Nestin-linked green fluorescent protein transgenic nude mouse for imaging human tumor angiogenesis

We report here a novel transgenic nude mouse for the visualization of human tumor angiogenesis. We have recently shown that the neural stem cell marker nestin is expressed in hair follicle stem cells and blood vessel networks in the skin of C57/B6 transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). Others have shown ND-GFP is expressed in the brain, pancreas, and testes in these mice. In the present study, the nestin ND-GFP gene was crossed into nude mice on the C57/B6 background to obtain ND-GFP nude mice. ND-GFP was expressed in the brain, spinal cord, pancreas, stomach, esophagus, heart, lung, blood vessels of glomeruli, blood vessels of skeletal muscle, testes, hair follicles, and blood vessel network in the skin of ND-GFP nude mice. Human lung cancer, pancreatic cancer, and colon cancer cell lines as well as a murine melanoma cell line and breast cancer tumor cell line expressing red fluorescent protein were implanted orthotopically, and a red fluorescent protein-expressing human fibrosarcoma was implanted s.c. in the ND-GFP nude mice. These tumors grew extensively in the ND-GFP mice. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, visualized by dual-color fluorescence imaging. Results of immunohistochemical staining showed that CD31 was expressed in the ND-GFP-expressing nascent blood vessels. The ND-GFP transgenic nude mouse model enables the visualization of nascent angiogenesis in human and mouse tumor progression. These results suggest that this model is useful for the imaging of the angiogenesis of human as well as rodent tumors and visualization of the efficacy of angiogenetic inhibitors.     

Transgenic nude mouse with ubiquitous green fluorescent protein expression as a host for human tumors

We report here the development of the transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives GFP expression in essentially all tissues. In crosses between nu/nu GFP male mice and nu/+ GFP female mice, the embryos fluoresced green. Approximately 50% of the offspring of these mice were GFP nude mice. Newborn mice and adult mice fluoresced very bright green and could be detected with a simple blue-light-emitting diode flashlight with a central peak of 470 nm and a bypass emission filter. In the adult mice, the organs all brightly expressed GFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum. The following systems were dissected out and shown to have brilliant GFP fluorescence: the entire digestive system from tongue to anus; the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart and major arteries and veins. The skinned skeleton highly expressed GFP. Pancreatic islets showed GFP fluorescence. The spleen cells were also GFP positive. Red fluorescent protein (RFP)-expressing human cancer cell lines, including PC-3-RFP prostate cancer, HCT-116-RFP colon cancer, MDA-MB-435-RFP breast cancer, and HT1080-RFP fibrosarcoma were transplanted to the transgenic GFP nude mice. All of these human tumors grew extensively in the transgenic GFP nude mouse. Dual-color fluorescence imaging enabled visualization of human tumor-host interaction by whole-body imaging and at the cellular level in fresh and frozen tissues. The GFP mouse model should greatly expand our knowledge of human tumor-host interaction.     

Application of Benchtop-magnetic resonance imaging in a nude mouse tumor model

Background

MRI plays a key role in the preclinical development of new drugs, diagnostics and their delivery systems. However, very high installation and running costs of existing superconducting MRI machines limit the spread of MRI. The new method of Benchtop-MRI (BT-MRI) has the potential to overcome this limitation due to much lower installation and almost no running costs. However, due to the low field strength and decreased magnet homogeneity it is questionable, whether BT-MRI can achieve sufficient image quality to provide useful information for preclinical in vivo studies. It was the aim of the current study to explore the potential of BT-MRI on tumor models in mice.

Methods

We used a prototype of an in vivo BT-MRI apparatus to visualise organs and tumors and to analyse tumor progression in nude mouse xenograft models of human testicular germ cell tumor and colon carcinoma.

Results

Subcutaneous xenografts were easily identified as relative hypointense areas in transaxial slices of NMR images. Monitoring of tumor progression evaluated by pixel extension analyses based on NMR images correlated with increasing tumor volume calculated by calliper measurement. Gd-BOPTA contrast agent injection resulted in a better differentiation between parts of the urinary tissues and organs due to fast elimination of the agent via kidneys. In addition, interior structuring of tumors could be observed. A strong contrast enhancement within a tumor was associated with a central necrotic/fibrotic area.

Conclusions

BT-MRI provides satisfactory image quality to visualize organs and tumors and to monitor tumor progression and structure in mouse models.     

Green fluorescent protein imaging of tumour growth,metastasis, and angiogenesis in mouse models

We have developed a way of imaging metastases in mice by use of tumour cells expressing green fluorescent protein (GFP) that can be used to examine fresh tissue, both in situ and externally. These mice present many new possibilities for research including real-time studies of tumour progression, metastasis, and drug-response evaluations. We have now also introduced the GFP gene, cloned from bioluminescent organisms, into a series of human and rodent cancer-cell lines in vitro, which stably express GFP after transplantation to rodents with metastatic cancer. Techniques were also developed for transduction of tumours by GFP in vivo. With this fluorescent tool, single cells from tumours and metastases can be imaged. GFP-expressing tumours of the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs have also been visualised externally by use of quantitative transcutaneous whole-body fluorescence imaging. GFP technology has also been used for real-time imaging and quantification of angiogenesis.     

The bisphosphonate olpadronate inhibits skeletal prostate cancer progression in a green fluorescent protein nude mouse model.

PURPOSE: Metastatic bone disease is one of the major causes of morbidity and mortality in prostate cancer patients. Bisphosphonates are currently used to inhibit bone resorption and reduce tumor-induced skeletal complications. More effective bisphosphonates would enhance their clinical value. EXPERIMENTAL DESIGN: We tested several bisphosphonates in a green fluorescent protein (GFP)-expressing human prostate cancer nude mouse model. The in vivo effects of four bisphosphonates, including pamidronate, etidronic acid, and olpadronate, on bone tumor burden in mice intratibially inoculated with PC-3-GFP human prostate cancer cells were visualized by whole-body fluorescence imaging and X-ray. RESULTS: The PC-3-GFP cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3-GFP cell growth was monitored by GFP fluorescence and the bone destruction was evaluated by X-ray. We showed that 3,3-dimethylaminopropane-1-hydroxy-1,1-diphosphonic acid (olpadronate) was the most effective bisphosphonate treatment in reducing tumor burden as assessed by GFP imaging and radiography. The GFP tumor area and X-ray score significantly correlated. Reduced tumor growth in the bone was accompanied by reduced serum calcium, parathyroid hormone-related protein, and osteoprotegerin. CONCLUSIONS: The serum calcium, parathyroid hormone-related protein, and osteoprotegerin levels were significantly correlated with GFP area and X-ray scores. Treatment with olpadronate reduced tumor growth in the bone measured by GFP and X-ray imaging procedures. Imaging of GFP expression enables monitoring of tumor growth in the bone and the GFP results complement the X-ray assessment of bone disease. The data in this report suggest that olpadronate has potential as an effective inhibitor of the skeletal progression of clinical prostate cancer.     

Characterization of two mouse models of metastatic pheochromocytoma using bioluminescence imaging

Pheochromocytoma is the most common tumor of the adrenal medulla in adults. The lack of sensitive animal models of pheochromocytoma has hindered the study of this tumor and in vivo evaluation of antitumor agents. In this study we generated two sensitive luciferase models using bioluminescent pheochromocytoma cells: an experimental metastasis model to monitor tumor spreading and a subcutaneous model to monitor tumor growth and spontaneous metastasis. These models offer a platform for sensitive, non-invasive and real-time monitoring of pheochromocytoma primary growth and metastatic burden to follow the course of tumor progression and for testing relevant antitumor treatments in metastatic pheochromocytoma.     

Real-time imaging of tumor progression in a fluorescent orthotopic mouse model of thyroid cancer

There is a need for a clinically relevant mouse model of thyroid cancer that enables real-time, non-invasive monitoring of tumor growth, progression, and drug response over time. Human thyroid cancer cell lines NPA (papillary) and KAK-1 (anaplastic) were stably transfected to express either red or green fluorescent protein. Cancer cells were injected into the thyroid glands of 8-week-old athymic mice. The animals were imaged with whole-body fluorescence imaging weekly and sacrificed when premorbid. At necropsy, the primary tumor was resected en bloc with the respiratory system for processing and analysis. Histology was performed on fixed tissue specimens for review of morphologic findings. Both anaplastic and papillary thyroid cancer cell lines led to robust development of orthotopic fluorescent tumors in nude mice. Injection of 5×10(5) cancer cells was sufficient for tumor development. Tumors were visualized for both cell lines via non-invasive imaging as early as 3 weeks post-implantation and were monitored over time. Time to premorbid condition varied between mice and was associated with a primary tumor growth pattern (early local compression of the esophagus vs. late metastatic disease) rather than tumor size. At necropsy, tumor fluorescence demonstrated metastases in the lungs, lymph nodes and vessels that were not visible under white light. Thus an orthotopic mouse model of thyroid cancer has been developed that replicates the major clinical features of thyroid cancer and enables real-time, non-invasive monitoring of tumor progression. This model should permit preclinical evaluation of novel thyroid cancer therapeutics.     

Necrosis avid near infrared fluorescent cyanines for imaging cell death and their use to monitor therapeutic efficacy in mouse tumor models

Quantification of tumor necrosis in cancer patients is of diagnostic value as the amount of necrosis is correlated with disease prognosis and it could also be used to predict early efficacy of anti-cancer treatments. In the present study, we identified two near infrared fluorescent (NIRF) carboxylated cyanines, HQ5 and IRDye 800CW (800CW), which possess strong necrosis avidity. In vitro studies showed that both dyes selectively bind to cytoplasmic proteins of dead cells that have lost membrane integrity. Affinity for cytoplasmic proteins was confirmed using quantitative structure activity relations modeling. In vivo results, using NIRF and optoacoustic imaging, confirmed the necrosis avid properties of HQ5 and 800CW in a mouse 4T1 breast cancer tumor model of spontaneous necrosis. Finally, in a mouse EL4 lymphoma tumor model, already 24 h post chemotherapy, a significant increase in 800CW fluorescence intensity was observed in treated compared to untreated tumors. In conclusion, we show, for the first time, that the NIRF carboxylated cyanines HQ5 and 800CW possess strong necrosis avid properties in vitro and in vivo. When translated to the clinic, these dyes may be used for diagnostic or prognostic purposes and for monitoring in vivo tumor response early after the start of treatment.     

THE ENHANCED GREEN FLUORESCENT PROTEIN AS A MARKER FOR HUMAN TUMOR CELLS LABELLED BY RETROVIRAL TRANSDUCTION

Efficient delivery of foreign genes into target cells is needed for gene marking and gene therapy. Since the inception of murine retroviral-mediated gene transfer, there has been an intense interest in gene marker systems that allow direct observation of transferred genes into living cells. Many selectable reporter molecules, such as the neomycin resistance gene NeoR, the bacterial b-galactosidase gene LacZ, the gene for human low-affinity nerve growth factor receptor (NGFR) as well as the …     

Primary transfection as a mechanism for transformation of host cells by human tumor cells implanted in nude mice

Establishment of cell lines in vitro from a human lung cancer xenograft in nude mice resulted in transformed mouse cell lines. The transformed mouse cell lines expressed both mouse-specific and human-specific histocompatibility antigens. Of 3 cell lines, 2 were tumorigenic in BALB/c nude mice but not in normal mice. Tumors formed by the transformed mouse cell lines were fibroblastoid and epithelioid by histology. In addition, tumors exhibited neuroepithelial differentiation by ultrastructural and immunohistochemical analysis. Phenotypically they were similar to the original patient and human xenograft tumor. These data suggest that previous reports of host cell transformation and induction of fibrosarcomas may not be true fibrosarcomas. Human DNA sequences were present in the tumorigenic cell lines, indicating that spontaneous transfection of human tumor DNA into host cells had occurred. The implication of these findings is that human genetic information has been transferred to primary mouse host fibroblasts, which resulted in a transformed as well as a differentiated phenotype.     

裸鼠模型在人膀胱癌中的实验研究现状及展望

人源性膀胱癌裸鼠模型与人类膀胱癌生长特性相似,能更好地模拟膀胱癌在人体内的自然生长过程及许多生物学行为.为深入研究人膀胱癌发生发展与转归机制,进行新型膀胱腔内生物免疫制剂和化疗药物临床前期评价,探索分子靶向治疗策略,建立人源性膀胱癌裸鼠模型具有重要的临床与科研意义.     

Effects of hyperthermia on primary and metastatic tumor growth and host immune response in rats

A hot water bath was used to heat locally a metastasizing carcinoma in Wistar/Furth rats. Applying heat such that intratumor temperature is maintained at a mean value of 42.3 degrees for two 90-min sessions results in a decreased growth rate of the primary tumor as well as distant metastases. Heating the primary tumor for only one 90-min session or heating the leg contralateral to the tumor-bearing limb has no effect on the growth rate of either the primary tumor or metastases. Heat therapy has no detrimental effect on the spleen cell-mediated tumor immune response of rats as tested by an in vitro lymphocytotoxicity assay 1 day later. However, heating isolated spleen cells to similar temperatures in vitro reduces their capacity for in vitro tumor cell killing.     

Selection of metastatic variants from heterogeneous tumor cell lines using the chicken chorioallantoic membrane and nude mouse

The chicken chorioallantoic membrane was used to select variant tumor cell subpopulations from the murine melanoma B16-BL6 and the rat glioma C6 cell lines. Tumor cells were deposited on the chicken chorioallantoic membrane of eggs 10 days postfertilization. Upon hatching, chickens were autopsied, and organs were removed, minced, and implanted s.c. in C57BL/6J mice (for melanoma) or nude mice (for glioma). A glioma growing s.c. from a chicken lung implant metastasized to the liver of the recipient nude mouse, and a melanoma growing s.c. from a chicken liver implant metastasized to the lung of its murine host. The s.c. melanoma contained distinct black and gray areas. Cell lines were established from the s.c. glioma (C6-V-1), from a metastasis of the C6-V-1 tumor (C6-V-2), and from the black and gray regions of the melanoma. Marked differences in lung colonization were seen 14 days after 1 X 10(5) parent BL6, Black, or Gray cultured cells were injected by tail vein into C57BL mice. In four separate experiments, fewer than 15 lung foci per mouse were found when BL6 cells were injected, whereas 100 to several hundred lung melanoma colonies per mouse were observed when Black or Gray cells were inoculated. Four of 18 nude mice bearing the s.c. C6-V-1 glioma developed liver metastases; no metastases have been observed in 15 nude mice bearing the s.c. parent C6 glioma. Significant differences in sensitivities to antineoplastic drugs were demonstrated between parent and variant glioma cell lines. The 33-fold increase in sensitivity to vincristine determined for C6-V-1 cells compared to parent C6 cells was particularly striking. Results suggest that the use of the chicken chorioallantoic membrane in situ, together with the nude mouse, might provide a method suitable for the selection and isolation of aggressive variants in heterogeneous human tumors.     

Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model

We report here whole-body optical imaging, in real time, of genetically fluorescent pancreatic tumors growing and metastasizing to multiple sites in live mice. The whole-body optical imaging system is external and noninvasive. Human pancreatic tumor cell lines, BxPC-3 and MiaPaCa-2, were engineered to stably express high-levels of the Aequorea victoria green fluorescent protein (GFP). The GFP-expressing pancreatic tumor cell lines were surgically orthotopically implanted as tissue fragments in the body of the pancreas of nude mice. Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions that developed in the spleen, bowel, portal lymph nodes, omentum, and liver. Intravital images in the opened animal confirmed the identity of whole-body images. The whole-body images were used for real-time, quantitative measurement of tumor growth in each of these organs. Intravital imaging was used for quantification of growth of micrometastasis on the liver and stomach. Whole-body imaging was carried out with either a trans-illuminated epi-fluorescence microscope or a fluorescence light box, both with a thermoelectrically cooled color CCD camera. The simple, noninvasive, and highly selective imaging made possible by the strong GFP fluorescence allowed detailed simultaneous quantitative imaging of tumor growth and multiple metastasis formation of pancreatic cancer. The GFP imaging affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals without the need for anesthesia, substrate injection, contrast agents, or restraint of animals required by other imaging methods. The GFP imaging technology presented in this report will facilitate studies of modulators of pancreatic cancer growth, including inhibition by potential chemotherapeutic agents.     

The ARF protein in tumor suppression: lessons from mouse models and human tumors

The ARF protein is a key mediator of the activation of the p53 tumor suppressor in response to excessive mitogenic signals. ARF is encoded in the INK4a/ARF locus, together with the cell-cycle inhibitor p16INK4a. Mice genetically deficient for ARF show a marked predisposition to tumor formation, supporting an important role for ARF in tumor protection. Alterations of the INK4a/ARF locus are a highly frequent event in human tumors. In some instances, the alterations in the locus result in specific inactivation of ARF. This review will summarise the current knowledge about the biological function and regulation of ARF, and will discuss the evidence from genetically modified murine models, and human tumors which supports a relevant role for ARF in tumor suppression.     

Effect of a mouse mammary tumor virus-derived protein vaccine on primary tumor development in mice.

The vaccines used in this study were derived from purified murine mammary tumor virus (MuMTV) preparations. Approximately 60% of the protein fractions consisted of the major viral membrane glycoprotein gp52. Inoculation sc of 10 microgram MuMTV-S-derived vaccine significantly delayed the appearance of primary mammary tumors in GR and BALB/cfC3H mice (strains with high incidences of mammary cancer); in BALB/c and C3Hf mice, which have a moderate tumor incidence at an advanced age, this treatment resulted in a slight and substantial acceleration, respectively, of primary tumor development. The induced cellular immune reactivity for vaccination, as measured with the in vivo Winn test and the in vitro leukocyte adherence inhibition assay, was strongest in the GR strain as compared to the BALB/c strain. The titer of antibodies to tumor cells, as estimated by membrane immunofluorescence, was also higher in the GR strain. In BALB/cfC3H mice, the influence of different vaccination schemes with an MuMTV-O-derived protein vaccine on primary tumor development was studied. Before sc injection, the vaccine was precipitated on alum. A dose of 10 microgram vaccine resulted in a 61% decrease in tumor incidence. Two or five additional booster injections with 1 microgram protein vaccine had no beneficial effect, although the amount of antibody measured was increased after boosting.     

神经母细胞瘤细胞系裸鼠转移模型及局部皮下荷瘤模型的建立

目的 建立人神经母细胞瘤(NB)裸鼠荷瘤模型,研究NB的侵袭、转移机制以及实验治疗的可能性.方法 体外培养人NB细胞系,取对数生长期瘤细胞,以1×107个/0.1ml细胞悬液接种于裸小鼠右侧前肢肋腹部皮下,观察倚瘤的生物学特性,并进行病理组织学检查及基因芯片分析,检测皮下荷瘤组织、转移瘤组织和患儿原发肿瘤组织的神经元特异性烯醇化酶(NSE)表达.结果 48只裸鼠中,荷瘤成功36只,总荷瘤率为75.0%.其中5只裸鼠肿瘤体积很大,甚至达到裸鼠自身体重的1/2,但未发生转移;4只裸鼠接种部位无肿瘤生长,却发生全身转移;6只裸鼠既有局部肿瘤生长,又发生转移.本组有10只裸鼠发生转移,转移率为20.8%(10/48).结论 成功建立人NB裸鼠荷瘤模型并稳定传代,肿瘤的移植生长率和转移率均较高,是NB体内研究理想的动物模型.NB具有异质性,可能是NB转移的重要原因.     

Development of a metastatic human colon cancer xenograft model in the nude mouse

The objective of our experimental protocols was to develop a metastatic model for a human colon carcinoma xenograft in congenitally athymic nude mice. This model would be useful in evaluating the efficacy of radiolabeled monoclonal antibodies for detection and treatment of regional and distant metastases. The LS-174T human colon carcinoma line was used to establish primary subcutaneous tumors in nude mice. Mice were killed at varying time intervals to establish the incidence of spontaneous metastases. Only lung micrometastases were observed during the 2-month observation period. To increase the metastatic rate, the site of primary implantation was varied and/or surgical manipulations were performed. Excision of small primary tumors resulted in a low incidence of local recurrence and no distant metastases. However, with excision of large primary tumors, a high local recurrence rate was noted and over 30% of mice had gross metastases. Mice bearing hind footpad tumors underwent excision when tumors were at least 1 cm in size. There were no local recurrences, but by 8 weeks over 40% had large pulmonary metastases. The LS-174T tumor was also established as a primary implant in the spleen from which 10 to 15% of the mice developed liver or lung metastases. The LS-174T tumor can metastasize in the nude mice and the latter two models may prove very useful in imaging and therapy studies.