# MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics

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## Abstract

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## Simple Summary

## Abstract

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Cell Culture

#### 2.2. Quantitative Real-Time Reverse Transcription PCR (qRT-PCR)

#### 2.3. Western Blot Analysis

#### 2.4. Atomic Force Microscopy

_{0}is the central indentation.

^{2}< 0.8.

#### 2.5. Single Cell Motility and Doubling Time

_{2}regulation (5% (v/v)). The experiments were conducted as described before [14,18]. From the obtained images, the contact area of the cells with the substrate, the mean speed, and the directionality were determined. Directionality was defined as the distance between the start and endpoint of a cell divided by the sum of incremental movements, and ranges between 0 and 1 for random or straight movements, respectively. To characterize the type of motion further, the Fürth formula was used to extract the persistence of motion and the diffusion coefficient from the mean squared displacement (MSD). The Fürth formula is defined as [19]:

_{0}is the time of persistent movement, and D is the diffusion coefficient. As the images displayed cell divisions, the doubling time for each cell line was determined assuming exponential growth.

#### 2.6. Measurement of the Properties of Collective Cell Migration

_{2}(5% (v/v)) control. Images were taken every 5 min for 20 h and filtered using block matching 3D transform [20]. To analyze the local velocity, particle image velocimetry (PIV) [21,22,23] was used with a cross-correlation window size of 32 × 32 pixels (pixel size: 0.48 µm).

#### 2.7. Measuring Colony Expansion

#### 2.8. Simulation of a Persistent Random Walker with Directional Constraints

_{1}and R

_{2}are random numbers between 0 and 1. To obtain the persistence of movement, the results of the single cell motility analysis were used. To model boarder cells in small cell clusters, the same approach, together with the previously obtained persistence, was used, but the movement was set to zero if the modeled cell moved in the negative x direction: $\u2206\overrightarrow{x\left(t\right)}=0if\mathrm{cos}\left(\alpha \left(t\right)\right)0$, as illustrated in Figure A1. This assumption was made to reflect the observation that cells did not migrate back into the cell cluster. As readout, the cellular displacement from its initial position per hour was determined, corresponding to the expansion speed of the cluster $dr\left(t\right)/dt$. For more details, see the Appendix A.

#### 2.9. Statistics

## 3. Results

#### 3.1. Effect of MACC1 Expression on Single Cell Properties

^{2}> 0.85), values for all cell lines were very similar, with diffusion coefficients in the order of 0.2 µm

^{2}/min and a persistence time of 5 min, which was the time difference between successive images. Furthermore, the contact area of SW480 cells with the substrate dropped from 2435 to 1530 µm

^{2}upon MACC1 overexpression (p < 0.0001). Silencing MACC1 in SW620 increased the contact area from 817 to 949 µm

^{2}(p = 0.025). From the live cell images, the doubling times were calculated, confirming the proliferative effect of MACC1 on these CRC cell lines. For SW480 cells, the doubling time decreased from 29 to 22 h upon MACC1 overexpression while silencing of MACC1 in SW620 cells increased the doubling time from 14 to 18 h.

#### 3.2. MACC1 Promotes Collective Migration in a Cell Line-Dependent Manner

#### 3.3. MACC1 Promotes Colony Expansion and Migration Dependent on Proliferation

^{2}> 0.99) and the difference in the cluster size between SW480/EV and SW480/MACC1 increased over time. When normalized to the number of cells (SW480 cells) or relative cell volume (SW620 cells), the curve reflected the average cell size or volume and all previously observed effects diminished and the curves of the high- and low-MACC1-expressing cells run parallel. The average cell size decreased for all populations and thus the rate of outward migration did not keep up with the rate of proliferation (Figure 6C,D).

^{2}/h) cell clusters expanded faster than SW480/MACC1 (slope: 326 ± 1 µm

^{2}/h) clusters, independent of the cell number (Figure 7D,E). Extrapolated to the size of a single cell, cluster expansion rates were calculated for SW480/EV (v = 3.09 ± 0.05 µm/h) and SW480MACC1 (v = 2.36 ± 0.01 µm/h). Notably, these values were approximately four to five times higher than the values obtained for the displacement speed of single SW480 cells (14 µm/24 h ≈ 0.58 µm/h). At the border of cell clusters, the movement of SW480 cells was bound to outward movements because on the side of the cluster, other confined cells inhibited motion into the cluster. Consequently, the degrees of freedom for directional choices of migration were approximately halved. This assumption was in line with our observations showing that no boarder cell moved into the cluster during the measurements.

## 4. Discussion

## 5. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## Appendix A

#### Persistent Random Walk Model

_{1}and R

_{2}are random numbers between 0 and 1. To obtain the persistence of movement, the results of the single cell motility analysis were used (Figure 1). The speed v was set to 10 µm/h and the persistence was varied from 0 to 1 in steps of 0.01 to identify the best matching displacement for the experimental data. We deemed this approach feasible as the Fürth formula fitted [19] the mean squared displacements of single cells very well, with R

^{2}> 0.85. To model boarder cells in small cell clusters, the same approach, together with the previously obtained persistence, was used. Additionally, the movement was set to zero if the modeled cell moved in the negative x direction: $\u2206\overrightarrow{x\left(t\right)}=0if\mathrm{cos}\left(\alpha \left(t\right)\right)0$. See Figure A1 for illustration of the model. Using this approach, it was assured that a cell could not move back into the cluster, as such phenomena were not observed in our experiment, leading to an effectively lowered degree of freedom for the directions of movement. Thus, it is implicitly assumed that each boundary cell moves independently of each other and no additional cell–cell interactions take place. The advantage of these assumptions is that the geometry of the cluster can be disregarded and the whole process can be modeled as persistent random walk of a single cell with constraints.

**Figure A1.**Illustration of the simulation model. The red dot corresponds to a cell moving at a constant speed v, in the direction determined by the angle α. Note that movements to the left, representing the cell cluster, are forbidden.

## Appendix B

**Figure A2.**mRNA expression of MACC1 (top) and representative Western blot (bottom) for SW480/EV and SW620/shCTL, including densiometric analysis.

**Figure A3.**Directionality of single cells. Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers), and outliers (red dots). Sample sizes: n

_{SW480/EV}= 66; n

_{SW480/MACC1}= 98; n

_{SW620/shMACC1}= 102; n

_{SW620/shCTL}= 111.

**Figure A4.**Plot of the smoothed cellular speed inside one representative cluster of SW480\EV cells together with some example images associated with peaks and dips. The curve was smoothed with a moving average filter of size 15. Please note the high number of cells showing the characteristic mitotic rounding at peaks (top images) and their absence in dips (bottom images). The scale bar corresponds to 50 µm.

**Figure A5.**Reorganization and coordination in small cell clusters. (

**A**,

**B**) Illustration of the conserved neighborhood as a function of time. (

**C**,

**D**) Velocity auto-correlation at a distance of 200 µm as a function of time. (

**E**,

**F**) Angular variance of the velocity field for both cell types. Error bars and shaded areas depict the standard error of the mean. Sample sizes: n

_{SW480/EV}= 14; n

_{SW480/MACC1}= 14; n

_{SW620/shMACC1}= 18; n

_{SW620/shCTL}= 17.

Parameters | SW480\EV | SW480\MACC1 | SW620\shMACC1 | SW620\shCTL |
---|---|---|---|---|

Young Modulus | 35 | 33 | 40 | 40 |

Cortical Tension | 33 | 31 | 25 | 26 |

Single Cell Speed/Contact Area | 66 | 98 | 102 | 111 |

Collective Migration | 75 | 75 | 32 | 32 |

Colony Expansion | 14 | 14 | 18 | 17 |

Colony Expansion + Mitomycin | 18 | 15 | --- | --- |

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**Figure 1.**Single cell properties of high- and low-MACC1-expressing colon carcinoma cells. (

**A**,

**B**) depict the results of the biomechanical measurements for the Young´s modulus and the cortex tension. (

**C**,

**D**) show the results of live cell imaging of single cells for the mean speed and the contact area with the substrate. Sample sizes: (

**A**) n

_{SW480/EV}= 35; n

_{SW480/MACC1}= 33; n

_{SW620/shMACC1}= 40; n

_{SW620/shCTL}= 40. (

**B**) n

_{SW480/EV}= 33; n

_{SW480/MACC1}= 31; n

_{SW620/shMACC1}= 25; n

_{SW620/shCTL}= 26. (

**C**,

**D**) n

_{SW480/EV}= 66; n

_{SW480/MACC1}= 98; n

_{SW620/shMACC1}= 102; n

_{SW620/shCTL}= 111. Asterisk depicts statistically significant results with p < 0.05. Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers), and outliers (red dots).

**Figure 2.**Collective migration of SW480 and SW620 cells. (

**A**) The left column shows a typical phase contrast image of SW480, overlaid with the vectors of the velocity fields, and the right column shows the magnitude of the velocity and its direction. The scale bar depicts 50 µm. (

**B**,

**C**) Graph of the mean speeds of SW480 and SW620 cells, respectively. (

**D**,

**E**) Mean of the movement speeds of SW480 and SW620 cells from data in (

**B**,

**C**). Sample sizes: n

_{SW480/EV}= 75; n

_{SW480/MACC1}= 75; n

_{SW620/shMACC1}= 32; n

_{SW620/shCTL}= 32. Asterisk depicts statistically significant results with p < 0.05. Error bars and shaded areas depict the standard error of the mean.

**Figure 3.**Collective migration properties of SW420 and SW620 cells. (

**A**,

**B**) Order parameter Q for both cell lines for the 20 h time window. (

**C**,

**D**) Four-point susceptibility as obtained from the velocity fields over the whole measurement time. The peak positions of the 4-point susceptibility represent the average life time of collectively moving packs of cells. (

**E**,

**F**) Analysis of the layer reorganization in terms of cells that did not make new neighbors during live-cell imaging. Sample sizes: n

_{SW480/EV}= 75; n

_{SW480/MACC1}= 75; n

_{SW620/shMACC1}= 32; n

_{SW620/shCTL}= 32. Shaded areas depict the standard error of the mean. Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers), and outliers (red dots). Asterisk depicts statistically significant results with p < 0.05.

**Figure 4.**Effects of cell division on local cellular velocities. This image collection depicts a single cell division, together with the associated local speeds as a function of time. Please denote the high speeds during mitotic rounding and subsequent high speeds during the expansion of the two daughter cells. The scale bar depicts 50 µm.

**Figure 5.**Expansion of small cell clusters. (

**A**) Illustration of cluster expansion for SW480 and SW620 cells during the measurement time, together with the associated velocity maps. The scale bar depicts 50 µm. (

**B**,

**D**) Plots of the cellular speed inside the clusters as a function of time for both cell types. (

**C**,

**E**) Temporal averages of the cell speed inside the clusters for SW480 and SW620 cells. Sample sizes: n

_{SW480/EV}= 14; n

_{SW480/MACC1}= 14; n

_{SW620/shMACC1}= 18; n

_{SW620/shCTL}= 17. Asterisk depicts statistically significant results with p < 0.05. Error bars and shaded areas depict the standard error of the mean.

**Figure 6.**Evolution of cluster and cell size as a function of time. (

**A**,

**B**) Average size of cell clusters of SW480 and SW620 cells as a function of time. (

**C**) Cluster size of SW480 cells normalized to the cell number, corresponding to the average cell size. (

**D**) Cluster size of SW620 cells normalized to the average cell volume at time point 0. Sample sizes: n

_{SW480/EV}= 14; n

_{SW480/MACC1}= 14; n

_{SW620/shMACC1}= 18; n

_{SW620/shCTL}= 17. Shaded areas depict the standard error of the mean.

**Figure 7.**Expansion of small cell clusters without proliferation. (

**A**) Illustration of cluster expansion for SW480 cells treated with mitomycin (Mito) during the measurement time, without proliferation. The scale bar depicts 50 µm. (

**B**,

**C**) Speed of cells in clusters of SW480 cells as a function of time or averaged over time. (

**D**,

**E**) Total cluster size of SW480 cells over time and normalized to the cell number. Sample sizes: n

_{SW480/EV+Mito}= 18; n

_{SW480/MACC1+Mito}= 15. Error bars and shaded areas depict the standard error of the mean.

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## Share and Cite

**MDPI and ACS Style**

Hohmann, T.; Hohmann, U.; Dahlmann, M.; Kobelt, D.; Stein, U.; Dehghani, F.
MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics. *Cancers* **2022**, *14*, 2857.
https://doi.org/10.3390/cancers14122857

**AMA Style**

Hohmann T, Hohmann U, Dahlmann M, Kobelt D, Stein U, Dehghani F.
MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics. *Cancers*. 2022; 14(12):2857.
https://doi.org/10.3390/cancers14122857

**Chicago/Turabian Style**

Hohmann, Tim, Urszula Hohmann, Mathias Dahlmann, Dennis Kobelt, Ulrike Stein, and Faramarz Dehghani.
2022. "MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics" *Cancers* 14, no. 12: 2857.
https://doi.org/10.3390/cancers14122857