Zebrafish imaging reveals hidden oncogenic-normal cell communication during primary tumorigenesis.

Oncogenic mutations drive tumorigenesis, and single cells with oncogenic mutations act as the tumor seeds that gradually evolve into fully transformed tumors. However, oncogenic cell behavior and communication with neighboring cells during primary tumorigenesis remain poorly understood. We used the zebrafish, a small vertebrate model suitable for in vivo cell biology, to address these issues. We describe the cooperative and competitive communication between oncogenic cells and neighboring cells, as revealed by our recent zebrafish imaging studies. Newly generated oncogenic cells are actively eliminated by neighboring cells in healthy epithelia, whereas oncogenic cells cooperate with their neighbors to prime tumorigenesis in unhealthy epithelia via additional mutations or inflammation. In addition, we discuss the potential of zebrafish in vivo imaging to determine the initial steps of human tumorigenesis.Key words: zebrafish, imaging, cell-cell communication, cell competition, EDAC, senescence, primary tumorigenesis.

Extracellular ATP facilitates cell extrusion from epithelial layers mediated by cell competition or apoptosis.

For the maintenance of epithelial homeostasis, various aberrant or dysfunctional cells are actively eliminated from epithelial layers. This cell extrusion process mainly falls into two modes: cell-competition-mediated extrusion and apoptotic extrusion. However, it is not clearly understood whether and how these processes are governed by common molecular mechanisms. In this study, we demonstrate that the reactive oxygen species (ROS) levels are elevated within a wide range of epithelial layers around extruding transformed or apoptotic cells. The downregulation of ROS suppresses the extrusion process. Furthermore, ATP is extracellularly secreted from extruding cells, which promotes the ROS level and cell extrusion. Moreover, the extracellular ATP and ROS pathways positively regulate the polarized movements of surrounding cells toward extruding cells in both cell-competition-mediated and apoptotic extrusion. Hence, extracellular ATP acts as an "extrude me" signal and plays a prevalent role in cell extrusion, thereby sustaining epithelial homeostasis and preventing pathological conditions or disorders.

Zebrafish imaging reveals TP53 mutation switching oncogene-induced senescence from suppressor to driver in primary tumorigenesis.

Most tumours are thought to arise through oncogenic cell generation followed by additional mutations. How a new oncogenic cell primes tumorigenesis by acquiring additional mutations remains unclear. We show that an additional TP53 mutation stimulates primary tumorigenesis by switching oncogene-induced senescence from a tumour suppressor to a driver. Zebrafish imaging reveals that a newly emerged oncogenic cell with the RasG12V mutation becomes senescent and is eliminated from the epithelia, which is prevented by adding a TP53 gain-of-function mutation (TP53R175H) into RasG12V cells. Surviving RasG12V-TP53R175H double-mutant cells senesce and secrete senescence-associated secretory phenotype (SASP)-related inflammatory molecules that convert neighbouring normal cells into SASP factor-secreting senescent cells, generating a heterogeneous tumour-like cell mass. We identify oncogenic cell behaviours that may control the initial human tumorigenesis step. Ras and TP53 mutations and cellular senescence are frequently detected in human tumours; similar switching may occur during the initial step of human tumorigenesis.

Calcium Wave Promotes Cell Extrusion.

When oncogenic transformation or apoptosis occurs within epithelia, the harmful or dead cells are apically extruded from tissues to maintain epithelial homeostasis. However, the underlying molecular mechanism still remains elusive. In this study, we first show, using mammalian cultured epithelial cells and zebrafish embryos, that prior to apical extrusion of RasV12-transformed cells, calcium wave occurs from the transformed cell and propagates across the surrounding cells. The calcium wave then triggers and facilitates the process of extrusion. IP3 receptor, gap junction, and mechanosensitive calcium channel TRPC1 are involved in calcium wave. Calcium wave induces the polarized movement of the surrounding cells toward the extruding transformed cells. Furthermore, calcium wave facilitates apical extrusion, at least partly, by inducing actin rearrangement in the surrounding cells. Moreover, comparable calcium propagation also promotes apical extrusion of apoptotic cells. Thus, calcium wave is an evolutionarily conserved, general regulatory mechanism of cell extrusion.

[Live-imaging Analyses Using Small Fish Models Reveal New Mechanisms That Regulate Primary Tumorigenesis].

Since the 1980s, zebrafish (Danio rerio) have been used as a valuable model system to investigate developmental processes because they: 1) grow outside their mothers; 2) are transparent during the embryonic stage; and 3) have organs similar to those in humans. Recently, zebrafish have emerged as a powerful model animal for studying not only developmental biology but also human diseases, especially cancer. Owing to the significant advantages of zebrafish, such as low-cost breeding, high efficiency of transgenesis, and ease of in vivo imaging and oncogenic/tumor cell induction, zebrafish offer a unique opportunity to unveil novel mechanisms of cancer progression, invasion, and metastasis. In addition, the small size of zebrafish larvae enables high-throughput chemical screening, and this advantage contributes to generating useful platforms for antitumor drug discovery. Owing to these various merits, which other model animals (such as fly, mouse, and rat) do not possess, zebrafish could achieve a unique status in cancer research. In this review, we discuss the availability of zebrafish for studying cancer and introduce recent cancer studies that have used zebrafish.