Differentiation of human testicular embryonal carcinoma and teratocarcinoma grown in nude mice and soft-agar cultures

The differentiation pattern of two human germ cell tumors, grown in nude mice and in vitro is described. Tumor A was an embryonal carcinoma (EC) of borderline histology with characteristics of yolk sac tumor and of seminoma; tumor B was a teratocarcinoma with yolk sac elements and syncytiotrophoblastic giant cells. The morphology of an EC as well as cytogenetic characteristics were maintained during 20 passages in nude mice from tumor A and over 11 passages from tumor B. Tumor A did not grow in vitro. Cell suspensions prepared from xenografted tumor B grew into cystic embryoid bodies in semi-solid tissue culture medium. These embryoid bodies showed cuboidal and flattened cells with microvilli, junctional complexes, peripheral microfilaments, and annulated lamellae, reminiscent of the 'inner cell mass' of a blastula and of endoderm, respectively. When such colonies were transplanted into nude mice, however, only tumors with the morphology found in the transplants appeared.     

Establishment of human colon carcinoma lines in nude mice.

After subcutaneous inoculation into nude mice of 24 human colon adenocarcinomas, growth, defined as histopathologically confirmed tumor growth which has been passed, was observed in 13 cases (54%). Tumors from metastatic sites showed higher take rates (58%) than tumors from primary sites or recurrent tumors (50%). Nine continuous tumor lines were established (69% of growing tumors) with metastatic tumors establishing more readily (100% of growing tumors) than primary tumors (40%). The average period in primary transplant was shorter for metastasis (8.3 weeks), than for primary tumors (18.5 weeks); total material 10.6 weeks. Average periods between passages were shorter than primary transplant times; these periods were shorter for metastases (6.6 weeks) than for primary tumors (9.4 weeks); total material 7.4 weeks. Of four growing tumors not established as continuous lines, three were primary and one a recurrent tumor, and the loss of tumor growth occurred in very early passages, not later than passage 3. All nude mouse-grown colon tumors were moderately well differentiated.     

骨髓间充质干细胞和部分肿瘤细胞中Nucleostemin基因的表达

以分离的人胚胎和大鼠骨髓间充质干细胞 (MSCs) ,6种肿瘤细胞株 ,裸鼠肿瘤和转移瘤组织为实验材料 ,以大鼠心肌组织和人胎盘组织为对照 ,探讨nucleostemin基因的表达情况 .RT PCR结果显示 ,nucleostemin基因在MSCs、肿瘤细胞和肿瘤组织中均有不同程度的表达 ,而大鼠心肌和人胎盘组织中无表达 .DNA测序结果证明 ,扩增的PCR产物与GenBank提供的DNA序列完全同源 .SCID裸鼠肿瘤动物模型定量PCR结果证实 ,nucleostemin的mRNA在裸鼠肿瘤组织和转移瘤组织中表达较高 .研究结果表明 ,在细胞中nucleostemin基因不同水平的表达可能与MSCs、肿瘤细胞的增殖和肿瘤的发生、发展与转移有关 .     

Cytostatic and cytolytic effects of human recombinant tumor necrosis factor on human renal cell carcinoma cell lines derived from a single surgical specimen

The purpose of this study was to determine whether human tumor cell lines derived from a single tumor exhibit heterogeneous responses to the anti-tumor effects of human recombinant tumor necrosis factor (h-r-TNF). Several cell lines with different metastatic propensities have been established in culture from a single surgical specimen of a human renal cell carcinoma following different selection procedures in nude mice. The cell lines exhibited significant differences in in vitro susceptibilities to cytotoxic effects of TNF. Kinetic analysis of the interaction of TNF with susceptible renal carcinoma cells demonstrated that a short 30 min interaction of TNF with the cells is sufficient to produce significant lysis 72 hr later. One cycle of exposure of sensitive cells to h-r-TNF did not produce cells resistant to it, nor did the degree of sensitivity to h-r-TNF vary after one passage of the cells in nude mice. Tumor cell resistance to the effects of TNF did develop spontaneously after prolonged cultivation in culture. We conclude that tumor cells derived from a single human renal cell carcinoma exhibit a heterogeneous response to the cytotoxic effects of h-r-TNF.     

An ascites form of human mammary carcinoma transplantable in nude mice

Mammary carcinoma cells from pleural effusion of a patient were inoculated into the peritoneal cavity of nude mice, and a large amount of ascites was produced about 120 days later. From the ascites, serial passages in the same form were successful in nude mice by intraperitoneal injection of 10(7) or more cells. The ascites cells retained the morphology almost similar to that of the patient tumor cells, whereas specific estrogen-binding proteins in the cytoplasm disappeared after growing in male nude mice. The results were compared with those of other established human cancer cell lines in nude mice.     

Human pancreatic adenocarcinoma: In vitro and in vivo morphology of a new tumor line established from ascites

Summary A human pancreatic tumor cell line has been established from the ascites of a patient with histopathologically confirmed adenocarcinoma of the head of the pancreas and maintained for more than 12 months in the laboratory. Epitheloid tumor cell colonies, which resulted from primary tissue cultures of the ascitic cell component, were mechanically isolated by needle micromanipulation. Tumorigenicity was proven in athymic nude mice. Morphologically the pancreatic tumor epithelial cells grew to confluency with moderately tight adhesion to the culture plastic surface and with free-floating cells in the medium. Upon re-establishment of the tumoral xenograft in tissue culture, the epithelial cells retained their original morphology. Histologically the tumor grown in nude mice exhibited prototypic characteristics of the primary adenocarcinoma in the patient, producing abundant mucin and displaying a broad spectrum of glandular differentiation, which ranged from well to poorly differentiated adenocarcinomas with occasionally localized lymphocytic infiltrations. Furthermore, the tumor expressed carcinoembryonic antigen and human pancreas cancer associated antigen. This tumor line, designated AsPC-1, has been cultured for at least 10 passages in vitro and 3 in vivo. It represents a new model for human pancreatic cancer. This work was supported in part by Research Grant CA-18410 awarded by the National Cancer Institute through the National Pancreatic Cancer Project.     

人卵巢癌裸鼠移植实体瘤模型的建立

目的建立人卵巢癌裸鼠移植实体瘤模型。方法将1例人卵巢癌组织移植裸鼠,建立人卵巢癌裸鼠原代移植实体瘤模型的基础上,再将实体瘤皮下移植、实体瘤原位移植、实体瘤细胞移植裸鼠。观察裸鼠实体瘤生长和转移情况,称量其体重、子宫卵巢重、瘤重及瘤的大小,并作病理、电镜、染色体检查。结果成功地建立人卵巢癌裸鼠移植实体瘤模型,并已传至第18代,传代移植成功率100％,组织学和超微结构形态均证明该实体瘤保留了原人卵巢癌特征,有人卵巢癌染色体特征,并出现肝、脾转移。结论本研究建立人卵巢癌裸鼠移植实体瘤模型与人相似,通过18代传代和实验观察方法稳定可靠。为人卵巢癌的研究提供了理想的动物模型。     

利用裸鼠建立人泌尿生殖系统肿瘤细胞系

目的建立人泌尿系肿瘤无限细胞系,为泌尿系肿瘤研究提供实验模型.方法无菌取下肿瘤标本后,将标本剪成大小约1.0mm3的组织块,在裸鼠右后肢皮下包埋,当皮下肿瘤块发生明显增殖并长到一定程度后,再行裸鼠体内传代两次,最后取下组织块进行原代培养.培养细胞传代超过20代后按建系标准[2]进行检测.结果共取40例标本,裸鼠体内传代F1代成功6例,F3代成功3例,该3例标本行原代培养后建成3个无限细胞系人肾透明细胞癌RCC-9863,人膀胱癌BC-6,人前列腺癌PC-98106,全部细胞传代1年以上,生长稳定,传代周期固定,其形态结构,分化程度与原发瘤保持一致,染色体形态仍为人类核型.结论裸鼠肿瘤皮下种植法是泌尿系肿瘤建系的一个较好方法.     

One method to establish Epstein‐Barr virus‐associated NK/T cell lymphoma mouse models

Novel nude mice model of human NK/T cell lymphoma were established by subcutaneously injecting two NK/T cell lymphoma cell lines into the right axillary region of mice and successful passages were completed by injecting cell suspension which was obtained through a 70‐μm cell strainer. These mice models and corresponding cell clones have been successfully developed for more than 8 generations. The survival rates of both resuscitation and transplantation in NKYS and YT models were 90% and 70% correspondingly. Pathologically, the tumour cells in all passages of the lymphoma‐bearing mice and cell lines obtained from tumours were parallel to initial cell lines. Immunologically, the tumour cells expressed the characteristics of the primary and essential NK/T lymphomas. The novel mice models maintained the essential features of human NK/T cell lymphoma, and they would be ideal tools in vivo for further research of human NK/T cell lymphoma.     

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells ability to exhibit a malignant potential in nude mice.     

Human lung cancer cell lines in our laboratory: establishment of three large cell carcinoma cell lines and their biological characterization]

Three large cell carcinoma cell lines were established from tumors of lung cancer patients. The cell lines were named NUTLC-2, NUTLC-4 and NUTLC-5 and they were found to have the following biological characterization. 1) By chromosomal analysis, the tumor cells of two of the cell lines (NUTLC-2 and NUTLC-5) were human-origin cells. Numbers of chromosomes of these cells ranged from 52 to 59 in NUTLC-2 and from 68 to 75 in NUTLC-5, with the modal numbers of 56 and 71, respectively. 2) Primary tumor resected from the patient with lung cancer was heterotransplanted into the subcutis of a nude mouse. NUTLC-4 cell line was established in vitro from the tumor in nude mouse and the tumor cells were found to be mouse-origin cells by chromosomal analysis. Human DNA was not detected by Alu analysis. 3) The tumor cells of three cell lines could be heterotransplanted subcutaneously into nude mice. However, no natural distant metastasis in nude mouse was observed. 4) Drug sensitivity to NUTLC-2, NUTLC-4 and NUTLC-5 tumor cells differed individually according to MTT colorimetric assay, and characteristic drug sensitivity was not noted in histological types of lung cancer.     

Character of a human cholangiocarcinoma CHGS, serially transplanted to nude mice

With the progress in surgical technique, remarkable improvement has been noted in the treatment of bile duct carcinoma. However, in the cholangiocarcinoma at porta hepatis or in the progressive carcinoma, many cases have been reported, for which radical surgery is not achievable. In recent years, discussion has been concentrated on the necessity of multidisciplinary treatment for the bile duct carcinoma, but fundamental research has not been done enough. In the present paper, the process for obtaining CHGS strain implantable to the nude mouse derived from a human cholangiocarcinoma as achieved in our department was discussed, and its biological characteristics-above all, the sensitivity to carcinostatic agents and to radiation-were evaluated. The doubling time of CHGS strain is 6.2 days, and nude mice showed stable proliferation with 100% viability. Histologically, it was tubular adenocarcinoma similar to the primary tumor. It has high mucin producing ability, and necrosis hardly occurs. The search for DNA ploidy by flow cytometry revealed the presence of two types of cells: The cells of diploid pattern and aneuploid pattern. In the tests to determine the sensitivity of CHGS strains to carcinostatic in MMC, ADR, 5-FU and CDDP groups, and to radiation according to the Battele Columbus Laboratories Protocol, the regression of tumor was observed in MMC, ADR, CDDP groups. Particularly, in MMC group, some of the tumors had disappeared. Recurrence was also noted in this case, but the survival, was still recognized nearly four years after the operation through the postoperative auxiliary therapy. This was regarded as the case, where the sensitivity test using the nude mouse implantable tumor strain was reflected well in clinical application.     

Establishment and characterization of a human malignant fibrous histiocytoma line serially transplanted in nude mice

Serial heterotransplantation of human malignant fibrous histiocytoma (MFH) derived from tibia was attempted in BALB/c nu/nu mice, and HKMFH-nu transplantable tumor line was established. This line had the following biological properties. (1) Eighteen serial passages were carried out in 41 months. (2) Morphological changes of the grafts occurred in nude mice with serial passages: During the first 6 passages, histiological picture was consistent with the common type of MFH similar to that of the original tumor, then after the 7th passage, the myxoid type coexisted with the common type, and finally the myxoid type occupied the entire grafts to form large cysts. (3) The common type grafts grew more rapidly than the myxoid type grafts. (4) Granulocytosis (neutrophilia) was observed in mice bearing the common type tumor, but not in mice bearing the myxoid type tumor.     

Functional characterization of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1)

We have successfully isolated a cell line (IEC-1) from an intraepidermal carcinoma of the skin of a patient and compared its behavior, in vitro, to normal human epidermal keratinocytes (HEK) and squamous cell carcinoma cell lines (SCCs). HEK differentiation comprises an initial growth arrest followed by an induction of squamous differentiation-specific genes such as transglutaminase type 1 (TG-1). Using thymidine uptake and TG-1 induction as markers of proliferation and differentiation, respectively, we were able to show that HEKs and the IEC-1 cells undergo growth arrest and induce TG-1 mRNA expression in response to various differentiation-inducing stimuli, while neoplastic SCC cell lines did not. However, differentiation in HEKs was an irreversible process whereas differentiation of the IEC-1 cells was reversible. Furthermore, growth of IEC-1 cells in organotypic raft cultures revealed differences in their ability to complete a squamous differentiation program compared with that of normal HEKs. The IEC-1 cells also exhibited a transitional phenotype with respect to replicative lifespan; HEKs had a lifespan of 4-6 passages, IEC-1 cells of 15-17 passages, and SCC cells were immortal. These alterations in IEC-1 cell behavior were not associated with functional inactivation or mutations of the p53 gene. These data indicate that the IEC-1 cells, derived from a preneoplastic skin tumor, exhibit differences in their ability to undergo terminal differentiation and have an extended replicative lifespan.     

Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines

Summary Five spontaneous canine mammary tumors were cultured in vitro and cell lines were established. The tumors included three frozen carcinomas, fine-needle aspirate from one fresh carcinoma, and one fresh atypical benign mixed tumor. The cell lines have so far been cultured for about 2 yr and passaged between 45 and 200 times. The cell lines expressed different types of intermediate filaments, including a heterogenous pattern. In some cases no intermediate filaments were expressed. Ultrastructure studies showed epithelial cells and cells intermediate between epithelial and myoepithelial types. Retrovirus associated A-particles were found in two carcinomas. The mixed mammary tumor cell line formed ductlike structures in collagen substrate. The cell lines grew when inoculated s.c. into male nude mice. Two carcinomas caused lymph node metastases in two mice and another carcinoma single lung metastases in one tested mouse. DNA hypodiploidy, studied by flow cytometry, in one of the primary carcinoma was retained in vitro, and this cell line showed polyploidy during later passages. The other cell lines had a more unstable DNA profile, although a tendency for polyploidy was found. These findings were also illustrated in chromosome studies.     

Establishment and characterization of new cell lines from human renal cell carcinoma

We have characterized seven human renal cell carcinoma cell lines established from primary sites of five patients between 1987 and 1989. Two lines, OUR-20P and OUR-20S, were derived from the OUR-20 cells by cloning with a dilution method 3 months after the primary culture. These three cell lines were tumorigenic in athymic nude mice when inoculated subcutaneously. Examined by a dye uptake method, OUR-20 was highly sensitive to interferon-alpha (IFN-alpha); OUR-20P, OUR-20S and OUR-30 showed slight sensitivities, while the other three cell lines were insensitive. All seven cell lines have been maintained for more than 2 years and over 50 passages in vitro. Cytogenetic analyses performed 1.5 to 3 years after the starts of primary cultures indicated that all seven cell lines, which exhibited different morphologies in phase-contrast micrographs, were aneuploid with modal chromosome numbers 41 to 89.     

Human cervical cancer transplantable to athymic mice

Pregnancy in nude mice proved to be a favourable period for a successful human cervical tumor transplantation. This approach made it possible to obtain human cervical tumors, serially transplanted to nude mice. After several passages the tumor acquired the ability to be transplanted to nonpregnant female mice. The tumor retained a histological pattern of an original human tumor.