Once the cell monolayer is scratched, the wound presents sharp straight boundaries that become blurred as the healing process progresses. Thus, the healing process occurs irregularly. The rate of cell migration can be quantified using two single metrics: the wound width as the average distance between the edges of the scratch and/or the wound area, which is calculated by the cell-free area in captured images. During this process, nearby leader and follower cells migrate in different directions, not just in the direction of the width of the wound. The cells disperse, making the contours of the wound less sharp and less straight.
Cells migrate when exposed to an empty space. Under normal conditions, the wound takes approximately 1.5 days to close for Panc-1 cancer cells. Figure 2 shows the average velocities of dozens of cells near the wound boundary in a movie of our experiments. They are drawn by green arrows overimposed in the filmed images. Each cell on the leading edge has a velocity vector pointing in the direction of migration. These measurements provide information about the polarity of cell clusters and dynamic cell flows and the degree of cell-cell rearrangement. Cell movements occur at low Reynolds numbers (Stokes regime), which means that viscous drag of their surrounding medium dominates inertial movements, slowing down the cell movements. In this way, cell dynamics could be assimilated to fluid dynamics. However, cell behaviors cannot be attributed to simple laminar flows. In contrast, cells often show coordinated rotational motions that span over dozens of cells near the edges of the wound in the monolayer, including swirling movements of cell groups and vortices, also observed in these processes (rotation motion described by green arrows in the images of Figure 2)17.
The leading cells at the border of the migrating tissue adhere and migrate in amaeboid motion on the substrate through the adhesion assembly and extensions of fillopodia that propagate perpendicular to the direction of migration, as described previously in the literature9-13. In addition, other cell biological processes could occur in parallel during collective cell migration, such as proliferation, which is limited under starving conditions. In our experiments, we found that leader cells enlarge their shapes and acquire higher velocity amplitudes than follower cells to scan the substrate free of cells, according to the literature.
However, after LICU irradiation, the leader cells acquire different unexpected behaviors, as described in the following.
Effects of LICUs on cell migration in PANC-1 monolayers. Long-term effects were observed on the cell motion. The cells do not undergo any individual or collective movement during sonication or during the following hours, but they show altered dynamics several hours later and two days after LICU treatment. Thus, this dynamic does not respond to Newtonian forces with immediate action but rather induces altered long-term dynamics during at least two days of culture after ICU treatment.
Figure 3 shows long-term trajectories described by dozens of cells bearing the faced edges of the monolayer wound. They were reconstructed in ImageJ freeware from different cells of the monolayer near the wound boundaries after 20 min irradiation of LICUs before culture.
Figure 4 shows three culture processes with different doses of ultrasonic irradiation. The healing process of the wound showed a delay dependent on the time of the LICU treatment. Figures 4.b and 4.c show different delays in wound closure after acoustic irradiation for 10 min and 20 min, respectively, with respect to the time required by the samples for wound closure (Figure 4.a). A supplementary video (Video MM1) shows a wound healing process without closure 48 hours after 20 min of ultrasonic irradiation. This inhibitory effect induced on cell migration was repeatedly observed in experiments.
On the other hand, we found that cancer cell monolayers behave as a single tissue rather than a succession of linked cells exposed to different acoustic conditions defined by their respective positions along the acoustic pressure pattern established in the chamber of treatment with high spatial variations in very short distances of quarter of wavelength between nodes and antinodes (close to 375 µm) (Figure 1.b).
Some leader cells showed complex trajectories described throughout the substrate, not only along the gap direction but also describing displacements in directions transverse to it, mainly the leader cells at the edge of a wound. Looking in detail at the motion of single cells in the wound area, erratic motion of some leader cells from faced wound edges was observed during the second day of culture after ultrasound irradiation.
A supplementary video MM2.mp4 shows two other compared movies with overimposed colours on each cell quantifying their individual displacements, including amplitude and direction of motion. Figure 5 shows an instantaneous snapshoot of the two movies included in the video without and with LICU irradiation at the same culture time. Colors in the images refer to the direction of displacement and are described in the figure. In particular, red refers to right-direction cell displacements, green refers to left-direction displacements, blue refers to vertical displacements and yellow refers to stagnant conditions.
The majority of cells from the left wound edge show red colors during their progression toward their faced wound edge and vice versa. However, several leader and follower cells behind the leaders experience complex reversible and rotational motions over time, showing different colors in the images. Thus, in a natural woudn helaing process, wihtout any external forcé applied, PANC-1 leader and follower cells in the first 6-8 rows behind the left edge (Figure 5.a) show large displacements toward the wound gap to find cells coming from the right edge of the gap which experience displacements in the opposed direction, coming both sets of cells together in relatively short times close to 1 day. In contrast, the cáncer cells in monolayers exposed before to 20 min-LICUs partially inhibited their motion, describing very slow displacements over long times (Supplementary video MM2.mp4). These samples require much longer times for wound closure, even 72 hours.
Influence of the wound width on the cell motion. Greater displacements and higher migration velocity amplitudes were found in samples with wider wound widths (Figure 5). Wider the wound, the wound closure process is faster until a certain wound width from which its closure process slows down. Figure 5.c shows the progression of five average velocity amplitudes measured from respective movies of wound healing starting from different wound widths. These graphs were obtained from five assays in which the dynamics of hundreds of cells were analyzed. In the experiments, average velocities higher than 8 µm/h were found on samples with initial wound widths close to 600 µm (graphs of Assays 2 and 4 in the figure), which decreased over time by approximately 60% until reaching a gap with widths close to 200 µms, showing a decrease of approximately 60%. Meanwhile, lower average cell velocities of narrower wounds were found in the experiments, between 2.5 and 6.0 µm/h for gaps ranging from 200-300 µm (graphs of Assays 1, 3 and 5, respectively), similar to those of wider wounds once this gap size was reached.
This slowdown in the wound closure process is associated with the approximation between the monolayer fronts, increasing cell density and decreasing the distance between cells. This finding may be indicative of a solid-state behavior of the cell monolayer rather than fluid-like behavior, often assumed in the literature22.
Attraction-repulsion processes between single leader cells. Attraction-repulsion processes were found in the experiments between pairs of leader cells separated from the opposed boundaries of the wound during part of the 2nd day of culture that was previously irradiated with 20 min LICUs (Figure 6). It is an unexpected behavior not reported before in the literature that takes almost ten hours of culture during the second day (between 26th —35th hours of culture). After 48 hours, cells A and B remained separated, and both joined back to their respective initial wound boundaries.
During their mutual attraction-repulsion process, the cells change their shape and volume in a process of dual expansion-retraction, spreading multiple fillopodia during their apparent erratic displacements throughout the substrate. After this process, the cells do not join to remain together, but they return to their initial wound edges, leaving the wound open at the end of the period of observation.
Influence of starvation on the wound healing processes after LICU irradiation. Four different types of experiments were carried out according to the combination of two variables or conditions: LICU irradiation treatment and serum starvation for 24 h before the treatment/culture. They provided different results that were analyzed and compared in the following.
Figure 8 shows quantified results of wound progression over time for these four test conditions referred to different treatments before the sample cultures. For this analysis, wounds with initial widths of 400 µm were made on PANC-1 monolayers to start under similar physical conditions.
Relative wound closure in control samples without LICU irradiation. Monolayers previously exposed to starvation showed a value of 0.5 relative to wound closure after 24 h of culture, as shown in Fig. 7a.). In contrast, the wound of samples not starved closed much more rapidly, probably due to a combined effect of cell migration and proliferation. An increase of approximately 0.9 was found after 24 hours in control cultures under nonstarving conditions (Figure 7b). However, after 48 hours, the relative wound closure value was similar in both cases (starving and nonstarving control cells).
Wound healing processes after 20 min of LICU treatment. Again, effects in samples previously exposed to starvation were not noticeable during the first 24 hours of culture (Figures 7.a). However, differentiated quantified effects were evident in the long term at 48 hours of culture.
In contrast, samples not exposed to starvation demonstrated a notable influence of LICUs on the relative wound closure at 24 hours (Figure 7b).
Some PANC-1 cell monolayers cultured under starving conditions prior to scratching were exposed to LICUs, but others were not. The relative wound closure measure and its posterior statistical analysis demonstrated that those cells irradiated with ultrasounds did not show any difference from control cells in the first 24 hours after the wound (Figure 7a). However, the effect of LICUS seems to begin in the next 24 hours, where the relative wound closure ratio was 0.2 points lower in treated cells than in control cells. Furthermore, statistical analysis confirmed the significant difference between the two groups at 48 h, *p≤0.05. As starvation is commonly used to ensure that only migration in wound healing assays is being assessed, this experiment confirms the effect of LICUS on migration inhibition of PANC-1 cancer cells.
To verify whether LICUS affected any other event apart from migration, a nonstarving wound healing assay was carried out (Figure 7.b). Surprisingly, both relative wound closure and statistical analysis revealed the important impact of LICUS in other cellular events. In this case, the difference in relative wound closure was overwhelming since the beginning of the assay. As shown in Figure 7b, at both 24 h and 48 h, the PANC-1 cells treated with LICUS showed a much lower relative wound closure ratio than their control homologs. The relative wound closure ratio was decreased by 0.6 points in the first 24 hours and 0.5 points in the posterior 24 hours compared to nontreated cells. This was supported by statistical analysis, which showed a **p≤0.01 in both cases. This result suggests an effect of LICUS in the inhibition of other cell events, such as proliferation or in the synergy of proliferation and migration.
Further research is necessary to elucidate which patients have been affected in this case. Regardless, as the wound closure ratio is decreased, it could be a game changer for future anticancer research, with cell proliferation and dissemination being one of the most important challenges in cancer treatment.
In summary, nonstarving cell monolayers show quantitatively remarkable effects from the first hours of culture when irradiated with ultrasound. However, the monolayers subjected to fasting prior to the experiments did not show the effects of LICUs so clearly during the first 24 h of cultivation, but rather they were manifested later, on the 2nd day, and they are not as prominent effects as those found in the experiments without starving.
Cell Viability after LICUs irradiation. Cell viability was analyzed after the experiments using Alamar BLUE assays. There was no significant change in viability in response to LIUS, as shown in Figure 8, in which data from 4 replica experiments are displayed.