Chie Kato

LGR5‐Positive Colon Cancer Stem Cells Interconvert with Drug‐Resistant LGR5‐Negative cells and are Capable of Tumor Reconstitution

The cancer stem cell (CSC) concept has been proposed as an attractive theory to explain cancer development, and CSCs themselves have been considered as targets for the development of diagnostics and therapeutics. However, many unanswered questions concerning the existence of slow cycling/quiescent, drug‐resistant CSCs remain. Here we report the establishment of colon cancer CSC lines, interconversion of the CSCs between a proliferating and a drug‐resistant state, and reconstitution of tumor hierarchy from the CSCs. Stable cell lines having CSC properties were established from human colon cancer after serial passages in NOD/Shi‐scid, IL‐2Rγnull (NOG) mice and subsequent adherent cell culture of these tumors. By generating specific antibodies against LGR5, we demonstrated that these cells expressed LGR5 and underwent self‐renewal using symmetrical divisions. Upon exposure to irinotecan, the LGR5+ cells transitioned into an LGR5− drug‐resistant state. The LGR5− cells converted to an LGR5+ state in the absence of the drug. DNA microarray analysis and immunohistochemistry demonstrated that HLA‐DMA was specifically expressed in drug‐resistant LGR5− cells, and epiregulin was expressed in both LGR5+ and drug‐resistant LGR5− cells. Both cells sustained tumor initiating activity in NOG mice, giving rise to a tumor tissue hierarchy. In addition, anti‐epiregulin antibody was found to be efficacious in a metastatic model. Both LGR5+ and LGR5− cells were detected in the tumor tissues of colon cancer patients. The results provide new biological insights into drug resistance of CSCs and new therapeutic options for cancer treatment. STEM CELLS 2012;30:2631–2644

The expression profile of glypican-3 and its relation to macrophage population in human hepatocellular carcinoma

Background: Glypican‐3 (GPC3) is frequently upregulated in hepatocellular carcinoma (HCC). Analysis of GPC3‐deficient mice implies GPC3 involvement in macrophage‐lineage cells.

Therapeutic antibodies: their mechanisms of action and the pathological findings they induce in toxicity studies

Antibodies can swiftly provide therapeutics to target disease-related molecules discovered in genomic research. Antibody engineering techniques have been actively developed and these technological innovations have intensified the development of therapeutic antibodies. From the mid-1990’s, a series of therapeutic antibodies were launched that are now being used in clinic. The disease areas that therapeutic antibodies can target have subsequently expanded, and antibodies are currently utilized as pharmaceuticals for cancer, inflammatory disease, organ transplantation, cardiovascular disease, infection, respiratory disease, ophthalmologic disease, and so on. This paper briefly describes the modes of action of therapeutic antibodies. Several non-clinical study results of the pathological changes induced by therapeutic antibodies are also presented to aid the future assessment of the toxic potential of an antibody developed as a therapeutic.

Spontaneous thymic lymphomas in the non-obese diabetic/Shi-scid, IL-2Rγnull mouse

The NOD/Shi-scid, IL-2Rγ null (NOG) mouse is a severely immunodeficient mouse used for the engraftment of human tissues and cells. In this study, 2406 mice (8–62 weeks old, 503 males and 1903 females) were subcutaneously engrafted with human tissues. In 16 mice (12–26 weeks old, 1 male and 15 females), a mass was seen in the anteroventralis of the thorax on gross examination with an incidence of 0.7%. Histologically, the masses were composed of sheets of lymphoblastic cells. A ‘starry sky’ pattern was observed with numerous mitoses. Immunohistochemically the lymphoblastic cells were positive for Thy 1. The lymphoblastic cells were also seen in the spleen, lung, liver, kidney and heart. The gross and histopathological findings led to the diagnosis of spontaneous thymic lymphoma in NOG mice.

Segmentation of the Pathophysiological Stages of Diabetic Changes in the db/db Mouse.

The db/db mouse is one of the diabetes mellitus animal models and if the pathophysiological stages of diabetic changes in the mouse model could simulate the stages in human diabetes, the db/db mouse could be used to better evaluate drug candidates. Blood insulin, HbA1c levels and morphological features of pancreatic islets in db/db mice were evaluated to determine the pathophysiological stage. At 6 weeks of age, db/db mice showed the highest level of plasma insulin and lowest level of HbA1c, and histopathological examination revealed enlarged islets with a circular shape and hypertrophic islet cells. By 9 and 12 weeks of age, the plasma insulin levels had decreased to mid levels and HbA1c had increased to mid to high levels; histopathological examination at this time revealed two types of islets coexisting, enlarged circular islets and small irregular-shaped islets. By 15 and 22 weeks of age, plasma insulin had decreased further to low levels and HbA1c was at its highest level; the histopathological examination at this time revealed an increase in irregular-shaped and small islets. Based on blood insulin levels, HbA1c levels and histopathology findings in the db/db mice in this study, the clinical staging of diabetic changes were recognized. The pathophysiological stages of diabetes mellitus in this animal model were similar to the stages in humans.

Anti-Thy-1 Antibody-mediated Complement-dependent Cytotoxicity is Regulated by the Distribution of Antigen, Antibody and Membrane Complement Regulatory Proteins in Rats.

Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs.

Expression of Membrane Complement Regulatory Proteins Crry and CD55 in Normal Rats

Some anticancer therapeutic antibodies are designed to act through complement-dependent cytotoxicity (CDC). It has been reported that there are many membrane complement regulatory proteins (mCRPs) that inhibit CDC. In the present study, we examined the expression of two mCRPs, the complement receptor 1-related gene/protein Y (Crry) and the decay-accelerating factor CD55, in three normal rats by immunohistochemistry. Crry and CD55 were detected widely in rat organs and tissues. Crry was found mainly in the urinary, digestive, respiratory, immunohematopoietic, circulatory and neuroendocrine systems. CD55 was found in the urinary, digestive and neuroendocrine systems. However, the two molecules were expressed in separate cells within the same organ. These results suggest that the distribution of mCRPs is related to the strict regulation of CDC activation in these organs and tissues and that the two molecules have a nonoverlapping expression pattern, a fact indicating specific roles in CDC regulation.

Stimulation of adrenal chromaffin cell proliferation by hypercalcemia induced by intravenous infusion of calcium gluconate in rats.

Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.