Establishment of a novel method to evaluate peritoneal microdissemination and therapeutic effect using luciferase assay

Peritoneal dissemination is a major cause of recurrence in patients with malignant tumors in the peritoneal cavity. Effective anticancer agents and treatment protocols are necessary to improve outcomes in these patients. However, previous studies using mouse models of peritoneal dissemination have not detected any drug effect against peritoneal micrometastasis. Here we used the luciferase assay to evaluate peritoneal micrometastasis in living animals and established an accurate mouse model of early peritoneal microdissemination to evaluate tumorigenesis and drug efficacy. There was a positive correlation between luminescence intensity in in vivo luciferase assay and the extent of tumor dissemination evaluated by ex vivo luciferase assay and mesenteric weight. This model has advantages over previous models because optimal luciferin concentration without cell damage was validated and peritoneal microdissemination could be quantitatively evaluated. Therefore, it is a useful model to validate peritoneal micrometastasis formation and to evaluate drug efficacy without killing mice.     

Characterization of a high-grade malignant murine B-cell lymphoma and a study of its dissemination pattern after intraperitoneal or intravenous inoculation

A high-grade malignant lymphoma that arose spontaneously in a C57BL mouse has been transplanted by intraperitoneal injection of a single cell suspension for over 20 years. In the current study it was characterized cytohistologically and immunologically and was found to be a high-grade malignant (immunoblastic) B-lymphoma. The distribution in the various organs and the peripheral blood was investigated after intraperitoneal and intravenous injection and the effect of splenectomy was studied. The findings suggest that the major initial proliferation of lymphoma cells occurs in the spleen both after intraperitoneal and intravenous injection. The second, final stage is characterized by leukemic infiltration throughout the body organs. The current observations are compared to those concerning the previously described, low-grade malignant murine BCL1-lymphoma.     

Orthotopic implantation is essential for the selection,growth and metastasis of human real cell cancer in nude mice

Human neoplasms are heterogeneous for a variety of biological properties that include invasion and metastasis. The presence of a small subpopulation of cells with a highly metastatic phenotype has important clinical implications for diagnosis and therapy of cancer. For this reason, it is important to develop an animal model for the selection and isolation of metastatic variants from human neoplasms and for testing the metastatic potential of human tumor cells.We have implanted human renal cell carcinoma (HRCC) cells (obtained from a surgical specimen) into different organs of nude mice and then recovered the tumors and established each in culture. The 5 established lines differed in their biological-metastatic properties and had a unique karyotype, indicating that growth at different organs selects for different subpopulations of HRCC. Moreover, the HRCC did not metastasize unless they were implanted orthotopically. These findings indicate that the appropriate nude mouse model for studying the biology and therapy of HRCC must be based on the orthotopic implantation of tumor cells.     

Development of a slow release system of anti-cancer drugs retained in calcium-hydroxyapatite ceramic

A new type of anticancer material with sustained release by enclosure of cisplatin (CDDP) into porous calcium hydroxyapatite ceramic (CDDP-CHA) was developed. A slow release of anticancer drug from CDDP-CHA was confirmed in in vitro experiments. When this material was implanted into normal thigh muscle of mouse, a sustained release of CDDP was observed during over 8 weeks after implantation. The diffusion of drug into blood and other organs from the implanted site was very small. CDDP-CHA placed into the implanted tumor of mouse allowed a slow release and high drug level from the material in the tumor. The concentration of the drug in other organs such as liver and kidney was very low compared with that of the tumor. These results suggest that this material has an important role for cancer chemotherapy, particularly in bone tumors for the reasons of calcium hydroxyapatite ceramic carrier having a mechanical strength.     

Mutagenesis induced by benzo[a]pyrene in lacZ mouse mammary and oral tissues: comparisons with mutagenesis in other organs and relationships to previous carcinogenicity assays.

Thus far, in vivo mutagenic assays have detected organ-specific effects of benzo[a]pyrene (B[a]P) in a number of organs, but not in oral tissues and breast. Previous studies have shown that the mouse tongue is a target for tumorigenesis induced by B[a]P when incorporated into feed, and polycyclic aromatic hydrocarbons are carcinogens in mouse mammary tissue. In order to evaluate the capacity of the lacZ mouse in vivo mutagenesis assay to detect mutations in these target tissues, we have measured mutagenesis induced by B[a]P in breast and oral tissues. The oral tissue consisted of either tongue or a mixture of oral tissues from several sites in the oral cavity. B[a]P was more mutagenic in breast tissue than in most other organs tested (liver, lung and kidney) when administered at relatively high dose by gavage, and more mutagenic than in liver, but not lung, at low dose. When administered in an emulsion in drinking water, B[a]P was more mutagenic in oral tissues than in liver, and somewhat less mutagenic than in lung. Regardless of dose, the mutagenic activity was greatest in colon where it was much higher than in other organs. A reasonable correlation was observed between mutagenesis observed here and carcinogenesis in previous studies although some differences were noted. To our knowledge, this represents the first report of in vivo mutagenesis in non-tumor mammary and oral tissue, and the results indicate these organs can efficiently metabolize B[a]P to genotoxic products, although some transport of active metabolites from the liver cannot be ruled out. The lacZ mouse mutagenesis assay may represent a shorter term alternative to carcinogenesis assays for investigations of factors affecting initiation of carcinogenesis in mammary and oral tissues. However, it is less predictive of actual tumor formation.     

Effect of chronically administered pentagastrin on the nude mouse

The nude mouse has been used to evaluate the effect of gastrin on xenografted tissues, but little is known about long-term actions of gastrin on native organs in this species. We investigated the impact of chronically administered synthetic pentagastrin on the nude mouse. Six groups of mice (eight animals each) received intraperitoneal injections twice daily for 14 days with saline or pentagastrin at 0.5, 5, 50, 500, or 5,000 micrograms/kg. Behavior, overall health, and body weight were unaffected by this treatment. Of the seven organs examined at necropsy, only the pancreas showed a weight gain in response to pentagastrin treatment, and this occurred only at the highest dose. Total DNA content of the pancreas decreased in a dose-related manner, indicating hypoplasia, whereas pancreatic RNA content increased, indicating hypertrophy. No effect on the stomach was observed. This work indicates that the nude mouse is less sensitive than other species to visceral growth regulation by pentagastrin, and that toxicity is low.     

Progression of tumor histiotype during mouse hepatocarcinogenesis associated with the viable yellow (Avy) gene

Increasing attention has been focused recently upon those factors in carcinogenesis that are responsible for the proliferation of initiated cells and the increasingly aberrant phenotype that they progressively manifest. The agouti locus allele Avy (viable yellow) has been shown to be associated with conditions which favor promotion of cells that have been initiated by a wide variety of causes, in many organs, but has not been previously associated with tumor progression in those systems. In the current study, the presence of the Avy gene in a strain of mice not normally predisposed to hepatocarcinogenesis, C57BL/6N was, for the first time, associated not only with much earlier appearance, but with progression of the histiotype of hepatic tumors, following neonatal administration of diethylnitrosamine. At 52 weeks, 28 C57BL/6N mice demonstrated 7 mouse liver tumors 0.5 cm or greater in diameter, all of more benign histiotype, without associated metastasis. The 31 C57BL/6N-Avy demonstrated 194 mouse liver tumors at that time, 22% of which were of malignant histiotype, 19% of which were associated with metastasis. This system would appear to offer the possibility of identifying the underlying mechanisms for components of the carcinogenic process. In addition, the C57BL/6N-Avy mouse appears to offer advantages as a test animal in bioassay procedures that use the liver as a target organ. Thus, it represents a mouse with little or no spontaneous predisposition to hepatocarcinogenesis, with a predicted short lag period toward response to hepatocarcinogens.     

Chloroform bioactivation leading to nucleic acids binding.

Chloroform was bound covalently to DNA, RNA and proteins of rat and mouse organs in vivo after i.p. injection. Covalent Binding Index values of rat and mouse liver DNA classify chloroform as a weak initiator. Labelings of RNA and proteins from various organs of both species were higher than that of DNA. In an in vitro cell-free system, chloroform was bioactivated by cytochrome P450-dependent microsomal fractions, by cytosolic GSH-transferases from rat and mouse liver, and particularly by the latter enzymes from mouse lung. This observation suggests that GSH plays a role in the binding of chloroform metabolites to DNA. The presence of both microsomal and cytosolic enzymatic systems in the standard incubation mixture generally led to an additive or synergistic bioactivating effect for rat and mouse, respectively.     

Singlet oxygen luminescence as an in vivo photodynamic therapy dose metric: validation in normal mouse skin with topical amino-levulinic acid

Although singlet oxygen ((1)O(2)) has long been proposed as the primary reactive oxygen species in photodynamic therapy (PDT), it has only recently been possible to detect it in biological systems by its luminescence at 1270 nm. Having previously demonstrated this in vitro and in vivo, we showed that cell survival was strongly correlated to the (1)O(2) luminescence in cell suspensions over a wide range of treatment parameters. Here, we extend this to test the hypothesis that the photobiological response in vivo is also correlated with (1)O(2) generation, independent of individual treatment parameters. The normal skin of SKH1-HR hairless mice was sensitised with 20% amino-levulinic acid-induced protoporophyrin IX and exposed to 5, 11, 22 or 50 J cm(-2) of pulsed 523 nm light at 50 mW cm(-2), or to 50 J cm(-2) at 15 or 150 mW cm(-2). (1)O(2) luminescence was measured during treatment and the photodynamic response of the skin was scored daily for 2 weeks after treatment. As observed by other authors, a strong irradiance dependence of the PDT effect was observed. However, in all cases the responses increased with the (1)O(2) luminescence, independent of the irradiance, demonstrating for the first time in vivo an unequivocal mechanistic link between (1)O(2) generation and photobiological response.     

Tumor cells are protected from NK-cell-mediated lysis by adhesion to endothelial cells

We have examined the effect of various cell monolayers on the ability of mouse spleen cells to lyse tumor cell targets (natural cytotoxicity reaction). Natural killer (NK) cell activity was reduced by as much as 75% depending on the cell substrate, with the greatest protection afforded by endothelial cells. Cell adhesion to the underlying cell monolayer was directly correlated with the degree of protection from lysis. Since it has previously been shown that tumor cells manifest selective adhesion to endothelial cells from those organs to which they are likely to metastasize, the experiments suggest a means by which natural surveillance mechanisms might be circumvented during the metastatic process.     

Reduction of human T-cell leukemia virus type-1 infection in mice lacking nuclear factor-kappaB-inducing kinase

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia and inflammatory disorders. Aberrant activation of nuclear factor-κB (NF-κB) has been linked to HTLV-1 pathogenesis and to various kinds of cancers, including adult T-cell leukemia. NF-κB-inducing kinase (NIK) is critical for non-canonical activation of NF-κB and for the development of lymphoid organs. HTLV-1 activates NF-κB by the non-canonical pathway, but examination of the role of NIK in proliferation of HTLV-1-infected cells in vivo has been hindered by lack of a suitable animal model. Alymphoplasia ( aly/aly ) mice bear a mutation of NIK, resulting in defects in the development of lymphoid organs and severe deficiencies in both humoral and cell-mediated immunity. In the present study we therefore used a mouse model of HTLV-1 infection with aly/aly mice. The number of HTLV-1-infected cells in the reservoir organs in aly/aly mice was significantly smaller than in the control group 1 month after infection. In addition, aly/aly mice did not maintain provirus for 1 year and antibodies against HTLV-1 were undetectable. These results demonstrate that the absence of functional NIK impairs primary HTLV-1 proliferation and abolishes the maintenance of provirus. Interestingly, clonal proliferation of HTLV-1-infected mouse cells was not detected in aly/aly mice, which is consistent with the lack of HTLV-1 persistence. These observations imply that the clonal proliferation of HTLV-1-infected cells in secondary lymphoid organs might be important for HTLV-1 persistence. ( Cancer Sci 2008; 99: 872–878)     

Establishment of animal model for the analysis of cancer cell metastasis during radiotherapy

ABSTRACT: BACKGROUND: Gamma-Ionizing radiation (IR) therapy is one of major therapeutic tools in cancer treatment. Nevertheless, gamma-IR therapy failed due to occurrence of metastasis, which constitutes a significant obstacle in cancer treatment. The main aim of this investigation was to construct animal model which present metastasis during radiotherapy in a mouse system in vivo and establishes the molecular mechanisms involved.Materials and methodsThe C6L transfectant cell line expressing firefly luciferase (fLuc) was treated with gamma-IR, followed by immunoblotting, zymography and invasion assay in vitro. We additionally employed the C6L transfectant cell line to construct xenografts in nude mice, which were irradiated with gamma-IR. Irradiated xenograft-containing mice were analyzed via survival curves, measurement of tumor size, and bioluminescence imaging in vivo and ex vivo. Metastatic lesions in organs of mice were further assessed using RT-PCR, H & E staining and immunohistochemistry. RESULTS: gamma-IR treatment of C6L cells induced epithelial-mesenchymal transition (EMT) and increased cell invasion. In irradiated xenograft-containing mice, tumor sizes were decreased dramatically and survival rates extended. Almost all non-irradiated xenograft-containing control mice had died within 4 weeks. However, we also observed luminescence signals in about 20.9 % of gamma-IR-treated mice. Intestines or lungs of mice displaying luminescence signals contained several lesions, which expressed the fLuc gene and presented histological features of cancer tissues as well as expression of EMT markers. CONCLUSIONS: These findings collectively indicate that occurrences of metastases during gamma-IR treatment accompanied induction of EMT markers, including increased MMP activity. Establishment of a murine metastasis model during gamma-IR treatment should aid in drug development against cancer metastasis and increase our understanding of the mechanisms underlying the metastatic process.     

Application of Benchtop-magnetic resonance imaging in a nude mouse tumor model

Background

MRI plays a key role in the preclinical development of new drugs, diagnostics and their delivery systems. However, very high installation and running costs of existing superconducting MRI machines limit the spread of MRI. The new method of Benchtop-MRI (BT-MRI) has the potential to overcome this limitation due to much lower installation and almost no running costs. However, due to the low field strength and decreased magnet homogeneity it is questionable, whether BT-MRI can achieve sufficient image quality to provide useful information for preclinical in vivo studies. It was the aim of the current study to explore the potential of BT-MRI on tumor models in mice.

Methods

We used a prototype of an in vivo BT-MRI apparatus to visualise organs and tumors and to analyse tumor progression in nude mouse xenograft models of human testicular germ cell tumor and colon carcinoma.

Results

Subcutaneous xenografts were easily identified as relative hypointense areas in transaxial slices of NMR images. Monitoring of tumor progression evaluated by pixel extension analyses based on NMR images correlated with increasing tumor volume calculated by calliper measurement. Gd-BOPTA contrast agent injection resulted in a better differentiation between parts of the urinary tissues and organs due to fast elimination of the agent via kidneys. In addition, interior structuring of tumors could be observed. A strong contrast enhancement within a tumor was associated with a central necrotic/fibrotic area.

Conclusions

BT-MRI provides satisfactory image quality to visualize organs and tumors and to monitor tumor progression and structure in mouse models.     

Dynamics of monoclonal antibody distribution and prolonged tumour localisation in nude mice bearing a human CEA-producing colon carcinoma xenograft

A new monoclonal anti-CEA antibody (1H12) has been raised which has localising characteristics in a human colon tumour xenograft which could make it suitable for human immuno-radiotherapy. The amount of 1H12 localising in tumour reached about 5% of the injected dose by 7 hours. This rate appeared to be related to the concentration of 1H12 in the blood pool since non-excretory normal organs such as colon and stomach accumulated similar amounts up to 4 hours. Whereas 1H12 was lost from normal organs after 4 hours, the amount in the tumour continued to increase slightly reaching a maximum concentration of 6.5% of the injected dose by day 9. Prolonged retention of 1H12 in tumour enabled increasing tumour: normal tissue ratios to be attained during the residence time of the antibody thus providing scope for maximising the dose of radiation delivered to tumour cells. Preliminary dose escalation showed that up to 500 micrograms of 1H12 could be administered with increasing concentrations of antibody localising in tumour. Saturation of the tumour site was evident in only one mouse where 1.09 micrograms of 1H12 actually localised--equivalent to 60 micrograms per gram of tumour.     

肿瘤类器官技术及其在耐药研究中的应用

类器官，作为自问世以来便飞速发展并在各大实验平台广泛应用的三维结构微器官，其相比于传统的实验模型如2D细胞、人源性组织异种移植（patient-derived xenografts,PDX）等，更具有与真实器官较为相似的复杂结构以及多种细胞形态，并能较大程度地契合来源组织或器官的生理机能。此外，随着类器官培养技术及分析手段在研究中的创新改进，日益丰富的培养介质方案及高通量筛选等方法的建立使得类器官的应用更趋完备化和多样化。同时，新兴的类器官技术在肿瘤研究中扮演重要角色，尤其在药物筛选、疾病建模等领域可作为可靠度极高的实验模型。本篇综述概括了近年来肿瘤类器官在培养、分析方法上的部分改进和细化，以及肿瘤类器官在发展应用中相比于传统实验模型的优势，最后总结了肿瘤类器官在耐药性应用中的研究进展。本文对肿瘤类器官的耐药性概述将有利于为肿瘤耐药研究提供较为可靠的理论依据和方法参考。     

Wide distribution of [3H](-)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue

The increasing recognition of green tea and tea polyphenols as cancer preventives has created a need for a study of their bioavailability. For this purpose, we synthesized [3H] (-)-epigallocatechin gallate ([3H]EGCG) with a specific activity of 48.1 GBq/mmol and directly administered the solution into the stomachs of CD-1 female or male mice. Radioactivity in the digestive tract, various organs, blood, urine and feces was measured with an oxidizer at various times after administration and significant radioactivity was found in the previously reported target organs of EGCG and green tea extract (digestive tract, liver, lung, pancreas, mammary gland and skin), as well as other organs (brain, kidney, uterus and ovary and testes) in both sexes. Incorporation of radioactivity in the cells was confirmed by microautoradiography. Within 24 h, 6.6 (females) and 6.4% (males) of total administered radioactivity was excreted in the urine and 37.7 and 33.1% in feces. HPLC analysis of urine from both sexes revealed that 0.03-0.59% of administered [3H]EGCG, along with at least five metabolites, was excreted. In addition, we found that a second, equal administration to female mice after a 6 h interval enhanced tissue levels of radioactivity in blood, brain, liver, pancreas, bladder and bone 4-6 times above those after a single administration. These results suggest that frequent consumption of green tea enables the body to maintain a high level of tea polyphenols and this paper is the first pharmacological evidence of a wide distribution of [3H]EGCG in mouse organs, indicating a similar wide range of target organs for cancer prevention in humans.      

In vivo and in vitro binding of benzene to nucleic acids and proteins of various rat and mouse organs

Benzene binds to macromolecules of various organs in the rat and mouse in vivo. Labelling of RNA and proteins is higher (1 order of magnitude) than DNA labelling, which is low in many organs (liver, spleen, bone marrow and kidney), and negligible in lung; no difference between labelling of rat and mouse organs was found. The covalent binding index (CBI) value was about 10, i.e. typical of genotoxic carcinogens classified as weak initiators. In vitro binding of benzene to nucleic acids and proteins is mediated by hepatic microsomes, but not by microsomes from kidney, spleen and lung, or by cytosol from whatever organ. Nucleic acid binding can be induced by pretreatment with phenobarbitone (PB) and suppressed in the presence of SKF 525-A, of cytosol and/or GSH or of heat-inactivated microsomes. Labelling of exogenous DNA is low and is similar in the presence of rat or mouse microsomes in agreement with the low interaction with DNA measured in vivo.     

Hepatic vascular tumors, angiectasis in multiple organs, and impaired spermatogenesis in mice with conditional inactivation of the VHL gene

von Hippel-Lindau (VHL) disease is a multisystem inherited cancer syndrome characterized by the development of highly vascular tumors including hemangioblastomas of the retina and central nervous system, pheochromocytomas, and clear cell renal carcinoma, which result from somatic inactivation of the wild-type VHL allele in cells harboring a germ-line VHL mutation. Homozygous inactivation of the VHL gene in mice resulted in embryonic lethality. To produce a mouse model that closely mimics human VHL disease and avoids embryonic lethality, we used Cre/lox site-specific recombination technology. We generated mice carrying conditional VHL alleles and a cre transgene under the control of the human beta-actin promoter, which directs cre expression in a mosaic pattern in multiple organs. VHL(f/d)/Cre mice developed multiple, hepatic hemangiomas that led to premature death, as well as angiectasis and angiogenesis in multiple organs. Interestingly, testes of male VHL(f/d)/Cre mice were unusually small with severely reduced sperm count resulting in infertility. Loss of pVHL function in this VHL conditional knockout mouse model results in an extensive abnormal vascular phenotype in multiple mouse organs, which will provide a useful animal model for testing potential antiangiogenic therapies for VHL disease treatment. Importantly, the phenotypic defects in sperm development observed in these mice support a novel role for VHL in spermatogenesis. This VHL conditional knockout mouse model will provide an in vivo system for studying the functional requirement of the VHL gene in reproductive biology.     

Evaluation of beta-absorbed fractions in a mouse model for 90Y, 188Re, 166Ho, 149Pm, 64Cu, and 177Lu radionuclides

Several short-lived, high-energy beta emitters are being proposed as the radionuclide components for molecular- targeted potential cancer therapeutic agents. The laboratory mice used to determine the efficacy of these new agents have organs that are relatively small compared to the ranges of these high-energy particles. The dosimetry model developed by Hui et al. was extended to provide realistic beta-dose estimates for organs in mice that received therapeutic radiopharmaceuticals containing (90)Y, (188)Re, (166)Ho, (149)Pm, (64)Cu, and (177)Lu. Major organs in this model included the liver, spleen, kidneys, lungs, heart, stomach, small and large bowel, thyroid, pancreas, bone, marrow, carcass, and a 0.025-g tumor. The study as reported in this paper verifies their results for (90)Y and extends them by using their organ geometry factors combined with newly calculated organ self-absorbed fractions from PEREGRINE and MCNP. PEREGRINE and MCNP agree to within 8% for the worst-case organ with average differences (averaged over all organs) decreasing from 5% for (90)Y to 1% for (177)Lu. When used with typical biodistribution data, the three different models predict doses that are in agreement to within 5% for the worst-case organ. The beta-absorbed fractions and cross-organ-deposited energy provided in this paper can be used by researchers to predict mouse-organ doses and should contribute to an improved understanding of the relationship between dose and radiation toxicity in mouse models where use of these isotopes is favorable.     

Adenoma multiplicity in irradiated Apc(Min) mice is modified by chromosome 16 segments from BALB/c

Ionizing radiation (IR) is a well-characterized carcinogen in humans and mice. The BALB/c mouse strain is unusually sensitive to IR-induced tissue damage and cancer development in a range of organs, suggestive of a partial defect in DNA damage response. This has been confirmed by finding BALB/c-specific functional polymorphism in Prkdc, a gene on mouse chromosome 16 that encodes the catalytic subunit of DNA-dependent protein kinase. Prkdc(BALB) has been associated with increased susceptibility to IR-induced mammary and lymphatic neoplasia. Here, we provide evidence that chromosome 16 segments from BALB/c interact with Apc(Min) (multiple intestinal neoplasia) and specifically enhance IR-induced adenoma development in the upper part of the small intestine.