**Abstract**

Majority of ovarian cancers are diagnosed at advanced stages with intra-peritoneal spread as the most common mode of disease metastasis. The formation of cancer spheroids is essential for the collective migration process, where shed tumour cells from the primary tumour form aggregates rather than disseminating as individual cells and seed within the peritoneal cavity. These cancer spheroids consist of leader cells (LC) and follower cells (FC), with the LC subset as key drivers of cellular movement and invasion. LCs have stem cell-like properties and are highly chemo-resistant with a specific survival addiction to several cell signalling pathways, such as the PI3K/AKT/mTOR pathway. We explore in this book chapter, the evidence supporting the role of LC in OC metastasis and the suppression of LC as an attractive therapeutic option for the treatment of advanced OC.

**Keywords:** Ovarian cancer, Leader Cells, KRT14, PI3K/AKT/mTOR, Collective migration

## **1. Introduction**

### **1.1 The majority of ovarian cancers disseminate passively within the intraperitoneal space via ascitic fluid**

The majority of ovarian cancers (OC), up to 70%, are diagnosed at advanced stages (stage III-IV) with intra-peritoneal spread as the most common mode of metastasis [1]. OC dissemination is often accompanied by the formation of ascitic fluid within the peritoneal cavity [2–4]. Under normal conditions, a small amount of fluid is secreted by the peritoneal capillaries into the cavity to lubricate the movement of abdominal organs which is normally re-absorbed by the lymphatic channels as a result of intrathoracic pressure [5]. However, in the presence of malignant cells, fluid can accumulate in large volumes in the peritoneum and facilitate passive cancer cell dissemination [6]. Whilst haematogenous spread may account for some ovarian tumour metastasis [7], it is largely the passive peritoneal dissemination of spheroids that results in ovarian cancer spread [8].

Prior to detachment from the primary tumour, OC cells are believed to exhibit a unique gene expression profile. This includes co-expression of both epithelial and mesenchymal markers and the acquisition of an epithelial-mesenchymal transition

### **Figure 1.**

*Ovarian cancer passive mode of metastasis. Ovarian cancer cells from the primary tumour are exfoliated into the peritoneal cavity. Exfoliated cancer cells aggregate to form compact multicellular spheroids and disseminate within the peritoneal cavity, where single cells are subject to anoikis. Spheroids further attach to and invade the perineal lining by displacing the mesothelial cell layer in a process mediated by ovarian cancer leader cells.*

(EMT)-like phenotype [9, 10]. The detached OC cells are then shed into the peritoneal cavity and simultaneously, E-cadherin expression is replaced by P-cadherin and N-cadherin, an event known as the global cadherin switch [11]. A fluctuation in E-cadherin levels is once again observed when detached cells form multicellular spheroids and E-cadherin levels are elevated [12], collectively demonstrating OC phenotypic plasticity is crucial for each step of the metastatic process [13].

## **1.2 OC spheroids play a key role in intra-peritoneal spread of malignant cells**

Detached tumour cells from the primary tumour aggregate as spheroids in the ascites to overcome anoikis [2]. We believe that these cancer cell spheroids "floating" in the ascites are a key component in OC passive dissemination and play a pivotal role in both invasion and metastasis [6]. Furthermore, OC spheroids exhibit remarkable chemoresistance and progenitor-like properties [14, 15].

The mesothelial monolayer covering all of the abdominal organs is the initial point of contact for the disseminating spheroids during the metastatic process [16]. This layer lies on top of basement membrane, which is composed of collagen I, IV, laminin and fibronectin and contains a milieu of macrophages and fibroblasts populating the extracellular matrix (ECM) space [17–19]. It was observed that transcriptional reprogramming occurred within the floating spheroids which transformed tumour cells from a proliferative to an invasive phenotype to facilitate invasion through the mesothelium via the ECM [6]. Studies have shown that α5β1-integrin expression by spheroids binds fibronectin expressed by mesothelial cells and is critical for spheroid adhesion to the mesothelial lining [20–24]. However, multiple preclinical studies targeting individual integrin complexes failed to prevent the adhesion of spheroids to the peritoneum, hence the role of non-integrin-based adhesion molecules, such as CD44 and L1CAM, may be crucial to the spheroid adhesion process [25]. The attachment of OC spheroids to the peritoneum initiates the process of infiltration and invasion. The process of passive dissemination is illustrated in **Figure 1**.
