In recent years, major technological advancements have led to a better understanding of the molecular mechanisms of TGCTs. However, this progress has had a slight impact on the cancer therapeutic approach, probably due to the limitation of experimental models to predict efficacy in clinical trials. In an effort to offset this limitation, the interest in the development of different TGCT models is increasing.

It remains a challenge for the clinic to investigate the molecular and genetic mechanisms involved in the development of cisplatin resistance in TGCTs, for which the frequency of recurrence is low and the availability of histological samples post-chemotherapy is scarce, because surgical resection of the tumor is the first line of treatment [99]. In general, cisplatin resistance in TGCTs is commonly studied in primary tumors of patients who may develop them at some point in the future, which is not ideal due to its naive relationship with chemotherapy [100].

Other strategies to investigate the mechanisms associated with the resistance acquired by tumor cells to cisplatin include the use of preclinical models, in vitro and in vivo, obtained from the cultivation and exposure of TGCT cell lines to incremental doses of the drug, for long periods of time [101], in addition to the use of animal models that reproduce the phenotypic properties of the human tumor [102]. Although in vitro cell culture systems have been used extensively for decades, they represent oversimplified models, which are characterized by the absence of heterogeneity and lack of microenvironment components [103]. Therefore, it is crucial to develop more accurate and clinically relevant mice models that genuinely represent TGCTs in adult and pediatric patients, according to their etiopathogenesis, histopathology, and metastatic progression, and the response of therapy. In this context, patient-derived xenograft (PDX) models have been used as an outstanding alternative [104,105]. PDX model development is generated via the transplant of primary tumor fragments or tumor-derived cancer cells from the patient into immunocompromised mice [106,107], and has been established in different types of tumors. In TGCTs, PDX models have been developed with a focus on mouse models of resistant disease, which may be established by injecting the cisplatin-resistant clones of TGCT cell lines or by implanting cisplatin-resistant human tumors [102,108,109].

Different models of chemoresistant TGCT cell lines have already been developed and studied for genotypic and phenotypic changes [99]. NTERA-2 and NCCIT cisplatin-resistant cell lines were injected into immunodeficient mice and disulfiram was used to examine chemosensitization of resistant cell lines. Disulfiram in combination with cisplatin showed synergy for NTERA-2 and NCCIT cisplatin-resistant cells and inhibited the growth of NTERA-2 (cisplatin-resistant) xenografts. High ALDH1A3 expression and increased ALDH activity were detected in both refractory cell lines. In addition, the upregulation of the ALDH isoform ALDH1A3 was confirmed in 216 patient samples with all histological subtypes of testicular tumors. These results suggest ALDH1A3 as a novel therapeutic treatment in TGCTs, and disulfiram represents a feasible treatment option for refractory TGCTs [109].

Our group developed an in vitro model of cisplatin resistance to identify new potential therapies for TGCT-resistant patients (data not published). We established a CDDP-resistance model using the NTERA-2 cell line (NTERA-2R), which was treated for approximately eight months with incremental doses of CDDP. We then performed a phenotypical characterization and NTERA-2R exhibited a significant increase in cell proliferation capacity, augmented clonogenic survival, and higher migration ability, suggesting an aggressive phenotype. To elucidate the molecular changes associated with CDDP-resistance, we analyzed the expression of genes related to damage and repair mechanisms. Compared to the parental cell line, NTERA-2R showed several differentially expressed genes related to DNA repair and cell cycle regulation. These results support the idea that the main change in NTERA-2R is possibly an increased DNA repair capacity and specific changes in cell cycle control, which may trigger apoptosis evasion and allow cells to proliferate, even in the presence of CDDP adducts.

Changes in the cell cycle (increase in G1 and decrease in the S phase), increase in the number of acquired mutations (mainly in the ATRX gene), changes in the gene expression pattern, and chromosomal variation (gain of 12p, 1, 17, 20, and 21 loss of X) were also observed in the resistant NCCIT strain [100].

To investigate cisplatin-resistance genetic basis in TGCT, Piulats et al. implanted a collection of matched cisplatin-sensitive and -resistant non-seminoma tumors in nude mice and compared the genomic hybridization (CGH). Comparative CGH analyses showed a gain at the 9q32-q33.1 region, and the presence of this chromosomal rearrangement was correlated with poorer overall survival (OS) in metastatic germ cell tumors. Moreover, POLE3 and AKNA genes were deregulated in resistant tumors harboring the 9q32-q33.1 gain. Therefore, the cisplatin-refractory orthoxenografts of TGCTs are potent models to test the efficiency of drugs, and identify prognosis markers and gene alterations [102].

An immunohistochemical study investigated xenograft models and NSGCT samples with a focus on OCT4-negative cells with undifferentiated EC morphology and their association with chemotherapy resistance [108]. Subcutaneous xenograft tumors of the NSGCT cell lines H12.1 (cisplatin-sensitive) and 1411HP (cisplatin-resistant) were established in athymic nude mice. The cisplatin-sensitive cell line H12.1 leads to xenografts in which EC structures are mainly composed of OCT4-positive cells, whereas xenografts from the resistant cell line 1411HP exclusively comprise OCT4-negative EC areas, suggesting that the growth of NSGCTs in patients with cisplatin-refractory disease may be determined by OCT4-negative EC cells [108]. In addition to this data, a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation was developed as a representative model of malignant TGCTs in men. The resulting mice developed malignant, metastatic TGCTs composed of teratomas and embryonal carcinomas, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4 [110].

The need for new therapeutic options for patients with natural or acquired resistance to cisplatin has led to the investigation of the activity of different compounds (kinase inhibitors directed at mTOR, EGFR, HER2, VEGFR, and IGF-1R), in sensitive (H12.1 and GCT72), and resistant (H12.1RA, H12.1D, 1411HP, and 1777NRpmet) cell lines of TGCT. Research has shown that these compounds have potential activity when used alone, but not when in combination with cisplatin [111].

Despite these recent advances in the use of mouse models to study TGCTs, such models must be developed for pediatric patients, and new molecular studies must be performed to provide powerful experimental tools to prioritize new therapeutic approaches for future clinical trials. Figure 2 summarizes the comparison of clinical and molecular differences between adult and pediatric patients with TGCTs as hallmarks of cancer.