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.     

Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression

We report here the genetic engineering of dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The parental HT-1080 and the derived dual-color clones had similar cell proliferation rates, suggesting that expression of GFP and/or RFP does not affect cell cycle progression. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse brain where extreme elongation of the cell body as well as the nucleus occurred. Dual-color cells in various positions of the cell cycle were visualized in excised mouse lungs after tail-vein injection of the dual-color cells. In the lung, the dual-color cells were observed frequently juxtaposing their nuclei, suggesting a potential novel form of cell-cell communication. The dual-color cells thus are a useful tool for visualizing living-cell dynamics in vivo as well as in vitro. Drugs that could specifically perturb these processes can now be readily screened in real time in vivo.     

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.     

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.     

Color-coded real-time subcellular fluorescence imaging of the interaction between cancer and host cells in live mice

Stromal cells are essential for tumor growth. Stromal cells interact with cancer cells during tumor growth and progression. We report here the development of a tri-color imageable mouse model to visualize the interaction between host cells and cancer cells. To observe subcellular cancer cell dynamics in vivo, HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-green fluorescent protein (GFP) and with retroviral red fluorescent protein (RFP) in the cytoplasm. HT-1080-GFP-RFP cells were sprinkled over a skin-flap in transgenic GFP immunocompetent mice. After 24 h, the mice were imaged with an Olympus IV100 laser scanning microscope. HT-1080-GFP-RFP cells were visualized surrounded by host-derived lymphocytes and macrophages both expressing GFP. It was possible to observe host GFP macrophages contacting, engulfing, and digesting dual-color HT-1080-GFP-RFP cells in real time. The dual-color cancer cells were readily visible after being engulfed in the GFP macrophages. Other cancer cells were visualized being killed by lymphocytes. The results of this study show that differentially labeling cells with spectrally-distinct fluorescent protein can allow subcellular-resolution imaging of cell-cell interactions between host and cancer cells.     

Whole-body subcellular multicolor imaging of tumor-host interaction and drug response in real time

To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of green fluorescent protein (GFP)-expressing mice transplanted with dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. The Olympus IV100 Laser Scanning Microscope, with ultra-narrow microscope objectives ("stick objectives"), is used for three-color whole-body imaging of the two-color cancer cells interacting with the GFP-expressing stromal cells. In this model, drug response of both cancer and stromal cells in the intact live animal is also imaged in real time. Various in vivo phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow. This new model system enables the first cellular and subcellular images of unperturbed tumors in the live intact animal. New visible real-time targets for novel anticancer agents are provided in this model, including the color-coded interacting cancer and stromal cells, tumor vasculature, and blood flow. This imageable model should lead to many new insights of in vivo cancer cell biology and to novel drug discovery.     

Gossypol inhibition of mitosis, cyclin D1 and Rb protein in human mammary cancer cells and cyclin-D1 transfected human fibrosarcoma cells.

The antiproliferative effects of gossypol on human MCF-7 mammary cancer cells and cyclin D1-transfected HT-1060 human fibrosarcoma cells were investigated by cell cycle analysis and effects on the cell cycle regulatory proteins Rb and cyclin D1. Flow cytometry of MCF-7 cells at 24 h indicated that 10 microM gossypol inhibited DNA synthesis by producing a G1/S block. Western blot analysis using anti-human Rb antibodies and anti-human cyclin D1 antibodies in MCF-7 cells and high- and low-expression cyclin D1-transfected fibrosarcoma cells indicated that, after 6 h exposure, gossypol decreased the expression levels of these proteins in a dose-dependent manner. Gossypol also decreased the ratio of phosphorylated to unphosphorylated Rb protein in human mammary cancer and fibrosarcoma cell lines. Gossypol (10 microM) treated also decreased cyclin D1-associated kinase activity on histone H1 used as a substrate in MCF-7 cells. These results suggest that gossypol might suppress growth by modulating the expression of cell cycle regulatory proteins Rb and cyclin D1 and the phosphorylation of Rb protein.     

miR-663通过靶向TGFB1对肺癌细胞A549增殖的调控

目的利用双荧光蛋白报告基因分析系统,验证miR-663的直接靶基因TGFB1,探讨miR-663促进肺癌细胞A549增殖的可能机制。方法实时定量RT-PCR检测10对肺癌组织和正常肺组织中miR-663的表达水平;利用细胞计数和集落形成实验来验证细胞转染miR-663 ASO后的A549细胞增殖。选取表达绿色荧光蛋白的质粒pcDNA3/EGFP,将TGFB1 3′UTR的一段特异性序列插入该质粒中,并与miR-663及表达红色荧光蛋白质pDsRed2-N1共同转染肺癌细胞系A549,转染后细胞提取的蛋白样品,荧光分光光度计进行定性和定量检测。结果 miR-663在肺癌组织中的表达高于在正常肺组织中的表达;miR-663表达明显促进了细胞A549的增殖;共转miR-663和pcDNA3/EGFP-TGFB13′UTR质粒后,绿色荧光蛋白的表达量明显低于pcDNA3和pcDNA3/EGFP-TGFB13′UTR共转组。结论 miR-663可能通过靶定靶基因TGFB1,促进了肺癌细胞A549的增殖。     

Use of fluorescent proteins and color-coded imaging to visualize cancer cells with different genetic properties

Fluorescent proteins are very bright and available in spectrally-distinct colors, enable the imaging of color-coded cancer cells growing in vivo and therefore the distinction of cancer cells with different genetic properties. Non-invasive and intravital imaging of cancer cells with fluorescent proteins allows the visualization of distinct genetic variants of cancer cells down to the cellular level in vivo. Cancer cells with increased or decreased ability to metastasize can be distinguished in vivo. Gene exchange in vivo which enables low metastatic cancer cells to convert to high metastatic can be color-coded imaged in vivo. Cancer stem-like and non-stem cells can be distinguished in vivo by color-coded imaging. These properties also demonstrate the vast superiority of imaging cancer cells in vivo with fluorescent proteins over photon counting of luciferase-labeled cancer cells.     

GFP-fluorescence-guided UVC irradiation inhibits melanoma growth and angiogenesis in nude mice

Melanoma cell lines that stably express green fluorescent protein (GFP) and nude mice that ubiquitously express red fluorescent protein (RFP) have previously been developed to study tumor-host interaction by color-coded imaging. In the present study, the efficacy of fluorescence-guided ultraviolet C (UVC) irradiation on the growth of murine melanoma expressing GFP in the ear of RFP mice was determined using a non-invasive ear-tumor imaging model developed previously. The GFP-expressing melanoma and RFP-expressing blood vessels from the transgenic mice expressing RFP used as hosts were readily visible using non-invasive imaging. The melanoma was treated under fluorescence guidance with UVC at 650 J/m2/minute for 3 minutes. The ears of the mice were observed before and 24 hours after irradiation with UVC. UVC inhibited melanoma growth and also damaged blood vessels in the tumor. Thus, UVC irradiation has a direct effect on melanoma growth as well as an anti-angiogenesis effect. This color-coded tumor-host model is useful for evaluation of treatment efficacy on melanoma growth and angiogenesis, which are readily discernable with non-invasive color-coded fluorescent protein imaging. These results suggest that fluorescence-guided UVC irradiation is a promising therapeutic strategy for melanoma.     

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.     

Detection of peritoneal micrometastases of gastric carcinoma with green fluorescent protein and carcinoembryonic antigen promoter.

The aim of this study was to specifically visualize micrometastases in the peritoneal cavity, which cannot be detected by conventional methods, by using enhanced Green Fluorescent Protein (EGFP) containing carcinoembryonic antigen (CEA) promoter in an upstream position. In in vitro experiments, two cell lines from human gastric cancer, MKN45 and MKN1, and a cell line from human fibrosarcoma, HT1080, were transduced with pCEA-EGFP, which contains the CEA promoter region. MKN45 and MKN1, which expressed CEA mRNA, showed positive fluorescence after transduction of pCEA-EGFP, whereas HT1080 did not. In in vivo experiments, 7 days after 10(7) MKN45 had been injected into the peritoneal cavity of BALB/c nude mice, pCEA-EGFP was transduced in the peritoneal cavity using a fusogenic liposome with the envelope protein of Hemagglutinating Virus of Japan on the surface. On the peritoneum of the abdominal wall, fluorescent nodules were detected by fluorescence stereomicroscopy. These nodules had a minimal size of approximately 0.15 mm and could not be detected by conventional stereomicroscopy or macroscopy. They were histologically confirmed to be cancer cells by H&E staining. The results suggest that visualization of peritoneal micrometastasis of gastric cancer using CEA promoter and EGFP can offer a new strategy for diagnosis of micrometastasis.     

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.     

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.     

In vitro and in vivo interaction between murine fibrosarcoma cells and natural killer cells

Murine fibrosarcoma cells were examined for sensitivity to killing by natural killer (NK) and natural cytotoxic lymphocytes from mouse spleens. These tumor cell lines were sensitive to killing by effector cells which were nonadherent to plastic or nylon wool, Thy-1 negative, asialo-GM1 negative, and present in the spleens of beige mice, nude mice, and A/J mice, as well as in the spleens of normal syngeneic and allogeneic control mice. This indicates that the cytotoxic effects were due to natural cytotoxic lymphocytes rather than to NK lymphocytes, T-cells, or macrophages. Although the fibrosarcoma cells were not killed in vitro by endogenous NK cells, these tumor cells were able to "cold target" compete for Yac-1 (an NK-sensitive target) killing and to bind to asialo-GM1-positive, nonadherent spleen lymphocytes in a target cell binding assay. This suggests that the fibrosarcoma cells were recognized by NK cells. In addition, these cell lines were killed in a 4-h NK cytotoxicity assay by polyinosinic-polycytidylic acid-activated effector lymphocytes. The interaction between NK cells and the murine fibrosarcoma cells may have in vivo significance. When syngeneic mice were treated with anti-asialo-GM1 serum to eliminate NK activity and then given i.v. injections of the fibrosarcoma cells, many more lung tumors developed than in control animals. The structural basis for the recognition of the murine fibrosarcoma cells by the NK effector cells is not known. However, laminin may be involved. When the fibrosarcoma cells, which have receptors for the laminin molecule, were preincubated with laminin, they were reduced in their ability to compete for the killing of Yac-1 cells by the NK effectors and had reduced capacity to bind to NK cells in a target cell binding assay.     

Site-dependent differences in response of the UV-2237 murine fibrosarcoma to systemic therapy with adriamycin

Murine fibrosarcoma UV-2237MM cells were implanted into different organs of syngeneic C3H/HeN mice. The resultant tumors were treated by i.v. administration of Adriamycin (ADR). Despite the high sensitivity of the fibrosarcoma cells to ADR in vitro, the established tumors growing in vivo exhibited marked differences in their responses to ADR. Tumors growing in the subcutis and the spleen were ADR-sensitive, whereas lung metastases were not. The resistance of lung metastases to ADR was not due to selection of a drug-resistant population since tumor cells isolated from lung metastases were highly sensitive to ADR under in vitro conditions. The responsiveness of skin and spleen tumors to ADR was due neither to increased blood supply nor to preferential accumulation of ADR, since both parameters were higher in lung metastases. Protein kinase C activity levels correlated with ADR resistance in the closely related murine fibrosarcoma cell line UV-2237 and its ADR-selected multidrug-resistant variants. However, nearly identical levels of protein kinase C activity were found in UV-2237MM tumors growing in the lung, spleen, and subcutis, indicating that protein kinase C activity levels did not account for the different responses to ADR. The present studies suggest that the organ environment influences the response of UV-2237MM to ADR administered systemically. This finding may have implications for the design of animal models for therapy of disseminated cancer.     

Functional in vivo optical imaging of tumor angiogenesis, growth, and metastasis prevented by administration of anti-human VEGF antibody in xenograft model of human fibrosarcoma HT1080 cells

Angiogenesis plays a crucial role in cancer progression and metastasis. Thus, blocking tumor angiogenesis is potentially a universal approach to prevent tumor establishment and metastasis. In this study, we used in vivo and ex vivo fluorescence imaging to show that an antihuman vascular endothelial growth factor (VEGF) antibody represses angiogenesis and the growth of primary tumors of human fibrosarcoma HT1080 cells in implanted nude mice. Interestingly, administering the antihuman VEGF antibody reduced the development of new blood vessels and normalized pre-existing tumor vasculature in HT1080 cell tumors. In addition, antihuman VEGF antibody treatment decreased lung metastasis from the primary tumor, whereas it failed to block lung metastasis in a lung colonization experiment in which tumor cells were injected into the tail vein. These results suggest that VEGF produced by primary HT1080 cell tumors has a crucial effect on lung metastasis. The present study indicates that the in vivo fluorescent microscopy system will be useful to investigate the biology of angiogenesis and test the effectiveness of angiogenesis inhibitors. ( Cancer Sci 2009)     

RACK1通过影响MCM7磷酸化促进肺癌细胞的增殖

目的:研究蛋白激酶C受体1(receptor for activated C kinase 1,RACK1)基因沉默或过表达对大细胞肺癌细胞系H460及肺腺癌细胞系A549细胞增殖的影响,并探讨其可能的作用机制.方法:采用脂质体转染法分别将针对RACK1基因的RACK1 siRNA和重组质粒pCMV-sport6-RACK1转入H460和A549细胞中.采用MTT法和集落形成实验分析RACK1基因对细胞增殖的影响,FCM检测其对细胞周期的影响;采用酵母双杂交法、免疫共沉淀法、激光共聚焦显微术及磷酸化蛋白免疫共沉淀法检测RACK1表达与肺癌细胞增殖之间的关系.结果:转染RACK1 siRNA后,H460和A549细胞中RACK1蛋白的表达量明显下调,细胞的增殖能力和集落生成数明显降低,S期细胞所占比例明显下降(P＜0.01);转染pCMV-sport6-RACK1后,H460和A549细胞中RACK1蛋白的表达量明显上调,细胞的增殖能力增强,倍增时间增加,集落生成数明显增多,S期细胞所占比例明显升高(P＜0.01).采用酵母双杂交法和免疫共沉淀法检测发现,RACK1与MCM7存在直接作用.RACK1进入细胞核内,与微小染色体维持蛋白7 (minichromosome maintenance protein 7,MCM7)特异性结合,促进MCM7蛋白磷酸化.结论:RACK1通过直接与MCM7结合促进MCM7蛋白磷酸化途径,继而促进肺癌细胞增殖.     

Determination of clonality of metastasis by cell-specific color-coded fluorescent-protein imaging

It has been thought that metastases are clonal and originate from rare cells in primary tumors that are heterogeneous in genotype and phenotype. Recent studies using DNA array analysis challenge this hypothesis and suggest the genetic background of the host is the important determinant of metastatic potential implying that metastases are not necessarily clonal. Previous methods to determine clonality of metastasis used karyotype or molecular analysis that were complicated, thereby limiting the number of metastatic colonies analyzed and the conclusions that could be drawn. We describe here the use of green fluorescent protein-labeled or red fluorescent protein-labeled HT-1080 human fibrosarcoma cells to determine clonality by simple fluorescence visualization 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. In a spontaneous metastasis model originating from footpad injection in severe combined immunodeficient mice, 95% of the resulting lung colonies were either pure green or pure red, indicating monoclonal origin, whereas 5% were of mixed color, indicating polyclonal origin. In an experimental lung metastasis model established by tail vein injection in severe combined immunodeficient mice, clonality of lung metastasis was dependent on cell number. With a minimum cell number injected, almost all (96%) colonies were pure red or green and therefore monoclonal. When a large number of cells were injected, almost all (87%) colonies were mixed color and therefore heteroclonal. We conclude that spontaneous metastasis may be clonal because they are rare events, thereby supporting the rare-cell clonal origin of metastasis hypothesis. The clonality of the experimental metastasis model depended on the number of input cells. The simple fluorescence method of determining clonality of metastases described here can allow large-scale clonal analysis in numerous types of metastatic models.     

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.