Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Establishment of human tumor strains from cell lines transplanted into athymic mice and rats

The authors describe human cancer strains established from 6 cell lines transplanted to nude mice and rats: cancer of the liver, colon, lung, bladder and Burkitt's lymphoma. In spite of a long history (for several years) of cell line transfers in vitro, on transplantation of tumor cells, nude animals established tumors histologically identical to the primary cancer.     

p53 mutations in tumors derived from irradiated human thyroid epithelial cells

A non-tumorigenic human thyroid epithelial cell line (HTori-3) has been transformed into tumorigenic cells by exposure in vitro to alpha particles or gamma-radiation. These transformants were tumorigenic in athymic nude mice and tumors were transplantable into other nude mice. To further characterize processes involved in neoplastic progression, the tumor cell lines derived from these radiation-induced primary tumors were screened for mutations in the p53 tumor suppressor gene. p53 mutation was detected by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 inclusive. Mutations detected by SSCP analysis were confirmed by sequencing. Mutations were detected in all four exons analysed, although there was no correlation between dose, LET or mutation position or frequency. Mutations in p53 exons 6 and 7 have been reported in the childhood papillary thyroid carcinomas in Belarus presumably as a result of radioiodine fall-out. Similarly here, p53 mutations are induced experimentally during the development of human thyroid tumors generated by irradiation of a human thyroid epithelial cell line in vitro.     

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.     

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.     

Growth of tumor cells with different sensitivities for murine natural killer cells in young and adult athymic nude mice

Of four tumor cell lines, the murine YAC lymphoma, the human K562 lymphoma, and the human prostatic carcinomas PC3 and PC93, the susceptibility to murine natural killer (NK) cells as well as the tumorigenicity in young (3.5-4 weeks old) and in adult (8-10 weeks old) nude mice were studied. In young nude mice, which exhibited a lower level of NK cell activity than adult nude mice, the formation of solid tumors after inoculation of tumor cell suspensions occurred more frequently and with a shorter time lag than in adult animals. These effects were observed not only with the NK-sensitive YAC cells, but also with the relatively NK-insensitive PC3 and PC93 cells, indicating that also factors other than NK cell susceptibility may influence the growth of tumor cells in nude mice. Therefore, the use of young nude mice may enhance the rate of success of heterotransplantation of human tumors, regardless of the NK cell susceptibility of the tumor cells.     

Induction of stromal cell transformation in xenografts of human colonic cancer]

Two transformed stromal cell lines FM-7 and FR-7 were obtained from human xenografts of colon tumor (HCT-7), propagated in nude mice and nude rats, respectively. These two cell lines were confirmed to be murine and rat's by a cytogenetic study and were tumourigenic in BALB/c nude mice with the morphology of fibrosarcoma. Human DNA sequences were not detected by hybridization with pBlur8 probe in DNA of FR-7 cell line grown in vitro. These results indicate that human cancer cells from xenograft HCT-7 can induce malignancy in adjacent normal cells of nude mice and nude rats in the absence of DNA transfer.     

Changes in histological differentiation of human tumors transplanted to athymic nude mice: a morphometric study

Differing conclusions have been reached regarding the phenotypic stability of human tumors transplanted to athymic nude mice. Since previous conflicting studies of tumor histology have been largely subjective, quantitative methodology was applied to an analysis of 13 human adenocarcinoma tumor lines that were originally derived directly from surgical specimens. Glandular differentiation was quantitated, both in the original human tumor (OHT) and in a minimum of 6 serial passages of the nude mouse-grown tumors (MGT), by means of point counting. A significant change in differentiation was noted in 12 lines, with 9 showing a decrease. Variance from the OHT was most commonly noted in the initial MGT, but additional changes were also noted in 8 lines during subsequent passages. Most of the lines also showed increased necrosis in the MGTs. Since histological differentiation and necrosis are related to tumor aggressiveness, it would appear that the predominant tendency was to evolve toward a more malignant phenotype. These changes may mimic those seen in human tumor metastases.     

Development and characterization of breast carcinoma cell lines as in vitro and in vivo models for breast cancer diagnosis and therapy

Summary To establish a model system for preclinical radioimmunotherapy studies, attempts were made to graft 16 different human breast carcinoma cell lines into BALB/c nu/nu (nude) mice. Nine produced serially transplantable tumors growing at a variable rate, whereas seven failed to do so. Conversely, three new cell lines were established in monolayer culture from transplantable human breast tumors in nude mice. Twelve selected tumors and their corresponding cell lines were characterized for DNA ploidy, % S-phase, and breast epithelial mucin expression by immunohistochemistry and flow cytometry. A wide diversity of these cellular characteristics were found in that each tumor was unique and distinct from the others. DNA ploidy differed among the tumors but was not affected by switching between in vitro to in vivo growth. Some tumors expressed similar levels of the breast mucin both in vitro and in vivo, whereas most expressed lower levels as transplantable tumors. There was a good correlation between immunohistochemical and flow cytometric determination of surface and cytoplasmic mucin expression, and with both techniques estrogen and progesterone receptor positive tumors had significantly higher levels of mucin expression than receptor negative tumors. These 12 transplantable breast tumors, with their corresponding cell lines, provide an excellent model system for testing radioimmunotherapy and other therapeutic reagents because they exhibit diverse phenotypic characteristics that represented a mini-population of breast cancer patients’ tumors, allowing assessment of the effect of therapy when confronted with different breast tumors’ genotype and phenotype.     

Retinoblastoma protein activation of interleukin 8 expression inhibits tumor cell survival in nude mice.

Loss of retinoblastoma protein (Rb) has been implicated in the formation of a variety of human malignancies. Restoration of Rb expression in the cell lines representing these tumors eliminates or significantly reduces tumorigenicity in nude mice, but the mechanism for this Rb effect is unknown. Results from this study indicated that Rb expression reduced tumor cell survival in nude mice by dramatically enhancing interleukin 8 (IL-8) secretion. IL-8 secreted by the Rb-transformed cells attracted neutrophils in vitro and tumor-infiltrating neutrophils in vivo, which is consistent with the Rb-mediated tumor regression being dependent on IL-8. The apparent, contradictory roles of IL-8 as a protumorigenic and antitumorigenic cytokine are discussed.     

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.     

Establishment of a Chinese bladder cancer cell line (T921) with high metastatic activity

Muscle-invasive bladder cancer is prone to metastasis without a standard organ preference. The current cell lines used to study bladder cancer have primarily been derived from individuals in Western populations, and no human bladder cancer cell line has been established from the Chinese population. A bladder cancer cell line was derived from a female Chinese patient with muscle-invasive bladder cancer, and these cells were then xenografted into the bladders of three nude mice. Five weeks later, these mice were killed to observe local invasion and distant metastasis. The metastatic tumors were also removed and analyzed to assess the metastatic mechanism. This bladder cancer cell line, named T921, was successfully established, as evidenced by karyotype and immunohistochemistry analyses. Multi-organ metastases were observed in all three of the nude mice 5 wk after the orthotopic transfer of the cell line. In addition, epithelial–mesenchymal transition (EMT)-related genes were involved in the tumor metastases. The T921 bladder cancer cell line was successfully established, and EMT was observed to play a role in bladder cancer metastasis.     

Cellular tumorigenicity in nude mice. Test of associations among loss of cell-surface fibronectin, anchorage independence, and tumor-forming ability

Fibronectin (FN; also called large external transformation-sensitive [LETS] protein or cell-surface protein [CSP]) is a large cell-surface glycoprotein that is frequently observed to be either absent or greatly reduced on the surfaces of malignant cells grown in vitro. Because FN may be a useful molecular marker of cellular malignancy, we have carried out an extensive screening to test the specific association among the degree of expression of FN, anchorage-independent growth, and tumorigenicity in the athymic nude mouse. A variety of diploid cell strains and established cell lines were tested for the expression of surface FN by indirect immunofluorescence using rabbit antisera against human cold insoluble globulin, rodent plasma FN, or chicken cell- surface FN. Concomitantly, the cells were assayed for tumor formation in nude mice and for the ability to form colonies in methylcellulose. Tumorigenic cells often showed very low surface fluorescence, confirming earlier reports. However, many highly tumorigenic fibroblast lines from several species stained strongly with all three antisera. In contrast, the anchorage-independent phenotype was nearly always associated with tumorigenicity in approximately 35 cell lines examined in this study. In another series of experiments, FN-positive but anchorage-independent cells were grown as tumors in nude mice and then reintroduced into culture. In five of the six tumor-derived cell lines, cell-surface FN was not significantly reduced; one such cell line showed very little surface FN. Our data thus indicate that the loss of cell-surface FN is not a necessary step in the process of malignant transformation and that the growth of FN-positive cells as tumors does not require a prior selection in vivo for FN-negative subpopulations.     

Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type.

Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell.     

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.