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.     

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.     

SIGNIFICANCE OF THE EXPRESSION OF GREEN FLUORESCENT PROTEIN ON DETECTION OF GLIOMA INVASION IN VIVO

The study on biological character of glioma is rapidly expanding day by day. However, there are some shortcomings in present experimental model and examination methods in vivo, which could not easily permit the visualization of tumor invasion, especially for the single invasive tumor cell. Identification of small numbers or individual invading tumor cells against background of host CNS cells may be impossible by standard HE staining. The deficiency of specific antibody against glioma cell de…     

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.     

Significance of the expression of green fluorescent protein on detection of glioma invasion in vivo

Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma. Foundation item: This work was supported by a grant from the National Natural science foundation of China (No. 39970752). Biography: LI Xia (1975–), master of neurosurgery, Xijing Hospital, the Fourth Military Medical University, majors in gene diagnosis and gene therapy of brain malignant tumor.     

Circulating human prostate cancer cells from an orthotopic mouse model rapidly captured by immunomagnetic beads and imaged by GFP expression

Circulating tumor cells (CTCs) are potential precursors of metastasis. They are also of use in diagnosing malignancy and for prognostic purposes. Our laboratory has previously isolated CTCs from orthotopic nude mouse models of human prostate cancer cells where the PC-3 cancer cells express green fluorescent protein (GFP). It was found that orthotopic tumors produced CTCs and not subcutaneous tumors, which may explain why orthotopic tumors metastasize and subcutaneous tumors do not. However, in this previous study, CTCs were observed only after culture. In the present study, using the GFP-expressing PC-3 orthotopic model and immunomagnetic beads coated with anti-epithelial cell adhesion molecule (EpCAM) and anti-prostate specific membrane antigen (PSMA), GFP-expressing CTC were isolated within 15 minutes and were readily visualized by GFP fluorescence. It was possible to immediately place the immunomagnetic-bead-captured GFP-expressing PC-3 CTCs in 3-dimensional sponge cell culture, where they proliferated. The combination of GFP expression and the use of immunomagnetic beads is a very powerful method to obtain CTCs for either immediate analysis or for biological characterization in vivo or in 3-dimensional culture.     

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.     

绿色荧光蛋白基因标记的胃癌细胞系的建立

目的 建立能连续传代稳定高表达绿色荧光蛋白（GFP）的胃癌细胞株,探讨胃癌的生长与转移特征。方法 利用脂质体将携带GFP-cDNA的真核表达载体质粒转染胃癌细胞株GC9811-P,经过G418筛选和克隆化培养,用荧光显微镜及流式细胞仪检测转染的EGFP基因在癌细胞体内外的表达,MTT比色法观察细胞生长,Western blot检测转染细胞和未转染细胞肿瘤相关抗原的表达。结果 携带EGFP的真核表达载体能有效地转染胃癌细胞GC9811-P,转染的细胞能稳定、高效和持久地表达EGFP,与未转染细胞比较,他们的生物学特性未改变（P〉0. 05）。在裸鼠皮下肿瘤中,EGFP也能稳定、高效的表达。结论 胃癌GC9811P-GFP的建立为研究肿瘤侵袭和转移的发生机制提供了理想的细胞株。     

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.     

Imageable fluorescent metastasis resulting in transgenic GFP mice orthotopically implanted with human-patient primary pancreatic cancer specimens

Tumors from pancreatic cancer patients were established in NOD/SCID mice immediately after surgery and subsequently passaged orthotopically in transgenic nude mice ubiquitously expressing green fluorescent protein (GFP). The primary patient tumors acquired GFP-expressing stroma. Subsequent liver metastases, and disseminated peritoneal metastases maintained the stroma from the primary tumor, and possibly recruited additional GFP-expressing stroma, resulting in their very bright fluorescence. The GFP-expressing stroma included cancer-associated fibroblasts and tumor-associated macrophages in both the primary and metastatic tumors. This imageable model of metastasis from a patient-tumor is an important advance over patient "tumorgraft" models currently in use, which are implanted subcutaneously, do not metastasize and are not imageable. The new imageable model of patient pancreatic cancer metastasis provides unique opportunities to identify current and novel antimetastatic therapeutics for individual patients.     

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.     

Complementary use of fluorescence and magnetic resonance imaging of metastatic esophageal cancer in a novel orthotopic mouse model

We describe the development of an aggressive orthotopic metastatic model of esophageal cancer, which is visualized in real time with combined magnetic resonance imaging (MRI) and fluorescence imaging. The aim of the study was to describe the development of a novel model of metastatic tumor disease of esophageal carcinoma and use this model to evaluate fluorescence and MRI in early detection of local and metastatic disease. The human esophageal adenocarcinoma cell line PT1590 was stably transfected with green fluorescent protein (GFP). Nude mice were orthotopically implanted with PT1590‐GFP cells. Orthotopic tumor growth as well as metastatic spread was examined by fluorescence imaging and high‐resolution MRI at defined intervals after orthotopic implantation. Highly aggressive novel fluorescent cell lines were isolated from metastatic tissues and put into culture. After implantation of these cells, 100% of the animals developed orthotopic primary tumors. In 83% of animals, metastatic spread to liver, lung and lymph nodes was observed. Primary tumor growth could be visualized with fluorescence imaging and with MRI with high correlation between the 2 methods. Fluorescence imaging allows fast, sensitive, and economical imaging of the primary and metastatic tumor without anesthesia. With MRI, anatomical structures are visualized more precisely and tumors can be more accurately localized to specific organs. This model should prove highly useful to understand esophageal carcinoma and to identify novel therapeutics for this treatment‐resistant disease.     

Genetically fluorescent melanoma bone and organ metastasis models.

We report here the establishment and metastatic properties of bright, highly stable, green fluorescent protein (GFP) expression transductants of the B16 mouse malignant melanoma cell line and the LOX human melanoma line. The highly fluorescent malignant melanoma cell lines allowed the visualization of skeletal and multiorgan metastases after i.v. injection of B16 cells in C57BL/6 mice and intradermal injection of LOX cells in nude mice. The melanoma cell lines were transduced with the pLEIN expression retroviral vector containing the GFP and neomycin resistance genes. Stable B16F0 and LOX clones expressing high levels of GFP were selected stepwise in vitro in levels of G418 of up to 800 microg/ml. Extensive bone and bone marrow metastases of B16F0 were visualized by GFP expression when the animals were sacrificed 3 weeks after cell implantation. Metastases for both cell lines were visualized in many organs, including the brain, lung, pleural membrane, liver, kidney, adrenal gland, lymph nodes, skeleton, muscle, and skin by GFP fluorescence. This is the first observation of experimental skeletal metastases of melanoma, which was made possible by GFP expression. These models should facilitate future studies of the mechanism and therapy of bone and multiorgan metastasis of melanoma.     

A green fluorescent protein-expressing murine tumour but not its wild-type counterpart is cured by photodynamic therapy

The ideal cancer treatment should both destroy the primary tumour and at the same time educate the immune system to recognise the tumour as foreign so that distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the i.v. administration of photosensitisers followed by illumination of the tumour with red light producing reactive oxygen species that eventually cause vascular shutdown and tumour cell death by apoptosis and necrosis. Anti-tumour immunity is stimulated after PDT due to the acute inflammatory response, generation of tumour-specific antigens, and induction of heat-shock proteins. Green fluorescent protein (GFP) is used as an optical reporter to noninvasively image the progression of mouse tumours, and in addition, may act as a foreign (jellyfish) antigen. We asked whether GFP-expressing tumours could be used to monitor the response of tumour-bearing mice to PDT, and whether the tumour response differed when a nonimmunogenic tumour cell line was transduced with GFP. We injected RIF-1 or RIF-1 EGFP (stably transduced with a retroviral vector) cells in the leg of C3H/HeN mice and both the cells and tumour grew equally well. We used PDT with benzoporphyrin derivative and a short drug-light interval. There were complete cures and 100% mouse survival of RIF-1 EGFP while RIF-1 wild-type tumours all recurred. Cured mice were resistant to rechallenge with RIF-1 EGFP cells and a rechallenge with wild-type RIF-1 cells grew significantly slower. There was also slower RIF-1 EGFP rechallenge growth but no rejection when RIF-1 EGFP tumours were surgically removed. There was a low rate of PDT cure of tumours when RIF-1 cells were transduced with an empty retroviral vector. The presence of antibodies against EGFP in mouse serum suggests EGFP can act as a foreign antigen and PDT can then stimulate a long-term memory immune response.     

Gene transfer in ovarian cancer cells: a comparison between retroviral and lentiviral vectors

Local gene therapy could be a therapeutic option for ovarian carcinoma, a life-threatening malignancy, because of disease containment within the peritoneal cavity in most patients. Lentiviral vectors, which are potentially capable of stable transgene expression, may be useful to vehicle therapeutic molecules requiring long-term production in these tumors. To investigate this concept, we used lentiviral vectors to deliver the enhanced green fluorescent protein (EGFP) gene to ovarian cancer cells. Their efficiency of gene transfer was compared with that of a retroviral vector carrying the same envelope. In vitro, both vectors infected ovarian cancer cells with comparable efficiency under standard culture conditions; however, the lentiviral vector was much more efficient in transducing growth-arrested cells when compared with the retroviral vector. Gene transfer was fully neutralized by an anti-VSV-G antibody, and in vitro stability was similar. In vivo, the lentiviral vector delivered the transgene 10-fold more efficiently to ovarian cancer cells growing i.p. in SCID mice, as evaluated by real-time PCR analysis of the tumors. Confocal microscopy analysis of tumor sections showed a dramatic difference at the level of transgene expression, because abundant EGFP(+) cells were detected only in mice receiving the lentiviral vector. Quantitative analysis by flow cytometry confirmed this and indicated 0.05 and 5.6% EGFP(+) tumor cells after administration of the retroviral and lentiviral vector, respectively. Injection of ex vivo transduced tumor cells, sorted for EGFP expression, indicated that the lentiviral vector was considerably more resistant to in vivo silencing in comparison with the retroviral vector. Finally, multiple administrations of a murine IFN-alpha(1)-lentiviral vector to ovarian carcinoma-bearing mice significantly prolonged the animals' survival, indicating the therapeutic efficacy of this approach. These findings indicate that lentiviral vectors deserve attention in the design of future gene therapy approaches to ovarian cancer aimed at achieving long-term expression of therapeutic genes.     

Differential expression of a recombinant adeno-associated virus 2 vector in human CD34+ cells and breast cancer cells

The use of autologous hematopoietic stem cell (HSC) grafts after high-dose chemotherapy protocols may be hampered by contamination of the grafts with tumor cells. Because epithelial cells seem to be the natural hosts of adeno-associated virus 2 (AAV-2), we speculated that epithelial tumor cells in HSC grafts might be selective targets for AAV-2-based vectors. To test this hypothesis, the breast cancer cell lines T47D and MCF-7 were infected with a recombinant AAV-2 vector expressing the green fluorescent protein (GFP) gene; in addition, human CD34+ mobilized peripheral progenitor cells were infected with the same vector. At a multiplicity of infection of 100, only 1.39% +/- 0.51% CD34+ cells expressed the GFP gene whereas, 36.06% +/- 6.53% of the infected T47D cells and 41.52% +/- 3.16% of the infected MCF-7 cells expressed the transduced GFP gene. After further optimizing the transduction procedure by using higher multiplicities of infection (100-500) and preincubation of samples with the tyrosine kinase inhibitor genistein, up to 82.52% and 85.35% GFP+ T47D and MCF-7 cells, respectively, were observed. The GFP fluorescence intensity in transduced mammary tumor cells was up to 3 logs higher than that of transduced CD34+ cells. The differential expression of recombinant AAV-2 vectors in hematopoietic and epithelial tumor cells warrants further research with this vector system, including the use of suicide genes for the purging of autologous HSC grafts.     

Negative Regulatory Role of TWIST1 on SNAIL Gene Expression

Epithelial-mesenchymal transition (EMT) is crucial for specific morphogenetic movements during embryonic development as well as pathological processes of tumor cell invasion and metastasis. TWIST and SNAIL play vital roles in both developmental and pathological EMT. Our aim in this study was to investigate the functional correlation between TWIST1 and SNAIL in human ESCC cell line (KYSE-30). The packaging cell line GP293T was cotransfected with either control retroviral pruf-IRES-GFP plasmid or pruf-IRES-GFP-hTWIST1 and pGP plasmid. The KYSE-30 ESCC cells were transduced with produced viral particles and examined with inverted fluorescence microscope. DNA was extracted from transduced KYSE-30 cells and analyzed for copy number of integrated retroviral sequences in the target cell genome. The concentration of retroviral particles was determined by Real-time PCR. After RNA extraction and cDNA synthesis, the mRNA expression of TWIST1 and SNAIL was assessed by comparative real-time PCR amplification. Ectopic expression of TWIST1 in KYSE-30, dramatically reduces SNAIL expression. Retroviral transduction enforced TWIST1 overexpression in GFP-hTWIST1 nearly 9 folds in comparison with GFP control cells, and interestingly, this TWIST1 enforced expression caused a ? 7 fold decrease of SNAIL mRNA expression in GFP-hTWIST1 compared to GFP control cells. Inverse correlation of TWIST1 and SNAIL mRNA levels may introduce novel molecular gene expression pathway controlling EMT process during ESCC aggressiveness and tumorigenesis. Consequently, these data extend the spectrum of biological activities of TWIST1 and propose that therapeutic repression of TWIST1 may be an effective strategy to inhibit cancer cell invasion and metastasis.     

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.     

A primary tumor promotes dormancy of solitary tumor cells before inhibiting angiogenesis.

Mechanisms that regulate the transition of micrometastases from clinically undetectable and dormant to progressively growing are critically important but poorly understood in cancer biology. Here we examined the effect of a primary tumor on the growth of solitary tumor cells in the mouse liver, as well as on the development of tumor angiogenesis in a dorsal skin-fold chamber. s.c. placement of a CT-26 (BALB/c-derived mouse colon carcinoma) primary tumor markedly inhibited development of liver metastasis in BALB/c mice after subsequent intraportal injection of tumor cells. Dorsal skin-fold chamber experiments showed that this growth inhibition paralleled a strong antiangiogenic effect by the primary tumor. Furthermore, intravital microscopy of the liver after intraportal injection of green fluorescent protein-expressing tumor cells showed that primary tumors promoted dormancy of single tumor cells for up to 7 days. Immunohistological staining for Ki-67 confirmed that these solitary cells were indeed dormant. In contrast, in the absence of a primary tumor, GFP-expressing tumor cells quickly developed into micrometastases. Thus, primary CT-26 tumor implants nearly abrogated tumor metastasis by inhibition of angiogenesis and by promoting a state of single-cell dormancy. Knowledge of the mechanism underlying this dormancy state could result in the development of new therapeutic tools to fight cancer.     

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.