Abstract:
The present invention relates to a LOX-GFP marker and methods of analyzing tumor burden in ascites or in lung. The invention also relates to a new LOX-GFP-LM cell line which demonstrates increased lung metastasis. The methods of the invention result in better quantitative tumor burden assessment and improved efficacy evaluation. These improved models provide a feasible alternative for ascites or experimental metastasis evaluation of novel cancer therapeutics.

Description:
PRIORITY TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application No. 60/788,250, filed Mar. 31, 2006, which is hereby incorporated by reference in its entirety. 
    
    
     BACKGROUND OF THE INVENTION 
     Several examples of utilizing peritoneal (ascites) tumor growth to assess the activity of chemotherapeutics have been reported in the literature, including one that utilized LOX melanoma cells. For example, R. H. Shoemaker et al.,  Proc. Am. Assoc. Cancer Res.,  26:330 (1985), reported that LOX melanoma cells could form ascites, and that the model could be used to assess cancer therapeutics by using a survival endpoint around day 20. In 2003, H. Nakanishi et al.,  Cancer Sci.,  94:112-118 (2003), reported a peritoneal model utilizing gastric cancer cells tagged with GFP. This model was used to study the chemosensitivity of peritoneal cell growth to an anti-cancer agent. Tumor burden was measured by harvesting GFP cells from the peritoneal cavity, homogenizing the cells, centrifuging cells at 10000 g, and then measuring the fluorescence of the supernatant using a fluorescence counter. In order to extrapolate the number of cells that produced the fluorescence, a calibration curve was used with a standard number of GFP cells. In this model, &gt;1 month was needed for ascites production in the peritoneum. 
     Several examples of utilizing metastatic tumor growth to assess the activity of chemotherapeutics have been reported in the literature. The LOX experimental metastasis model was reported by O. Fodstad et al.,  Int. J. Cancer,  41:442-449 (1988), by R. H. Shoemaker et al. in 1991, and by M. Yeng et al.,  Clin. Cancer Res.,  5:3549-3559, (1999) with GFP-tagged cells. 
     For example, Fodstad et al. reported that LOX cells injected into the tail vein of immunocompromised mice were able to metastasize to lung with nearly 100% frequency. The size and number of colonies differed from one animal to another however, and thus the authors found it was not possible to establish an accurate relationship between the cell number injected and resulting colony number. For this reason, they used animal survival as an endpoint rather than counting metastatic colonies on the lungs. 
     In a report by R. H. Shoemaker et al. 1991, LOX-L cells were generated by 16 cycles of subcutaneous (sc) tumor transplantation, followed by removal of a lung metastasis for growth in vitro. Unlike the parental cell line LOX, the LOX-L cell line was able to metastasize to lung from sc tumor implantation, whereas LOX cells could only metastasize from iv implantation. LOX-L sc tumors were utilized to study the effects of chemotherapeutics on metastasis, however the authors went through the very arduous procedure of transplanting metastatic lungs into new mice for evaluation of pulmonary metastases. In subsequent studies (Wang X et al.,  Int. J. Cancer,  112:994-1002, 2004) the LOX-L model was implanted iv, however metastases were evaluated simply by counting colonies and utilizing a survival endpoint. 
     In a report by M. Yeng et al.,  Clin. Cancer Res.,  5:3549-4559 (1999), metastasis models were established utilizing GFP tagged LOX or B16 melanoma cells. For the LOX-GFP model, tumors were implanted orthotopically (transdermally), whereas for B16 GFP model, cells were implanted iv. GFP was used to identify lung metastases, however the authors failed to quantify the lung metastatic tumor burden, and instead used a subjective (qualitative) endpoint. They simply visualized metastases in live animals or upon necropsy by utilizing a fluorescent microscope to establish the presence or absence of metastases. 
     SUMMARY OF THE INVENTION 
     The human melanoma cell line LOX can induce either ascites when tumor cells are implanted intra-peritoneally, or lung metastasis when inoculated intravenously. The ascites model can be used as a fast drug-screening model, whereas the lung metastasis model may be useful to evaluate anti-metastatic agents. In both models, quantitative analysis of tumor growth and efficacy has been a challenge due to difficulties in assessing tumor burden. To resolve this issue, the present invention provides LOX cells transfected with GFP (called LOX-GFP), and this marker was utilized to analyze tumor burden in ascites or in lung. 
     For the ascites model, 10×10 6  LOX-GFP cells were inoculated intra-peritoneally in Nu/Nu mice, and ascites were harvested after 7 days. Ascites was visualized under a fluorescence microscope and relative fluorescence was quantitated utilizing Acumen Explorer. Anti-proliferative efficacy in this model was validated using a cytotoxic agent, Taxol, as well as some development compounds. 
     For the lung metastasis model, a new cell line called LOX-GFP-LM was established; this cell line was isolated from a lung metastasis colony in mice which was induced through intravenous inoculation of LOX-GFP cells. The LOX-GFP-LM cell line reproducibly colonizes lung 25-30 days post IV inoculation of 2×10 6  cells. Lungs were harvested and visualized under a fluorescence microscope, and the relative fluorescence of homogenized lung suspension was assessed utilizing Acumen Explorer. Anti-metastatic efficacy was validated in this model utilizing two development compounds previously shown to have broad and potent anti-tumor activity in traditional subcutaneous xenograft studies. To compare two new cell lines (LOX-GFP and LOX-GFP-LM) with the parental cell line (LOX), gene array analysis and tumor histopathology were characterized. 
     The present invention provides application of GFP to two human melanoma LOX models in mice, resulting in better quantitative tumor burden assessment and improved efficacy evaluation. These improved models should provide a feasible alternative for ascites or experimental metastasis evaluation of novel cancer therapeutics. 
     The present invention provides a method of evaluating whether a tumor metastasizes which comprises injecting GFP-expressing tumor cells intravenously into an athymic mouse, such as a nude or SCID mouse, followed by sacrificing the mouse and removing one or more tissues to be evaluated. The removed tissue is homogenized, and the level of GFP in the homogenized sample quantified using laser-scanning fluoroscopy, e.g. an Acumen Explorer. 
     The present invention also provides a method for evaluating a candidate drug or protocol for the inhibition of metastasis of a tumor which comprises injecting an athymic mouse intravenously with GFP-expressing tumor cells and administering a candidate drug or protocol to the mouse. The mouse is then sacrificed and one or more tissues removed for evaluation of metastasis inhibition. The removed tissue is homogenized and the level of GFP in a homogenized sample of the tissue quantified using laser-scanning fluoroscopy. The GFP level is compared to the level of GFP in a homogenized sample from a control animal which has not been treated with the candidate drug or protocol. A decreased level of GFP in the treated sample as compared to the control sample denotes inhibition of metastasis. 
     The present invention further provides a method for evaluating a candidate drug or protocol for the treatment of a tumor which comprises injecting an athymic mouse intraperitoneally with GFP-expressing tumor cells and administering a candidate drug or protocol to the mouse. Ascites or an organ containing the tumor is removed for evaluation and the level of GFP in a sample of the ascites of homogenized tissue is quantified using laser-scanning fluoroscopy. The level of GFP in the ascites or homogenized sample is then compared to that from a control animal which has not been treated with the candidate drug or protocol. A decreased level of GFP in the treated sample as compared to the control sample denotes that the candidate drug or protocol is useful in the treatment of said tumor. 
     The present invention provides a method of enhancing the propensity of a tumor cell line to metastasize to a particular tissue which comprises injecting an athymic mouse intraperitoneally with tumor cells that express GFP, removing the ascites formed in the mouse and injecting it intravenously into another athymic mouse. The mouse is sacrificed, and the tissue to which metastasis is to be enhanced is removed. GFP-expressing tumor cells are then recovered from the removed tissue, cultured in vitro, and injected into an athymic mouse, where the cultured tumor cells metastasize to the tissue from which the cells were recovered to a greater degree than the original GFP-expressing tumor cells. 
     The present invention further provides a LOX-GFP-LM cell line which metastasizes to lung to a greater degree than the parental LOX-GFP cell line. This cell line provides advantages in the assays described herein. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1  illustrates the morphology of LOX, LOX-GFP, and LOX-GFP-LM tumors in SCID beige mice. 
         FIGS. 2A through 2M  show Affymetrix microarray data isolated from LOX, LOX-GFP, and LOX-GFP-LM cells demonstrating the effect of these cells on the indicated genes. 
         FIG. 3  indicates the relative fluorescence units (RFU) of ascites samples for [4-Amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Compound A) run on an Acumen Explorer. 
         FIG. 4  depicts the relative fluorescence units (RFU) of ascites samples for 4-[4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound B) and 5-(4-Ethoxy-quinolin-6-ylmeth-(Z)-ylidine)-2-(2-hydroxy-1-(R)-phenyl-ethylamino)-thiazol-4-one (Compound C) run on an Acumen Explorer. 
         FIG. 5  depicts the relative fluorescence units (RFU) of ascites samples for 4-[(4S,5R)-4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound D) and for the combination of Taxol and Compound D run on an Acumen Explorer. 
         FIG. 6  illustrates the percent fluorescent intensity of ascites samples from Nu/Nu mice treated with [4-Amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Compound A) as compared to Vehicle control group. 
         FIG. 7  illustrates the percent fluorescent intensity of ascites samples from Nu/Nu mice treated with 4-[4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound B) or 5-(4-Ethoxy-quinolin-6-ylmeth-(Z)-ylidine)-2-(2-hydroxy-1-(R)-phenyl-ethylamino)-thiazol-4-one (Compound C) as compared to Vehicle control group. 
         FIG. 8  illustrates the percent fluorescent intensity of ascites samples from Nu/Nu mice treated with 4-[(4S,5R)-4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound D) or with the combination of Taxol and Compound D as compared to Vehicle control group. 
         FIG. 9  provides photographs of lung homogenate sample wells of mice treated with 3-methyl-5-(2-chlorophenyl)-7-amino-pyrazolo[3,4][1,4]benzodiazepine (Compound E). Lungs were harvested at day 25 post-implantation (2×10 6  cell/mouse iv) and homogenized and determined run on an Acumen Explorer. 
         FIG. 10  provides photographs of lung homogenate sample wells of mice treated with 4-[(4S,5R)-4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound D). Lungs were harvested at day 26 post-implantation (2×10 6  cell/mouse iv) and homogenized and determined run on an Acumen Explorer. 
         FIG. 11  depicts the relative fluorescence units (RFU) of metastatic lung tissue from SCID beige mice treated with 3-methyl-5-(2-chlorophenyl)-7-amino-pyrazolo[3,4][1,4]benzodiazepine (Compound E) as compared to Vehicle group. 
         FIG. 12  depicts the relative fluorescence units (RFU) of metastatic lung tissue from SCID beige mice treated with 4-[(4S,5R)-4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound D) as compared to Vehicle group. 
         FIG. 13  provides Kaplan-Meier survival curves of SCID beige mice, implanted with LOX-GFP-LM cells, that were treated with 3-methyl-5-(2-chlorophenyl)-7-amino-pyrazolo[3,4][1,4]benzodiazepine (Compound E). 
         FIG. 14  provides Kaplan-Meier survival curves of SCID beige mice, implanted with LOX-GFP-LM cells, that were treated with 4-[(4S,5R)-4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound D). 
         FIG. 15  is a simple schematic of the crucial portions of a pCMV-tag 5A plasmid containing GFP and Neo. 
         FIG. 16  ( a - i ) depicts the restriction map for the GFP expression vector. 
         FIG. 17  provides photographs of lung homogenate sample wells of mice injected either with LOX-GFP or with LOX-GFP-LM tumor lines. Lungs were harvested at 14, 21, and 28 days post-injection. 
         FIG. 18  compares LOX-GFP-LM and LOX-GFP-induced experimental lung metastasis in SCID mice. Samples were measured on a 96 well plate with Acumen Explorer. 
         FIG. 19  provides Kaplan-Meier survival curves both LOX-GFP and LOX-GFP-LM tumor lines in SCID beige mice. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides several key modifications over the LOX peritoneal (ascites) models previously used to produce a model having a much shorter duration than those previously employed. The present model allows for rapid quantification of cell number as an endpoint. Although the duration for previous studies was fairly short at only 20 days, the current model does not rely on survival as the sole endpoint, and can be completed in as little as 7 days. 
     The current model utilizes cells tagged with GFP, however the method of ascites quantification has been improved by eliminating the homogenization and centrifugation steps. The process of the invention directly measures cell number from ascites using the Acumen Explorer. 
     The present invention provides significant improvements to the LOX experimental metastasis models described previously by stably transfecting GFP into the cells, so that lungs can be removed and metastatic tumor burden can be accurately quantified by measuring fluorescence. This method reduces reliance on survival as an endpoint (although it may sometimes be monitored when scientifically relevant). The model of the present invention utilizes an experimental metastatic model using iv implantation of LOX cells rather than using LOX-L cells. Additionally, metastases is quantified using GFP-tagged cells rather than relying on colony counts and survival, as those methods are not accurate enough to discern small differences in metastatic tumor burden. 
     In the model of the invention, the metastatic tumor burden is quantified in order to accurately assess the anti-metastatic capability of experimental therapeutics using various treatment schedules. Therefore the step of visualizing metastases in vivo was omitted in favor of removing the lungs, homogenizing them, and measuring the relative fluorescence of lungs from vehicle treated Vs. therapeutic treated mice. 
     In one aspect, the present invention provides a stable clone of the LOX melanoma cell line expressing green fluorescent protein (GFP) and an assay for evaluating the anti-tumor efficacy of potential clinical candidate therapeutics, i.e. drugs and protocols, in vivo. In particular, the invention provides two different in vivo models; 1) a short (7-10 day) peritoneal (ascites) model for rapid screening of compounds for efficacy, and 2) an experimental metastasis model for assessment of the anti-metastatic capabilities of novel cancer therapeutics. 
     In another aspect, the present invention provides a peritoneal (ascites) model for rapid screening of the anti-tumor efficacy of potential cancer clinical candidate therapeutics in vivo. The model is unique in that it provides efficacy data in as little as 7 days and provides quantitative rather than qualitative data. 
     In still another aspect, the present invention provides an experimental metastasis model for evaluating the anti-metastatic efficacy of potential cancer clinical candidate therapeutics in vivo. The model is unique in that it provides quantified data regarding metastatic tumor burden rather than relying on survival as the sole endpoint. 
     Bicistronic construct denotes a mammalian expression vector containing two genes inserted into expression vector. Bicistronic GFP construct denotes a mammalian expression vector, preferably pCMV-tag 5A (Stratagene, Genbank accession number AF076312), which has been modified to contain a nucleotide sequence encoding a GFP molecule, preferably GFP from  Renilla mullerei , and a nucleotide sequence encoding Neo (Neomycin resistant gene) for G418 selection. 
     A spontaneous metastasis model is one in which a primary tumor is established in an animal and is allowed to grow and spread to secondary sites without any manipulation or intervention. This process requires that the cells from the primary tumor gain entry into the circulatory system through their natural capability, and then seed and grow in distant sites. 
     An experimental metastasis model is one in which a primary tumor is not established. Cells are directly injected into the circulatory system to mimic the seeding and growth process of metastasis to distant sites. 
     Green Fluorescent Protein (GFP) is a luminescent protein produced by species of soft coral. GFP can be obtained from a variety of different sources, including  Renilla mullerei, Renilla reniformis, Renilla kollikeri Aeruorea victoria . While any GFP molecule can be used in the present invention, the preferred GFP is from  Renilla mullerei.    
     LOX-GFP cell line is a cell line created by transfection of LOX melanoma cells obtained from National Cancer Institute (NCI) with the bicistronic GFP construct described above. 
     LOX-GFP-LM cell line is a cell line created by injecting athymic mice intraperitoneally with LOX-GFP cells, removing ascites containing LOX-GFP cells from the mice, injecting said ascites intravenously into mice, collecting the resultant metastatic lungs from the mice, and culturing colonies of GFP-expressing tumor cells recovered from the metastatic lung tissue in vitro. 
     Tumor cells that stably express green fluorescent protein can be prepared in the following manner. Tumor cells from an established tumor cell line can be transfected in a conventional manner with a bicistronic GFP construct prepared in accordance with the procedure in Example 1. For example, Fugene, a multi-component lipid-based (non-liposomal) transfection reagent (Roche Molecular Diagnostics) can be added to a serum free medium, such as RPMI1640, followed by addition of the bicistronic GFP construct. While the ratio of Fugene to construct can vary, the ratio is advantageously 3:1. The sample is incubated, for example at room temperature, for a period of about 30 minutes, and the mixture transferred to a flask of tumor cells. The tumor cells are present in culture at a ration of about 80%. The cells are incubated for a period of about 6 hours, followed by removal of the incubation medium and addition of a selection medium containing 1% Geneticin (G418). 
     Selection for G418 resistance will take a few weeks, for example 3 to 6 weeks, after which the cells are sorted for those which show the greatest GFP expressions. The top 5% GFP expressing cell population are then selected, isolated, grown up, and further sorted to obtain cells having 100% GFP expression. 
     Tumor cells that metastasize to a particular tissue more aggressively than the corresponding parental tumor cell line can be prepared in the following manner. Athymic mice are implanted, preferably intra-peritoneally (ip), with approximately 10 to 20 million GFP-expressing tumor cells each. After about 10 to 14 days, ascites fluid containing the GFP-expressing tumor cells is harvested from the mice, and the ascites diluted with PBS. The ascites/PBS solution is filtered through a 40 μm nylon cell strainer and centrifuged at about 1500 rpm. Pelleted cells are resuspended in PBS and counted to achieve the desired cell concentration. 
     Athymic mice are then transplanted intravenously (iv), for example via the tail vein, with the GFP-expressing tumor cells isolated from ascites (above). The cells are preferably transplanted at a concentration between about 1×10 6  cell/mouse and about 2×10 6  cell/mouse. Once the mice are moribund or dead, the tissue of interest, for example lung or breast tissue, is isolated and examined under a fluorescence stereomicroscope to see potential micro-metastases. 
     Colonies of GFP-expressing tumor cells which show very strong expression are recovered from the tissue, for example by gentle dissection. The colonies can then be ground on sterile metal gauze (#40), washed with 2-3 ml culture medium, and centrifuged at about 1500 rpm. The cell pellets can then be washed in a serum free medium, such as RPMI1640 culture medium which preferably contains about 10% penicillin/streptomycin and about 10% fetal bovine serum (FBS). The cells are seeded into flasks of RPMI1640 medium containing about 10% FBS and about 2% penicillin/streptomycin. The cells are routinely passaged in selection medium containing G418 to remove any mouse cell contaminants. After several, preferably 2-3, passages, cultures can be scaled up and frozen down for future use. The cells will metastasize to the tissue from which they were isolated to a greater extent than the original GFP-expressing tumor cells. This ability can be shown by the following assay. Hereinafter, these will be referred to as enhanced metastatic tumor cells (EMTC). 
     Athymic mice are implanted iv with approximately 1×10 6  to about 5×10 6  EMTC into the tail vein. At approximately 25 to 30 days post implantation, moribundity or mortality is assessed, and the mice are euthanized. The tissue to which the cells are expected to metastasize is isolated and homogenized in PBS. A sample, e.g. 0.1 ml, of the homogenized tissue is transferred to a 96 well plate and fluorescence is measured via laser-scanning fluoroscopy, for example using an Acumen Explorer. 
     The cell lines prepared and evaluated by the above procedures are valuable for the evaluation of candidate therapies, in particular clinical drug candidates and protocols, for the treatment of cancer and/or inhibition of metastasis. For evaluation of candidate therapies against tumors, GFP-expressing tumor cells in PBS are injected intra-peritoneally (ip) into athymic mice. Preferably about 10 million cells in a volume of about 500 μl PBS are injected. The mice are divided into control and treatment groups, and the treatment groups are treated with the candidate drug or protocol. It should be understood that the GFP-expressing tumor cells can be injected into the mice first, followed by treatment with the candidate drug or protocol, or the candidate drug or protocol can be administered followed by injection of the mice with GFP-expressing tumor cells. 
     The ascites is harvested by euthanizing the mice and aspirating the ascites fluid from the peritoneum. The peritoneal cavity is rinsed with saline, which is then recovered. The ascites and the recovered saline are transferred to a tube, filtered through a 40 μm nylon filter to obtain a single cell suspension, and centrifuged at about 1500 rpm for a period of about 10 minutes. The supernatant is removed, and the cell pellet resuspended in fresh saline. A sample, e.g. 0.1 ml, from each mouse is transferred into a 96 well plate to evaluate cell number (reported as relative fluorescence units) utilizing laser-scanning fluoroscopy, for example, an Acumen Explorer. If the treated mice show a lower relative fluorescence than the control group, the candidate therapy is useful for the treatment of that type tumor. 
     For evaluation of candidate therapies for inhibiting metastasis, enhanced metastatic tumor cells maintained in RPMI 1640 medium plus 10% FBS and 1% Geneticin (G418) are injected intravenously into athymic mice via the tail vein. Preferably about 2 million cells in a volume of about 200 μl serum free RPMI1640 are employed. The mice are randomized into control and treatment groups, and the treatment groups are treated with the candidate drug or protocol. It should be understood that the EMTCs can be injected into the mice first, followed by treatment with the candidate drug or protocol, or the candidate drug or protocol can be administered followed by injection of the mice with EMTCs. When the control mice are moribund or when they die, their metastatic tumor burden evaluated. 
     In addition to survival, a quantitative evaluation of anti-metastatic efficacy can be made using the present invention by measuring the fluorescence intensity of tissue homogenates. Live mice are euthanized, and tissue to be evaluated is removed and homogenized in saline. A sample of the tissue homogenate, e.g. 0.2 ml, from each mouse is transferred to a 96 well plate, and fluorescence is then read using laser-scanning fluoroscopy, e.g. an Acumen Explorer. Using this method, the amount of metastasis as compared to the control group can be determined. If the treated my show a lower metastatic tumor burden, the candidate drug inhibits metastasis. 
     EXAMPLES 
     Example 1 
     Preparation of Bicistronic GFP Construct 
     The bicistronic GFP construct was prepared and provided by Anne Chua and Ueli Gubler, and contained genes for both  Renilla mulleri  GFP (Prolume Ltd., Pinetop, Ariz.) and the Neomycin phosphotransferase (Neomycin resistant marker) for G418 selection. The sequence of the  R. mulleri  GFP was engineered into a mammalian expression vector as follows. The vector “pCMV-tag 5A” (Stratagene, Genbank accession number AF076312) was first modified by removing the sequence fragment between the single NotI and BstBI sites. This leaves a plasmid backbone consisting of the ColEl origin of replication, the HSV-TK polyA sequence and the CMV promoter. The deleted fragment was then replaced with a fragment encoding an [IRES-Neomycin phosphotransferase resistance marker]. The IRES-sequence was disabled based on the principle described by e.g. Rees et al, Biotechniques 20:102, 1996, incorporated by reference herein. The disabling fragment was chosen to represent the bacterial beta-lactamase (“bla”) promoter; this strategy allowed for the use of the neomycin-phosphotransferase marker for plasmid selection in  E. coli  (Kanamycin). In a third step, the ORF for the  R. mulleri  GFP was inserted upstream of the IRES sequence, in between two SfiI sites. The HSV-TK sequence that is located downstream of the NEO-resistance gene serves as a polyA signal sequence for expression in mammalian cells. A simple schematic of the crucial portions of this plasmid is shown in  FIG. 15 . 
     Example 2 
     Preparation of Bicistronic GFP Construct 
     The sequence of  R. mulleri  GFP was engineered for stable expression in mammalian cells using a specifically designed modular vector. The construct was prepared and provided by Ann Chua and Ueli Gubler, and contained genes for both  Renilla mulleri  GFP (Prolume Ltd., Pinetop, Ariz.) and the Neomycin phosphotransferase (Neomycin resistant marker) for G418 selection. The sequence of the  R. mulleri  GFP was engineered into a mammalian expression vector as follows. 
     Step 1 
     The vector “pCMV-tag 5A” (Stratagene, Genbank accession number AF076312) was first modified by removing the sequence fragment between the single NotI and BstBI sites. This leaves a plasmid backbone consisting of the ColEl origin of replication, the HSV-TK polyA sequence and the CMV promoter. 
     Step 2 
     By overlap-PCR, a module of having the general makeup 5′-AscI-IRES-Neomycin phosphotransferase-BstB1-3′ was generated. Within this module, the IRES-sequence was disabled based on the principle described by e.g. Rees et al, Biotechniques 20:102, 1996, incorporated by reference herein. The disabling fragment was chosen to represent the bacterial beta-lactamase (“bla”) promoter (Seq ID No. 1); this strategy allowed for the use of the neomycin-phosphotransferase marker for plasmid selection in  E. coli  (Kanamycin) as well as selection of mammalian cells in G418. It also eliminated the need for an extra transcription unit for plasmid selection in  E. coli , making the final plasmid smaller. 
     Step 3 
     The plasmid-derived NotI/BstbI module from step 1 and the AscI-IRES-Neo-BstbI module from step 2 were subsequently ligated and circularized by addition of a synthetic short AscI to NotI-linker. DNA was transformed and single isolates were checked for proper assembly of the three fragments. A properly assembled plasmid clone was selected for the last modification. 
     Step 4 
     The cloning sites for the gene of interest (GFP) were subsequently introduced into the plasmid via a short synthetic linker of the structure EcoRV-SfiIa-stuffer-SfiIb-NotI. This linker was cloned into the plasmid derived in step 3 via ligation in between the OliI-NotI sites, thus placing it upstream of the IRES-NEO module. OliI and EcoRV are both blunt-end cutters, making them compatible for ligation without recreating the sites. The rationale behind using SfiI sites for cloning the gene of interest was twofold: SfiI is an 8-base cutter and thus occurs very infrequently as internal sites in ORFs chosen for expression in this vector. The site has the recognition sequence ggccnnnnnggcc (Seq ID No. 2), allowing the design of two different sites at either end of an ORF for directional cloning. The sequence 5′-ggccattatggcc-3′ (Seq ID No. 3) was chosen as the SfiI-a (upstream) site, while the SfiI-b (downstream) site has the sequence 5′-ggccgcctcggcc-3′ (Seq ID No. 4). 
     Step 5 
     The ORF for the  R. mulleri  GFP engineered to have the appropriate SfiI sites was inserted upstream of the IRES sequence, in between two SfiI sites, resulting in a plasmid of 4196 bp length (Seq ID No. 5). The restriction map for the GFP Expression Vector is provided in  FIG. 16 . 
     Example 3 
     Establishment of LOX-GFP Cells 
     Cell Transfection 
     Cells from the human melanoma cell line LOX (National Cancer Institute) were cultured in RPMI1640 medium RPMI1640 medium with 10% fetal bovine serum (FBS). All culture medium and related reagents were purchased from Gibco (Invitrogen Corporation, Carlsbad, Calif.). Cells were transfected using Fugene (Roche Molecular Diagnostics) transfecting reagent at a ratio of 3:1 (Fugene:DNA). The bicistronic GFP construct was kindly prepared and provided by Ann Chua and Ueli Gubler in accordance with Example 1. The construct contained genes for both  Renilla mullerei  (Prolume Ltd., Pinetop, Ariz.) and Neo (Neomycin resistant gene) for G418 selection. 
     100 μl of RPMI1640 serum free medium was added to a small sterile tube, and then 9 μl pre-warmed Fugene was added. Finally, 3 μl GFP DNA construct was added to the bottom of the tube, mixed, and incubated at room temperature for 30 min. The entire Fugene/DNA mixture was added to one T-25 flask of 80% confluent LOX cells, and the cells were incubated for 6 hrs. Following incubation, the medium in the flask was removed and replaced with selection medium containing 1% Geneticin (G418). 
     Selection for G418 resistance took about four weeks, after which approximately 30% of cells expressed GFP at various levels. To further select for the most highly GFP expressing cells, the cells were sorted at the Department of Pathology and Pediatrics, UMDNJ. Cells were sorted to collect the top 5% GFP expressing cell population. Cells isolated and grown up from the first sort were subsequently sorted a few weeks later, so that the resulting cells achieved 100% GFP expression. These LOX-GFP cells were then frozen down for future in vivo use. 
     Example 4 
     Establishment of LOX-GFP-LM Cells 
     Five female Nu/Nu mice (Charles River) were implanted intra-peritoneally (ip) with 10 million LOX-GFP cells each. After 13 days, ascites fluid containing LOX-GFP cells was harvested from the mice, and the ascites was diluted 1:4 with PBS. The ascites/PBS solution was then filtered through a 40 μm nylon cell strainer and centrifuged at 1500 rpm. Pelleted cells were resuspended in PBS and counted to achieve the desired cell concentration. 
     Twenty female Nu/Nu mice (10 mice/group) were implanted intravenously (iv) via the tail vein with the LOX-GFP tumor cells isolated from ascites (above) at either 2×10 6  cell/mouse or 1×10 6  cell/mouse. After a few mice in the group were found moribund or dead, the remaining mice in the group were euthanized. Lungs were isolated and examined under a fluorescence stereomicroscope to see potential micro-metastases. The resultant iv LOX-GFP lung metastases are listed in Table 1. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 LOX-GFP ascites implantation into Nu/Nu mice. 
               
             
          
           
               
                 Days post- 
                   
                   
               
               
                 implantation 
                 2 × 10 6  cell/mouse 
                 1 × 10 6  cell/mouse 
               
               
                   
               
               
                 Day 36 
                 5 mice dead 
                   
               
               
                   
                 2 mice with lung 
               
               
                   
                 metastases with 
               
               
                   
                 moderate GFP 
               
               
                   
                 expression. 
               
               
                   
                 3 mice had no signs 
               
               
                   
                 of metastasis 
               
               
                 Day 59 
                   
                 2 mice dead 
               
               
                   
                   
                 2 mice with lung metastases with no 
               
               
                   
                   
                 GFP expression. 
               
               
                   
                   
                 1 mouse (No. 10) had lung 
               
               
                   
                   
                 metastases with strong GFP 
               
               
                   
                   
                 expression 5 mice had no signs 
               
               
                   
                   
                 of metastasis 
               
               
                   
               
             
          
         
       
     
     It appeared that the rate of metastasis to lung was not as high as reported in the literature, which might cause difficulty for quantitative analysis. Some metastatic colonies lost GFP expression, suggesting the cell line was not stable in vivo. One mouse (No. 10) from the 1×10 6  cell group had very strong GFP expression in the lung metastatic colonies. 
     Four colonies of LOX-GFP cells (about 2×3 mm) were recovered from the lung of mouse No. 10 (see above), by gentle dissection. Each of the colonies was ground separately on sterile metal gauze (#40), washed with 2-3 ml culture medium and centrifuged at 1500 rpm. Cell pellets were washed in RPMI1640 culture medium containing 10% penicillin/streptomycin and 10% FBS and were seeded into T-25 flasks containing 10 ml of RPMI1640 medium containing 10% FBS and 2% penicillin/streptomycin. The cells were routinely passaged in selection medium containing G418 to remove any mouse cell contaminants. After 2-3 passages, cultures from colony numbers 1 and 2 were discarded due to weak GFP expression and poor growth. Cultures from colony numbers 3 and 4 were scaled up and frozen down for future use. Cells from colony number 4 were deemed superior in terms of GFP expression and growth and were named LOX-GFP-LM (LM for Lung Metastasis). 
     Example 5 
     Metastasis of LOX-GFP-LM Cells In Vivo 
     Thirty female SCID beige mice (Charles River) were implanted iv with one, two, or five million LOX-GFP-LM cells into the tail vein. At day 29 post implantation, moribundity or mortality from each group up to that point was recorded, and the remaining mice were euthanized. Lungs were isolated and homogenized in 3 ml of PBS per sample. 0.1 ml per sample of lung homogenate was transferred into a 96 well plate and fluorescence was measured using an Acumen Explorer. After 29 days post-implantation, the incidence of morbidity or mortality was directly related to the cell number implanted, with the highest morbidity and mortality rate observed in mice implanted with 5 million cells (Table 2.) All mice had GFP expressing lung metastatic colonies, however the number and density of the lung metastases varied greatly. 
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 LOX-GFP-LM induced experimental lung metastases 
               
               
                 in SCID beige mice. 
               
             
          
           
               
                   
                   
                   
                   
                 Relative 
               
               
                   
                   
                   
                   
                 Fluorescence 
               
               
                   
                 Morbidity/ 
                 Lung 
                 GFP Expression 
                 Units of Lung 
               
               
                   
                 Mortality 
                 Metastases 
                 Observed in Lung 
                 Homogenates 
               
               
                 Group 
                 Day 29 
                 Present 
                 Metastases 
                 (mean ± SD) 
               
               
                   
               
               
                 5 × 10 6  cells/ 
                 7/10 
                 3/3 
                 3/3 
                 Not assessed 
               
               
                 mouse 
               
               
                 2 × 10 6  cells/ 
                 4/10 
                 6/6 
                 6/6 
                 58457423 ± 
               
               
                 mouse 
                   
                   
                   
                 52009858 
               
               
                 1 × 10 6  cells/ 
                 2/10 
                 8/8 
                 8/8 
                 45712175 ± 
               
               
                 mouse 
                   
                   
                   
                 30253653 
               
               
                   
               
             
          
         
       
     
     Example 6 
     Characterization of LOX-GFP-LM, LOX-GFP, and LOX Cells 
     Morphology of LOX, LOX-GFP, and LOX-GFP-LM Tumors In Vivo: 
     Nine female SCID beige mice (Charles River) were implanted subcutaneously (sc) with either LOX or LOX-GFP cells, or were implanted iv with LOX-GFP-LM cells. LOX and LOX-GFP tumors were allowed to grow until they reached a volume of ˜300-400 mm 3  (about 10-14 days post implantation) and were then collected and fixed in 10% formalin. LOX-GFP-LM cells were allowed to develop lung metastases over 29 days, and then portions of the lung were harvested and fixed in 10% Formalin. Both tumor and lung samples were stained with H &amp; E and morphology was assessed. No difference in morphology between the tumors derived from the three different LOX tumor cell lines (LOX, LOX-GFP and LOX-GFP-LM) was observed ( FIG. 1 ). 
     Example 7 
     Gene Microarray Analysis of LOX, LOX-GFP, and LOX-GFP-LM Cell Lines 
     Cells were plated in 6 well culture plates with RPMI-1640, 10% FBS, and 1% Penicillin/Streptomycin (plus 0.5% G418 for LOX-GFP and LOX-GFP-LM cells), and incubated for 48 hours. After removing medium, the cells were washed once with PBS, 0.8 ml of RLT buffer was added per well, and the plate was shaken for 2 min at room temperature. Cell suspensions from each well were transferred into separate tubes and were frozen at −80° C. for future microarray analysis. Four separate samples from each tumor line were run in the microarray assay using Affymetrix U133plus2 chips. Unique gene signatures were shown for both LOX-GFP and LOX-GFP-LM cells as compared to the LOX parental cell line. ( FIG. 2 ) In LOX-GFP-LM cells, 124 genes were found to be altered overall, with 67 genes up-regulated and the remaining 57 genes down-regulated, as compared to LOX-GFP cells. Among the genes with at least 4 fold up-regulation, a series of genes (at least 7 genes, marked in bold) were recognized to be related adhesion, matrix degradation, or angiogenesis. Another category of genes (marked in underline) were recognized as related to growth factors or differentiation. Both series of genes comprise the type of genes that might be expected to be enriched in a cell population with a more aggressive and invasive phenotype. 
     Example 8 
     LOX-GFP Peritoneal (Ascites) Model 
     Human melanoma LOX-GFP cells, prepared in accordance with the procedure of Example 3, were maintained in RPMI 1640 medium plus 10% FBS, and 1% Geneticin (G418). Female Nu/Nu mice were injected intra-peritoneally (ip) with 10 million LOX-GFP cells in a volume of 500 μl PBS, randomized into groups, and treated as shown in Tables 3, 4, and 5 with a variety of doses and/or dose schedules. 
     Compounds Tested 
     
         
         [4-Amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Compound A) 
       
    
     
       
                 
         
             
             
         
      
         
         4-[4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound B) 
       
    
     
       
                 
         
             
             
         
      
         
         5-(4-Ethoxy-quinolin-6-ylmeth-(Z)-ylidine)-2-(2-hydroxy-1-(R)-phenyl-ethylamino)-thiazol-4-one (Compound C) 
       
    
     
       
                 
         
             
             
         
      
         
         4-[(4S,5R)-4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound D) 
       
    
     
       
                 
         
             
             
         
      
     
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Treatment groups for LOX-GFP Ascites model. 
               
             
          
           
               
                   
                 iv cell 
                   
                   
                   
                 Day of 
               
               
                   
                 injection 
                   
                 Number 
                 Days dosed (after 
                 ascites 
               
               
                 Group 
                 Day 0 
                 Treatment 
                 of mice 
                 cell injection) 
                 harvest 
               
               
                   
               
               
                 1 
                 10 × 10 6   
                 Vehicle for Taxol 
                 2 
                 Days 4, 5, &amp; 6 
                 Day 7 
               
               
                 2 
                 cells/mouse 
                 Taxol 10 mg/kg iv, 0.2 ml, 
                 2 
                 Days 4, 5, &amp; 6 
                 Day 7 
               
               
                   
                   
                 3 doses 
               
               
                 3 
                   
                 Taxol 10 mg/kg iv, 0.2 ml, 
                 2 
                 Days 5 &amp; 6 
                 Day 7 
               
               
                   
                   
                 2 doses 
               
               
                 4 
                   
                 Taxol 10 mg/kg iv, 0.2 ml, 
                 2 
                 Day 6 
                 Day 7 
               
               
                   
                   
                 single dose 
               
               
                 5 
                   
                 Vehicle for Compound A 
                 2 
                 Days 4, 5, &amp; 6 
                 Day 7 
               
               
                 6 
                   
                 Compound A 40 mg/kg 
                 2 
                 Days 4, 5, &amp; 6 
                 Day 7 
               
               
                   
                   
                 po, 0.2 ml, 3 doses 
               
               
                 7 
                   
                 Compound A 40 mg/kg 
                 2 
                 Days 5 &amp; 6 
                 Day 7 
               
               
                   
                   
                 po, 0.2 ml, 2 doses 
               
               
                 8 
                   
                 Compound A 40 mg/kg 
                 2 
                 6 Day 6 
                 Day 7 
               
               
                   
                   
                 po, 0.2 ml, single dose 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Treatment groups for LOX-GFP Ascites model. 
               
             
          
           
               
                   
                 Tumor cell 
                   
                   
                   
                 Day of 
               
               
                   
                 implanted 
                   
                 Number 
                 Days dosed (after 
                 ascites 
               
               
                 Group 
                 (day 0) 
                 Treatment 
                 of mice 
                 cell injection) 
                 harvest 
               
               
                   
               
               
                 1 
                 10 × 10 6   
                 Vehicle for Compound B 
                 5 
                 Days 2, 3, 4.5, 6 &amp; 7 
                 Day 8 
               
               
                 2 
                 cells/mouse 
                 Compound B 40 mg/kg 
                 5 
                 Days 2, 3, 4.5, 6 &amp; 7 
                 Day 8 
               
               
                   
                   
                 sc, 0.2 ml, 6 doses 
               
               
                 3 
                   
                 Vehicle for Compound B 
                 5 
                 Days 2, 3, 4.5, 6 &amp; 7 
                 Day 8 
               
               
                 4 
                   
                 Compound C 200 mg/kg 
                 5 
                 Days 2, 3, 4.5, 6 &amp; 7 
                 Day 8 
               
               
                   
                   
                 po bid, 0.2 ml, 12 doses 
               
               
                 5 
                   
                 Vehicle for Taxol 
                 5 
                 Days 5, 6 &amp; 7 
                 Day 8 
               
               
                 6 
                   
                 Taxol 15 mg/kg iv, 0.2 ml, 
                 5 
                 Days 5, 6 &amp; 7 
                 Day 8 
               
               
                   
                   
                 3 doses 
               
               
                   
               
             
          
         
       
     
                                               TABLE 5                   Treatment groups for LOX-GFP Ascites model.                Tumor cell               Day of           implanted       Number   Days dosed (after   ascites       Group   (day 0)   Treatment   of mice   cell injection)   harvest                   10 × 10 6     Vehicle for Compound D   4   Days 4, 5 &amp; 6   Day 7       1   cells/mouse   Compound D 100 mg/kg   4   Days 4, 5 &amp; 6   Day 7               po bid, 0.2 ml, 6 doses       2       Compound D 50 mg/kg   4   Days 4, 5 &amp; 6   Day 7               po bid, 0.2 ml, 6 doses       3       Compound D 25 mg/kg   4   Days 4, 5 &amp; 6   Day 7               po bid, 0.2 ml, 6 doses       4       Taxol 15 mg/kg iv, 0.2 ml,   4   Days 5 &amp; 6   Day 7               2 doses       5       Taxol 15 mg/kg iv, 0.2 ml,   4   Days 5 &amp; 6   Day 7               2 doses + Compound       (Taxol)               D 100 mg/kg po bid, 0.2 ml,       Days 4, 5 &amp; 6               6 doses       (Compound D)       6       Taxol 15 mg/kg iv, 0.2 ml,   4   Days 5 &amp; 6   Day 7               2 doses + Compound       (Taxol)               D 50 mg/kg po bid, 0.2 ml,       Days 4, 5 &amp; 6               6 doses       (Compound D)       7       Taxol 15 mg/kg iv, 0.2 ml,   4   Days 5 &amp; 6   Day 7               2 doses + Compound       (Taxol)               D 25 mg/kg po bid, 0.2 ml,       Days 4, 5 &amp; 6               6 doses       (Compound D)                    
Ascites Harvesting Procedure (at Day 7 or 8 Post Implantation):
 
     Mice were euthanized, and then a small incision was made along the midline of the abdomen through the skin and peritoneum. A glass Pasteur pipet was utilized to aspirate and remove ascites fluid from the peritoneum, and the ascites was transferred to a 15 ml tube. 3 ml saline was used to rinse the peritoneal cavity, and all of the saline was recovered and transferred into the 15 ml tube containing the ascites fluid. The ascites cell suspension was filtered through a 40 μm nylon filter to obtain a single cell suspension and centrifuged at 1500 rpm for 10 min. The supernatant was removed, and the cell pellet was resuspended in 2 ml of fresh saline. 0.1 ml from each sample was transferred into a 96 well plate to evaluate cell number (reported as relative fluorescence units) utilizing an Acumen Explorer. 
     Results 
     Seven or eight days was a sufficient duration for adequate ascites to form in mice implanted ip with LOX-GFP cells, and additionally was sufficient to measure the growth inhibitory properties of cancer therapeutics administered systemically. Both Taxol and Compound A demonstrated inhibitory effects on LOX-GFP ascites growth that was directly dependent on the number of treatments. A single dose did not inhibit cell growth, whereas two doses reduced cell growth, and three doses reduced cell growth maximally. ( FIGS. 3 and 6 ). Both Compound B and Compound C demonstrated inhibitory effects on LOX-GFP ascites growth. ( FIGS. 4 and 7 ). Compound D inhibited LOX-GFP ascites growth at several doses, however the effect was not dose-dependent. With regard to ascites growth inhibition, there was no added benefit to combining Compound D with Taxol as compared to Taxol alone, however the combination was not antagonistic. ( FIGS. 5 and 8 ). 
     Example 9 
     LOX-GFP-LM Metastasis Model 
     Human melanoma LOX-GFP-LM cells, prepared in accordance with the procedure of Example 4, were maintained in RPMI 1640 medium plus 10% FBS and 1% Geneticin (G418). Female SCID beige mice were injected iv via the tail vein with 2 million cells in a volume of 200 μl serum free RPMI1640, randomized into groups, and treated as shown in Tables 6 and 7 with a variety of doses and/or dose schedules. When &gt;3 mice in the Vehicle treated group were found moribund, five mice per treatment group were removed to evaluate metastatic lung tumor burden. The remaining mice from each group were monitored for survival benefit until they were moribund. 
     Compounds Tested 
     
         
         4-[(4S,5R)-4,5-Bis-(4-chloro-phenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one (Compound D) 
       
    
     
       
                 
         
             
             
         
      
         
         3-methyl-5-(2-chlorophenyl)-7-amino-pyrazolo[3,4][1,4]benzodiazepine (Compound E) 
       
    
     
       
                 
         
             
             
         
      
     
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Treatment groups for LOX-GFP-LM experimental metastasis 
               
               
                 model (Compound E Study) 
               
             
          
           
               
                   
                 Tumor 
                   
                   
                   
                 Day of lung 
               
               
                   
                 cells 
                   
                   
                 Days of 
                 harvest 
               
               
                   
                 injected 
                   
                 Number of 
                 dosing after 
                 (5 
               
               
                 Groups 
                 (day 0) 
                 Treatment 
                 mice 
                 cell injection 
                 mice/group) 
               
               
                   
               
               
                 1 
                 2 × 10 6   
                 Vehicle 
                 20 
                 Day −1 to 21 
                 25 
               
               
                 2 
                 cells/ 
                 Compound E 
                 15 
                 Day −1 to 21 
                 25 
               
               
                   
                 mouse 
                 5 mg/kg po, 
                   
                 (7+/4− 
               
               
                   
                   
                 bid 
                   
                 schedule) 
               
               
                 3 
                   
                   
                 15 
                 Day −1 to 7 
                 25 
               
               
                 4 
                   
                   
                 15 
                 Day 3 to 21 
                 25 
               
               
                   
                   
                   
                   
                 (4+/3− 
               
               
                   
                   
                   
                   
                 schedule) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Treatment groups for LOX-GFP-LM experimental metastasis model 
               
               
                 (Compound D Study) 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Day of 
               
               
                   
                 Tumor 
                   
                   
                 Days of 
                 lung 
               
               
                   
                 cell 
                   
                 Number 
                 dosing 
                 harvest 
               
               
                   
                 implanted 
                   
                 of 
                 after cell 
                 (5 mice/ 
               
               
                 Groups 
                 (day 0) 
                 medication 
                 mice 
                 injection 
                 group) 
               
               
                   
               
               
                 1 
                 2 × 10 6   
                 Vehicle 
                 20 
                 Day −1 to 21 
                 26 
               
               
                 2 
                 cells/mouse 
                 Compound D 
                 15 
                 Day −1 to 21 
                 26 
               
               
                 3 
                   
                 200 mg/kg po, 
                 15 
                 Day −1 to 7 
                 26 
               
               
                 4 
                   
                 bid 
                 15 
                 Day 3 to 21 
                 26 
               
               
                   
               
             
          
         
       
     
     Two parameters were assessed for quantitative evaluation of anti-metastatic efficacy: 1) Fluorescence intensity of lung homogenates and 2) Survival. 
     Fluorescence Intensity of Lung Homogenates: 
     5 mice per treatment group were removed from the study at Day 25 or 26 for evaluation of lung metastatic tumor burden. Mice were euthanized, and lungs were removed, placed in 3 ml saline, and homogenized. 0.2 ml of lung homogenate was transferred to a 96 well plate, and fluorescence was read using Acumen Explorer. ( FIGS. 9 and 10 ). Fluorescence was reported in relative fluorescence units (RFU). (Tables 8 and 9,  FIGS. 11 and 12 ). Statistical analysis was determined by Student-test or Mann-Whitney U test, and statistic differences between groups were considered to be significant when the probability value (p) was ≦0.05. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Relative Fluorescence Units (RFU) of lung homogenate samples run on Acumen 
               
               
                 Explorer (Compound E Study) 
               
             
          
           
               
                   
                 P values 
               
             
          
           
               
                   
                   
                   
                   
                 Vs 
                 Vs 
                 Vs 
               
               
                   
                 RFU 
                   
                 *TGI % 
                 Vehicle 
                 Day −1-d23 
                 Day −1-d7 
               
               
                 Treatment 
                 (mean ± SD) 
                 CV 
                 At day 25 
                 Group 
                 Group 
                 Group 
               
               
                   
               
             
          
           
               
                 Vehicle 
                 48793363 ± 2922819  
                 47 
                   
                   
                   
                   
               
               
                 Compound E 
                 12738540 ± 5426672  
                 43 
                 73.9 
                 0.022 
               
               
                 5 mg/kg po, bid 
               
               
                 Day −1-23 
               
               
                 Compound E 
                 51670506 ± 29321586 
                 57 
                 −5.9 
                 0.87 
                 0.040 
               
               
                 5 mg/kg po, bid 
               
               
                 Day −1-7 
               
               
                 Compound E 
                 8618508 ± 3198528 
                 37 
                 82.3 
                 0.017 
                 0.19 
                 0.030 
               
               
                 5 mg/kg po, bid 
               
               
                 Day 3-23 
               
               
                   
               
               
                 *TGI = Tumor growth inhibition relative to Vehicle control group. 
               
             
          
         
       
     
                                                             TABLE 9                   Relative Fluorescence Units (RFU) of lung homogenate samples run on Acumen       Explorer (Compound D Study)                P value                RFU       *TGI %       Vs   Vs       Treatment   (mean ± SD)   CV   At day 26   Vs vehicle   Day −1-d21   Day −1-d7               Vehicle   62091178 ± 16262491   26                       Compound D   13230372 ± 11960417   90   78.7   0.001       200 mg/kg po, bid       Day −1-21       Compound D   21092887 ± 10460489   50   66.0   0.002   0.301       200 mg/kg po, bid       Day −1-7       Compound D   2359191 ± 526586    22   96.2   0.001   0.077   0.004       200 mg/kg po, bid       Day 3-21               *TGI = Tumor growth inhibition relative to Vehicle control group.            
Survival
 
     Survival represented overall metastatic status either due to lung metastasis or metastasis to other organs. Moribundity due to labored breathing or hind limb paralysis was monitored and recorded as the surrogate endpoint for survival. For survival assessment, results were plotted as the percentage survival against days after tumor implant. The Increased lifetime-span (% ILS) was calculated as: ILS %=100×[(median survival day of treated group−median survival day of control group)/median survival day of control group]. Median survival or (50% survival time) was determined utilizing Kaplan Meier survival analysis. ( FIGS. 13 and 14 ). Differences in survival were analyzed by the log-rank test. Statistic differences between groups were considered to be significant when the probability value (p) was ≦0.05. Similar to the initial characterization of the LOX-GFP-LM metastasis model in Example 5, cells metastasized to lung in 100% of mice when injected iv, and the time frame for observing lung metastasis was also similar (40% survival @ 29 days in the previous study vs. 50% survival @ 24 or 28 days in the present two studies). (Tables 10 and 11;  FIGS. 13 and 14 ) 
     Compound E had equivalent anti-metastatic activity with either late intervention (Dosed Day 3 through 23) or full length intervention (Day −1 through 23) as assessed by fluorescence intensity of lung homogenates. 
     Compound D had superior anti-metastatic activity with late intervention (Dosed Day 3 through 23) as compared to Vehicle, as assessed by fluorescence intensity of lung homogenates. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Survival of groups treated with Compound E as compared to Vehicle control group. 
               
             
          
           
               
                   
                 P values 
               
             
          
           
               
                   
                 50% 
                   
                 Vs 
                   
                   
               
               
                   
                 survival 
                   
                 Vehicle 
                 Vs 
                 Vs 
               
               
                 Treatment 
                 days 
                 ILS % 
                 Group 
                 Day −1-d23 
                 Day −1-d7 
               
               
                   
               
             
          
           
               
                 Vehicle 
                 24 
                   
                   
                   
                   
               
               
                 Compound E 5 mg/kg po, bid 
                 28 
                 16.7 
                 &lt;0.0001 
               
               
                 Day −1-d23 
               
               
                 Compound E 5 mg/kg po, bid 
                 25 
                 4.2 
                 0.0001 
                 0.02 
               
               
                 Day −1-d7 
               
               
                 Compound E 5 mg/kg po, bid 
                 28 
                 16.7 
                 &lt;0.0001 
                 0.83 
                 0.03 
               
               
                 Day 3-d23 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Survival of groups treated with Compound D as compared to Vehicle control group. 
               
             
          
           
               
                   
                 P values 
               
             
          
           
               
                   
                 50% 
                   
                 Vs 
                   
                   
               
               
                   
                 survival 
                   
                 Vehicle 
                 Vs 
                 Vs 
               
               
                 Treatment 
                 days 
                 ILS % 
                 Group 
                 Day −1-d23 
                 Day −1-d7 
               
               
                   
               
             
          
           
               
                 Vehicle 
                 28 
                   
                   
                   
                   
               
               
                 Compound D 200 mg/kg po, bid 
                 31 
                 10.7 
                 0.0074 
               
               
                 Day −1-d21 
               
               
                 Compound D 200 mg/kg po, bid 
                 28 
                 0 
                 0.649 
                 0.03 
               
               
                 Day −1-d7 
               
               
                 Compound D 200 mg/kg po, bid 
                 36 
                 29 
                 &lt;0.0001 
                 0.15 
                 0.002 
               
               
                 Day 3-d21 
               
               
                   
               
             
          
         
       
     
     Example 10 
     Comparison of LOX-GFP-LM and LOX-GFP on Experimental Lung Metasis 
     Previously generated human melanoma LOX-GFP and LOX-GFP-LM cells were maintained in RPMI 1640 medium plus 10% FBS, and 1% Geneticin (G418). Female SCID beige mice (25 mice each tumor line) were injected iv via the tail vein with either the LOX-GFP or LOX-GFP-LM, 2 million cells in a volume of 200 μl serum free RPMI1640. Lungs were harvested from five mice for each time point (day 14, 21 and 28 after implantation, total 15 mice, see Table 12). The rest of the mice, 10 mice per group, were monitored for survival benefit until they were moribund. Two parameters were assessed for quantitative evaluation of tumor growth: 1) fluorescence intensity of lung homogenates and 2) survival. 
                                           TABLE 12                   Implantation of LOX-GFP and LOX-GFP-LM into SCID beige mice                Tumor cell           Day of lung           implanted           harvesting       Groups   (day 0)   medication   Mice No.   (5 mice/group)               1   2 × 10 6     LOX-GFP-LM   25   14, 21 and 28       2   cell/0.2 ml/   LOX-GFP   25   14, 21 and 28           mouse, iv               Total           50                    
Fluorescence Intensity of Lung Homogenates:
 
     Five (5) mice were removed from each group for evaluation of lung metastatic tumor burden. The mice were euthanized, and their lungs were removed, placed in 3 ml saline, and homogenized. Lung homogenate, 0.2 ml, was transferred to a 96 well plate, and fluorescence was read using Acumen Explorer. ( FIG. 17 ). The fluorescence was reported in relative fluorescence units (RFU). ( FIG. 18  and Table 13). Statistical analysis was determined by Student-test or Mann-Whitney U test and statistic differences between groups were considered to be significant when the probability value (p) was ≦0.05. 
                                                                               TABLE 13                   Summary table of tumor lines (LOX-GFP-LM and LOX-GFP       induced experimental metastasis                Lung metastasis: Relative Fluoresecence   50%           Unit (RFU) (mean ± SD)   Survival            Tumor line   Day 14   Day 21   Day 28   Days                    LOX-GFP-   1375463 ± 461906   38173338 ±   NA   25       LM       26458094       LOX-GFP   1323534 ± 465272    1908836 ±   13848195 ±   31               376654   22888238       P value   0.86   0.037       &lt;0.001                    
Survival Assessment
 
     For survival assessment, moribund mice due to difficulty of breathing or hind limb paralysis as end point were recorded, and results are plotted as percent survival against days after tumor implant. Median survival was determined utilizing Kaplan Meier survival analysis. Differences in survival curves were analyzed by the log-rank test and statistic differences between groups were considered to be significant when the probability value (p) was ≦0.05. ( FIG. 19 ) 
     Results and Discussion: 
     SCID beige mice injected with LOX-GFP-LM, as compared to the same strain (SCID beige) of mice injected with LOX-GFP, exhibited a much higher lung metastasis rate (100%) at day 21 and a shorter survival time (all mice were dead in 25 days) with stable GFP transfection in vivo (100%). In the LOX-GFP group, at day 21, two out of five mice were found to have lung metastasis without showing GFP signals, suggesting a lower metastasis rate and non-stable GFP transfection in vivo. 50% survival time in the LOX-GFP group was 6 days delay versus LOX-GFP-LM group (31 days vs 25 days). 
     Both groups did not show any lung metastasis at day 14. However, in the LOX-GFP-LM group, from day 21 to day 25, the mice either showed strong lung metastasis or were moribund. For the LOX-GFP groups, mice were dead or moribund from day 26 to over day 39. It appeared that there is no plateau time period in terms of tumor burden in lungs; in other words, when the lungs developed extensive lung metastasis, mice will quickly become moribund or dead in a short time period. 
     CONCLUSION 
     LOX-GFP-LM causes more lung metastasis with stable GFP signal, as compared to LOX-GFP in the same strain of mice. Both tumor lines showed dynamic tumor burden growth in lungs over time.