Human RT‐4 bladder carcinoma is highly metastatic in nude mice and comparable ras‐H‐transformed RT‐4 when orthotopically onplanted as histologically intact tisuee-猫眼课题宝

人 RT-4 膀胱在裸鼠癌中具有高度转移性，并且当作为组织学完整的组织原位移植时，ras-H 转化的 RT-4 具有相当的转移性

DOI：

发表时间：

1992

期刊：

International Journal of Cancer

影响因子：

6.4

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

X. Fu;Hoffman Rm

据估计，5-10% 的人类膀胱移行细胞癌 (TCC) 含有突变的 ras 基因（Theodorescu 等，1990）。这就提出了一个问题：激活的 ras 基因是否与膀胱肿瘤转移程序的获得有关。西奥多雷斯库等人。 (1990) 观察到 RT-4 人膀胱癌细胞系在裸鼠中不具有侵袭性，即使在原位注射分解细胞后也是如此。然而，当将突变的人H-ras基因转染到RT-4中，使得该基因在选定的细胞系（例如RT-4-mr-10（RT-lo））中发生过度表达时，在经尿道原位接种分解的细胞后，选定的细胞系能够局部侵入膀胱。然而，在其他器官中未发现 RT-10 造成的邻近或转移性扩散。亲代细胞系和 ras 转染子在皮下接种时均产生肿瘤。然而，肿瘤在皮下部位生长为假包膜肿块，没有任何组织侵袭的证据（Theodorescu 等，1990）。我们开发了一种在裸鼠体内进行人类肿瘤原位移植的完整组织移植方法（Fu et al., 1991qb）。这种方法最初是针对结肠癌开发的（Fu et al., 1991a），允许将手术患者组织直接移植到裸鼠的原位器官上，从而产生局部生长和随后的广泛区域转移、淋巴结和肝转移（Fu et al., 1991a）。该方法利用收获的、皮下生长的 RT-10 组织作为组织来源，并表明原位种植的 RT-10 对裸鼠的许多器官具有高度转移性（Fu 等，1991b）。这些结果与通过注射 RT-10 分解细胞获得的结果形成对比，后者具有侵袭性但非转移性（Theodorescu 等，1990）。我们现在已将原位移植方法应用于亲本 RT-4 本身的组织学完整组织，发现它也具有高度转移性。 RT-4 人膀胱细胞获自 ATCC（马里兰州罗克维尔），并在 4 周龄远交雌性裸鼠皮下培养。将 S.C. 生长的肿瘤切除并切成 2 mm3 的块，并按先前所述进行原位种植（Fu 等人，1991qb）。简而言之，用异芴吸入麻醉裸鼠，并用碘酒和酒精拭子消毒下腹部。进行一个小的中线切口并暴露膀胱。将手术粘合剂 2-氰基丙烯酸酯涂在 2-mm3 肿瘤异体的一侧。随后将组织和肿瘤块粘在膀胱顶部。腹部切口用7-0手术丝线缝合一层。然后将动物饲养在无菌环境中。当这些动物濒临死亡时，它们会被处死并进行全面尸检。尸检时，对所有主要器官进行了仔细检查。将每个器官放入10％福尔马林中，脱水，包埋在石蜡中，切片并用苏木精和曙红染色。将RT-4原位移植到裸鼠膀胱后，出现广泛的局部生长（超过2厘米×2厘米），并侵袭膀胱并转移至淋巴结、膈肌、腹壁、大网膜、胰腺和肝脏（表I）。这些结果与完整组织原位移植后 ras-H 转化的 RT-4 肿瘤 RT-10 的结果相似（Fu 等人，1991b）。图1a显示了裸鼠膀胱，其中RT-4已原位生长83天。可以看出，肿瘤在腹部区域形成了大肿块，并且变得比膀胱本身大得多（也显示）。图1b显示了正常人膀胱组织原位移植后的裸鼠膀胱，其没有导致肿瘤形成，从而强调了RT-4的肿瘤形成能力。图1c显示了裸鼠局部生长的RT-4肿瘤的病理组织学，其是典型的移行细胞癌。图1d显示了与肿瘤相关的极大增大的裸鼠髂淋巴结。图1e显示了裸鼠肿瘤相关淋巴结的病理组织学，表明是典型的移行细胞癌。图1f显示了人RT-4膀胱肿瘤已转移至裸鼠的胰腺、肝浆膜和淋巴结。肝浆膜上生长的转移瘤未侵犯实质组织。因此，不能排除跨体腔传播的可能性。图1g显示了裸鼠胰腺转移的组织病理学。图1h显示肿瘤涉及的胃淋巴结。图1i显示了裸鼠肿瘤相关胃淋巴结的病理组织学，提示为移行细胞癌。图lj显示了切除的裸鼠隔膜，完全涉及膀胱肿瘤。图1k显示裸鼠涉及肿瘤的膈肌的病理组织学，表明移行细胞癌。整个隔膜被替换为

It has been estimated that 5-10% of human transitional-cell carcinomas (TCCs) of the bladder contain a mutated ras gene (Theodorescu et al., 1990). This raises the question of whether activated ras genes are causal to acquisition of the metastatic program of bladder tumors. Theodorescu et al. (1990) observed that the RT-4 human bladder carcinoma line is not invasive in nude mice, even after orthotopic injection of disaggregated cells. However, when a mutated human H-ras gene was transfected into RT-4 so that over-expression of the gene occurred in selected cell lines such as RT-4-mr-10 (RT-lo), the selected cell line was able to locally invade the bladder after transurethral orthotopic inoculation of disaggregated cells. However, no contiguous or metastatic spread by RT-10 was found in other organs. The parental cell lines and the ras-transfectants all produced tumors when inoculated S.C. However, the tumors grew in the S.C. site as pseudoencapsulated masses without any evidence of tissue invasion (Theodorescu et al., 1990). We have developed an intact-tissue onplant method of orthotopic transplantation of human tumors in nude mice (Fu et al., 1991qb). This method, first developed with colon cancer (Fu et al., 1991a), allows surgical patient tissue to be directly grajled onto the orthotopic organ of the nude mouse with resulting local growth and subsequent extensive regional metastases, lymph-node and liver metastases (Fu et al., 1991a). The method utilized harvested, s.c.-grown tissue of RT-10 as a tissue source, and showed that the orthotopically-onplanted RT-10 was highly metastatic to many organs in the nude mouse (Fu et al., 1991b). These results contrasted with findings obtained by injection of disaggregated cells of RT-10, which were invasive but not metastatic (Theodorescu et al., 1990). We have now applied the orthotopic onplant method to histologically intact tissue of the parental RT-4 itself and found that it, too, was highly metastatic. RT-4 human bladder cells were obtained from the ATCC (Rockville, MD) and grown S.C. in 4-week-old outbred female nude mice. The S.C. grown tumors were excised and cut into 2-mm3 pieces and orthotopically onplanted as previously described (Fu et al., 1991qb). Briefly, the nude mice were anesthetized with isofluorene inhalation and the lower abdomen was sterilized with iodine and alcohol swabs. A small midline incision was made and the urinaly bladder was exposed. The surgical adhesive 2-cyanoacrylic acid ester was applied on one side of the 2-mm3 tumor xeenograj? tissue and the piece of tumor was subsequently glued on top of the urinary bladder. The abdominal incision was closed with 7-0 silk surgical sutures in one layer. The animals were then kept in a sterile environment. When the animals wepe moribund they were sacrijiced and full autopsies were performed. At autopsy all major organs were grossly examined. Each organ was Jived in 10% formalin, dehydrated, embedded in parafin, sectioned and stained with hematoxylin and eosin. After orthotopic transplantation of RT-4 to the nude-mouse bladder, extensive local growth occurred (over 2 cm x 2 cm) along with invasion of the bladder with metastases to the lymph nodes, diaphragm, abdominal wall, omentum, pancreas and liver (Table I). These results are similar to those with the ras-H-transformed RT-4 tumor RT-10 after orthotopic onplantation of intact tissue (Fu et al., 1991b). Figure l a shows a nude mouse bladder in which RT-4 had grown orthotopically for 83 days. As can be seen, the tumor formed large masses in the abdominal area and became much larger than the bladder itself (also shown). Figure I b shows the nude mouse bladder after orthotopic onplantation of normal humanbladder tissue which did not result in tumor formation, thus emphasizing the tumor-forming ability of RT-4. Figure l c shows the pathohistology of the locally-growing RT-4 tumor in the nude mouse which is a typical transitional-cell carcinoma. Figure I d shows a greatly enlarged nude-mouse iliac lymph node involved with tumor. Figure 1 e shows the pathohistology of the tumor-involved lymph node in the nude mouse, indicating typical transitional-cell carcinoma. Figure 1 f shows the human RT-4 bladder tumor which has metastasized to the pancreas, liver serosa and lymph nodes in the nude mouse. The metastatic tumor growth on the liver serosa did not invade the parenchymal tissue. Therefore, the possibili& of transcoelomic spread cannot be ruled out. Figure 1 g shows the histopathology of the pancreatic metastasis in the nude mouse. Figure l h shows tumor-involved gastric lymph nodes. Figure 1 i shows the pathohistology of the tumor-involved gastric lymph node of the nude mouse, indicating transitional-cell carcinoma. Figure lj shows the excised nude mouse diaphragm fully involved with bladder tumor. Figure l k shows the pathohistology of the tumor-involved diaphragm of the nude mouse indicating transitional-cell carcinoma. The entire diaphragm was replaced by

Overexpression of normal and mutated forms of HRAS induces orthotopic bladder invasion in a human transitional cell carcinoma.