**2. Collective migration and leader cells**

### **2.1 Collective migration occurs during epithelial cancer metastasis**

During embryonic development, tissue homeostasis and also cancer invasion, cells migrate as multicellular clusters with a directed and coordinated movement – this

**141**

*Targeting Leader Cells in Ovarian Cancer as an Effective Therapeutic Option*

process is called collective migration [26]. Collective migration is characteristic of metastatic tumours in transit, particularly cancers with epithelial origin [27, 28] including pancreatic cancer [29], colon cancer [30], sebaceous cancer [31], melanoma [32], breast cancer [33–35], lung cancer [36] and OC [37, 38]. There are three key features that define the collective phenomenon; (i) the preservation of the physical connections and cell–cell junctions to orchestrate collective movement; (ii) the shared cytoskeletal dynamics within the cell clusters, allowing groups of cells to proceed as a single unit and maintain multicellular polarity; and (iii) the interactions with other cells and ECM along the migration path [26, 39, 40]. Interestingly, not all cells within the collective invading cell cluster are invasion competent [26] and it is now understood that the complex cohesive movement of collective invasion is orchestrated by a

*DOI: http://dx.doi.org/10.5772/intechopen.98689*

subset of cells called "leader cells" (LCs) [37, 41–44].

actively influence LC function.

invasion into these spaces [62].

**2.2 Cancer leader cells are the key drivers in cancer cell migration**

The LCs have been well characterised in the context of collective migration in normal physiological events such as wound healing [41], nephric ducts growth [45], angiogenesis [46], and mammary branching [47]. More recently, cancer LCs have been identified in bladder [48–50], breast [34, 35, 51], prostate [50], pancreatic [52], small cell lung cancer (SCLC) [53], and now in metastatic OC [37]. These cells have a distinct front-rear polarity and membrane protrusions to sense environmental cues in order to direct the invading cluster [28, 54]. Studies have shown that within a collectively migrating cancer cluster the cancer LCs will be situated at the invasive front, followed by follower cells (FCs) in a packed morphology [28, 54, 55]. It has been shown that the removal of the LCs from an invading cluster of kidney epithelial cells results in the loss of orientation and speed in movement of the FCs this highlighted the importance of LCs in the organisation of collective movement [44]. However, the dynamic interaction between the LCs and FCs is required to ensure the success of collective movement. Therefore, the FCs play a critical role in LCs polarisation, gradient sensing, and chemotaxis [54, 56, 57], and thus in return

**2.3 Leader cells exhibit remarkable ability to alter their surrounding tissue micro-environment, which is crucial in their role as cell migration drivers**

Within the collective migration process, LCs are able to lose or rearrange their baso-apical polarity during cellular elongation, while maintaining attachment to FCs by retaining molecular plasticity through the expression of epithelial markers such as *CDH1,* which encodes for E-cadherin [34, 55, 58]. LCs can mediate cytoskeletal organisation by displaying front-to-rear polarisation [28, 59]. Activation of phosphoinositide 3-kinase (PI3K) [60], GTPase proteins, cell division cycle 42 (Cdc42) and Ras-related C3 botulinum toxin substrate (Rac) [54] at the front of the spheroid induces actin polymerisation and integrin-based interactions with ECM components [61], while the expression of matrix metalloproteinases (MMPs) by LCs generates a track within the ECM and the basement membrane allowing for cell

In the absence of a known LC marker, earlier studies have focused on the physical positioning of LCs within a collectively invading cluster to investigate the LCs profile. Carey et al., shed light on heterogeneous tumour subpopulations within 3D spheroids and showed different invasion and ECM remodelling capacities with LCs driving malignant protrusions [63]. Later, Yamaguchi et al., used the same approach and showed that by removing the LCs from a collectively invading cluster of epithelial kidney cells, the follower population movement lost direction [44].

*Targeting Leader Cells in Ovarian Cancer as an Effective Therapeutic Option DOI: http://dx.doi.org/10.5772/intechopen.98689*

process is called collective migration [26]. Collective migration is characteristic of metastatic tumours in transit, particularly cancers with epithelial origin [27, 28] including pancreatic cancer [29], colon cancer [30], sebaceous cancer [31], melanoma [32], breast cancer [33–35], lung cancer [36] and OC [37, 38]. There are three key features that define the collective phenomenon; (i) the preservation of the physical connections and cell–cell junctions to orchestrate collective movement; (ii) the shared cytoskeletal dynamics within the cell clusters, allowing groups of cells to proceed as a single unit and maintain multicellular polarity; and (iii) the interactions with other cells and ECM along the migration path [26, 39, 40]. Interestingly, not all cells within the collective invading cell cluster are invasion competent [26] and it is now understood that the complex cohesive movement of collective invasion is orchestrated by a subset of cells called "leader cells" (LCs) [37, 41–44].

### **2.2 Cancer leader cells are the key drivers in cancer cell migration**

The LCs have been well characterised in the context of collective migration in normal physiological events such as wound healing [41], nephric ducts growth [45], angiogenesis [46], and mammary branching [47]. More recently, cancer LCs have been identified in bladder [48–50], breast [34, 35, 51], prostate [50], pancreatic [52], small cell lung cancer (SCLC) [53], and now in metastatic OC [37]. These cells have a distinct front-rear polarity and membrane protrusions to sense environmental cues in order to direct the invading cluster [28, 54]. Studies have shown that within a collectively migrating cancer cluster the cancer LCs will be situated at the invasive front, followed by follower cells (FCs) in a packed morphology [28, 54, 55]. It has been shown that the removal of the LCs from an invading cluster of kidney epithelial cells results in the loss of orientation and speed in movement of the FCs this highlighted the importance of LCs in the organisation of collective movement [44]. However, the dynamic interaction between the LCs and FCs is required to ensure the success of collective movement. Therefore, the FCs play a critical role in LCs polarisation, gradient sensing, and chemotaxis [54, 56, 57], and thus in return actively influence LC function.

## **2.3 Leader cells exhibit remarkable ability to alter their surrounding tissue micro-environment, which is crucial in their role as cell migration drivers**

Within the collective migration process, LCs are able to lose or rearrange their baso-apical polarity during cellular elongation, while maintaining attachment to FCs by retaining molecular plasticity through the expression of epithelial markers such as *CDH1,* which encodes for E-cadherin [34, 55, 58]. LCs can mediate cytoskeletal organisation by displaying front-to-rear polarisation [28, 59]. Activation of phosphoinositide 3-kinase (PI3K) [60], GTPase proteins, cell division cycle 42 (Cdc42) and Ras-related C3 botulinum toxin substrate (Rac) [54] at the front of the spheroid induces actin polymerisation and integrin-based interactions with ECM components [61], while the expression of matrix metalloproteinases (MMPs) by LCs generates a track within the ECM and the basement membrane allowing for cell invasion into these spaces [62].

In the absence of a known LC marker, earlier studies have focused on the physical positioning of LCs within a collectively invading cluster to investigate the LCs profile. Carey et al., shed light on heterogeneous tumour subpopulations within 3D spheroids and showed different invasion and ECM remodelling capacities with LCs driving malignant protrusions [63]. Later, Yamaguchi et al., used the same approach and showed that by removing the LCs from a collectively invading cluster of epithelial kidney cells, the follower population movement lost direction [44].

This study further showed that LCs express high level of proteins involved in cell migration and polarisation, such as Rac, integrin β1 and PI3K [44]. Konen and colleagues established a novel image-guided manipulation technique to isolate the LCs from collectively invading lung cancer spheroids [64]. The spatiotemporal genomic and cellular analysis (SaGA) technique involved labelling cells within the spheroid with a green-to-red photoconvertible fluorescent protein. Invasive cells at the front were tagged with a laser beam which converted the fluorescence to red allowing the isolation of the invasive LCs by fluorescent activated cell sorting (FACS) [64, 65]. Using SaGA, transcriptomic analysis of lung cancer LCs identified 788 differentially expressed genes comparing LCs and FCs. Among them, genes involved in VEGF signalling, focal adhesion and RNA polymerase II transcription were significantly over-expressed in the LCs population [64]. The authors further demonstrated that although LC function was not dependent on VEGF signalling, it was necessary to drive the collective movement of FCs [64]. In SCLC, a distinctive mutation profile between LCs and FCs showed that mutations in the actin related protein-3 (*ARP3*) gene enhanced LCs function [53]. Further, introducing this mutation into the noninvasive follower population promoted invasion and collective movement [53].
