Liver Micrometastases Research Articles

Shorter Hepatic Transit Time Can Suggest Coming Metastases

The aim of this study was to assess the value of the hepatic transit time in suggesting coming liver metastases by contrast-enhanced ultrasonography (CEUS). Fifty patients with identified liver metastasis (metastasis group [Gmet]), 26 patients without liver metastasis (unclear group [Gunc]) who had proven extrahepatic malignant tumors, and 27 healthy control volunteers (control group [Gcon]) were included in this study. The Gmet group was divided into small and large subgroups. The Gmet group was also divided into pauci and multi subgroups. Every patient was examined by CEUS. The hepatic artery and hepatic vein arrival times were measured, and the difference between them was calculated as the hepatic artery-vein transit time (HAVTT). Patients in Gunc were given a CEUS examination and an enhanced computed tomography or magnetic resonance imaging examination 3 to 4 months later. The HAVTTs in Gmet were significantly shorter than those in Gcon (P < .05), but there were no statistical differences among the subgroups. A normal cutoff point of 8 seconds in the HAVTTs could distinguish Gmet and Gcon with accuracy, sensitivity, and specificity of 97.40%, 92.59%, and 100%, respectively. As for Gunc, when an HAVTT shorter than 8 seconds was used to predict liver metastasis, the accuracy, sensitivity, and specificity were 92.30%, 100%, and 91.67%. The HAVTT may be a useful tool in monitoring liver micrometastases. If a patient with a primary malignant tumor has a shorter HAVTT, it suggests that an extra examination and additional therapy are needed.

Abstract 2271: Dabigatran etexilate blocks breast cancer progression in vitro and in a 4T1 breast cancer tumor model in mice

Abstract Introduction: Coagulation proteases and the generation of thrombin are increased in tumors. Thrombin can modify tumor cell behavior directly through the activation of protease-activated receptors (PAR) or indirectly by generating fibrin matrices. Aim: To test the hypothesis that dabigatran etexilate (Pradaxa®), an oral direct thrombin inhibitor, can prevent the progression of tumor cells in experimental models. Materials and Methods: The effect of dabigatran on invasiveness of MDA-MB-231 breast carcinoma cells was evaluated in vitro by invasion across Matrigel-coated membranes and in vivo using a tracheal xenotransplant invasion assay in athymic nude mice. To evaluate the effect of dabigatran on the development of metastatic foci, 4T1 tumor cells were injected orthotopically in the mammary fat pads of syngeneic Balb/c mice. Tumor growth and metastasis was monitored weekly. Dabigatran etexilate was administered twice daily at a dose of 45 mg/kg over 4 weeks in all in vivo models. Results: Whereas dabigatran had no effect on the proliferation index of cultured tumor cells, it inhibited invasiveness of MDA-MB-231 cells across Matrigel-coated membranes. In vivo evaluation of invasiveness of MDA-MB-231 cells in tracheal xenotransplants in mice administered dabigatran for 3 and 4 weeks demonstrated significantly less penetration of tumor cells through the tracheal wall. Dabigatran treatment had a modest inhibitory effect on growth of the primary 4T1 mammary carcinoma (∼50% reduction in tumor volume at 4 weeks) in syngeneic Balb/c mice, and it reduced both 4T1 tumor cells in the blood and liver micrometastases by 50-60%. Dabigatran treatment resulted in no weight loss in mice. Conclusion: Oral administration of the direct thrombin inhibitor, dabigatran etexilate, inhibits both invasion and metastasis of malignant breast tumors, suggesting that dabigatran may be beneficial in not only preventing thrombotic events in cancer patients, but also as adjunct therapy to treat malignant tumors. (Supported by funds from Boehringer Ingelheim Pharma) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2271.

Abstract 2304: Stem features associated with EMT are driven by TGFβ1 in liver-metastatic cells

Abstract The liver is a major target organ for metastasis. Development of metastases requires an early release of tumour-initiating-cells from the primary tumour and adhesion of metastatic-cells into a permissive niche. We hypothesized that Epithelial-Mesenchymal Transition (EMT) underlies not only the early events, but also the promotion of the most advanced stages of a successful dissemination process to the liver. It is believed that TGFβ1 might allow tumour-initiating-cells to acquire cancer stem cell properties via EMT as a mechanism that modulates the microenvironment contributing to primary and also metastatic tumor progression. However, the mechanism of EMT induction by TGFβ1 and its involvement in liver metastases remain largely unknown. Aiming to recapitulate the hepatic metastases, we established a murine model of highly metastatic colon carcinoma cells in the context of TGFβ1-signaling. To this end, luciferase-expressing cells (MC-38luc) were pre-treated with TGFβ1 (100 pM, 48 h), before intrasplenic injection of 106 cells in C57BL/6J mice. Larger primary tumours were consistently observed and liver metastasis was achieved more rapidly after injection of MC-38luc+TGFβ1 cells, than after injection of untreated MC-38luc cells. Histological examination of liver micrometastasis also revealed slightly increased staining of Ki67, α-SMA and F4/80 markers in MC-38luc+TGFβ1 tumours. Splenic co-injection of these liver-metastatic-stem cells with non transformed hepatocytes (AML-12 cells) accelerated the incidence of liver metastasis, indicating the supportive role of the normal hepatic parenchyma. Intraperitoneal injection with a novel TGFβ1-inhibitory peptide P17 (obtained from a peptide library binding to TGFβ1) resulted in reduced liver metastasis and decrease of both CECs and CEPs. We isolated liver-metastatic cells that showed higher cellular proliferation as compared to the parental counterparts. These cells posses increased tumorogenicity in vivo, a complete lack of E-cadherin expression, and increased TGFβ1/EMT signalling properties that all relate to stem cell features. Our results provide an insight into the molecular events leading to TGFβ1-mediated malignant progression and indicate that targeting TGFβ1/EMT might represent an optimal therapeutic strategy by impairing tumor growth, stemness and further liver metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2304.

R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors.

The role of 18F-FDG PET/CT in detecting metastatic deposits of recurrent medullary thyroid carcinoma: A prospective study

Aim To assess the diagnostic role of 18F-FDG PET/CT performed with a hybrid tomograph in the detection of tumoral deposits of recurrent medullary thyroid carcinoma (MTC). Methods Nineteen MTC patients with elevated serum calcitonin levels (58–1350 pg/ml) after first treatment were enrolled (11 F, 8 M, mean age 53.4 years, 14 sporadic MTC, 5 MEN-related MTC). All patients had previously undergone total thyroidectomy and lymphoadenectomy. When referred to us, they were studied with ultrasound (US), 18F-FDG PET/CT, 111In-pentetreotide scan, and contrast-enhanced whole-body CT (c.e. CT). In 4 patients with equivocal abdominal findings at 18F-FDG PET/CT and/or at c.e. CT, laparoscopy was also performed. Results 18F-FGD PET/CT depicted metastases in 15 patients, 111In-pentetreotide in 8, c.e. CT in 11, US in 6. In 2 patients, liver micrometastases were detected at laparoscopy only. At a lesion-by-lesion analysis, 18F-FDG PET/CT visualized a total of 26 metastatic deposits, c.e. CT 18, 111In-pentetreotide 12, US 8. Final diagnosis was obtained by cytological or surgical findings. Four patients with evidence of limited metastatic spread to neck/upper mediastinum were re-operated, and in 2 of them serum calcitonin levels normalized. Conclusions In our study, 18F-FDG PET/CT was the most sensitive imaging modality in detecting metastases in recurrent MTC patients with increased serum calcitonin levels. Moreover, 18F-FDG PET/CT was useful in some patients to plan a more accurate re-operation. From a diagnostic point of view, a multimodality imaging approach is recommended in recurrent MTC, especially based on the combination of c.e. CT and 18F-FDG PET/CT.

Improved Visualization of Vessels and Hepatic Tumors by Micro-Computed Tomography (CT) Using Iodinated Liposomes

The goal of this study was to determine whether iodinated liposomes are a suitable tracer for mice microvessel and liver imaging by preclinical computed tomography (CT). Iodinated liposomes were evaluated for vessel and liver imaging. A first group of nude mice was imaged by micro-CT after i.v. injection of liposomes at 1 or 2 gI/kg body weight (b.w.) for intervals up to 24 hours. A second group of mice bearing liver micrometastases was imaged after injection of liposomes at 2 gI/kg b.w. for intervals up to 24 hours. Vascular enhancements of 120 +/- 8 and 322 +/- 20 Hounsfield unit (HU) were obtained after injection of liposomes at 1 or 2 gI/kg b.w., respectively. This enhancement decreased with a blood half-life of 135 +/- 10 and 86 +/- 9 minutes, respectively. Liver enhancement of 157 +/- 5 and 235 +/- 23 HU were obtained after injection of iodinated liposomes at 1 and 2 gI/kg b.w., respectively. Liver micrometastases (250 microm) were detectable after injection of iodinated liposomes at 2 gI/kg b.w. Iodinated liposomes are a suitable contrast agent for vessels and liver imaging by micro-CT allowing clear vascular enhancement and detection of small liver metastases.

Major hepatectomy in patients with synchronous colorectal liver metastases: whether or not a contraindication to simultaneous colorectal and liver resection?

Synchronous hepatic lesions account for 15-25% of newly diagnosed colorectal cancer and its optimal timing to surgery is not completely defined, but simultaneous colorectal and liver resection is recently gaining acceptance, at least in patients with a right colonic primary and liver metastases that need a minor hepatectomy to be fully resected. From September 2002 to December 2004, 16 patients underwent simultaneous resection as treatment of synchronous colorectal liver resection; in 10 patients (62.5%) a major hepatectomy was performed. The mean duration of intervention was 322.5 +/- 59.5 min, operative mortality and morbidity rates was 0% and 25% respectively; the hospitalization was 14.4 (range 8-60) days on average. Mean follow-up was 14 months and actuarial survival was 76.5% at 1 year and 63.5% at 2 years. We concluded that simultaneous colonic and liver resection should be undertaken in selected patients with synchronous colorectal liver resection regardless of the extent of hepatectomy; major liver resection, in fact, seems capable of providing better oncological results, allowing resection of liver micrometastases that, in almost one-third of the patients, are located in the same liver lobe of macroscopic lesions, without increased morbidity rates.

Dormant liver metastases: an experimental study

Experimental work was undertaken to evaluate whether intrahepatic recurrences, observed after resection of colorectal liver metastases in humans, could be due to the activation of dormant cancer cells already present within the liver at liver resection. About 250 cell aggregates (DHDK12 colon carcinoma cell line) were injected into the portal vein of 70 BD IX rats. Eight weeks later, 43 rats with no apparent liver metastases were divided randomly into three groups: group 1 (n = 15) served as control; group 2 (n = 15) were given cyclosporin A (10 mg kg body-weight-1 day-1) for 28 days; and group 3 (n = 13) underwent a 70 per cent hepatectomy. Twelve weeks after the injection of cells, when the animals were killed, 20 per cent of rats in group 1 had liver metastases, 80 per cent in group 2 (P less than 0.01) and 62 per cent in group 3 (P less than 0.05). Undetectable liver micrometastases may have been present at 8 weeks and had not developed until stimulation by cyclosporin A-induced immunosuppression or by liver regeneration after hepatectomy. A similar mechanism may occur clinically and explain some of the recurrences observed after resection of liver metastases.

Characterization of Ocular and Metastatic Uveal Melanoma in an Animal Model

To characterize, in detail, tumor development, malignant cell dissemination, and metastasis in a 10-week animal model of uveal melanoma. One million 92.1 human primary uveal melanoma cells were injected into the suprachoroidal space of the right eye of 27 immunosuppressed albino rabbits. Intraocular tumor growth was monitored weekly by fundoscopy and by ultrasonography at the end of the experiment. To document the progression of the disease, one animal per week was killed. The enucleated eyes, lungs, and livers were macroscopically examined and histopathologically studied by hematoxylin and eosin, periodic acid-Schiff, and immunohistochemistry. Mononuclear layers isolated from the rabbits' blood samples were cultured. Histopathology showed intraocular tumors in 89% of the animals. Tumor growth was found 1 week after cell inoculation, and by the end of the experiment large tumor masses were observed. Microscopic pulmonary metastatic foci were first observed 4 weeks after cell injection. By the end of the experiment, all the animals had metastasis to the lungs. Interestingly, 18% of the animals also had micrometastasis to the liver. Viable adherent uveal melanoma cells were successfully isolated from peripheral blood and grown in vitro. In this study, most rabbits developed intraocular tumors followed by lung metastasis, and some of these rabbits later developed liver micrometastases. This novel source of research material warrants a follow-up longer than 10 weeks to further explore the pathophysiologic bases of liver involvement commonly encountered in humans. The success in the isolation and culture of circulating malignant cells in this animal model suggests that it might be worthwhile to explore the application of this technique to the management of patients with primary uveal melanoma.

An in vivo tumor model expressing green fluorescent protein for the investigation of metastasis.

Although strong evidence is available suggesting that microenvironmental parameters play a role in lymphogenic or hematogenic metastasis, the underlying mechanisms are still unclear and further investigations of this topic are needed. For such a study however, an appropriate model of metastasis for in vivo analysis of this process would be required. An in vivo model of a solid tumor (rat DS sarcoma) has therefore been established to enable monitoring of the steps involved in tumor metastasis. Rat DS sarcoma cells were transfected with the pTracer-SV40 plasmid, containing the super-GFP and zeocin resistance genes. DS sarcoma cells showing high and stable expression of GFP (DSGFP cells) were selected by cell sorting and in vitro culturing with zeocin. To establish in vivo growth, DSGFP cells were subsequently injected intraperitoneally (i.p.) without additional selection by zeocin and GFP expression was monitored by flow cytometry. Using DSGFP ascites cells, solid tumors were implanted subcutaneously into the hind foot dorsum of rats. The expression of GFP was assayed by fluorescence microscopy. The detection of circulating DSGFP sarcoma cells in the blood was performed using the PCR technique. GFP expression in vitro was stable for more than 40 passages. Cell sorting, however, did not enable selection of a DSGFP cell population with a higher long-term stable GFP expression. After i.p. cell implantation, GFP expression in DSGFP ascites cells was maintained over at least 19 passages. Solid tumors implanted by injection of DSGFP ascites cells showed stable GFP expression. The growth rate of solid DSGFP sarcomas was slightly slower compared to that of non-transfected cell lines. The detection limit for circulating DS sarcoma cells in blood was 100 DSGFP cells/ml whole rat blood. Micrometastases in loco-regional lymph nodes, lung and liver were detectable by immunohistology and real-time PCR. This in vivo model showing stable expression of GFP could be useful for analyzing the mechanisms of metastasis, particularly where micrometastases or circulating tumor cells are to be identified.

Oral administration of uracil-tegafur (UFT) inhibits liver micrometastasis of human colon cancer in an orthotopic nude mouse model and its early detection system.

To evaluate the effect of UFT (an oral antineoplastic drug combining uracil and tegafur) as an adjuvant chemotherapy. We examined whether UFT inhibits micrometastasis of the liver from colon cancer implanted into the cecum of nude mice in an orthotopic model. Moreover, we studied whether our early detection system using a polymerase chain reaction (PCR) of the human beta-globin gene would be useful in this model. The administration of 20 mg/kg UFT p.o., which is a relatively small dose compared with 65 mg/kg of the maximum tolerated dose of this drug in mice, inhibited liver metastasis completely when started immediately after a cecectomy (micrometastasis present at this time), but did not inhibit liver metastasis significantly when started at 4 weeks after a cecectomy (gross tumor present at this time). There were no severe toxicities at this dose. In our PCR study, all livers in 10 mice to which therapy was given immediately after a cecectomy and without liver metastasis showed no PCR-amplified fragment, while 7 of 10 livers in the nontreatment group in which gross liver metastases were not observed demonstrated this fragment. These findings indicate that UFT is useful for either adjuvant chemotherapy or the inhibition of micrometastasis, and our system to detect micrometastasis by examining the human beta-globin gene is useful for the early evaluation of the efficacy of these drugs.

Synthesis and physicochemical characteristics of a liver-targeted conjugate of fluorodeoxyuridine monophosphate with lactosaminated human albumin.

In previous experiments fluorodeoxyuridine monophosphate (FUdRMP) was conjugated with lactosaminated human albumin (L-HSA). Fluorodeoxyuridine (FUdR) is an anticancer agent and L-HSA is a hepatotropic carrier of drugs obtained by the covalent linkage of lactose residues to the albumin molecule. The conjugate was synthesised via the imidazolide of FUdRMP at alkaline pH. Peripheral venous administration of L-HSA-FUdRMP produced enhanced FUdR levels in hepatic blood and might accomplish a non-invasive loco-regional chemotherapy of liver micrometastases. In the present paper some physicochemical characteristics of L-HSA-FUdRMP are reported. Polyacrylamide gel electrophoresis indicated that the coupling reaction did not cause covalent aggregation of the L-HSA molecules. 31P NMR spectra of the conjugate showed that FUdRMP was linked to L-HSA by phosphoamide bonds to lysine and histidine residues, and the area of the peak due to the lysine bond represented more than 80% of the spectrum of L-HSA-FUdRMP. MALDI analysis revealed a partial degradation of the peptide backbone of the conjugate which could not be detected using other methods of analysis. The degradation was not caused by the coupling of lactose molecules to albumin, but rather a consequence of FUdRMP conjugation with L-HSA. This fragmentation was dependent on the pH of the medium used for the FUdRMP coupling reaction. By decreasing the pH to 7.5, conjugates were obtained with a lower drug load but with a substantially reduced fragmentation, which should be preferred for a clinical use of L-HSA-FUdRMP.

Floxuridine coupling with lactosaminated human albumin to increase drug efficacy on liver micrometastases.

Conjugates of nucleoside analogues with galactosyl terminating peptides selectively enter hepatocytes through the asialoglycoprotein receptor. After intracellular release from the carrier, the drugs partly exit from hepatic cells into hepatic blood. To establish whether administration of a conjugate of floxuridine with lactosaminated human albumin selectively enhances drug concentrations in hepatic blood. Floxuridine is a fluoropyrimidine active on human colorectal cancer, a tumour which metastasises first to the liver. In rats injected with free or conjugated floxuridine, plasma levels of the drug were determined in hepatic veins and in inferior vena cava, in order to measure drug concentrations in hepatic blood and in the systemic circulation, respectively. Ratios between floxuridine levels in hepatic veins and those in systemic circulation were found to be seven times higher in rats injected with the conjugate (p=0.000). The present results suggest that coupling to lactosaminated albumin might improve the effect of floxuridine in adjuvant chemotherapy of colorectal cancer by exposing the cells of liver micrometastases (nourished by hepatic sinusoids) to enhanced drug concentrations.

Conjugates of nucleoside analogs with lactosaminated human albumin to selectively increase the drug levels in liver blood: requirements for a regional chemotherapy.

Nucleoside analogs (NAs) conjugated with galactosyl terminating peptides selectively enter hepatocytes via the asialoglycoprotein receptor and, after intracellular release from the carrier, partly exit from these cells into the bloodstream, resulting in higher concentrations in liver blood than in systemic circulation. Therefore, conjugates of anticancer NAs can be exploited to accomplish a loco-regional noninvasive treatment of liver micrometastases. In the present experiments we studied whether the enhancement of drug levels in liver blood achieved when NAs are given in the coupled form depends on the rate of drug elimination from the bloodstream. Three NAs, adenine arabinoside (ara-A), 5-fluoro-2'-deoxyuridine (FUdR), and 2',2'-difluorodeoxycytidine, were coupled with lactosaminated human albumin, a galactosyl terminating carrier. In rats that received an intravenous bolus injection of these conjugates, we compared the drug concentrations in liver blood to those in the systemic circulation. We found that enhanced levels of NAs in liver blood were only achieved by administering the conjugates of the drugs (ara-A and FUdR), which are rapidly cleared from the bloodstream. Increased drug levels also were obtained when ara-A and FUdR conjugates were slowly infused (a way of administration often used for anticancer drugs). The experiments also showed that galactosyl terminating conjugates of NAs might have the potential to produce a therapeutic effect only when the coupled drugs are active at low blood concentrations, since the amounts of drugs introduced into hepatocytes and released by these cells in the bloodstream cannot be increased when the receptor for the hepatic uptake of galactosyl terminating peptides is saturated.

Dynamic Micro-MRI of Liver Micrometastasis with a Novel Liver Macromolecular MR Contrast Agent DAB-Am64-(1B4M-Gd) 64

DAB-Am64-(1B4M-Gd)64 is a novel macromolecular liver MRI contrast agent consisting of a polypropylenimine diaminobutane (DAB) dendrimer conjugated with a bifunctional DTPA derivative complexing Gd(III) . The characteristics of DAB-Am64-(1B4M-Gd)64, including a rapid liver accumulation, have recently been reported. In this study, dynamic micro-MRI with DAB-Am64-(1B4MGd)64 was obtained in a liver metastasis-model in mice using colon carcinoma cells to evaluate visualization of micrometastatic tumors as compared to Gd-DTPA. The dynamic micro-MRI with DAB-Am64-(1B4M-Gd)64 was able to homogeneously enhance normal liver parenchyma and visualize micrometastatic tumors of 0.3 mm diameter in the liver of the mice with better contrast than with GdDTPA. In conclusion, DAB-Am64-(1B4M-Gd)64, a new liver MRI contrast agent, is potentially useful for diagnosis of liver micro-metastasis. The presence of hydrophobic groups on Gadolinium chelates can induce hepatocellular uptake and excretion into the bile ducts, gall bladder, and intestines, resulting in visualization of hyperintense/bright liver in a T1weighted image. This type of liver MRI contrast agents, containing Gadolinium and enhancing the signal of normal liver parenchyma, have actively been investigated for many years, and are currently in clinical use. The first Gadolinium-based agent of this class, [Gd(BOPTA)(H2O)] (MultiHance), has been approved in Europe. A related chelate, [Gd(EOB-DTPA)(H2O)] (Eovist), is currently in Phase III clinical trials. This agent is excreted to a greater extent via the liver, roughly 50% for [Gd(EOB-DTPA)(H2O)] versus 2-4% for [Gd(BOPTA)(H2O)] , resulting in significantly greater liver enhancement. DAB-Am64-(1B4M-Gd)64 was found to accumulate in the liver at approximately 50 % of the injected dose (%ID) within 15 min post-injection, and homogeneously enhanced the hepatic parenchyma [Kobayashi, Magn Reson Med 2001; 46: 795-802]. Thus, DAB-Am64-(1B4M-Gd)64 appears to be a comparable agent to [Gd(EOB-DTPA)(H2O)] . Herein, imaging of experimental liver micrometastases of LS174T tumors, which were not visualized by other imaging modalities in living animals, was studied using dynamic micro-MRI with DAB-Am64-(1B4M-Gd)64 to evaluate its imaging qualities as compared to Gd-DTPA. To the best of our knowledge, no study has been performed relating serial imaging of metastatic liver tumors in mice.

Green fluorescent protein — a word of caution

In vitro GFP expression in LS174T cells decreases with time despite various attempts at enhancing overall expression. In vivo studies confirm this phenomenon as GFP expression was completely lost in the primary tumour model. Fluorescence was found to be variable in liver micrometastases depending on the time of FACScan analysis.

Tracking prostate carcinoma micrometastasis to multiple organs using histochemical marker genes and novel cell systems.

Studies of human prostate carcinoma (PCA) have been hampered by only a few cell systems from already-metastatic human disease. We have developed a novel cell system by using tissue cultured CWR22R cells from a xenograft of a primary tumor from a human patient. These cells were transfected with the bacterial lacZ gene to maximize their detection during progression and metastasis in nude mice. LZ-CWR22R cells are extremely stable for lacZ expression over 25 passages and metastasize to lung, liver, and bone from the subcutis - major sites of metastasis of the human disease. A matrigel vehicle facilitated development of primary tumors and micrometastases in all organs. While some micrometastases developed into overt metastases, others remained as micrometastases for long periods of time, possibly providing a model of latency of metastatic disease. An experimental metastasis model (tail vein injection) also generated micrometastases in lung, liver, and bone with differing kinetics of formation and stability. Serial sections of many individual lung micrometastases within one hour of injection indicated considerable heterogeneity in cellular composition (from 1 to 19 cells/site) while liver sites at later times were comprised of only 1 or 2 cells (the size of bone sites were comparable to those of liver). By combining use of these histochemically-tagged PCA cell systems with high resolution molecular analyses (laser-capture microdissection), it will now be possible to analyze gene expression patterns characteristic of micrometastases developing in several different organs.

Early changes in liver perfusion caused by occult metastases in rats: detection with quantitative CT.

To determine whether computed tomography (CT) can depict liver hemodynamic changes caused by occult hepatic micrometastases in rat. Liver micrometastases (mean diameter, 500 micrometer +/- 300) were produced in seven BD IX rats by injecting 10(7) DHDK12 PROb colorectal carcinoma cells into the spleen. Macrometastases (mean diameter, 7 mm +/- 3) were produced in four other rats. Five normal rats were studied as controls. CT images were obtained every 300 msec for 30 seconds during the injection of 1 mL per kilogram of body weight of contrast medium. The time-attenuation curves of the aorta, portal vein, and liver were used to calculate liver perfusion with a deconvolution model designed for the dual blood supply. Micrometastases in an apparently normal liver caused a 34% decrease in portal blood flow and a 25% increase in the mean transit time for the blood to pass through the liver. These findings suggest increased resistance in the sinusoidal capillaries. Similar but greater changes were found in the macrometastases. Occult liver micrometastases in rats generate changes in liver perfusion that can be detected with CT.

Coupling of 5-fluoro 2′-deoxyuridine to lactosaminated poly-l-lysine: an approach to a regional, non-invasive chemotherapy of liver micrometastases

Nucleoside analogs conjugated with galactosyl-terminating peptides selectively enter liver cells and after intracellular release from the carrier partly exit into bloodstream, resulting in higher concentrations in liver blood than in systemic circulation. The aim of the present experiments was to ascertain whether, in mice injected with non-toxic doses of a 5-fluoro 2′-deoxyuridine (FUdR) conjugate with lactosaminated poly- l-lysine ( l-poly(LYS)), the drug was released by hepatic cells in high enough amounts to be pharmacologically active on neoplastic cells infiltrating the liver. We observed that l-poly(LYS)–FUdR inhibited the growth of hepatic metastases induced by intrasplenic administration of murine colon carcinoma C-26 cells. l-poly(LYS)–FUdR was not toxic for C-26 cells in vitro, was selectively taken up by mouse liver, and was stable in mouse blood, indicating that the effect on the metastases was due to FUdR (and/or its active metabolites) released in liver blood after the conjugate was taken up by the hepatic cells. These results suggest that l-poly(LYS)–FUdR might be useful in adjuvant chemotherapy of tumors giving liver metastases. The drug released from hepatic cells into liver blood following conjugate administration via the peripheral venous route might accomplish a locoregional, non-invasive treatment of micrometastases nourished by liver sinusoids.

A novel, clinically relevant animal model of metastatic pancreatic adenocarcinoma biology and therapy.

In this study, we report a metastatic model of Panc02 murine pancreatic adenocarcinoma. Parental Panc02 cells were orthotopically implanted into the pancreas of syngeneic C57BL/6 mice. Tumor cells were isolated from liver micrometastases 90 d after tumor implantation and established as a culture (Panc02-H1). The Panc02-H1 cells were then implanted into the pancreas of mice. Liver metastases were then collected and established as Panc02-H2 cells. This process was repeated until the Panc02-H7 cell line was established. These cells were extremely aggressive after implantation as manifested by progressive growth in the pancreas, peritoneal dissemination, and distant metastasis to multiple organs, including the liver and lungs. Moreover, Panc02-H7 cells expressed the inducible nitric oxide synthase gene at a very low level in culture and produced highly vascularized tumors having a large number of infiltrating macrophages. Collectively, this model system should be a valuable tool for investigating the molecular mechanisms governing pancreatic cancer growth and metastasis and exploring potential treatment modalities for this disease.