Orthotopic implantation is essential for the selection,growth and metastasis of human real cell cancer in nude mice

Human neoplasms are heterogeneous for a variety of biological properties that include invasion and metastasis. The presence of a small subpopulation of cells with a highly metastatic phenotype has important clinical implications for diagnosis and therapy of cancer. For this reason, it is important to develop an animal model for the selection and isolation of metastatic variants from human neoplasms and for testing the metastatic potential of human tumor cells.We have implanted human renal cell carcinoma (HRCC) cells (obtained from a surgical specimen) into different organs of nude mice and then recovered the tumors and established each in culture. The 5 established lines differed in their biological-metastatic properties and had a unique karyotype, indicating that growth at different organs selects for different subpopulations of HRCC. Moreover, the HRCC did not metastasize unless they were implanted orthotopically. These findings indicate that the appropriate nude mouse model for studying the biology and therapy of HRCC must be based on the orthotopic implantation of tumor cells.     

In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinomas implanted into nude mice

The purpose of these studies was to select and isolate cells with increased liver-metastasizing potential from heterogeneous primary human colon carcinomas (HCCs). Cells derived from a primary HCC classified as Dukes' stage B2 were directly established in culture or were injected into the subcutis, cecum, or spleen of nude mice. Progressively growing tumors were excised, dissociated, and established in culture. Subsequent to implantation into the cecum or spleen of nude mice, cells from all four lines produced only a few liver tumor foci. HCC cells from the few liver metastases were expanded in culture and then injected into the spleen of nude mice to provide a source for further cycles of selection. With each successive in vivo selection cycle, the metastatic ability of the isolated propagated cells increased. Four cycles of selection yielded cell lines with a very high metastatic efficiency in nude mice. In parallel studies using another primary HCC classified as Dukes' stage D, we isolated cell lines that were highly metastatic in nude mice. Successive selection cycles for growth in the liver increased the metastatic properties of the HCC cells, albeit to a lesser extent than it did those of the Dukes' B2 stage HCC. The ability of the HCC cells to produce liver metastases was not due to simple trapping in the liver. In vivo distribution studies using [125I] iododeoxyuridine-labeled tumor cells revealed that, shortly after injection into the spleen, a comparable number of cells with either low or high metastatic properties arrested in the liver. The differences between the low- and high-degree metastatic cells became apparent by 24 h after injection and, by 72 h, only highly metastatic cells survived in the liver. These results demonstrate that hepatic metastasis by HCC cells is a selective process and that the nude mouse model can be useful for isolating highly metastatic HCC cells and for studying the relevant host organ factors that regulate the pathogenesis of metastasis.     

7th Jan Waldenstr?m Lecture. The biology of human cancer metastasis.

The process of cancer metastasis is dynamic and consists of sequential, interrelated steps. Malignant cells that produce metastases have survived a series of potentially lethal interactions that are regulated by both the intrinsic properties of the tumor cells and host factors. Although some of the steps in this process contain stochastic elements, metastases develop from the nonrandom survival of a few subpopulations of cells that preexist within the parent neoplasm. Metastases can have a clonal origin, and different metastases can originate from the proliferation of different cells. The orthotopic implantation of human cancer cells derived from surgical specimens into nude mice provides a biological model of metastasis. Using this model, clonal analysis of a human renal carcinoma, colon carcinoma, and melanoma has revealed that these tumors are heterogeneous for metastatic properties. Damage to an organ's environment is followed by inflammation and repair, and these homeostatic processes facilitate the proliferation of normal (physiology), and in some cases, tumor cells (pathology). Accelerated growth of human colon cancer cells was found in hepatectomized nude mice, whereas accelerated growth of human renal cancer cells was found in nephrectomized nude mice. These data suggest that systemic physiological signals can be usurped by neoplastic cells. Collectively, the factors that regulate metastasis are the intrinsic properties of metastatic cells and host factors involved in homeostasis.     

Studies of human breast cancer metastasis using nude mice

Athymic nude mice have been used in recent years to study the biology of human tumors and to assess therapeutic responses in vivo rather than just in vitro. Some human tumors metastasize in nude mice, providing model systems for analyzing various aspects of the metastatic phenotype of human neoplasms. For breast carcinomas, however, the tumor-take rate of surgical specimens is low, and only a limited number of cell lines proliferate in nude mice. The site of injection of the breast carcinoma cells is important; tumors grow at a lower inoculum dose and with shorter latent intervals after implantation in the mammary fatpad of nude mice than after injection in the subcutis. One breast carcinoma cell line, MDA-MB-435, metastasizes from mammary fatpad tumors to lymph nodes, lungs, and other visceral organs. In contrast, two other cell lines show lower metastatic ability. Intravenous injection and injection of tumor cells into the internal carotid artery of nude mice produces lung and brain metastases, respectively, thus simulating the arrest and organ colonizing steps of the metastatic cascade. These different techniques demonstrate the potential of experimental studies of human breast cancer growth and metastasis using nude mice.     

Characterization of variants of a human breast-cancer cell-line isolated from metastases in different organs of nude-mice

The MDA-MB-435 human breast cancer cell line metastasizes from tumors growing in the mammary fatpad of nude mice; metastases are found in the lymph nodes and lungs of 75 to 100% of tumor-bearing mice, and at a lower incidence in other organs such as the heart, skeletal muscle, adrenal gland and brain. Variants of this breast cancer cell line were established from metastases in different organs, including the lungs and brain of nude mice; these lines were designated 435-Lung2 and 435-Br1, respectively. Karyotypic analysis of the new variants suggested a clonal origin for the different organ metastases. The lung-derived cells were as metastatic as the original cell line when re-injected in nude mice, yet the cells from the brain metastasis produced few metastases. Analyses of the invasive potential of the cell lines showed that they did not differ in expression of mRNA for 72-kDa type IV-collagenase, or in gelatinase activity (by zymography), or in ability to invade through a Matrigel-coated filter. The poorly metastatic 435-Br1 cells showed reduced binding to cultured monolayers of mouse lung-endothelial cells, compared with the metastatic 435-Lung2 and MDA-MB-435 cells. When the breast cancer cells were artificially arrested in the lungs of nude mice, by injecting Cytodex beads coated with cells, all three cell lines grew equally well, suggesting that the deficiency found in the metastatic potential of the 435-Br1 cells may be associated with interactions with endothelial cells, rather than growth potential in the lungs. Further comparisons of these two metastasis-derived variants of a heterogeneous cell line will lead to greater understanding of the metastatic phenotype of human breast cancer.     

Correlation of growth capacity of human tumor cells in hard agarose with their in vivo proliferative capacity at specific metastatic sites

The purpose of this study was to determine whether the degree of anchorage-independent growth of human tumor cells in increasing concentrations of agarose correlated with the capacity of the cells to produce experimental metastases in nude mice. Human melanoma, breast carcinoma, and colon carcinoma cells from parental lines and variants selected in vivo for metastasis and in vitro cloned lines were plated into medium containing 0.3%, 0.6%, 0.9%, or 1.2% of agarose. These cells were also injected into nude mice: intravenously for melanoma, into the mammary fat pad for breast carcinoma, and into the spleen for colon carcinoma. Production of tumor cell colonies in dense agarose (greater than 0.6%) correlated with production of experimental metastases in the lung (melanoma, breast carcinoma) or liver (colon carcinoma). We conclude that the degree of anchorage-independent growth of tumor cells can predict their biological behavior and metastatic potential in vivo. Thus, this technique may be useful for the isolation of metastatic cells from heterogeneous human neoplasms.     

Expression level of the nm23 gene in clonal populations of metastatic murine and human neoplasms.

The purpose of this study was to determine whether nm23 steady-state mRNA expression levels correlate with metastatic potential of mouse K-1735 melanoma cells, human KM12 colon cancer cells, and human SN12 renal cancer cells. Since neoplasms are heterogeneous and contain subpopulations of cells with different metastatic potentials, we analyzed multiple sets of nonmetastatic and metastatic clones isolated from each neoplasm. In addition, we also examined nine somatic cell hybrids produced by the fusion of nonmetastatic and metastatic K-1735 clones. In the mouse melanoma, we found heterogeneity in nm23-1 steady-state expression levels among the clones and hybrids that did not correlate with their metastatic phenotype. Clones isolated from human colon or renal carcinomas expressed similar levels of nm23-HI regardless of metastatic potential in nude mice. All of the human tumor cells were heterozygous for the nm23-HI-specific allelic DNA fragments, with no allelic deletions or gross alterations detected. Since the failure of tumor cells to produce metastasis can be due to multiple deficiencies, these data stress the importance of using independent clones with different metastatic potentials for the analysis of gene regulation of this process.     

Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture.

The process of metastasis is not random. Rather, it consists of a series of linked, sequential steps that must be completed by tumor cells if a metastasis is to develop. Although some of the steps in this process contain stochastic elements, as a whole, metastasis favors the survival and growth of a few subpopulations of cells that preexist within the parent neoplasm. Moreover, metastases can have a clonal origin, and different metastases can originate from the proliferation of single cells. The outcome of metastasis depends on the interaction of metastatic cells with different organ environments. Organ-specific metastases have been demonstrated in a variety of experimental tumor systems. Moreover, we have found tumor growth that is specific to a particular site within one organ. Whether the same conclusions can be reached for human cancers remained unanswered until very recently. Studies from our laboratory and from others have shown that the implantation of human cancer cells derived from surgical specimens into correct anatomical sites of nude mice can provide a suitable model of metastasis of human tumors. Clonal analysis of a human renal carcinoma, colon carcinomas, and melanomas has revealed that these tumors are indeed heterogeneous for metastatic properties, an observation made only after orthotopic implantation. Thus, growth in the environment of specific organs can be selective and the environment per se influences this process. While it is clear that vascularity and local immunity can facilitate or retard tumor growth, we have concentrated on understanding how damage to an organ and the subsequent repair process can facilitate tumor cell proliferation. Accelerated growth of human colon cancer cells was found in hepatectomized nude mice, whereas accelerated growth of human renal cancer cells was found in nephrectomized nude mice. These data suggest that systemic physiological signals can be recognized by neoplastic cells presumably by mechanisms similar to those shared by their normal cell counterparts. In summary, the critical factors that regulate metastasis are the intrinsic properties of metastatic cells and host factors involved in homeostasis. The recent increase in our understanding of metastasis should provide important leads for developing more effective approaches to the treatment of disseminated cancer.     

Establishment of highly metastatic human tumor cell line in nude mice

A total of 22 adult nude mice (7 approximately 17 weeks old) of BALB/cA origin were inoculated subcutaneously (SC) with human tumor cell lines derived from rhabdomyosarcoma and lung carcinomas, including squamous cell carcinoma, bronchiolo-alveolar carcinoma, small cell carcinoma, giant cell carcinoma and 2 types of adenocarcinoma. 12 neoplasms (54.5%) was confirmed at the initial transplantation and 7 serially transplantable tumors were established in 201 nude mice. Metastatic behavior of 7 human tumor cell lines grown in nude mice was judged histologically after sacrifice and SC transplanted tumors could either be highly metastatic (giant cell carcinoma), moderately metastatic (adenocarcinoma PAb), poorly metastatic (squamous cell carcinoma and adenocarcinoma PAa) or non-metastatic (small cell carcinoma and bronchiolo-alveolar carcinoma). The tumor from SC inoculated giant cell carcinoma (PG) could produce regional and/or distant lymph node metastases (12/12) and lung metastases (9/12) in BALB/cA nude mice maintained under semi-barrier system conditions. Six NIH nude mice developed metastatic tumor nodules both in the lymph node and lung (6/6) under the same condition. The highly metastasizing property of PG transplanted tumors remained after passage for as many as 18 times. The limitations of nude mice as an in vivo model for the study of tumor biology are discussed, eg. malignant neoplasms rarely metastasize when transplanted into nude mice. The use of such model (PG) could lead to a better understanding of the biology of metastasis, thus contributing to the development of new approaches to the therapy of metastasis.     

In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice

We determined whether the implantation of human pancreatic cancer cells into the pancreas of nude mice can be used to select variants with increasing metastatic potential. COLO 357 line fast-growing cells were injected into the spleen or pancreas of nude mice. Hepatic metastases were harvested, and tumor cells were reinjected into the spleen or pancreas. This cycle was repeated several times to yield cell lines L3.6sl (spleen to liver) and L3.6pl (pancreas to liver). The variant cells produced significantly higher incidence and number of lymph node and liver metastases than the parental cells. Their increased metastatic potential was associated with increased expression (mRNA and protein) of the proangiogenic molecules basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8. The metastatic cells also exhibited increased motility and invasiveness, which were associated with increased expression of collagenase type IV (MMP-9) and decreased expression of E-cadherin. Collectively, the data show that the orthotopic implantation of human pancreatic cancer cells in nude mice is a relevant model with which to study the biology of pancreatic cancer metastasis and to select variant cell lines with enhanced metastatic potential.     

Isolation of a metastatic ascitic subpopulation from a human lung adenocarcinoma cell line in nude mice and studies on its biological properties

An ascitic subpopulation (Anip) was isolated from human lung adenocarcinoma cell line AGZY-83-a through serial intraperitoneal passages in nude mice. Anip manifested a much higher metastatic potential than the parent line. Spontaneous lung metastases occurred in 95% of the mice xenografted intraperitoneally, and the diffuse pattern of the metastases in the lung was very impressive. Anip also differed more or less from the parent line in morphology, growth, rate, electrophoretic mobility, platelet aggregating activity and noticeably in the chromosome histogram. The present model may be used in the study of tumor invasion and metastasis, as large amounts of well dispersed metastatic human carcinoma cells could be reproduced by ascites and much information about the biology of invasion and metastasis of human tumor cells in nude mice could be obtained.     

The importance of orthotopic implantation to the isolation and biological characterization of a metastatic human clear cell renal-carcinoma in nude-mice

We established a new human renal cell carcinoma system to study some properties of metastatic renal cancer cells and the influence of the organ environment on their metastatic potential. Renal cell carcinoma obtained from a surgical specimen was dissociated enzymatically. Cells were injected into the subcutis, kidney, cecal wall, and spleen of nude mice. Tumors grew in the subcutis and kidney. Only kidney tumors produced distant metastasis. Subcutaneous tumors were avascular and encapsulated, whereas kidney tumors were highly vascularized and invaded the kidney parenchyma. Cell lines were also established from several spontaneous lung metastases. The most metastatic cell line (LM-6) expressed higher levels of basic fibroblast growth factor, gelatinase, and urokinase activity. These results show that human neoplasms are heterogeneous for biologic properties, that orthotopic implantation is essential for the selection, growth, and metastasis of human renal cell carcinoma cells, and that metastatic cells must possess multiple properties to enable them to complete the process.     

Reproducible metastatic growth of K-562 human myelogenous leukemia cells in nude mice.

A reproducible metastatic growth of K-562 human myelogenous leukemia cells occurred in immunodeficient athymic (nude) mice. Although previous studies have shown that K-562 cells grow as local subcutaneous myelosarcomas which continuously release leukemia cells into the systemic circulation in adult mice, metastases were not observed. However, the subcutaneous "priming" of newborn nude mice resulted in the metastatic proliferation of leukemia cells in the lungs, kidneys, brain, and lymph nodes. Three sc injections of 5 X 10(6) K-562 cells on days 1, 7, and 14 of life produced metastases in 51% of the mice. When the initial series of injections was followed by iv injections on days 35 and 42, the incidence of metastases increased to 67%. Karyotypes demonstrated that the tumor cells retained the same human chromosome markers as those in the human patient and tissue culture. These procedures may provide a model for study of the mechanisms of metastases and for chemotherapeutic and immunotherapeutic trials against metastases of neoplasms of human origin.     

Clonal selection within metastatic SP1 mouse mammary tumors is independent of metastatic potential

Our previous studies using randomly integrated plasmid DNA as unique clonotypic markers of SPI mouse mammary tumor cells transplanted into syngeneic CBA/J or nude mice demonstrated reproducible selection and eventual overgrowth of the primary transplant tumors by genotypically distinct metastatic subclones. Two independent metastatic SPI clones, neo5 and ras1, were shown to exhibit "clonal dominance" relative to the non-metastatic SPI tumor-cell population. These results suggested that the capacity for preferential growth within the tumors may be related to cellular properties associated with metastatic ability. To investigate the clonal interactions of metastatic SPI clones present within the same tumor mass, we have analyzed tumors composed of paired mixtures of neo5 and ras1. The tumors were monitored for the relative proportion of each clone by Southern blot analysis. The ras1 clone was found to dominate over the neo5 clone in the majority of tumors examined, even when present as 1% of the mixed inoculum. This represents a 20- to 50-fold enrichment of ras1, while the proportion of neo5 within the tumors was reduced at least 5-fold. No evidence for selection of either clone was seen during co-culture in vitro. Neo5 and ras1 are indistinguishable with respect to tumorigenic and metastatic potential when inoculated separately into different mice, suggesting that clonal dominance is independent of metastatic ability. Analysis of the metastases resulting from mixed inocula indicates that it is possible for a subpopulation representing less than 1% of the primary tumor mass to give rise to metastases. This also suggests that the process of metastasis within metastatic tumors is independent of clonal dominance.     

Environmental and genetic factors determine the level of NK activity of nude mice and affect their suitability as models for experimental metastasis

In these studies we have evaluated the effects of age, genetic background, and housing conditions on the NK-cell activity of nude mice measured in vitro and resistance to tumor metastasis in vivo. BALB/cAnN nude mice exhibited lower levels of NK-cell-mediated cytotoxicity than age-matched N:NIH(S) nude mice maintained under similar housing conditions. The stronger response of N:NIH(S) nude mice was observed also after experimental activation of NK cells by Corynebacterium parvum. Nude mice maintained under barrier conditions have weaker NK-cell activity than mice maintained under conventional conditions. The incidence of experimental pulmonary metastases of allogeneic tumors injected into nude mice was inversely correlated with the levels of NK-cell-mediated cytotoxicity. Thus, 3-week-old BALB/cAnN nude mice raised under barrier conditions were more sensitive to development of experimental metastasis than age-matched N:NIH(S) nude mice maintained under barrier conditions or nude mice of either strain maintained under conventional conditions. In both strains, however, the relative differences in metastatic potential among the tumor cell lines observed in syngeneic recipients were maintained. We conclude that young BALB/cAnN nude mice raised under barrier conditions may provide a valuable in vivo model for studying metastasis of neoplasms.     

Early tumor growth in metastatic organs influenced by the microenvironment is an important factor which provides organ specificity of colon cancer metastasis

We have previously demonstrated that liver metastases in nude mice and lung metastases in nude rats occurred specifically, when KM12SM human colon carcinoma cells were inoculated orthotopically into the cecal wall of nude mice and rats. To clarify the relationship between the tumor growth potential in the metastatic organs and the metastatic organ preference in these two metastatic models, we have evaluated the in vitro cell growth activities affected by the organ conditioned medium (CM) from the liver and lung, and the in vivo growth activities of the ectopic implanted tumors in the liver and lung. The tumorigenicity of the ectopic implanted tumors was 100% in mouse liver, 33% in rat liver, 50% in mouse lung, and 75% in rat lung. The crude liver CM of the animals showed inhibitory activities for KM12SM cell growth in a dosage-dependent manner, and the crude lung CM stimulated KM12SM cell growth. The liver CM of nude mice inhibited the KM12SM cell growth more strongly compared with the CM of nude rats, and the lung CM of nude rats was more strongly stimulated compared with the CM of nude mice. The liver CM of nude mice had non-heparin binding factors, which stimulated or inhibited KM12SM cell growth, in a molecular weight range of 50 to 100 kDa. By contrast, the liver CM of nude rats showed no growth stimulating activity for KM12SM cells. These results suggest that the metastatic organ specificity of KM12SM cells may depend on the early tumor growth influenced by the microenvironment in metastatic organs.     

人胃癌组织块裸鼠原位移植/转移模型的建立

 目的　用肿瘤组织块原位移植 ,建立人胃癌裸小鼠原位移植 /转移模型。方法　以人胃低分化腺癌细胞系接种于裸小鼠皮下 ,形成稳定传代的皮下移植瘤 ,再取该肿瘤组织块原位移植于裸鼠胃壁 ,观察移植肿瘤的生长状况、移植成功率和自发转移的发生率。结果　原位移植成功率 (成瘤率 )为 1 0 0 %、局部淋巴结转移率 1 0 0 %、远处淋巴结转移率 90 %、肝转移发生率为 75%。荷瘤鼠的中位生存期为 1 4周 ,晚期出现消瘦和全身衰竭。结论　该裸小鼠原位移植 /转移模型的生物学行为与人胃癌自然生长和转移过程相似 ,可作为一种有价值的工具用于胃癌转移机理和抗转移实验治疗的研究。     

Evidence that intravenously derived murine pulmonary melanoma metastases can originate from the expansion of a single tumor cell

The purpose of these studies was to determine whether hematogenous clonal pulmonary melanoma metastases originate from the expansion of a single cell and if so, by extrapolation, metastasis can be considered a cloning process. Three different cell lines of murine K-1735 melanoma with different metastatic properties and unique karyotypes were injected i.v. into syngeneic C3H/HeN mice as multicell aggregates of individual cell lines or combinations of cell lines. Resultant solitary lung metastases were isolated in culture as individual lines and then karyotyped. Even when heterogeneous clumps of tumor cells were injected, the individual metastases exhibited a karyotype unique to one metastatic cell type. Furthermore, when cellular aggregates were composed of metastatic cells admixed with cells that were tumorigenic but nonmetastatic, the resultant metastases exhibited only the karyotype of the metastatic cells. This finding suggests that the presence of metastatic cells did not change the inability of nonmetastatic cells to proliferate in a distant organ. Collectively, the results indicate that the resultant metastases were of clonal origin owing to the expansion of a single metastatic tumor cell in the lung parenchyma.     

Development of a metastatic model for human endometrial carcinoma using orthotopic implantation in nude-mice

We have developed an orthotopic model for human endometrial carcinoma in nude mice. The human serous papillary endometrial carcinoma cell line SPEC-2 was injected into the subcutis (ectopic site) or uterine wall (orthotopic site) of athymic mice. Tumors grew in both locations locally. However, only uterine wall tumors produced metastases in regional and distant lymph nodes and to the lungs and liver. Cell lines were established in culture from these uterine tumors and from lung and liver metastases, and then these cells were injected into the uteri of additional mice. The metastatic potential of the lines subsequently established from tumors growing in vivo was not significantly higher than the already highly metastatic parental culture cells. All SPEC-2 cell lines expressed high levels of both 72-kDa and 92-kDa collagenase type IV activity. mRNA for transforming growth factor-alpha, basic fibroblast growth factor, and epidermal growth factor-receptor was constant among the cell lines. These data support the concept that the orthotopic implantation of human endometrial carcinoma cells into the uteri of nude mice provides a valuable model for studying the biology of human endometrial adenocarcinoma.     

Intranasal therapy with an adenoviral vector containing the murine interleukin-12 gene eradicates osteosarcoma lung metastases.

The purpose of these studies was to determine the effect of adenovirus-mediated interleukin-12 (IL-12) gene transfer on the growth and development of osteosarcoma (OS) lung metastases in nude mice. A nude mouse model was produced by repetitive cycling of human SAOS OS cells through the lung. The resultant SAOS-LM6 cell line produced microscopic lung metastases by 5-6 weeks after i.v. injection of the tumor cells, with visible lung metastases present 8 weeks after injection. Transfection of SAOS-LM6 cells with a plasmid containing the murine IL-12 gene resulted in a decrease in metastatic potential. Animals injected with IL-12-transfected clones had fewer metastases compared with mice injected with SAOS-LM6 cells transfected with a control plasmid. Furthermore, nasal delivery of an adenoviral vector containing the murine IL-12 gene resulted in the inhibition of pulmonary metastases. Together, these data indicate that IL-12 may be an effective agent against OS and that nasal delivery may offer a unique way to deliver the gene to the local tumor environment, potentially decreasing systemic toxic effects.