3.2.5. Hi/Lo-Myc

Two plasmids having a rat probasin (PB) promoter alone (PB-Mycfor lo-Myc) and PB coupled with a sequence of the ARR2 (ARR2PB for hi-Myc) were used to achieve prostate-specific overexpression of c-Myc. The ARR2PB promotor contained two additional androgen response elements that forced the development of invasive adenocarcinoma from prostatic intraepithelial neoplasia (mPIN) in about 26 weeks [27,176,177]. Hi-Myc mice also displayed a decreased expression of Nkx3.1 at both mRNA and protein levels [27]. The PB-Myc mice showed similar pathological changes, but a slower progression of 30 weeks (time to invasive PCa development from PIN lesions) [27]. The main differences between these two models are their androgen responsiveness. The Hi-Myc is androgen-responsive, while the Lo-myc model displays no such sensitivity [27]. The mice model generated by non-viral oncogene ARR2PB-Myc and PB-Myc develop invasive adenocarcinoma and offer advantages over those expressing SV40. However, they do not develop metastasis, which is a major drawback of this model. Hubbard et al. in 2016 showed that the combination of *Myc* overexpression and *Pten* loss in mice resulted in the development of lethal prostatic adenocarcinoma with distant metastases [29]. Moreover, homeobox protein Hox-B13 (HOXB13) was suggested to participate in the *MYC* activation and *Pten* loss genomic instability and aggressive prostate cancer [29,178].

#### 3.2.6. MPAKT

The mouse prostate Akt (MPAKT) model is useful in studying the role of protein kinase B (Akt) in the transformation of prostate epithelial cells and in developing the biomarkers relevant to human PCa. This mouse model was developed by the introduction of Akt1 along with a myristoylation sequence (myr) and a hemagglutinin (HA) epitope in the form of the linearized rPb-myr-HA-Akt1. This insert was injected into the pronuclei of fertilized oocytes, and the friend leukemia virus B (FVB) mice founders were verified [179]. These mice exhibited the formation of PIN by 8 weeks. Immunohistochemistry analysis of the PIN lesions of MPAKT demonstrated numerous important findings such as Akt results in the activation of p70S6K and is associated with the development of PIN in MPAKT mice and Akt-induced PIN might be linked to neovascularization. Histological evaluation revealed that MPAKT mice had distinct phenotypic characteristics, including disorganized epithelial layers, loss of cell polarity, intraepithelial lumen formation, and nuclear atypia and apoptotic bodies. However, the MPAKT did not develop invasive carcinoma even after 78 weeks [180].

#### *3.3. Patient Tumor-Derived Models*

Patient-derived models are useful tools for translational research as they mimic human tumors. They are instrumental in studying the response of various therapies undergoing preclinical evaluation since they carry intrinsic tumor factors and microenvironmental presence involved in disease progression and therapy resistance.

#### 3.3.1. Three-Dimensional (3-D) Organoid Cultures

The transition from monolayer PCa cultures to the three-dimensional (3-D) cultures is a remarkable breakthrough in cancer research. Although culturing cancer cell lines is cost-effective and easy to handle, established cell lines do not carry the heterogeneity and genetic makeup of tumors from which they were initially derived [181,182]. These limitations are mostly overridden by the establishment of 3-D organoid culture models from the patient-derived tumors [183]. Dong et al. established the first PCa 3-D organoid culture from the biopsy of a patient in 2014 [184]. This organoid culture maintained the molecular signature of PCa, including *TMPRSS2-ERG* fusion, *SPOP* mutation, Chromodomain Helicase DNA Binding Protein 1 (*CHD1)* loss, and serine protease inhibitor Kazal-type 1 (*SPINK1)* overexpression. Further, whole-exome sequencing revealed mutations in several other genes, as well as the loss of the p53 and RB tumor suppressor pathway function [184]. Puca and colleges developed patient-derived organoids from needle biopsies of metastatic lesions from patients with neuroendocrine CRPC. These organoids showed genomic, epigenomic, and transcriptomic association with corresponding patient tumors [185]. 3-D models are thus beneficial for drug discovery and preclinical evaluation of therapeutic drugs for efficacy under in vitro setting that mimics the complex in vivo environment.

#### 3.3.2. Patient-Derived Xenografts (PDX)

Patient-derived xenografts (PDXs) are essential tools in cancer research as the results obtained from these resources more accurately predict clinical responses in patients (Table 2). The reason is that these models retain the genetic diversity of patient tumors and maintain a closely resembling tumor microenvironment [186]. PDX grown in immunocompromised mice carry essential histological and molecular features of the patient tumors, including gene expression programs, mutations, epigenetic regulators, and structural genomic events that ultimately drive their 3D growth [187,188]. Recent technical advancements, including the co-injection of PCa tissues with extracellular matrix (ECM) and transplantation into renal capsules, have increased the success rate of PDX establishment in mice [189–191]. The first androgen-dependent PCa xenograft model, designated as PC-82, was developed in 1977 by Schröder and colleagues at Erasmus University Rotterdam [192]. For this, the patient prostatic tumor tissue was grafted into the shoulder of nude mice. Later, two more androgen-independent in vivo models, designated as PC-133 and PC-135, were developed [192]. In 1996, seven other PDX models were established [193]. During 1991-2005, numerous other PDX models were developed that carried the *TMPRSS-ERG* rearrangement, *RB1* loss, AR amplification, *PTEN* deletion, *SPOP* mutation, *Tp53* deletion and mutation, and *BRCA2* loss [132,194,195]. The success rate of the localized PDX model has been increased in recent years due to the implantation of the chimeric graft with neonatal mouse mesenchyme. This method improved the survival rate and doubled the proliferation index of xenografted cancer cells [196]. The PDX models, however, have two significant limitations, i.e., the absence of functional human immunity and the lack of orthotopic modeling in the mice [197]. Further, the model takes a long time (about 8 months) for validation of detectable tumor growth in mice that limits its utility for the high-throughput drug screening [198].

**Table 2.** The advantages and limitations of patient-derived xenograft models.



**Table 2.** *Cont*.
