3.4.2. Zebrafish Model

The zebrafish model for cancer research has been utilized by many to acquire information that is traditionally obtained by mice and cell culture systems, although there are limited studies on zebrafish in an in vivo model for PCa research. The zebrafish model is suitable for visual observation of labeled tumor cells through the imaging technique since they are transparent. Nevertheless, the limitation of orthotopic transplantation could be the hurdle owing to the anatomical difference between zebrafish and the human body such as the breast, prostate, or lung [202]. The cancer cells can be injected into a different site in the zebrafish embryos, such as the blastodisc region, the yolk sac, the hindbrain ventricle, and into the circulation via the duct of Cuvier [203,204]. Melong et al. inoculated androgen-sensitive LNCaP cells into zebrafish and observed the effect of testosterone on the growth. Administration of exogenous testosterone increased the proliferation of PCa cells [205]. Further, the growth-promoting effect of testosterone was reversed by the anti-androgen receptor drug, enzalutamide. The invasive potential of PC3 cells overexpressing the calcitonin receptor (CTR) has also been evaluated in the zebrafish model [206]. The zebrafish model has several advantages, including the fact that zebrafish are small and can generate a large number of offspring in a short time, and they are easy to maintain and observe owing to their transparency. Moreover, humans and zebrafish have 71% protein similarity, and, most importantly, zebrafish absorb molecules from water providing an additional route for drug administration.

#### **4. Conclusions and Future Outlook**

In the past years, understanding of PCa pathobiology paired with mechanistic studies has remarkably advanced the field of PCa research. This insight has only been possible because of the availability of several types of research models. These models have been extremely helpful in improving our knowledge of PCa etiology, development, and metastatic progression. The cell line models have offered an easy and inexpensive platform to study the functions of aberrantly-expressed genes and various types of genetic alterations including gene mutations, splice variants, gene rearrangements, etc. Furthermore, cell lines serve as a primary model for screening of newer drugs or drug combination and provide us data on the molecular mechanisms of therapy resistance that is crucial for drug development. Since cell lines do not completely capture the tumor heterogeneity and are not grown in a complex microenvironment that tumor cells encounter in vivo, other in vivo models play an important role in further evaluation of gene functions and drug efficacies. The 3D-tissue culture model mimics the in vivo system under in vitro settings and has proven very useful in drug screening. Further, as the field of precision medicine is developing, these models could be of great significance in patient-tailored treatment planning based on preliminary assessment. Patient-derived xenografts (PDXs) grown in mice are useful as they more closely mimic a human tumor in vivo microenvironment. Genetically engineered mouse models (GEMs) are useful as they capture the complete progression of PCa from initiation to metastatic spread under a non-immunocompromised environment. Further, these models also develop a variety of PCa tumor types although they do not have the complete molecular diversity of human tumors (Figure 3). Regardless of limitations, each model has its own importance and these models often complement each other and are often utilized in progressive sets of experiments. There is, however, a need to develop models representing PCa of different racial and ethnic groups considering racial health disparities in incidence and clinical outcomes. Our refined knowledge of tumor genetics and awareness of health disparities and technologically advances will help us make further progress and we would continue to add to our list of PCa tumor models.

**Figure 3.** Application of the prostate cancer model in basic and preclinical cancer research. To develop the novel drugs or biomarkers, the prostate cancer models are required for in vitro and in vivo studies. The prostate cell lines, 3D-organiods, and patient-derived tumor xenografts (PDXs) can be generated from prostate tumor tissue from human patients. Patient tumor tissues can be also used to create genetically engineered mouse models (GEMMs). The results from research and preclinical studies are validated through several techniques such as whole genome sequencing, cell and molecular-based assays, high-throughput screening, metabolomics analysis, and ELISA. The promising drugs or biomarkers that emerge from those works will subsequently progress to preclinical and clinical studies.

**Author Contributions:** Conceptualization: A.P.S., S.S. (Seema Singh), S.D., S.P., S.S. (Sirin Saranyutanon), and S.K.D.; Supervision; A.P.S., S.S. (Seema Singh), and S.D.; Resources: A.P.S. and S.S. (Seema Singh); Writing, review and editing: A.P.S., S.S. (Seema Singh), S.D., S.P., S.S. (Sirin Saranyutanon), and S.K.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Institutes of Health/National Cancer Institute (CA185490, CA224306 (to AP Singh) and CA204801, CA231925 (to S Singh)) and the University of South Alabama Mitchell Cancer Institute.

**Acknowledgments:** Sirin Saranyutanon would also like to acknowledge the financial support provided by the Royal Thai Government Scholarship.

**Conflicts of Interest:** The authors declare no conflict of interest.
