Development of Genetically Flexible Mouse Models of Sarcoma Using RCAS-TVA Mediated Gene Delivery

Sarcomas are a heterogeneous group of mesenchymal malignancies and unfortunately there are limited functional genomics platforms to assess the molecular pathways contributing to sarcomagenesis. Thus, novel model systems are needed to validate which genes should be targeted for therapeutic intervention. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS-TVA) system would result in sarcomagenesis. We also sought to determine if the cell type transformed (mesenchymal progenitors vs. terminally differentiated tissues) would influence sarcoma biology. Cells transduced with RCAS vectors directing the expression of oncoproteins KrasG12D, c-Myc and/or Igf2 were injected into the hindlimbs of mice that expressed the retroviral TVA receptor in neural/mesenchymal progenitors, skeletal/cardiac muscle or ubiquitously (N-tva, AKE and BKE strains respectively). Disrupting the G1 checkpoint CDKN2 (p16/p19−/−) resulted in sarcoma in 30% of p16/p19−/−xN-tva mice with a median latency of 23 weeks (range 8–40 weeks). A similar incidence occurred in p16/p19−/−xBKE mice (32%), however, a shorter median latency (10.4 weeks) was observed. p16/p19−/−xAKE mice also developed sarcomas (24% incidence; median 9 weeks) yet 31% of mice also developed lung sarcomas. Gene-anchored PCR demonstrated retroviral DNA integration in 86% of N-tva, 93% of BKE and 88% of AKE tumors. KrasG12D was the most frequent oncogene isolated. Oncogene delivery by the RCAS-TVA system can generate sarcomas in mice with a defective cell cycle checkpoint. Sarcoma biology differed between the different RCAS models we created, likely due to the cell population being transformed. This genetically flexible system will be a valuable tool for sarcoma research.

肉瘤是一类异质性的间叶源性恶性肿瘤，遗憾的是当前可用于解析肉瘤发生相关分子通路的功能基因组学平台十分有限。因此，亟需构建新型模型系统，以验证哪些基因可作为治疗干预的靶点。我们提出假说：利用逆转录病毒（RCAS-TVA）系统将癌基因递送至小鼠骨骼肌内，可诱导肉瘤发生。同时，我们旨在探究转化的细胞类型（间叶祖细胞与终末分化组织）是否会影响肉瘤的生物学特性。将携带癌蛋白KrasG12D、c-Myc及/或Igf2表达元件的RCAS载体转导的细胞，注射至三类小鼠的后肢：分别是在神经/间叶祖细胞、骨骼肌/心肌或全身范围内表达逆转录病毒TVA受体的小鼠（对应品系分别为N-tva、AKE及BKE）。敲除G1检查点相关基因CDKN2（p16/p19−/−）后，30%的p16/p19−/−xN-tva小鼠出现肉瘤，中位潜伏期为23周（范围8~40周）。p16/p19−/−xBKE小鼠的肉瘤发生率与之相近（32%），但中位潜伏期更短，仅为10.4周。p16/p19−/−xAKE小鼠同样可发生肉瘤（发生率24%，中位潜伏期9周），但其中31%的小鼠还并发了肺部肉瘤。基因锚定PCR检测显示，86%的N-tva小鼠肿瘤、93%的BKE小鼠肿瘤及88%的AKE小鼠肿瘤中均存在逆转录病毒DNA整合。KrasG12D是最常分离到的癌基因。通过RCAS-TVA系统递送癌基因，可在细胞周期检查点缺陷的小鼠体内诱导肉瘤生成。我们构建的不同RCAS模型的肉瘤生物学特性存在差异，这可能与转化的细胞群不同有关。这一具备遗传灵活性的系统将成为肉瘤研究领域的宝贵工具。