance at p < 0.05.

Article Snippet: The microstructural features of acellular collagen networks were imaged using a Bruker Ultima Investigator multiphoton microscope (MPM) and a 60x oil-immersion objective.

Techniques: Single Cell, Migration, Concentration Assay, Imaging, Control

Journal: bioRxiv

Article Title: Tumor invasion as non-equilibrium phase separation

doi: 10.1101/2020.04.28.066845

Figure Lengend Snippet: (A) The hybrid computational model of tumor invasion into ECM is characterized by cancer cells (orange particles) that can move in random directions with varying degrees of self-propulsion (Supplementary Methods). At the beginning of each simulation, cancer cells are organized to form a circular collective that is surrounded by collagen (green particles), arranged randomly and with varying spatial densities (Supplementary Methods). At the end of each simulation, the 95 th percentile of the radial cell positions ( p 95th ) with respect to the centroid of the collective is used as a read-out of the degree of invasion. (B) A diagram is generated by gradually incrementing two state variables: cell motility and collagen density, both expressed in arbitrary units (A.U.). Data points are color-coded according to the mean value of p 95th over n = 10 simulations, each corresponding to randomly assigned positions of the collagen particles and orientations of the cell motility vectors. Three notable regions can be distinguished in the diagram and qualitatively correspond to solid-, liquid-, and gas-like behaviors at the invasive front (Movies S2-4). (C) These three regions can be distinguished from distinct elbow regions in the cumulative probability distribution of p 95th generated from all simulations. We identified the 34 th and 64 th percentiles as robust thresholds (cf. ) to separate solid from liquid and liquid from gas phases, respectively. (D) The resultant map represents a jamming phase diagram, now color-coded to indicate solid-like (blue squares), fluid-like (orange circles), and gas-like (yellow triangles) material phases. In analogy with equilibrium thermodynamic systems, here cell motility is replaced with an effective temperature ( T eff , Box 1) while collagen density is replaced with a confinement pressure ( P conf , Box 2). By tuning only two state variables, the model recapitulates much of the experimentally observed behaviors. For each material phase on the diagram, representative multiphoton images from experiments are shown in comparison to representative computational snapshots (insets). In the solid-like phase (blue area), a non-invasive MCF-10A spheroid in high collagen density (4 mg/ml) is shown in comparison to the result of a simulation parameterized with low cell motility (0.2) and high collagen density (0.82). In the fluid-like phase (orange area), an MDA-MB-231 spheroid collectively invading in high collagen density (4 mg/ml) is shown in comparison to the result of a simulation parameterized with high cell motility (1.0) and high collagen density (0.82). Finally, in the gas-like phase (yellow area), an MDA-MB-231 spheroid scattering into single cells in low collagen density (2 mg/ml) is shown in comparison to the result of a simulation parameterized with high cell motility (1.0) and low collagen density (0.21). Overall, we observe that at low cell motility, and thus low T eff , the system is homogeneously “cold” and the spheroid shows a non-invasive, solid-like behavior regardless of collagen density. However, at higher T eff the collagen density, and hence P conf , determines fluid-like or gas-like behaviors. Phase boundaries (black lines) on the jamming phase diagram are obtained as best-fit curves that separate data points belonging to different material phases. Unlike traditional thermodynamic phase transitions, where the boundary lines mark clear transitions between material phases, in our cellular systems material transitions are continuous and smeared. Thus, the boundary lines mark regions of coexistent phases, where near each phase boundary, the material phases become indistinguishable. The proposed diagram also predicts the existence of a “triple point” where solid-, liquid-, and gas-like phases coexist, and below which direct solid-to-gas transitions occur. (E) To test the plausibility of such prediction we ran an invasion assay in graded collagen concentrations (1 to 4 mg/ml) using MCF-10A spheroids. which, according to the phase diagram, are characterized by a lower T eff with respect to their MDA-MB-231 counterparts. The periphery of MCF-10A spheroids was found to remain solid-like and non-invasive in 4 mg/ml, to fluidize and invade collectively in 3 and 2 mg/ml and, more importantly, to separate directly into individual gas-like cells in 1 mg/ml. These findings support the direct individualization of cancer cells from a nearly jammed tumor as predicted by our jamming phase diagram.

Article Snippet: The microstructural features of acellular collagen networks were imaged using a Bruker Ultima Investigator multiphoton microscope (MPM) and a 60x oil-immersion objective.

Techniques: Generated, Comparison, Invasion Assay