裸小鼠原位移植筛选高转移性人骨肉瘤细胞系及其生物学特性分析

目的　在裸小鼠体内筛选人骨肉瘤高肺转移性细胞亚群 ,为实验研究提供模型。方法　利用原位移植方法 ,将人骨肉瘤细胞系SOSP 96 0 7皮下移植瘤接种于裸鼠胫骨骨髓腔。筛选到第 2代时 ,有 1例发生肺转移 ;以皮下扩增转移灶 ,再次原位移植 ,第 3代肺转移率达 83% ( 5 / 6 )。用组织块法培养收集 ,比较其与母系在形态结构、细胞核型 ,vimentin ,actin ,NSE抗原表达及体积倍增时间等方面的差别。结果　同母系相比较 ,该转移细胞保持了人骨肉瘤细胞的组织学特征 ,生物学特性也有较大差别。结论　该细胞亚群是一株新建立的人骨肉瘤细胞高转移性亚系     

A novel orthotopic murine model provides insights into cellular and molecular characteristics contributing to human osteosarcoma

As a reliable model for osteosarcoma is lacking, three human cell lines (SaOS-2, U2OS and 143B) were evaluated in cell-based assays for proliferation, adhesion, migration, invasion, anchorage-independent growth, angiogenesis, mineralised nodule formation, plasmid transfection and oligonucleotide transfection. Tumor take and metastasis after orthotopic injection of the three cell lines into mice was monitored. The levels of expression of typical bone markers were determined with semi-quantitative RT-PCR in cultured cells, primary tumors, and for the SaOS-2 cell line, the metastases. Tumors grew and spread to the lungs within 3 and 5 weeks respectively, mimicking the clinical progression of the disease as analysed by x-ray. Expression of molecular markers in SaOS-2 indicated a mostly differentiated cell type at the primary and secondary sites. The ability of osteosarcoma cells to interact with collagen-1 and to form mineralised deposits correlated positively with tumor aggression in vivo. Expression of alkaline phosphatase was a common theme in both tumor models at the primary site. The newly established SaOS-2 model should allow the testing of candidate anti-osteosarcoma agents as well as dissection of more intricate mechanisms involved in human osteosarcoma.     

A nude mouse model of human osteosarcoma lung metastases for evaluating new therapeutic strategies

The purpose of these studies was to develop a metastatic osteosarcoma nude mouse model to evaluate the in vivo efficacy of new therapeutic compounds. Human SAOS-2 osteosarcoma cells (10⁶ cells) were injected i.v. into nude mice. Cells isolated from a rare pulmonary metastases 6 months later were established (SAOS-LM1) in culture and re-injected. This procedure was repeated 5 additional times to produce the SAOS-LM6 cell line. Visible pulmonary nodules were present 8 weeks following i.v. injection of 10⁶ SAOS-LM6 cells as compared to 17 weeks using SAOS-LM2 cells. Microscopic SAOS-LM6 pulmonary metastases were demonstrated at 6 weeks. Administration of adriamycin on week 9 resulted in regression of macroscopic SAOS-LM6 lung tumors. The ability of the model to be used to evaluate the effectiveness of a biologic agent against microscopic disease was also verified. It was concluded that this model can assess therapeutic efficacy and therefore, may have a role in investigating the potential of novel approaches aimed at eliminating pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

A patient-like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer

Clinically relevant animal models of human cancer are important for studies of cancer biology, invasion and metastasis, and for investigating new forms of prognostic diagnosis and therapy. An ovarian tumor line (RMG-1: human clear cell carcinoma of the ovary) previously grown subcutaneously was implanted ortho-topically as intact tissue into the ovarian capsule of 22 nude mice. The tumors showed progressive growth at the orthotopic site in all animals. Tumor-associated serum galactosyltransferase (GAT) tended to be posi-tive in all nude -mice. The tumors invaded or metastasized to the contralateral ovary, retroperitoneum, mesentery and peritoneum, and omentum, and metastasized to the subcutaneous tissue, lymph nodes and distant organs including the liver, kidney, pancreas, and diaphragm. In striking contrast, subcutaneous trans-plantation of this tumor resulted in growth in only 2 of 5 animals with local lymph node and kidney involve-ment but no retroperitoneal or peritoneal involvement. These findings suggest that orthotopic implantation provides a suitable micro-environment in which ovarian cancer can express its intrinsic clinically-relevant properties. This approach is relevant to the clinical features of ovarian cancer and is thought to be a useful model for studies of therapy for this cancer.© Kluwer Academic Publishers 1998     

Reproduction of occult metastasis of head and neck cancer in nude mice

The presence of occult metastasis is the most important factor that influences the prognosis in patients with head and neck cancer. To reproduce occult metastasis of oral cancer cells, we serially resected the primary focus in an orthotopic implantation model to examine when metastasis of cancer cells occurs. Human squamous cell carcinoma was implanted into the tongue of nude mice divided into two groups, non-surgery and surgery groups. Mice in the non-surgery group were sacrificed, and the tongue cancer and cervical lymph nodes were resected simultaneously. In the surgery-group, resection of the tongue cancer was performed, and the cervical lymph nodes were resected on day 28. For the non-surgery-group, the incidences of metastasis were 0%, 9%, 36%, 91% and 100% on days 3, 7, 14, 21 and 28, respectively. For the surgery-group, resection of the tongue cancer was performed on days 3, 7 and 14, and the incidence of metastasis on day 28 was 0%, 82% and 91%, respectively. The occult metastasis was reproduced using resected primary cancer on day 7. This time-based model may be useful to clarify the mechanism of metastasis and to develop new treatments.     

An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential

To provide an investigative tool for the study of osteosarcoma (OSA) biology we have developed a syngeneic (balb/c) murine model of OSA, using cell lines derived from a spontaneously occurring murine OSA (Schmidt et al. Differentiation 1988; 39: 151-60). This model is characterized by orthotopic primary tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and clonally related variants (K7M2 and K12) that differ in pulmonary metastatic potential. Primary tumor and pulmonary metastasis histology was consistent with OSA in human patients. Expression of bone sialoprotein, biglyan, decorrin, and osteopontin was suggestive of bone lineage cells. The development and use of a more aggressive OSA cell line (K7M2) resulted in spontaneous metastasis to the lungs in over 90% of mice, whereas metastases were seen in only 33% of mice when a less aggressive OSA cell line (K12; Schmidt et al. Differentiation 1988; 39: 151-60) was used. Death from metastasis occurred at a median of 76 days using K7M2 whereas no median was achieved after 140 days using K12. Angiogenic potential, characterized by CD31 and factor VIII staining of primary tumors and pulmonary metastases, was greater in the K7M2 model compared to the K12 model. No significant differences in the in vitro or in vivo expression of angiogenesis associated genes (flt1, flt4, TIE1, TIE2, and VEGF) was found between K7M2 and K12. This well characterized and relevant model of OSA will be a valuable resource to improve our understanding of the biology and treatment of metastasis in OSA. This revised version was published online in July 2006 with corrections to the Cover Date.     

Importance of orthotopic implantation for human tumors as model systems: relevance to metastasis and invasion

Transplantation of human tumors into immunodeficient athymic nude mice has become an important experimental approach to study the biology and the treatment of human cancer. Most human tumor xenograft experiments have employed subcutaneous injection procedures, but the main limit of this technique is the lack of metastasis from the subcutaneous site. The possibility of producing experimental metastasis by intravenous injection of cells in the animals has been known for a long time, and it has been recently reported that tumorigenic properties and metastatic ability of human cancer can be altered by transplantation of the tumor into its organ or tissue of origin in the recipient animals (orthotopic transplantation). In this paper we review (1) the principal techniques of orthotopic injection of most solid tumors, (2) the most recent techniques to achieve experimental metastases, and (3) the methods for preparing tumor cell suspensions from human surgical specimens suitable for transplantation into animals. These animal models should be used for a more appropriate evaluation of new antitumor treatments including the ones targeted to inhibit metastatic spread.     

Characterization of spontaneous metastasis in an aggressive breast carcinoma model using flow cytometry

Studies of metastasis can be accelerated and provide more mechanistic information using cell lines which reproducibly and aggressively metastasize, and which are accurately and easily detected in tissues at all stages of the metastatic process. Although reporter proteins such as green fluorescent protein (GFP) and β-galactosidase have improved the tracking of tumor cells in vivo, their measurement has often been limited to visual observation and manual counting. In this study, we exploited the highly sensitive and objective quantitation provided by flow cytometry to characterize, in detail, the sequence of events associated with orthotopic metastasis in a highly aggressive mouse model. Following stable transfection of the MDA-MB-435 breast carcinoma cell line with GFP, we utilized an in vivo selection process to isolate a variant exhibiting increased primary tumor growth and metastasis. As few as one fluorescent tumor cell per 200,000 host cells could be accurately detected in dissociated tissues by flow cytometry, allowing us to demonstrate that metastatic cells migrate to the lungs of SCID mice very early after orthotopic implantation. Tumor burden in lungs increased in a smooth continuous manner, until death approximately eight weeks later. Levels of circulating tumor cells in blood were also detectable at an early timepoint, but remained relatively low throughout the course of secondary tumor development in the lungs. Surgical removal of the primary tumor at various times after inoculation significantly affected lung tumor burden, supporting the concept that circulating tumor cells in blood inefficiently initiate distal metastases. Furthermore, the continuing contribution to metastasis by the primary tumor was independent of tumor mass. The combined characteristics of enhanced orthotopic metastasis and quantitative detection in blood and tissues will make this a useful new model for the characterization of the multi-stage progression of cancer, and the preclinical evaluation of anti-neoplastic therapies. This revised version was published online in July 2006 with corrections to the Cover Date.     

An ultra-metastatic model of human colon cancer in nude mice

An ultra-high metastatic model of human colon cancer was developed in order to represent highly malignant patient disease for which there is no current model. Surgical orthotopic implantation (SOI) of a histologically intact liver metastasis fragment derived from a surgical specimen of a patient with metastatic colon cancer was initially implanted in the colon, liver and subcutaneously in nude mice. This tumor did not metastasize for the first 10 passages. At the eleventh passage, the tumor exhibited metastasis from the colon to the liver, spleen, and lymph nodes. At this time, two selective passages were carried out by transplanting resulting liver metastases in the nude mice to the colon of additional nude mice. After these two passages, the tumor became stably ultra-metastatic and was termed AC3488UM. One-hundred percent of mice transplanted with AC3488UM with SOI to the colon exhibited local growth, regional invasion, and spontaneous metastasis to the liver, lymph nodes, and spleen. While the maximum size of the primary tumor was 0.9 g, the metastatic liver was over 9 times the weight of the normal liver with the maximum weight of the metastatic liver over 12 g. Liver metastases were detected by the tenth day after transplantation in all animals. Half the animals died of metastatic tumor 25 days after transplantation. Histological characteristics of AC3488UM tumor were poorly differentiated adenocarcinoma of colon. Mutant p53 is expressed heterogeneously in the primary tumor and more homogeneously in the liver metastasis suggesting a possible role of p53 in the liver metastasis. The human origin of AC3488UM was confirmed by positive fluorescence staining for in situ hybridization of human DNA. The AC3488 human colon-tumor model with its ultra-high metastatic capability in each transplanted animal, short latency and a short median survival period is different from any known human colon cancer model and will be an important tool for the study of and development of new therapy for highly metastatic human colon cancer.     

Magnetic resonance imaging for the evaluation of a novel metastatic orthotopic model of human neuroblastoma in immunodeficient mice

Neuroblastoma is the second most common solid tumor in children. So far few tumor models for this cancer have been reported in mice. We have created a murine tumor model for studying human neuroblastoma based on surgical orthotopic implantation in scid mice. Small fragments of subcutaneous tumors of SK-N-BE(2) human neuroblastoma cells expressing enhanced green fluorescent protein were surgically implanted near the left adrenal gland of scid mice. One hundred percent of the animals (n=21) successfully implanted developed a large retroperitoneal tumor and became moribund between 22 and 57 days after implantation (mean survival time = 41 days). At the time of sacrifice the presence of bone marrow metastasis was detected by RT-PCR for green fluorescent protein in 95% of the cases. The growth of small tumor implants could be easily visualized and quantified by surveillance MR imaging, with a resolution of 117×117×750 μm in two orthogonal planes allowing accurate volume measurements, as well as assessment of necrosis and tissue invasion. This novel model should be a valuable tool to study the biology and therapeutic approaches to neuroblastoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel orthotopic model of breast cancer metastasis to bone

Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.     

人胰腺癌裸鼠移植瘤浸润和转移方式的研究

为了探讨胰头癌浸润及转移规律，我们用自建的三株人胰头癌裸鼠移植瘤，接咱在鼠背部、腹腔及抓垫皮下。结果表明：三株移植瘤背部接种均以局部浸润为主，可经淋巴疲乏转移至局部淋巴结（ＰＣＴ－３转移率为３３％；ＰＣＴ－４为４０％；ＰＣＴ－５为３３％），无远处淋巴结和（或）脏器转移；腹腔接种及抓垫接种与背部皮下接种结果相似。肿瘤的生长速度和浸润及转移的能力与肿瘤分化程度有一定关系。     

Development of a high metastatic orthotopic model of human renal cell carcinoma in nude mice: benefits of fragment implantation compared to cell-suspension injection

In this study we compared the metastatic rate of human renal cell carcinoma SN12C in two orthotopic nude mouse models: surgical orthotopic implantation (SOI) of histologically intact tumor tissue and cellular orthotopic injection (COI) of cell suspensions in the kidney. The primary tumors resulting from SOI were larger and much more locally invasive than primary tumors resulting from COI. SOI generated higher metastatic expression than COI. The differences in metastatic rates in the involved organs (lung, liver, and mediastinal lymph nodes) were 2–3 fold higher in SOI compared to COI (P<0.05). Median survival time in the SOI model was 40 days, which was significantly shorter than that of COI (68 days) (P<0.001). Histological observation of the primary tumors from the SOI model demonstrated a much richer vascular network than the COI model. Lymph node and lung metastases were larger and more cellular in the SOI model compared to COI. We conclude that the tissue architecture of the implanted tumor tissue in the SOI model plays an important role in the initiation of primary tumor growth, invasion, and distant metastasis. This study directly demonstrates that the implantation of histologically intact tumor tissue orthotopically allows accurate expression of the clinical features of human renal cancer in nude mice. This model should be of value in cancer research and antimetastatic drug discovery for renal cancer, a currently very poorly responding malignancy.     

共接种淋巴管内皮细胞对乳腺癌细胞和骨肉瘤细胞在裸鼠体内生长和转移的影响

目的分析淋巴管生成对肿瘤生长和转移的影响。方法从人淋巴结中分离纯化淋巴管内皮细胞(HLyECs),将人乳腺癌细胞系和人骨肉瘤细胞系分别单独或与HLyECs共同接种于裸鼠皮下,比较肿瘤生长和肺转移的差别。用伊文氏蓝显示瘤周淋巴管;用人和小鼠PDPN的免疫组织化学显示肿瘤组织中的淋巴管。用MTT法分析淋巴管内皮细胞的增殖。结果与单纯接种组相比,共接种HLyECs促进乳腺癌细胞的生长和转移,瘤周和瘤内淋巴管密度增加(P<0.01),存在人和鼠PDPN阳性的淋巴管;而共接种HLyECs对骨肉瘤的生长和转移无影响,未见瘤内和瘤周淋巴管。与骨肉瘤细胞相比,乳腺癌细胞的条件培养基明显促进淋巴管内皮细胞增殖(P<0.01)。结论共接种淋巴管内皮细胞可以促进乳腺癌细胞的生长及癌组织中淋巴管生成。     

Establishment of lymph node metastatic model for human gastric cancer in nude mice and analysis of factors associated with metastasis

The actual mechanisms responsible for lymph node metastasis in gastric cancer are still unclear. To investigate the mechanisms of lymph node metastasis in gastric cancer, we established a lymph node metastatic model for human scirrhous gastric carcinoma. Lymph node metastasis had frequently developed after orthotopic implantation of OCUM-2M LN derived from a scirrhous gastric cancer cell line, OCUM-2M, which had low capacity for lymph node metastasis. We elucidated the different characteristics including binding ability, migratory capacity and immunoresponses induced by the cell surface molecules of these two cell lines. The binding ability to Matrigel and migratory capacity of OCUM-2M LN cells were significantly greater than those of OCUM-2M cells. On flow cytometric analysis, both OCUM-2M and OCUM-2M LN cells strongly expressed HLA-I (99.5 and 97.1%) and LFA-3 (76.6 and 99.2%) in level of expression between the two cell lines, but neither cell line expressed HLA-II (0.0 and 0 .0%), B7-1 (0.0 and 0.0%) or B7-2 (0.4 and 0.3%). ICAM-1 expression in OCUM-2M LN cells was weaker (0.7%) than that in OCUM-2M cells (36.8%). Strong adhesiveness and cytotoxicity of mononuclear lymphocytes for OCUM-2M cells were observed in adhesion and cytotoxic assays, both of which were significantly decreased by the addition of anti-ICAM-1 antibodies. On the other hand, the adhesiveness and cytotoxicity of OCUM-2M LN cells were significantly less than those of OCUM-2M cells, and were not affected by the addition of anti-ICAM-1 antibodies. These findings suggest that decreased ICAM-1 expression in a new gastric cancer cell line with a high rate of lymph node metasta-sis may in turn decrease immune responses mediated through LFA-1-dependent effector cell adhesion, and that this escape from the immunosurveillance system may be one of the factors inducing lymph node metas-tasis. In conclusion, we established a gastric cancer cell line, OCUM-2M LN, with a high rate of lymph node metasta sis. An in vivo lymph node-metastatic model with this cell line should be useful for analysing the mech-anism and therapeutic approach of lymph node metastasis. © Rapid Science Ltd.     

Invasion-inhibiting factor 2-albumin conjugate inhibits invasion and spontaneous metastasis of MKL-4 human breast cancer cells transplanted into female nude mice

Invasion-inhibiting factor 2 (IIF-2) and its albumin conjugate have been reported to inhibit spontaneous metastasis of highly metastatic cancer cells with no effect on primary tumor growth. To confirm the inhibitory effects of the IIF-2 conjugate on tumor invasion and spontaneous metastasis, we administered the conjugate intra-peritoneally (i.p.) to female nude mice bearing transplanted tumors with MKL-4 cells, which are MCF-7 human breast cancer cells cotransfected with fibroblast growth factor 4 and lacZ. Neither 10 nor 20 mg/kg doses of the conjugate caused any inhibition of primary tumor growth, but 20 mg/kg significantly inhibited tumor invasion and spontaneous metastasis. Tumor invasion was measured by a novel computer-assisted image analysis. Spontaneous microscopic metastases into lymph nodes and distant organs were measured by whole organ staining for ß-galactosidase activity and observed with a dissecting microscope. The dose of 10 mg/kg significantly inhibited tumor invasion but not metastasis. Interestingly, the number of factor VIII-positive microvessels in the tumors was not reduced by treatment at either dose level. These findings suggest that the anti-invasive effect of the IIF-2 conjugate may reduce both lymphatic and hematogenous metastases in this MKL-4 metastasis model without affecting angiogenesis.     

临床PET-CT在裸鼠鼻咽癌移植瘤动物模型中的应用

目的探讨临床PET-CT在裸鼠鼻咽癌移植瘤放疗模型中的应用。方法①18只裸鼠鼻咽癌移植瘤动物模型分别于放疗前测量瘤体大小并行18F-FDGPET-CT显像。②按瘤体大小分别将裸鼠分成3组,显像结果与组织病理学结果进行比较;③取其中3只荷瘤鼠,分别按每床位3min、5min、8min时间采集,图像处理后由两位以上有经验的核医学科医师判断图像质量。结果①放疗前裸鼠鼻咽癌移植瘤18F-FDGPET-CT显像瘤体处均为阳性。②组1,瘤体平均T/N=1.203±0.189;组2,瘤体平均T/N=2.086±0.456;组3,瘤体平均T/N=1.535±0.160。③不同采集时间对图像质量影响不大。结论①临床PET-CT可用于裸鼠鼻咽癌移植瘤动物模型的研究,对于裸鼠鼻咽癌移植瘤动物模型,3min/床位采集是可行的。②瘤体直径在1.0～1.4cm进行PET-CT显像比较适宜。     

人巨细胞肺癌裸鼠移植瘤中mts1和nm23-H1表达及其与肺癌淋巴结转移关系

目的 探讨肿瘤转移相关基因mts1和nm23-H1在人巨细胞肺癌裸鼠移植瘤中的表达及其与啼癌淋巴结转移的关系。方法 用高转移的人巨细胞肺癌瘤株PLA-801—D和低转移的人巨细胞肺癌瘤株PLA-801—C在裸鼠中建立转移模型,观察瘤细胞在裸鼠淋巴结及肺中的转移情况;原位杂交检测mts1和nm23-H1mRNA在移植瘤中的表达。结果 PLA-801—D在12只移植裸鼠中均有淋巴结转移,转移率100%,2只裸鼠中有肺转移,转移率17％;PLA-801—C在12只移植的裸鼠中均无淋巴结及肺转移。mts1mRNA在PLA-801—D、PLA-801-C裸鼠移植瘤中阳性表达分别为12例、2例;nm23-H1mRNA在PLA-801—D、PLA-801—C裸鼠移植瘤中阳性表达分别为12例、11例。结论 mts1 mRNA表达与人巨细胞啼癌裸鼠移植瘤淋巴结转移呈正相关,nm23-H1 mRNA表达与人巨细胞肺癌裸鼠移植瘤淋巴结转移不相关。