Study of collective cell migration and rheological properties of epithelial cell monolayer

Abstract

this work, we studied the collective cell migration as well as the rheological properties of the cell monolayer. The collective cell motion is observed in many biological processes such as wound healing, embryogenesis, and cancer metastasis. In collective cell migration, cells move together as a cohesive group, such that each cell is connected to its neighbouring cell via cell-cell adhesion and actin cytoskeleton1. Therefore, the direction of cell migration is influenced by the neighbouring cells. It indicates that intercellular forces are vital for collective cell motion. Despite extensive theoretical and experimental studies on collective cell motion, there is no unified mechanism to explain it. Here, we experimentally report the collectively growing cell colonies found in the sub-marginal region of freely expanding epithelial Madin Darby Canine Kidney (MDCK) cell monolayer. The cells of the colonies grow at a higher rate compared to the remaining tissue, which may lead to the generation of localized mechanical stress. This may trigger some of the marginal cells to transform into leader cells, and these newly formed leader cells start pulling their immediate follower cells that are being pushed by the expanding cell colonies. Thus, both leader cells and growing colonies work together to displace the front cell rows, resulting in a highly aligned velocity field or collective cell migration observed in the front cell rows towards the leader cells. The displacement of the front cell rows may also relieve the mechanical stress from the sub-marginal region. These colonies are dynamic, as they can emerge at different locations with time. Their formation at a different location in the sub-marginal region with time leads to the generation of localized stress, which might get relived by the displacement of the front cell rows towards the leader cells. It is a plausible mechanism that could be responsible for the collective cell migration observed in the front cell rows of freely expanding monolayer. In literature, most of the experimental studies on collective cell migration used an artificial wound model to form the monolayer. Where cells are seeded in physical confinement, this physical confinement is removed after forming a fully confluent cell monolayer. The removal of the physical confinement provides the availability of free space, which is sufficient to trigger the collective cell migration towards the free space2. However, these monolayers possess high cell number density compared to physiological scenarios such as wound healing (cells damage due to applied cut) and cancer metastasis (small group of cells detach from tumour site and move to other location in the body). Therefore, these artificial wound model does not relate to physiological scenarios3. To study this literature gap, we formed two different types of monolayer based on their initial boundary conditions. The first type of monolayer was formed by using the initial free boundary condition, where cells were seeded at the centre of a circular glass coverslip and allowed to form the confluent monolayer. Since most cells of this type of monolayer prefer to grow, this is called Growth-Dominant Monolayer (GDM). The second type of monolayer was formed by using initial fixed boundary condition, where cells were seeded inside the wells of the culture insert attached to the glass coverslip. This insert was peeled off after the formation of a highly confluent monolayer. Since most cells of this type of monolayer prefer to migrate, this is called Migration-Dominant Monolayer (MDM). We observed less coordinated motion and less cell number density at the leading site of the GDM than MDM. The collective cell dynamics originate because cells are active material with fascinating mechanical properties. They can remodel their cytoskeleton (CSK) to perform various functions like spreading, growth, migration, division, contraction, and metastasis4. The deformability is one of the crucial features of the cells and tissues that are well-acknowledged but poorly understood5. Thus, micro and macro-rheology have been used to measure the mechanical properties of single cells and cell monolayer, respectively6. In literature, the macro-rheology is performed on a large number of single cells without cell-cell contact7,8. However, it does not represent the actual monolayer having cell-cell contacts. Additionally, it is mentioned in the literature that serum is the only source of micronutrients (proteins and minerals) in the culture media, which are essential for regulating cell viability, homeostasis, and DNA metabolic pathways9,10. Its absence can cause genomic instability, protein expression variations, disruptions in signalling pathways, and deprivation of growth factors in the cells9,10. However, the effect of serum starvation on the mechanical properties of the monolayer has not been addressed. In our study, the mechanical properties of a fully confluent epithelial cell monolayer were measured using an oscillatory rheometer. We explored the effect of serum starvation on the rheological properties of the cell monolayer maintained in the media with 0% FBS (Fetal Bovine Serum) and compared it to the healthy cell monolayer maintained in media with 10% FBS. We found that the dynamic processes halt in the absence of serum. Thus, cells could not recover or remodel the broken biopolymer after the removal of strain, and permanent deformation was observed. In contrast, the healthy monolayer could recover or remodel the broken biopolymers after the removal of the strain. It indicates the vital role of serum in remodelling of the cytoskeleton in the cells earlier subjected to the deformation. Altogether, this thesis presents the plausible mechanism responsible for the collective cell migration in freely expanding epithelial cell monolayer, distinct modes of tissue expansion depending upon initial boundary condition and the effect of serum starvation on the mechanical properties of the epithelial monolayer. This thesis concludes the vital role of follower cells in collective cell migration, the fundamental link between collective cell migration & cell number density, and the mechanical properties of the monolayer depends upon its activity.