The cyan fluorescent protein nude mouse as a host for multicolor-coded imaging models of primary and metastatic tumor microenvironments

The tumor microenvironment (TME) has an important influence on tumor progression. For example, we have discovered that passenger stromal cells are necessary for metastasis. In this report, we describe six different cyan fluorescent protein (CFP) multicolor TME nude mouse models. The six different implantation models were used to image the TME using multiple colors of fluorescent proteins: I) Red fluorescent protein (RFP)- or green fluorescent protein (GFP)-expressing HCT-116 human colon cancer cells were implanted subcutaneously in the CFP-expressing nude mice. CFP stromal elements from the subcutaneous TME were visualized interacting with the RFP- or GFP-expressing tumors. II) RFP-expressing HCT-116 cells were transplanted into the spleen of CFP nude mice, and experimental metastases were then formed in the liver. CFP stromal elements from the liver TME were visualized interacting with the RFP-expressing tumor. III) RFP-expressing HCT-116 cancer cells were transplanted in the tail vein of CFP-expressing nude mice, forming experimental metastases in the lung. CFP stromal elements from the lung were visualized interacting with the RFP-expressing tumor. IV) In order to visualize two different tumors in the TME, GFP-expressing and RFP-expressing HCT-116 cancer cells were co-implanted subcutaneously in CFP-expressing nude mice. A 3-color TME was formed subcutaneously in the CFP mouse, and CFP stromal elements were visualized interacting with the RFP- and GFP-expressing tumors. V) In order to have two different colors of stromal elements, GFP-expressing HCT-116 cells were initially injected subcutaneously in RFP-expressing nude mice. After 14 days, the tumor, which consisted of GFP cancer cells and RFP stromal cells derived from the RFP nude mouse, was harvested and transplanted into the CFP nude mouse. CFP stromal cells invaded the growing transplanted tumor containing GFP cancer cells and RFP stroma. VI) Mouse mammary tumor (MMT) cells expressing GFP in the nucleus and RFP in the cytoplasm were implanted in the spleen of a CFP nude mouse. Cancer cells were imaged in the liver 3 days after cell injection. The dual-color dividing MMT cells and CFP hepatocytes, as well as CFP non-parenchymal cells of the liver were imaged interacting with the 2-color cancer cells. CFP-expressing host cancer-associated fibroblasts (CAFs) were predominantly observed in the TME models developed in the CFP nude mouse. Thus, the CFP nude mouse adds another color to the pallet of the TME, allowing multiple types of color-coded cancer and stromal cells to be imaged simultaneously. The multi-colored models described in this report provide new opportunities to study the cellular interactions in the live primary and metastatic TME.     

Tumor imaging with multicolor fluorescent protein expression

Imaging with fluorescent proteins has been revolutionary and has led to the new field of in vivo cell biology. Many new applications of this technology have been developed. Green fluorescent protein (GFP)-labeled or red fluorescent protein (RFP)-labeled HT-1080 human fibrosarcoma cells were used to determine clonality of metastasis by imaging of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. Dual-color fluorescent cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were engineered. The dual-color cancer cells enable real-time nuclear–cytoplasmic dynamics to be visualized in living cells in vivo, including mitosis and apoptosis. The nuclear and cytoplasmic behavior of dual-color cancer cells in real time in blood vessels was observed as they trafficked by various means or extravasated in an abdominal skin flap. Dual-color cancer cells were also visualized trafficking through lymphatic vessels where they were imaged via a skin flap. Seeding and arresting of single dual-color cancer cells in the lung, accumulation of cancer-cell emboli, cancer-cell viability, and metastatic colony formation were imaged in real time in an open-chest nude mouse model using assisted ventilation. Novel treatment was evaluated in these imageable models. UVC irradiation killed approximately 70% of the dual-color cancer cells in a nude mouse model. An RFP-expressing glioma was transplanted to the spinal cord of transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). In ND-GFP mice, GFP is expressed in nascent blood vessels and neural stem cells. ND-GFP cells staining positively for neuronal class III-β-tubulin or CD31 surrounded the tumor, suggesting that the tumor stimulated both neurogenesis and angiogenesis. The tumor caused paralysis and also metastasized to the brain. The Salmonella typhimurium A1-R tumor-targeting bacterial strain was administered in the orthotopic spinal cord glioma model. The treated animals had a significant increase in survival and decrease in paralysis. S. typhimurium A1-R was effective against primary bone tumor and lung metastasis expressing RFP in a nude mouse model. S. typhimurium A1-R was effective against both axillary lymph and popliteal lymph node metastases of human dual-color pancreatic cancer and fibrosarcoma cells, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. Imaging with fluorescent proteins will reveal mechanisms of cancer progression and provide visual targets for novel therapeutics.     

Dual-color imaging of nuclear-cytoplasmic dynamics, viability, and proliferation of cancer cells in the portal vein area

We used dual-color in vivo cellular imaging to visualize trafficking, nuclear-cytoplasmic dynamics, and the viability of cancer cells after their injection into the portal vein of mice. For these studies, we used dual-color fluorescent cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and retroviral red fluorescent protein (RFP) in the cytoplasm. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT) cells were HCT-116-GFP-RFP in the portal vein of nude mice. The cells were observed intravitally in the liver at the single-cell level using the Olympus OV100 whole-mouse imaging system. Most HCT-116-GFP-RFP cells remained in sinusoids near peripheral portal veins. Only a small fraction of the cancer cells invaded the lobular area. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The number of apoptotic cells rapidly increased within the portal vein within 12 hours of injection. Apoptosis was readily visualized in the dual-color cells by their altered nuclear morphology. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, dual-color MMT-GFP-RFP cells injected into the portal vein mostly survived in the liver of nude mice 24 hours after injection. Many surviving MMT-GFP-RFP cells showed invasive figures with cytoplasmic protrusions. The cells grew aggressively and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells.     

Experimental nude mouse model of human colorectal cancer liver metastases

A nude mouse model (BALB/c) was established for investigating the production of hepatic metastases by human colorectal carcinoma (HCC) cells. The malignant potentials of 3 different HCCs derived from a primary tumor (HCC-P4733), a lymph node metastasis (HCC-M14328), and a hepatic metastasis (HCC-M1410) were investigated following implantation into the spleens of athymic nude mice. Cells of the HCC-M1410 line produced extensive liver tumors in all mice by 30 days after injection. Cells of the HCC-M14328 and HCC-P4733 lines produced few liver tumors in the inoculated mice and then only after a 90-day period. Isozyme and karyotype analyses ascertained the human origin of all the tumors. Studies with [125I]IdUrd-labeled HT-29 carcinoma cells suggested that tumor cells reached the liver shortly after injection into the spleen. Thus the production of HCC tumors in livers of nude mice was determined by the ability of HCC cells to proliferate in the liver parenchyma rather than by the ability of the cells to reach the liver. The results suggest that the intrasplenic injection of HCC cells can provide a valuable model for the study of the biology and therapy of the liver metastases.     

结肠癌转移相关基因1对结肠癌细胞肝转移的影响

目的:研究结肠癌转移相关基因1(metastasis-associated in colon cancer1,MACC1)的表达对结肠癌肝转移的影响。方法:将融合绿色荧光蛋白(green fluorescent proteins,GFP)的慢病毒表达载体分别感染具有不同转移潜能的人结肠癌细胞株SW1116和HCT116。给予BALB/c-nu/nu裸鼠脾脏注射感染后的结肠癌细胞,构建结肠癌肝转移模型。注射后35d,在荧光解剖显微镜下观察肝脏内结肠癌细胞的转移情况,计算肝转移率。应用蛋白质印迹法检测SW1116和HCT116细胞中MACC1蛋白的表达水平,评估MACC1的表达水平与肝转移率的关系。结果:SW1116细胞的肝转移率明显高于HCT116细胞。MACC1蛋白在HCT116细胞中呈低表达,而在SW1116细胞中呈高表达,差异有统计学意义(P<0.01)。结论:MACC1蛋白的表达水平可能与结肠癌的肝转移能力呈正相关。     

Tumor-targeting Salmonella typhimurium A1-R arrests growth of breast-cancer brain metastasis

Brain metastasis is a morbid, treatment-resistant, end-stage frequent occurrence in breast cancer patients. The aim of this study was to evaluate the efficacy of tumor-targeting Salmonella typhimurium A1-R on breast cancer brain metastases. High brain-metastatic variants of murine 4T1 breast cancer cells expressing red fluorescent protein (RFP) were injected orthotopically in the mammary fat pad in non-transgenic nude mice or in the left ventricle of non-transgenic nude mice and transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). ND-GFP mice express GFP in nascent blood vessels. In the orthotopically-injected mice, the primary tumor was surgically-resected in order to allow brain metastasis to develop. At various time points, the tumors and vasculature in the brain were imaged by confocal and stereo fluorescence microscopy. Some of the breast cancer cells that reached the brain extravasated and grew perivascularly and some of the cells proliferated within the vasculature. S. typhimurium A1-R significantly inhibited brain metastasis in both metastatic models and increased survival of the orthotopically-transplanted, primary-tumor-resected mice (p<0.05). The results of the present study suggest the clinical potential of bacterial therapy of breast cancer brain metastasis.     

A fluorescent orthotopic bone metastasis model of human prostate cancer

Here, we report a fluorescent spontaneous bone metastatic model of human prostate cancer developed by surgical orthotopic implantation of green fluorescent protein (GFP)-expressing prostate cancer tissue. Human prostate cancer PC-3 cells were transduced with the pLEIN expression retroviral vector containing the enhanced GFP and neomycin resistance genes. Stable GFP high-expression PC-3 clones were selected in vitro with G418, which were then combined and injected s.c. in nude mice. For metastasis studies, fragments of a single highly fluorescent s.c. growing tumor were implanted by surgical orthotopic implantation in the prostate of a series of nude mice. Subsequent micrometastases and metastases were visualized by GFP fluorescence throughout the skeleton, including the skull, rib, pelvis, femur, and tibia The central nervous system, including the brain and spinal cord, was also involved with tumor, as visualized by GFP fluorescence. Systemic organs, including the lung, plural membrane, liver, kidney, and adrenal gland, also had fluorescent metastases. The metastasis pattern in this model reflects the bone and other metastatic sites of human prostate cancer. Thus, this model should be very useful for the study and development of treatment for metastatic androgen-independent prostate cancer.     

Comparison of cancer-cell seeding, viability and deformation in the lung, muscle and liver, visualized by subcellular real-time imaging in the live mouse

The comparison of cancer cell seeding, deformation and viability in the lung, muscle and liver of nude mice in real-time is reported here. The mice were intubated to support ventilation with positive end-respiratory pressure (PEEP) for imaging on the lung. Human fibrosarcoma cells with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (dual-color HT-1080 cells) were injected into the tail vein for lung imaging, the portal vein for liver imaging or the abdominal aorta for muscle imaging which was performed with an Olympus OV100 Small Animal Imaging System. The length of the cytoplasm and nuclei in 20 seeded cancer cells were measured. A large number of cells initially arrested in the lung capillaries and many cells formed aggregates. The cell number decreased rapidly at 6 and 24 h. There was no significant difference in cancer cell survival when immunocompetent C57BL/6 mice were used in place of the nude mice, suggesting that T cell reaction is not very important in the first 24 h after seeding of cancer cells in the lung. In the lung and liver, little cancer cell deformation occurred. In contrast in the muscle, the cytoplasm and nuclei of the seeded cells were highly deformed and many fragmented cells were observed. The rate of cancer cell death was highest in the lung and lowest in the muscle. In each organ, single disseminated cells tended to die earlier than aggregated cells. The results of this study suggest that the early steps of metastasis are different in the lung, liver and muscle.     

Imaging the inhibition by anti-β1 integrin antibody of lung seeding of single osteosarcoma cells in live mice

Integrins play a role in tumor growth and metastasis. However, the effect of integrin inhibition has not been visualized on single cancer cells in vivo. In this study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti-integrin antibody affects seeding and growth of osteosarcoma cells on the lung. The 143B human osteosarcoma cell line, expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus, was established. Such double-labeled cells enable imaging of apoptosis and mitosis and other nuclear-cytoplasmic dynamics. Using the double-labeled osteosarcoma cells, single cancer-cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged. The anti-β1 integrin monoclonal antibody, AIIB2, greatly inhibited the seeding of cancer cells on the lung (experimental metastasis) while a control antibody had no effect. To image the efficacy of the anti-integrin antibody on spontaneous metastasis, mice with orthotopically-growing 143B-RFP cells in the tibia were also treated with AIIB2 or control anti-rat IgG1 antibody. After 3 weeks treatment, mice were sacrificed and primary tumors and lung metastases were evaluated with fluorescence imaging. AIIB2 significantly inhibited spontaneous lung metastasis but not primary tumor growth, possibly due to inhibition of lung seeding of the cancer cells as imaged in the experimental metastasis study. AIIB2 treatment also increased survival of mice with orthotopically growing 143B-RFP.     

Lymph node metastasis of oral cancer visualized in live tissue by green fluorescent protein expression

Here, we report the establishment of a stably transfected cell line which expresses high levels of green fluorescent protein (GFP), thus permitting the detection and visualization of developing tumors and lymph node metastases after injection into nude mice. Cells of the human oral squamous carcinoma cell line (SAS-L1) were transfected with an expression vector containing a cDNA encoding humanized GFP and the neomycin resistance gene. A clone with stable high-level expression of GFP was selected in vitro using G418. To study metastasis formation, GFP-expressing cells were injected orthotopically into the tongue of nude mice. The resultant tumor growth in the tongue and micrometastases in the lymph nodes could be visualized by GFP fluorescence. Therefore a useful model has been developed for the study of oral cancer, firstly to understand the metastatic process and secondly for the evaluation of potential treatments.     

In vivo tumor delivery of the green fluorescent protein gene to report future occurrence of metastasis

The green fluorescent protein (GFP) gene was administered to intraperitoneally (i.p.) growing human stomach cancer in nude mice to visualize future regional and distant metastases. GFP retroviral supernatants were injected i.p. from day 4 to day 10 after i.p. implantation of the cancer cells. Tumor and metastasis fluorescence was visualized every other week with the use of fluorescence optics via a laparotomy on the tumor-bearing animals. At 2 weeks after retroviral GFP delivery, GFP-expressing tumor cells were observed in gonadal fat, greater omentum, and intestine, indicating that these primary i.p. growing tumors were efficiently transduced by the GFP gene and could be visualized by its expression. At the second and third laparotomies, GFP-expressing tumor cells were observed to have spread to lymph nodes in the mesentery and other regional sites. At the fourth laparotomy, widespread tumor growth was visualized by GFP expression, inducing liver metastasis. No normal tissues were found to be transduced by the GFP retrovirus. Thus, reporter gene transduction of the primary tumor enabled detection of its subsequent metastasis. This gene therapy model could be applied to primary tumors before resection or other treatment to have a fluorescent early detection system for metastasis and recurrence.     

Real-time imaging of tumor-cell shedding and trafficking in lymphatic channels

In the present report, we show real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with both green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm or with GFP only or RFP only were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real time at the cellular level until they entered the axillary lymph node. The bright fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 small-animal imaging system enabled imaging of the trafficking cancer cells in the lymphatics. Using this imaging strategy, two different cancer cell lines, one expressing GFP and the other expressing RFP, were simultaneously injected in the inguinal lymph node. Fluorescence imaging readily distinguished the two color-coded cell lines and their different abilities to survive in the lymphatic system. Using this imaging technology, we also investigated the role of pressure on tumor-cell shedding into lymphatic vessels. Pressure was generated by placing 25- and 250-g weights for 10 s on the bottom surface of a tumor-bearing footpad. Tumor cell fragments, single cells, and emboli shed from the footpad tumor were easily distinguished with the labeled cells and OV100 imaging system. Increasing pressure on the tumor increased the numbers of shed cells, fragments, and emboli. Pressure also deformed the shed emboli, increasing their maximum major axis. Imaging lymphatic trafficking of cancer cells can reveal critical steps of lymph node metastasis.     

Non-invasive Fluorescent-protein Imaging of Orthotopic Pancreatic-cancer-patient Tumorgraft Progression in Nude Mice

In order to individualize and therefore have more effective treatment for pancreatic cancer, we have developed a multicolor, imageable, orthotopic mouse model for individual patients with pancreatic cancer by passaging their tumors through transgenic nude mice expressing green fluorescent protein (GFP) and red fluorescent protein (RFP). The tumors acquired brightly fluorescent stroma from the transgenic host mice, which was stably associated with the tumors through multiple passages. In the present study, pancreatic cancer patient tumor specimens were initially established in NOD.CB17-Prkdc(scid)/NcrCrl (NOD/SCID) mice. The tumors were then passaged orthotopically into transgenic nude mice ubiquitously expressing GFP and subsequently to nude mice ubiquitously expressing RFP. The tumors, with very bright GFP and RFP stroma, were then orthotopically passaged to non-transgenic nude mice. It was possible to image the brightly fluorescent tumors non-invasively longitudinally as they progressed in the non-transgenic nude mice. This non-invasive imageable tumorgraft model will be valuable to screen for effective treatment options for individual patients with pancreatic cancer, as well as for the discovery of improved agents for this treatment-resistant disease.     

Characterization of the tumorigenic and metastatic potential of a poorly differentiated human colon cancer cell line

We characterized the tumorigenic and metastatic potential of a poorly differentiated, non-CEA-producing colon cancer cell line, MIP-101, after injection at different sites in athymic mice. After subcutaneous and intrasplenic injection tumor grew locally in 100 and 50%, respectively, but no metastases were found, even after intravenous injection. Intraperitoneal implantation, however, resulted in a high tumor take (10/10) and subsequent liver colonization (8/10 mice). Exogenous CEA prior to intrasplenic injection induced metastasis in 7/8 mice (in 2 mice to the liver and in 5 mice to the lung). Intrasplenic injection of CX-1, a good CEA producer, resulted in hepatic metastases in 100% of the animals. These data suggest a direct or indirect role of CEA in the metastatic process. We conclude that MIP-101 has a high tumorigenic and invasive potential but a low metastatic proclivity, except when grown in the peritoneum, and that pretreatment of tumor-bearing animals with CEA affects the metastatic proclivity.     

Experimental cancer chemotherapy using A liver metastatic model of human colon cancer transplanted into the spleen of severe combined immunodeficient mice

We have developed a liver metastatic model of human colon cancer using severe combined immunodeficient (SCID) mice. Liver metastases were observed in all the SCID mice on day 28 after intrasplenic injection with 5 × 10⁶ dissociated tumor cells of COL-2-JCK, a human colon cancer strain serially transplanted in nude mice. When this model was applied for chemotherapeutic experiments, 5-fluorouracil (5-FU) demonstrated significant antitumor effects in preventing liver metastases, whereas the efficacy of 5-FU was limited in the currently used sc-ip chemosensitivity assay in nude mice. When the human LDH-5 isozyme was evaluated in the homogenized metastatic liver tissue of SCID mice, a good correlation was obtained between the liver tumor weights and LDH-5 isozyme, suggesting that it could be a promising quantitative indicator for metastases. This model would be useful for further studies on the treatment of liver metastases of colon cancer. © 1993 Wiley-Liss, Inc.     

Adjuvant treatment with tumor-targeting Salmonella typhimurium A1-R reduces recurrence and increases survival after liver metastasis resection in an orthotopic nude mouse model

Colon cancer liver metastasis is often the lethal aspect of this disease. Well-isolated metastases are candidates for surgical resection, but recurrence is common. Better adjuvant treatment is therefore needed to reduce or prevent recurrence. In the present study, HT-29 human colon cancer cells expressing red fluorescent protein (RFP) were used to establish liver metastases in nude mice. Mice with a single liver metastasis were randomized into bright-light surgery (BLS) or the combination of BLS and adjuvant treatment with tumor-targeting S. typhimurium A1-R. Residual tumor fluorescence after BLS was clearly visualized at high magnification by fluorescence imaging. Adjuvant treatment with S. typhimurium A1-R was highly effective to increase survival and disease-free survival after BLS of liver metastasis. The results suggest the future clinical potential of adjuvant S. typhimurium A1-R treatment after liver metastasis resection.     

树突状细胞疫苗治疗肺癌血源性转移的实验研究

目的 探索应用树突状细胞肿瘤疫苗治疗肺癌血源性转移的可能性。方法 将Ｌewis肺癌细胞经尾静脉注射给Ｃ５７ＢＬ/６j纯系小鼠建立肺癌血源性转移模型,应用树突状细胞负载合成的抗原多肽ＭＵＴ－１,作为肿瘤疫苗进行免疫治疗。存活的小鼠再用１０倍数量的癌细胞皮下攻击,观察其免疫保护效应。这些小鼠的脾脏Ｔ淋巴细胞经纯化后进行体外抗原致敏,然后测定其特异性细胞毒效应。结果 尾静脉注射Ｌewis肺癌细胞可引起多脏器肿瘤播     

Carcinoembryonic antigen: enhancement of liver colonisation through retention of human colorectal carcinoma cells.

Carcinoembryonic antigen (CEA) is an oncofetal antigen whose function in the progression of colorectal carcinoma remains unclear although recent studies suggest it participates in homotypic cellular adhesion. We have previously shown that 40 micrograms of CEA injected intravenously into athymic nude mice enhances experimental metastasis in liver and lung by two human colorectal carcinoma cell lines that are injected intrasplenically 30 min later. The metastatic potential of another three moderately to highly metastatic colorectal carcinoma cell lines and of one weakly metastatic line has now been analysed in this model. CEA pretreatment only enhanced colony formation by cell lines that were weakly metastatic in untreated nude mice; it did not affect experimental metastasis by highly metastatic lines. CEA pretreatment enhanced the retention of 125I Idudr-labelled weakly metastatic tumour cells within the liver and lungs 4 h after intrasplenic injection but not the retention of highly metastatic tumour cells or inert latex beads. A significant correlation existed between the formation of experimental metastases and the early retention of tumour cells within the liver after intrasplenic injection. Aggregation did not appear to be important for retention in liver because CEA did not aggregate colorectal carcinoma cells in vitro. Also CEA did not alter natural host effector cell function in a cytolysis assay in vitro. We suggest that CEA facilitates liver colonisation by three of eight human colorectal carcinomas in athymic nude mice by increasing the hepatic retention of tumour cells. The potential mechanisms by which CEA may increase the retention of tumour cells in the liver are discussed.     

CD44v6对实验性大肠癌肝转移的影响

目的：研究ＣＤ４４ｖ６在大肠癌肝转移中的作用，探索一种有效的抗肿瘤转移方法。方法：应用大肠癌肝转移模型来研究ＣＤ４４ｖ６单抗对大肠癌ＨＴ２９、Ｌｏｖｏ细胞裸小鼠实验性肝转移的影响，分析ＣＤ４４ｖ６单抗在抗大肠癌肝转移中的作用。结果：ＨＴ２９细胞对照组肝转移率为４／８，实验组肝转移率为２／８；Ｌｏｖｏ细胞对照组肝转移率为８／８，实验组肝转移率为０。结论：ＣＤ４４ｖ６单抗能有效阻断裸小鼠实验性大肠癌肝转移的发生。     

Orthotopic Transplant Mouse Models with Green Fluorescent Protein-expressing Cancer Cells to Visualize Metastasis and Angiogenesis

There have been major efforts in metastasis research in recent years, especially on the role of angiogenesis in the metastatic process. Much of the information in this area has been obtained from model systems that are not representative of clinical cancer. The technique of surgical orthotopic implantation (SOI) has allowed the development of clinically relevant metastatic models of human cancer in immunodeficient rodents such as the nude and SCID mouse. In order to allow direct visualization of the metastatic process, we took advantage of the green fluorescent protein (GFP) of the jellyfish, Aequorea victoria. A series of cancer cell lines have been stably transfected with vectors containing humanized GFP cDNA. To utilize GFP expression for metastasis studies, fragments of subcutaneously growing tumor, which were comprised of GFP-expressing cells, were implanted by SOI in nude mice. Subsequent metastases were visualized in systemic organs by GFP fluorescence in the lung, liver, bones, brain and other organs down to the single-cell level. With this fluorescent tool, we detected and visualized for the first time tumor cells at the microscopic level in fresh viable tissue in their normal host organs even in the live animal. Angiogenesis is readily visualized in the transplanted GFP-expressing tumors in real time in situ in the live animal using simple laparotomy and fluorescent techniques. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance to visualize and study cancer metastasis and the role of angiogenesis and other factors in the metastatic process. This revised version was published online in July 2006 with corrections to the Cover Date.