Several p53-deficient animal models have been developed to mimic sarcomagenesis. In particular, mice with
Tp53, inactivated by Cre-loxP-mediated recombination, develop spindle cell sarcomas and pleomorphic sarcomas. Additionally, mesenchymal sarcoma stem cells (Sca-1low) have been isolated from these animals [
23]. The rat however is also a feasible model for imaging studies, easier in surgical handling and imaging than mouse. Unlike the
Tp53 knockout mouse that often develop lymphomas first, the
Tp53-knockout rats most often develop sarcomas, which favors the use of the rat model in preclinical studies of sarcoma, including novel drug testing [
14,
24].
The first
Tp53-deficient rat—Dark Agouti rat (subsequently referred to as the
Tp53-deficient DA rat)—was created via homologous recombination in the rat embryonic stem cells. Homozygous
Tp53-deficient DA rats live no longer than six months and develop angiosarcomas and lymphomas. Heterozygous
Tp53-deficient DA rats survive up to 12 months of age and demonstrate a wide variety of sarcomas in both males and females, and also develop mammary carcinomas in about 20% of female rats [
25,
26]. On the contrary, in Fischer-344 (F344)’s rat-based 344-
Tp53tm1(EGFP-Pac)Qly/Rrrc (F344-
Tp53) model, the tumor spectrum is shifted towards the primary tumor types—osteosarcomas and meningeal sarcomas. The incidence of osteosarcomas is 57% and 36% in F344-
Tp53 homozygous and heterozygous animals, respectively. In this model, tumors are highly representative of human disease radiographically and histologically. They typically localize on long bones and are characterized with frequent pulmonary metastases [
7]. At the same time
Tp53 knockout rat in the Sprague Dawley background was generated using Zinc Finger Nuclease (ZFN) technology with target site located in the 22-bp exon 3 of the gene. A homozygous null rat—
Tp53Δ11/Δ11—with a complete loss of p53 protein has a shortened disease-free lifespan due to early onset of cancers. The tumor spectrum in these null mutant rats includes both sarcomas and carcinomas, with a predominance of nervous system tumors. The
Tp53Δ11/+ rats experience a later onset of tumorigenesis and develop skin and endocrine cancers in addition to the cancer types recognized in the null homozygote [
27]. Finally, the p53 TGEM Rat model that we used in this study was developed in a Wistar Han background (referred to subsequently as the
Tp53-deficient Wistar rat). At the Hubrecht Institute, after N-ethyl-N-nitrosourea (ENU)-driven mutagenesis, a target-selected screen was performed in the outbred Wistar background. Adult male rats were administered intraperitoneal ENU injections that target spermatogonial stem cells (SSCs) in rat gonads. Animals with a nonsense mutation at amino acid position 273 (Cys to stop) within the DNA binding domain of the p53 protein were selected. This mutation functionally resulted in a full knockout
Tp53 mutation. For model development systematic generation of these knockout rats was carried out by random mutagenesis of Wistar rats followed by PCR amplification and capillary sequencing check-up, referred to as TGEM
® technology by Transposagen. ENU-driven target-selected mutagenesis is an effective approach for artificial introduction of point mutations. ENU, as an alkylating agent, transfers its ethyl group to oxygen or nitrogen in nucleophilic groups of nucleobases, which results in nucleotide substitutions such as A–T base transversions [
28]. Wistar background homozygous mutant
Tp53C273X/C273X rats predominantly develop sarcomas with an onset at four months of age and with a high frequency of pulmonary metastases. In our study, MRI and PET examinations revealed metabolically active tumors in several locations, including the brain, head and neck, extremities and abdomen. These sites were consistently similar to those previously described in this model [
21]. Heterozygous rats develop sarcomas starting at eight months of age.
Tp53C273X/+ rats predominantly develop osteosarcomas, therefore this model may be generally used for soft tissue and bone sarcoma research and should be referred in appropriate papers, including imaging/PET-oriented manuals [
28,
29,
30]. In the TGEM Rat model the introduced DNA mutation finally truncates the protein at the DNA binding domain, eliminating functionally essential domains including the nuclear localization domain and the homo-oligomerization domain of the translated protein, which results in rapid degradation of residual non-functional peptide. A genetic analysis confirmed that developed sarcomas in heterozygotes exhibit a loss-of-heterozygosity of the wild-type
Tp53 allele [
14].
With regard to sarcoma-oriented studies, it must be pointed out that sporadic angiosarcomas (SA) and radiation-associated angiosarcomas (RAA) are similar in histology, immunohistochemical markers, and DNA mutation profiles and share a similar prognosis [
31]. In angiosarcomas, most of abnormalities are found in the p53 and MAPK pathways. More than 50% of angiosarcomas presented MAPK pathway activation. Simultaneously, angiosarcoma genome analyses revealed mutations and amplifications of
VEGF,
MDM2,
TP53,
CDKN2A,
KRAS and
MYC.
TP53 was reported as mutated in 35% of the lesions and
CDKN2A lost in 26%. Activating mutations were found in
KRAS,
HRAS,
NRAS,
BRAF,
MAPK1, while inactivating mutations in
NF1 and
PTPRB1. In particular,
MYC gene amplifications are more common in RAA [
32]. In our study, the most upregulated genes included Rho GTPase Activating Protein 42 (
Arhgap42), mitochondrial Propionyl-CoA Carboxylase Subunit Alpha (
Pcca), LOC294154 (similar to chromosome 6 open reading frame 106 isoform a) with ubiquitin binding activity, Spindle and kinetochore-associated protein 3 (
Ska3) and Solute Carrier Family 16, Member 3 (Monocarboxylic Acid Transporter 4—
Slc16a3). At the same time, the most upregulated pathways in angiosarcoma vs all other tissues were related to cell cycle with mitosis and meiosis; chromosome, nucleosome and telomere maintenance; as well as DNA replication and recombination. On the other hand, downregulated genes were responsible for metabolism, including respiratory chain electron transport, TCA cycle, fatty acid metabolism and amino-acid catabolism. Thus, the rat model that we studied here represents a novel interesting pre-clinical model that may easily be used for novel drug testing applications that surpasses a lack of tumor–host interactions and no immune response. This model has native microenvironment of an animal and also enables to conduct studies on an intact immune system response, including immunotherapy and cancer vaccines [
33,
34,
35].