Stem-like and non-stem human pancreatic cancer cells distinguished by morphology and metastatic behavior.

We report here that XPA1 human pancreatic cancer cells are dimorphic. After injection in the spleen, XPA1 cells isolated from the primary tumor in the spleen were predominantly round; while cells isolated from the resulting liver metastasis and ascites were comprised of both round- and spindle-shaped cell types. Cancer cells previously grown in the spleen and re-implanted in the spleen developed large primary tumors in the spleen only. Cancer cells isolated from liver metastasis and re-transplanted to the spleen resulted in a primary tumor in the spleen and liver metastasis. Cancer cells derived from ascites and re-transplanted to the spleen developed primary tumors in the spleen and distant metastasis in the liver, lung, and diaphragm in addition to ascites formation. Spindle and round cells were differentially labeled with fluorescent proteins of different colors. After co-injection of the two cell types in the spleen, cells were isolated from the primary tumors, liver metastasis, and ascites and analyzed by color-coded fluorescence microscopy and fluorescence-activated cell sorting (FACS). No significant differences between the percentages of spindle-shaped and round cancer cells in the primary tumor and the liver metastasis were observed. However, spindle-shaped cancer cells were enriched in the ascites. One hundred percent of the spindle-shaped and round cancer cells expressed CD44, suggesting that morphology and metastatic behavior rather than CD44 expression can distinguish the stem-like cells of the XPA1 pancreatic cancer cell line. The spindle-shaped cancer cells had the greater capability for distant metastasis and ascites formation, suggesting they are stem-like cells, which can be readily targeted for therapy.

Fluorescent LYVE-1 antibody to image dynamically lymphatic trafficking of cancer cells in vivo.

BACKGROUND: The lymphatic system is a major route for cancer cell dissemination, and a potential target for antitumor therapy. Despite ongoing interest in this area of research, the real-time behavior of cancer cells trafficking in the lymphatic system is poorly understood due to lack of appropriate tools to image this process. MATERIALS AND METHODS: We have used monoclonal-antibody and fluorescence technology to color-code lymphatic vessels and the cancer cells inside them in a living animal. Monoclonal anti-mouse LYVE-1 antibody was conjugated to a green fluorophore and delivered to the lymphatic system of a nude mouse, allowing imaging of mouse lymphatics. Tumor cells engineered to express red fluorescent protein were then imaged traveling within the labeled lymphatics in real time. RESULTS: AlexaFluor-labeled monoclonal anti-mouse LYVE-1 created a durable signal with clear delineation of lymphatic architecture. The duration of fluorescent signal after conjugated LYVE-1 delivery was far superior to that of fluorescein isothiocyanate-dextran or control fluorophore-conjugated IgG. Tumor cells engineered to express red fluorescent protein delivered to the inguinal lymph node enabled real-time tracking of tumor cell movement within the green fluorescent-labeled lymphatic vessels. CONCLUSIONS: This technology offers a powerful tool for the in vivo study of real-time trafficking of tumor cells within lymphatic vessels, for the deposition of the tumor cells in lymph nodes, as well as for screening of potential antitumor lymphatic therapies.

Unusual tumours of the pancreas.

UNLABELLED: All pancreatic masses are not necessarily the dismal pancreatic ductal adenocaricoma (PDA) and do not necessarily deserve a gloomy prognosis or a nihilistic attitude. We review a rarer group of pancreatic lesions and discuss their pathogenesis, diagnosis and treatment. A tumour specific selective surgical approach is recommended. The outcome is dependent on the tumour histology and the biological behaviour. The degree of malignancy is variable and ranges across benign, borderline and malignant entities. The prognosis is generally better than that of PDA. BACKGROUND: The advent of more sophisticated and ever widely employed imaging modalities has identified the presence of many unsuspected unusual masses in the pancreas. These lesions display natural histories and biological behaviours distinct from adenocarcinoma of the pancreas (PDA). Though the list is long they include neuroendocrine tumours, cystic tumours, primary pancreatic lymphoma, solid pseudopapillary tumours, connective tissue tumours, metastatic lesions to the pancreas and many others.

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.

Sodium taurocholate modifies the bile acid-independent fraction of canalicular bile flow in the rhesus monkey.

Bile acid-independent secretion and the choleretic response to taurocholate were determined in rhesus monkeys fitted with indwelling silastic cannulas in the common bile ducts. Bile acids were infused intravenously in random order at 3.5, 7.0, or 10.5 mumol/min for 1.5 h each. When data were analyzed with a single regression line, bile flow increased in proportion to the level of bile acid secretion, although the y-intercepts (the conventional measurement of bile acid-independent secretion) varied widely (77.9+/-40.9 ml/24 h). The variation in y-intercepts was observed between animals and with repeated studies in the same animal and could not be explained by sex differences or the effects of the indwelling silastic cannulas, but seemed to be related to the order of bile acid infusion. With only two taurocholic acid infusion rates (7.0 and 3.5 mumol/min), [(14)C]erythritol clearance was greater per mole of secreted bile acid when the initial bile acid infusion was at the high level, but approached zero at low bile acid secretion rates, which suggests that so-called bile acid-independent canalicular flow is closely related to bile acid secretion or is small in size. The augmentation in [(14)C]erythritol clearance when the high infusion rate was given first was also associated with an increase in biliary clearance of [(3)H]inulin, which indicates that the premeability to inulin was also enhanced. Identical experiments which substituted equimolar infusions of a nonmicelle-forming bile acid (taurodehydrocholate) for taurocholate failed to demonstrate any difference in choleretic response or biliary clearance of [(3)H]inulin with the order of bile acid infusion. These experiments demonstrate that a micelleforming bile acid, taurocholate, can increase the permeability of the biliary system to large molecular weight solutes and simultaneously modify the y-intercept and the volume of bile secreted in response to the transported bile acid. Taurocholate may, therefore, modify its own choleretic response, perhaps by altering the structure or function of bile secretory membranes, and appears to be a major determinant of so-called bile acid-independent flow in rhesus monkeys.

The role of the liver in glucagon metabolism.

Total plasma immunoreactive pancreatic glucagon (IRG) was measured in samples taken simultaneously from the proximal portal vein and superior vena cava of 26 healthy rats. The portal-peripheral ratio of IRG was 2.80+/-0.25, the portal-peripheral difference (Delta) 124+/-15 pg/ml, and percentage extraction 58+/-3. Gel filtration of paired portal and peripheral vein samples showed that reduction in the 3,500-dalton IRG component (glucagon) in peripheral samples accounted for almost all the differences, there being minimal and inconsistent changes in the high molecular weight (>40,000) fraction. The portal-peripheral ratio of the 3,500-dalton glucagon was 5.24+/-1.10, the portal-peripheral difference 130+/-33 pg/ml, and the percentage extraction 81+/-5. To study the transhepatic differences in the 9,000-dalton "proglucagon-like" material, the experiment was repeated in nine rats 24 h after bilateral nephrectomy, a procedure which increases plasma levels of this fraction. The portal-peripheral ratio for plasma IRG in these rats was 1.48+/-0.12, the portal-peripheral difference 140+/-29 pg/ml, and percentage extraction 28+/-5. Gel filtration revealed no consistent differences between portal and peripheral concentrations of the 9,000- and >40,000-dalton components, which comprised 40 and 13%, respectively, of the mean IRG level of 492+/-35 pg/ml. In contrast, there were marked differences between portal and peripheral levels of the 3,500-dalton component the ratio being 3.42+/-0.63, the portal-peripheral difference 182+/-32 pg/ml, and percentage extraction 64+/-5. Similar studies in a healthy dog, in which species there are significant circulating levels of the 9,000-dalton IRG component, confirmed the selective hepatic extraction of the 3,500-dalton fraction. We conclude that the various IRG fractions are metabolized differently by the liver, and that portal-peripheral ratios based on direct assay of plasma IRG will vary depending on the percentage glucagon immunoreactivity in each fraction; the greater the combined contribution of fractions other than the 3,500-dalton component to total plasma IRG, the lower will be the ratio. Because of the heterogeneity of circulating IRG and significant differences in the metabolism of its various components, gel filtration of plasma samples is necessary for precise quantitation of the hepatic uptake of each particular fraction.

Metabolism of C-peptide in the dog. In vivo demonstration of the absence of hepatic extraction.

The in vivo hepatic metabolism of connecting peptide (C-peptide) in relation to that of insulin has not been adequately characterized. A radioimmunoassay for dog C-peptide was therefore developed and its metabolism studied in conscious mongrel dogs, with sampling catheters chronically implanted in their portal and hepatic veins and femoral artery. The hepatic extraction of endogenous C-peptide under basal conditions was negligible (4.3 +/- 4.5%) and was similar to the hepatic extraction of C-peptide measured during the constant exogenous infusion of C-peptide isolated from dog pancreas. Simultaneously measured hepatic extraction of endogenous and exogenously infused insulin were 43.8 +/- 7.6 and 47.5 +/- 4.4%, respectively. The metabolic clearance rate of infused C-peptide was 11.5 +/- 0.8 ml/kg per min and was constant over the concentration range usually encountered under physiological conditions. In additional experiments, the effect of parenteral glucose administration on the hepatic extraction of C-peptide and insulin was investigated. The hepatic extraction of C-peptide (6.2 +/- 4.0%) was again negligible in comparison with that of insulin (46.7 +/- 3.4%). Parenteral glucose administration did not affect the hepatic extraction of either peptide irrespective of whether it was infused peripherally, intraportally, or together with an intraportal infusion of gastrointestinal inhibitory polypeptide. The fasting C-peptide insulin molar ratio in both the portal vein (1.2 +/- 0.1) and femoral artery (2.1 +/- 0.3) was also unaffected by the glucose stimulus. These results therefore indicate that, since the hepatic extraction of C-peptide is negligible and its clearance kinetics linear, the peripheral C-peptide concentration should accurately reflect the rate of insulin secretion. New approaches to the quantitation of hepatic extraction and secretion of insulin by noninvasive techniques are now feasible.

Mechanisms of insulin resistance in experimental hyperinsulinemic dogs.

This study was undertaken to characterize the insulin resistance and the mechanism thereof caused by chronic hyperinsulinemia produced in dogs by surgically diverting the veins of the pancreas from the portal vein to the vena cava. Pancreatic venous diversion (PVD, n = 8) caused a sustained increase in arterial insulin and decrease in portal insulin concentration compared with the control group (n = 6). Hyperinsulinemic euglycemic clamps were conducted 4 wk after surgery. The increase in the glucose disposal rate (GDR) was significantly less in the PVD group (39.0+/-5.0 vs. 27.9+/-3.2 micromol/kg/min, P < 0.01) compared with the control group, but the suppression of hepatic glucose production by insulin was similar for both groups. Muscle insulin receptor tyrosine kinase activity (IR-TKA) increased from 6.2+/-0.4 to 20.3+/-2.7 in the control group, but from 5.8+/-0.5 to only 12.7+/-1.7 fmol P/fmol IR in the PVD group (P < 0.01). With respect to the periphery, the time to half-maximum response (t1/2a) for arterial insulin was the same for both groups, whereas the t1/2a for lymph insulin (30+/-3 vs. 40+/-4 min, P < 0.05) and GDR (29+/-3 vs. 66+/-10 min, P < 0.01) were greater for the PVD group. Chronic hyperinsulinemia led to marked peripheral insulin resistance characterized by decreased insulin-stimulated GDR, and impaired activation of GDR kinetics due, in part, to reduced IR-TKA. Transendothelial insulin transport was impeded and was responsible for one third of the kinetic defect in insulin-resistant animals, while slower intracellular mechanisms of GDR were responsible for the remaining two thirds.

Real-time in vivo dual-color imaging of intracapillary cancer cell and nucleus deformation and migration.

The mechanism of cancer cell deformation and migration in narrow vessels is incompletely understood. In order to visualize the cytoplasmic and nuclear dynamics of cells migrating in capillaries, red fluorescent protein was expressed in the cytoplasm, and green fluorescent protein, linked to histone H2B, was expressed in the nucleus of cancer cells. Immediately after the cells were injected in the heart of nude mice, a skin flap on the abdomen was made. With a color CCD camera, we could observe highly elongated cancer cells and nuclei in capillaries in the skin flap in living mice. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells and nuclei in the capillaries elongated to fit the width of these vessels. The average length of the major axis of the cancer cells in the capillaries increased to approximately four times their normal length. The nuclei increased their length 1.6 times in the capillaries. Cancer cells in capillaries over 8 microm in diameter could migrate up to 48.3 microm/hour. The data suggests that the minimum diameter of capillaries where cancer cells are able to migrate is approximately 8 microm. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

Visualization of nascent tumor angiogenesis in lung and liver metastasis by differential dual-color fluorescence imaging in nestin-linked-GFP mice.

Nestin regulatory-element-driven green fluorescent protein (ND-GFP) transgenic mice highly express GFP in proliferating endothelial cells and nascent blood vessels. In the present study, we visualized angiogenesis in experimental lung and liver metastases by GFP imaging in the ND-GFP transgenic mice. The murine melanoma cell line, B16F10 expressing red fluorescent protein (RFP), was injected i.v. in ND-GFP mice. ND-GFP was highly expressed in proliferating nascent blood vessels in the tumors that developed in the lung after tail vein injection, and in the tumors that developed in the liver after portal vein injection of RFP-expressing melanoma cells. Liver metastasis and angiogenesis were imaged intravitally. Doxorubicin significantly decreased metastatic angiogenesis in the liver. These results demonstrate a new imageable model of angiogenesis in metastasis in the liver and the lung. This new model should enable further understanding of the onset of angiogenesis in metastasis and its effect on metastatic growth. The model will serve as a unique screen for inhibitors of angiogenesis of metastatic tumors. The fact that liver-metastasis angiogenesis can be imaged in the live animal enables real-time studies of the effect of angiogenesis inhibitors.

Dual-color imaging of nascent angiogenesis and its inhibition in liver metastases of pancreatic cancer.

As previously shown, the stem cell marker nestin is expressed in nascent blood vessels in transgenic nestin-driven green fluorescent protein (ND-GFP) nude mice. This mouse model was recently utilized to evaluate angiogenesis in primary tumors in an orthotopic model of pancreatic cancer. In the present study, nascent angiogenesis of pancreatic cancer liver metastasis in the ND-GFP transgenic nude mice after splenic injection of low-passage xPA-1 human pancreatic cancer cells expressing red fluorescent protein (RFP) was visualized by dual-color fluorescence imaging. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing liver metastasis. Immunohistochemical staining showed that CD31 co-localized in ND-GFP-expressing nascent blood vessels. The density of nascent blood vessels in the tumor was readily quantitated. Gemcitabine significantly decreased the mean nascent blood vessel density in the pancreatic liver metastases. In conclusion, the dual-color model of the ND-GFP nude mouse with RFP-expressing pancreatic cancer liver metastases, enabled the simultaneous visualization and quantitation of nascent angiogenesis and its response to angiogenesis inhibitors. This model will be useful for understanding the mechanism of angiogenesis of pancreatic cancer liver metastasis and for the discovery of effective new inhibitors of this process.

[Surgical treatment of benign lesions and strictures of the bile ducts]. / Chirurgische Behandlung benigner Läsionen und Strikturen der Gallenwege.

Benign strictures of the biliary ducts are treated surgically in 90% of cases. Usually they are caused by trauma to the choledochous duct during gallbladder operations. Younger patients are frequently affected and, particularly if the strictures go untreated, can suffer from secondary complications such as cholangitis or secondary biliary cirrhosis with the serious dangers of portal hypertension and even hepatic failure and death. Although immediate treatment by end-to-end anastomosis has sometimes been described, this method is reasonable only for smooth cuts to the choledochous duct. Good long-term results have been achieved in 86% of cases with Roux-en-Y hepaticojejunostomy. In general, the best way to avoid complications is the all-important surgical maxim of correct indication for the primary operation. The best course is to limit the decision for surgery to symptomatic gallstones.

Host organ specifically determines cancer progression.

In order to further understand the role of the host organ in tumor progression, we have transplanted into nude mice histologically intact human colon cancer tissue on the serosal layers of the stomach (heterotopic site) and the serosal layers of the colon (orthotopic site). Xenograft lines Co-3, which is well differentiated, and poorly differentiated COL-3-JCK were used for transplantation. After orthotopic transplantation of the human colon tumors on the nude mouse colon, the growing colon tumor resulted in macroscopically extensive invasive local growth in 4 of 10 mice, serosal spreading in 9 of 10 mice, musclaris propria invasion in 1 of 10 mice, submucosal invasion in 3 of 10 mice, mucosal invasion in 3 of 10 mice, lymphatic duct invasion in 4 of 10 mice, regional lymph node metastasis in 4 of 10 mice, and liver metastasis in 1 of 10 mice. In striking contrast, after heterotopic transplantation of the human colon tumor on the nude mouse stomach, a large growing tumor resulted but with only limited invasive growth and without serosal spreading, lymphatic duct invasion, or regional lymph node metastasis. It has become clear from these studies that the orthotopic site, in particular the serosal and subserosal transplant surface, is critical to the growth, spread, and invasive and metastatic capability of the implanted colon tumor in nude mice. These studies suggest that the original host organ plays the same critical role in tumor progression.

Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression.

We report the establishment of stable, high-level green fluorescent protein (GFP)-expressing cell lines in vitro that permit the detection and visualization of distant micrometastases when they are implanted orthotopically in nude mice. Chinese hamster ovary cells were transfected with the dicistronic expression vector containing the humanized GFP cDNA. A stable GFP-expressing clone was selected in 1.5 microM methotrexate in vitro and injected s.c. in nude mice. Stable high-level expression of GFP was maintained in the s.c. growing tumors. To use GFP expression for metastasis studies, fragments of s.c. growing tumor, which are comprised of GFP-expressing cells, were implanted by surgical orthotopic implantation in the ovary of nude mice. Subsequent micrometastases were detected in systemic organs and could be visualized by GFP fluorescence in the lung, liver, 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 organ. Confocal microscopy further enabled us to study physiologically relevant patterns of invasion and micrometastasis.

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.

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.

Widespread skeletal metastatic potential of human lung cancer revealed by green fluorescent protein expression.

To understand the skeletal metastatic pattern of non-small cell lung cancer, we developed a stable high-expression green fluorescent protein (GFP) transductant of human lung cancer cell line H460 (H460-GFP). The GFP-expressing lung cancer was visualized to metastasize widely throughout the skeleton when implanted orthotopically in nude mice. H460 was transduced with the pLEIN retroviral expression vector containing the enhanced GFP and the neomycin (G418) resistance gene. A stable high GFP-expressing clone was selected in vitro using 800 microg/ml G418. Stable high-level expression of GFP was maintained in s.c.-growing tumors formed after injecting H460-GFP cells in nude mice. To use H460-GFP for visualization of metastasis, fragments of s.c.-growing H460-GFP tumors were implanted by surgical orthotopic implantation in the left lung of nude mice. Subsequent micrometastases were visualized by GFP fluorescence in the contralateral lung, plural membrane, and widely throughout the skeletal system including the skull, vertebra, femur, tibia, pelvis, and bone marrow of the femur and tibia. The use of GFP-expressing H460 cells transplanted by surgical orthotopic implantation revealed the extensive metastatic potential of lung cancer in particular to widely disseminated sites throughout the skeleton. This new metastatic model can play a critical role in the study of the mechanism of skeletal and other metastasis in lung cancer and in screening of therapeutics that prevent or reverse this process.

Methioninase cancer gene therapy with selenomethionine as suicide prodrug substrate.

In this study, we report a novel approach to gene-directed enzyme prodrug therapy for cancer. This gene therapy strategy exploits the toxic pro-oxidant property of methylselenol, which is released from selenomethionine (SeMET) by cancer cells with the adenoviral-delivered methionine alpha,gamma-lyase (MET) gene cloned from Pseudomonas putida. In MET-transduced tumor cells, the cytotoxicity of SeMET is increased up to 1000-fold compared with nontransduced cells. A strong bystander effect occurred because of methylselenol release from MET gene-transduced cells and uptake by surrounding tumor cells. Methylselenol damaged the mitochondria via oxidative stress and caused cytochrome c release into the cytosol, thereby activating the caspase cascade and apoptosis. Adenoviral MET-gene/SeMET treatment also inhibited tumor growth in rodents and significantly prolonged their survival. Recombinant adenovirus-encoding MET gene-SeMET treatment thereby offers a new paradigm for cancer gene therapy.

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.

Methioninase gene therapy of human cancer cells is synergistic with recombinant methioninase treatment.

Results obtained over the past 40 years have demonstrated that tumor cells of all types tested have an elevated growth requirement for methioninase compared with normal cells. Recombinant methioninase (rMETase) cloned from Pseudomonas putida has been found previously to be an effective antitumor agent attributable to deprivation of the extracellular methionine source of the tumor. To degrade intracellular methioninase, we have now developed an adenoviral vector inserted with the P. putida methioninase (MET) gene (rAd-MET). The in vitro efficacy of rAd-MET was tested on the OVCAR-8 human ovarian cancer cell line, the HT1080 human fibrosarcoma cell line, and human normal fibroblasts. rAd-MET transduction of OVACAR-8 and HT1080 resulted in high levels of methioninase expression up to 10% or more of the total protein of the cells, depending on the multiplicity of infection. The IC50 of rAd-MET for OVCAR-8 cells in 96-well plates was approximately 2 x 106 plaque-forming units (pfu)/well. The IC50 of control adenovirus (control-rAd) was 4 x 10(7) pfu/well, 20 times higher than rAd-MET. In the presence of the IC50 of 2 x 10(6) pfu/well of rAd-MET, the addition of 0.025 units/ml of rMETase, which is 25% of the IC50, resulted in a 90% inhibition of tumor cell number. This indicated that rAd-MET enhanced the efficacy of rMETase. In contrast, 2 x 10(6) pfu/well of control-rAd in combination with 0.025 units/ml of rMETase had an efficacy of only 10% inhibition of cell number. The synergistic effect of the combination of rMETase and rAd-MET was quantitated by calculating the combination index (CI). The CIs for all combinations of rAd-MET and rMETase tested on OVCAR-8 were <0.7 with a mean of 0.5, indicating synergy. Similar synergy of rAd-MET and rMETase was seen on HT1080 human fibrosarcoma cells with a mean of 0.74. In contrast, the CIs of all combinations of rMETase and control adenovirus concentrations tested on both cell lines had a mean CI of approximately 1, which indicated that this combination had only an additive effect. The normal fibroblasts, on the other hand, appeared relatively resistant to the MET gene because in the presence of rMETase, 2.5 x 10(7) pfu/well of rAd-MET or control rAd had almost an identical effect on cell survival. The selectively strong synergy of rAd-MET and rMETase on cancer cells allows reduced levels of each agent to be used, thus decreasing potential side effects.