Bevacizumab suppresses the growth of established non-small-cell lung cancer brain metastases in a hematogenous brain metastasis model.

Brain metastases are common in patients with non-small-cell lung cancer (NSCLC). The efficacy of bevacizumab, an anti-vascular endothelial growth factor (VEGF) humanized antibody, has been demonstrated in patients with nonsquamous NSCLC. We established a transplantable NSCLC cell line (Nluc-H1915) that stably expresses NanoLuc® reporter and confirmed the correlation between total Nluc activity in tumor and tumor volume in vivo. SCID mice inoculated with these cells through the internal carotid artery formed reproducible brain metastases, in which human VEGF was detected. Next, after metastases were established in the model mice (15-17 days), they were intraperitoneally administered weekly doses of human immunoglobulin G (HuIgG) or bevacizumab. Nluc activity in the brain was significantly lower in bevacizumab-treated mice than in HuIgG-treated mice. Additionally, bevacizumab concentration in the brain was higher in mice with brain metastasis than in normal mice, and bevacizumab was primarily observed in brain metastasis lesions. The microvessel density in brain metastasis was lower in bevacizumab-treated mice than in HuIgG-treated mice. We believe bevacizumab's anti-proliferative effect on brain metastasis is due to anti-angiogenic activity achieved by its penetration into brain metastases; this suggests that a bevacizumab-containing regimen may be a promising treatment option for patients with NSCLC brain metastasis.

In vivo effects of mutant RHOA on tumor formation in an orthotopic inoculation model.

Ras homolog family member A (RHOA) mutations are driver genes in diffuse­type gastric cancers (DGCs), and we previously revealed that RHOA mutations contribute to cancer cell survival and cell migration through their dominant negative effect on Rho­associated kinase (ROCK) signaling in vitro. However, how RHOA mutations contribute to DGC development in vivo is poorly understood. In the present study, the contribution of RHOA mutations to tumor morphology was investigated using an orthotopic xenograft model using the gastric cancer cell line MKN74, in which wild­type (WT) or mutated (Y42C and Y42S) RHOA had been introduced. When we conducted RNA sequencing to distinguish between the genes expressed in human tumor tissues from those in mouse stroma, the expression profiles of the tumors were clearly divided into a Y42C/Y42S group and a mock/WT group. Through gene set enrichment analysis, it was revealed that inflammation­ and hypoxia­related pathways were enriched in the mock/WT tumors; however, cell metabolism­ and cell cycle­related pathways such as Myc, E2F, oxidative phosphorylation and G2M checkpoint were enriched in the Y42C/Y42S tumors. In addition, the gene set related to ROCK signaling inhibition was enriched in the RHOA­mutated group, which indicated that a series of events are related to ROCK inhibition induced by RHOA mutations. Histopathological analysis revealed that small tumor nests were more frequent in RHOA mutants than in the mock or WT group. In addition, increased blood vessel formation and infiltration of macrophages within the tumor mass were observed in the RHOA mutants. Furthermore, unlike mock/WT, the RHOA­mutated tumor cells had little antitumor host reaction in the invasive front, which is similar to the pattern of mucosal invasion in clinical RHOA­mutated DGC. These transcriptome and pathological analyses revealed that mutated RHOA functionally contributes to the acquisition of DGC features, which will accelerate our understanding of the contribution of RHOA mutations in DGC biology and the development of further therapeutic strategies.

Difference in morphology and interactome profiles between orthotopic and subcutaneous gastric cancer xenograft models.

In xenograft models, orthotopic (ORT) engraftment is thought to provide a different tumor microenvironment compared with subcutaneous (SC) engraftment. We attempted to characterize the biological difference between OE19 (adenocarcinoma of the gastroesophageal junction) SC and ORT models by pathological analysis and CASTIN (CAncer-STromal INteractome) analysis, which is a novel method developed to analyze the tumor-stroma interactome framework. In SC models, SCID mice were inoculated subcutaneously with OE19 cells, and tumor tissues were sampled at 3 weeks. In ORT models, SCID mice were inoculated under the serosal membrane of the stomach wall, and tumor tissues were sampled at 3 and 6 weeks after engraftment. Results from the two models were then compared. Histopathologically, the SC tumors were well circumscribed from the adjacent tissue, with scant stroma and the formation of large ductal structures. In contrast, the ORT tumors were less circumscribed, with small ductal structures invading into abundant stroma. Then we compared the transcriptome profiles of human tumor cells with the mouse stromal cells of each model by species-specific RNA sequencing. With CASTIN analysis, we successfully identified several interactions that are known to affect the tumor microenvironment as being selectively enhanced in the ORT model. In conclusion, pathological analysis and CASTIN analysis revealed that ORT models of OE19 cells have a more invasive character and enhanced interaction with stromal cells compared with SC models.

Innate Response to Human Cancer Cells with or without IL-2 Receptor Common γ-Chain Function in NOD Background Mice Lacking Adaptive Immunity.

Immunodeficient hosts exhibit high acceptance of xenogeneic or neoplastic cells mainly due to lack of adaptive immunity, although it still remains to be elucidated how innate response affects the engraftment. IL-2R common γ-chain (IL-2Rγc) signaling is required for development of NK cells and a subset of dendritic cells producing IFN-γ. To better understand innate response in the absence of adaptive immunity, we examined amounts of metastatic foci in the livers after intrasplenic transfer of human colon cancer HCT116 cells into NOD/SCID versus NOD/SCID/IL-2Rγc (null) (NOG) hosts. The intravital microscopic imaging of livers in the hosts depleted of NK cells and/or macrophages revealed that IL-2Rγc function critically contributes to elimination of cancer cells without the need for NK cells and macrophages. In the absence of IL-2Rγc, macrophages play a role in the defense against tumors despite the NOD Sirpa allele, which allows human CD47 to bind to the encoded signal regulatory protein α to inhibit macrophage phagocytosis of human cells. Analogous experiments using human pancreas cancer MIA PaCa-2 cells provided findings roughly similar to those from the experiments using HCT116 cells except for lack of suppression of metastases by macrophages in NOG hosts. Administration of mouse IFN-γ to NOG hosts appeared to partially compensate lack of IL-2Rγc-dependent elimination of transferred HCT116 cells. These results provide insights into the nature of innate response in the absence of adaptive immunity, aiding in developing tumor xenograft models in experimental oncology.

The reconstituted 'humanized liver' in TK-NOG mice is mature and functional.

To overcome the limitations of existing models, we developed a novel experimental in vivo platform for replacing mouse liver with functioning human liver tissue. To do this, a herpes simplex virus type 1 thymidine kinase (HSVtk) transgene was expressed within the liver of highly immunodeficient NOG mice (TK-NOG). Mouse liver cells expressing this transgene were ablated after a brief exposure to a non-toxic dose of ganciclovir (GCV), and transplanted human liver cells are stably maintained within the liver (humanized TK-NOG) without exogenous drug. The reconstituted liver was shown to be a mature and functioning "human organ" that had zonal position-specific enzyme expression and a global gene expression pattern representative of mature human liver; and could generate a human-specific profile of drug metabolism. The 'humanized liver' could be stably maintained in these mice with a high level of synthetic function for a prolonged period (8 months). This novel in vivo system provides an optimized platform for studying human liver physiology, including drug metabolism, toxicology, or liver regeneration.

Reduced CD73 expression and its association with altered purine nucleotide metabolism in colorectal cancer cells robustly causing liver metastases.

Liver metastases of colorectal cancers significantly affect the prognoses of patients. To further understand the biological aspects of the metastatic phenotypes, we established the highly liver-metastatic human colorectal cancer cell subline SW48LM2. The subline was established through the serial intrasplenic transfer of cells derived from poor but visible hepatic tumor foci formed by parental SW48 cells and transferred to NOD/SCID/IL-2Rγc(null) mice. The growth, both under monolayer culture conditions and during the formation of subcutaneous tumors, was similar between the two cell lines, although there were morphological differences in the in vitro spheroid formation. Of 41 molecules reportedly associated positively or negatively with tumor progression, four were overexpressed and four were underexpressed in SW48LM2 cells. Notably, this liver-metastatic cell subline exhibited a strongly reduced expression of the ecto-5'-nucleotidase CD73 as well as an altered metabolism of purine nucleotides. Previous studies showed a positive correlation between CD73 expression and metastatic cancer phenotypes. A reduced CD73 expression in tumor cells, however, may contribute to obtaining insight into the mechanisms of liver metastases.

Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice.

Severely immunodeficient NOD/Shi-scid IL2Rg(null) (NOG) mice are used as recipients for human tissue transplantation, which produces chimeric mice with various types of human tissue. NOG mice expressing transgenic urokinase-type plasminogen activator in the liver (uPA-NOG) were produced. Human hepatocytes injected into uPA-NOG mice repopulated the recipient livers with human cells. The uPA-NOG model has several advantages over previously produced chimeric mouse models of human liver: (1) the severely immunodeficient NOG background enables higher xenogeneic cell engraftment; (2) the absence of neonatal lethality enables mating of homozygotes, which increased the efficacy of homozygote production; and (3) donor xenogeneic human hepatocytes could be readily transplanted into young uPA-NOG mice, which provide easier surgical manipulation and improved recipient survival.

Effects of mitemcinal (GM-611), an orally active erythromycin-derived prokinetic agent, on delayed gastric emptying and postprandial glucose in a new minipig model of diabetes.

AIMS: This study was conducted to evaluate the suitability of a new minipig model for investigating aspects of diabetes such as delayed gastric emptying and glucose metabolism abnormalities, and to test the effects of mitemcinal (GM-611), an orally active erythromycin-derived motilin receptor agonist, on gastric emptying and postprandial glucose in normal and diabetic minipigs. METHODS AND RESULTS: Intravenous injection of 300 mg/kg streptozotocin (STZ) to 5-week-old minipigs induced moderate hyperglycemia (about 200 mg/dl) for >80 weeks without insulin treatment. Decreased insulin production (P<.05), increased area under the glucose curve (P<.05), and slower glucose disappearance (P<.05) were demonstrated, and there was no severe inhibition of body weight gain, liver failure, or renal failure. Gastric emptying was significantly delayed in diabetic minipigs (P<.05) at 80 weeks, but not at 40 weeks, post-STZ. Oral administration of mitemcinal (5 mg/kg) at 80 weeks accelerated gastric emptying and induced a similar postprandial glucose profile in normal and diabetic minipigs with delayed gastric emptying. CONCLUSIONS: The new diabetic minipig model showed suitability for investigating diabetes, gastric emptying, and plasma glucose excursions. Since delayed gastric emptying and irregular plasma glucose excursions are characteristic of diabetic gastroparesis, the accelerating and regulating effects of mitemcinal on this model add to the existing evidence that mitemcinal is likely to be useful for treating diabetic gastroparesis.

Mitemcinal (GM-611), an orally active motilin receptor agonist, improves delayed gastric emptying in a canine model of diabetic gastroparesis.

1. The aim of the present study was to evaluate the effects of mitemcinal (GM-611), an orally active motilin receptor agonist, on delayed gastric emptying in a canine model of diabetic gastroparesis and to compare these effects with those of cisapride. 2. Moderate hyperglycaemia was induced by a single intravenous injection of a mixture of streptozotocin (30 mg/kg) and alloxan (50 mg/kg). Dogs that maintained moderate hyperglycaemia (fasting plasma glucose 200-300 mg/dL) without insulin treatment were selected and gastric emptying in these dogs was determined by the paracetamol method. 3. One year after the onset of diabetes, there was no difference in the gastric emptying of normal and diabetic dogs. However, after 5 years, the diabetic dogs showed delayed gastric emptying. The motor nerve conduction velocity of the tibial nerve was significantly lower in diabetic dogs compared with normal dogs at both time points. 4. Histopathological examination at the end of the study showed that there were fewer nerve fibres in both dorsal vagal and tibial nerves of diabetic dogs compared with normal dogs. The onset of delayed gastric emptying is thought to have occurred gradually, in parallel with abnormal autonomic nerve function induced by the long period of moderate hyperglycaemia. 5. Oral administration of mitemcinal (0.125, 0.25 or 0.5 mg/kg) dose-dependently accelerated delayed gastric emptying, significant at 0.5 mg/kg, in diabetic dogs, whereas cisapride (1, 3 or 10 mg/kg) had no significant effect. These results add to the existing evidence that mitemcinal is likely to be useful for treating diabetic gastroparesis.

Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shi-scid IL2Rgammanull (NOG) mice.

To examine the drug efficacy of a novel farnesyltransferase inhibitor (FTI), CH4512600, in vivo, we developed a reliable liver metastasis model of human colon cancer using NOD/Shi-scid IL2Rgamma(null) (NOG) mice. Eleven human colon cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOG mice. When inoculated with 10(4) COLO320DM, HCT 116, HT-29, WiDr, LoVo and LS174T cells, liver metastasis was evident in 100% (6/6), 100% (6/6), 88.9% (8/9), 87.5% (7/8), 83.3% (5/6) and 50.0% (3/6) of the NOG mice, respectively. CaCo2, COLO201, LS123, SW48 and SW1417 showed no metastasis when seeded at 10(4) cells even in NOG mice. The mRNA expression levels and genetic mutations of N, H and K-RAS genes, which directly affect the levels of cellular RAS protein that would be molecular target for FTI, were also examined in these six metastatic human colon cancer cell lines for molecular biological and genotypic characteristics. Only three cell lines had a point mutation in the RAS oncogene. LS174T cell line had a point mutation of the K-RAS gene at codon 12 (gly12 --> asp; G12D), and HCT 116 and LoVo cell lines had a point mutation of the K-RAS gene at codon 13 (gly13 --> asp; G13D). Relative gene expression levels of N, H and K-RAS genes in the HCT 116 cell line were 2.6-5.0-fold lower than that of LS174T and LoVo cell lines. We selected HCT 116 cell line from our liver metastasis model for evaluation of FTI CH4512600 efficacy in vivo. Using the NOG mouse liver metastasis model, we demonstrated the effectiveness of FTI CH4512600 to suppress tumor growth in vivo and to prolong mouse survival significantly from 36.9+/-2.9 to 50.3+/-9.4 days.

Identification of a key molecular regulator of liver metastasis in human pancreatic carcinoma using a novel quantitative model of metastasis in NOD/SCID/gammacnull (NOG) mice.

We developed a reliable new model system for assaying liver metastasis using NOD/SCID/gamma(c)(null) (NOG) mice. Seven human pancreatic cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOD/SCID and NOG mice. Capan-2 and PL45 showed no metastasis when seeded at up to 10(5) cells in both strains, and no BxPC-3 metastasis was observed in NOD/SCID mice. The NOD/SCID mouse model detected liver metastasis only in the AsPC-1 cell line when inoculated with >10(3) cells. In contrast, when inoculated with only 10(2) MIA PaCa-2, AsPC-1 and PANC-1 cells, liver metastasis was evident in 71.4% (5/7), 57.1% (4/7) and 37.5% (3/8) of the NOG mice, respectively. Capan-1 and BxPC-3 cells metastasized when seeded at 10(3) cells in 50% (5/10) and in 12.5% (1/8) of the mice, respectively. Using the NOG mouse model system, we established a highly metastatic cell line, liver metastasized-BxPC-3 (LM-BxPC-3), from liver metastatic foci formed by the relatively poorly metastatic parental BxPC-3 cell line. The gene expression profiles of parental and LM-BxPC-3 cells were compared, and we identified forty-five genes that were either upregulated or downregulated >4-fold in the LM-BxPC-3 cell line. We validated 9 candidate protein-coding sequences, and examined the correlation between their expression pattern and the in vivo liver metastatic potential of all 7 pancreatic cancer cell lines. Only S100A4 expression correlated with the ability to form liver metastases, as evaluated in our quantitative model of metastasis in NOG mice. These results suggested that S100A4 is a key regulator of liver metastasis in pancreatic cancer, and demonstrated the feasibility of using the quantitative metastasis model to search for and develop new anti-cancer therapies and novel drugs against this and other key molecules.

Altered cytokine expression in mesenteric lymph nodes in a rat strain (Matsumoto Eosinophilic Shinshu) that spontaneously develops hypereosinophilia.

The Matsumoto Eosinophilic Shinshu (MES) rat is an inbred mutant strain that spontaneously develops systemic hypereosinophilia with eosinophilic inflammatory lesions similar to those associated with hypereosinophilic syndrome in humans and other mammals. To elucidate the pathogenic mechanisms that underlie these features of MES rats, we examined the pattern of cytokine gene expression in mesenteric lymph nodes (MLNs), the thymus, and peripheral blood mononuclear cells as well as the blood clinicopathology and MLN lymphocytic subsets of these animals. MES rats exhibited both leucocytosis, attributable in large part to hypereosinophilia and neutrophilia, and immunoglobulin M (IgM) and IgA gammaglobulinaemia, with increased titres of IgM autoantibodies to nuclear antigens. Reverse transcription and polymerase chain reaction analysis revealed that the amounts of interleukin (IL)-5, IL-4, eotaxin, and interferon-gamma mRNAs were increased in the MLN lymphocytes of MES rats compared with the corresponding values for Sprague-Dawley rats. Intraperitoneal administration of a monoclonal antibody specific for IL-5 resulted in an immediate suppression of hypereosinophilia and a delayed suppression of neutrophilia in MES rats. Flow cytometry revealed an increased percentage of CD3+ CD4- CD8- T lymphocytes in MLNs of MES rats. Our results suggest that the hypereosinophilia of MES rats results from an increased production of IL-5, and that the eosinophilic inflammatory lesions of these animals, which are largely restricted to the gut, may be related both to cytokine overexpression in MLNs and to T helper 1 and 2 immunological responses.

Establishment of a new model of human multiple myeloma using NOD/SCID/gammac(null) (NOG) mice.

We developed a new experimental animal model of human multiple myeloma using immunodeficient NOD/SCID/gammac(null) (NOG) mice. A human myeloma cell line, U266, was intravenously inoculated into 20 NOG mice, all of which developed hind leg paralysis and distress around 6 weeks after transplantation. Pathological studies showed that only the bone marrow was infiltrated with U266 cells, and no cells were present in other organs. Osteolytic lesions in cortical bones and loss of trabecular bones were prominent in U266-transplanted NOG mice. In contrast, U266 cells were not detected in CB17scid or NOD/SCID mice 6 weeks after intravenous inoculation. Human IgE, produced by U266 cells, was detected in the serum of U266-transplanted NOG mice by ELISA. The results indicated that this hu-myeloma NOG model might be useful for studying the pathogenesis of myeloma and related osteolytic lesions, and are suggestive of its applicability to the future development of new drugs.