Atsuhiko 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

Distribution of IL-31 and its receptor expressing cells in skin of atopic dermatitis

BACKGROUND To understand the clinical segments of IL-31 signaling blockade therapy in pruritus of atopic dermatitis (AD), direct detection of the target proteins in the diseased tissues will provide crucial information. There is a lack of direct evidence concerning the cellular origin of IL-31 in AD skins, and data on the expression of IL-31RA in AD are inconsistent. Also, there is no available information regarding IL-31RA protein expression in human dorsal root ganglia (DRG), which mediates the sensation of itch and is the long-suspected source of the protein. OBJECTIVE We sought to obtain direct evidence concerning the distribution of IL-31- and IL-31RA-protein expressing cells and their characteristics in AD skin samples and in human DRG. METHODS IL-31 was detected immunohistochemically in AD skins, and representative sections were double stained with IL-31 and several immune-markers. IL-31RA was stained immunohistochemically in AD skins and normal human DRG, and representative AD skins were double stained with IL-31RA and PGP9.5 (a nerve marker). RESULTS IL-31-positive cells were observed as mononuclear infiltrating cells and as CD11b co-expressing cells in severe AD samples. As for IL-31RA, positive reactions were detected in keratinocytes and nerve fibers in the dermis of AD and in the neurons of normal DRG. CONCLUSION The detection of IL-31 in infiltrating cells of severe AD skin and of IL-31RA in nerve fibers of AD dermis and normal DRG indicates IL-31 signaling may be a contributing factor in the persistence and exacerbation of AD skin lesions.

Prognostic significance of circumferential cell surface immunoreactivity of glypican‐3 in hepatocellular carcinoma

Background: GC33 is a recently developed monoclonal antibody against human glypican‐3 (GPC3), which is significantly upregulated in hepatocellular carcinoma (HCC). GC33 recognizes a GPC3 ectodomain and shows significant antitumour activity in vivo. Thus, humanized GC33 antibody may be a promising tool for treating HCC having cell surface GPC3 expression.

Involvement of glypican-3 in the recruitment of M2-polarized tumor-associated macrophages in hepatocellular carcinoma.

Previously, we demonstrated that membrane expression of glypican-3 (GPC3) stimulates the recruitment of macrophages into human hepatocellular carcinoma (HCC) tissues. However, functional polarization of the macrophages and the chemoattractant factors related to the recruitment has yet to be determined. In this study, to clarify the polarization (M1 or M2) of the macrophages and provide a clue as to the factors involved in the recruitment, we used xenograft models of SK-HEP-1 and SK03 cell lines with undetectable and high-level membrane expression of GPC3, respectively and analyzed the expression profiles of the relevant genes in both xenografts mainly using microarray techniques. Clustering analyses with mouse genome arrays revealed that the SK-HEP-1 and SK03 xenografts showed different expression profiles for M2 macrophage-related genes but not for M1 macrophage-related genes. Many of the M2 macrophage-related genes were upregulated in the SK03 xenografts compared to the SK-HEP-1 xenografts. Additionally, most of the macrophages infiltrating into the SK03 xenografts were positive for M2 macrophage-specific markers. Regarding the chemoattractant factors, the microarray and quantitative real-time PCR analyses revealed that, of the genes reportedly related to macrophage recruitment, CCL5, CCL3 and CSF1 were significantly upregulated in the SK03 xenograft. These findings suggest that the macrophages recruited into GPC3-overexpressing (with membrane expression) HCC are M2-polarized ones and, more specifically, M2 tumor-associated macrophages which are known to promote tumor progression and metastasis, and CCL5, CCL3 and CSF1 are possible candidate genes for the recruitment of macrophages.

AG-041R, a novel indoline-2-one derivative, induces systemic cartilage hyperplasia in rats.

AG-041R (3R-1-(2,2-diethoxyethyl)-3-((4 methylphenyl)aminocarbonylmethyl)-3-((4-methylphenyl) ureido)-indoline-2-one) is a novel small compound synthesized as a cholecystokinin-2 (CCK(2))/gastrin receptor antagonist. In the course of the development of this compound, we discovered unexpectedly that oral administration of a high dose for 4 weeks markedly induced systemic cartilage hyperplasia. This change was histologically observed in the auricles, the trachea, the marginal region of the femoral condyle, the xiphoid process and intervertebral disks in rats. Daily intraarticular injections of AG-041R into rat knee joints for 3 weeks also caused cartilage hyperplasia in the marginal region of the femoral condyle, but no hyperplasia was observed in any other cartilage. We have confirmed that chondrogenic activity of AG-041R is an intrinsic property of the compound, and is not due to its CCK(2)/gastrin receptor antagonistic actions. These results indicate that AG-041R is a novel stimulator of chondrogenesis, and can be expected to be a potent therapeutic agent for cartilage disorders.

Early effects of tocilizumab on bone and bone marrow lesions in a collagen-induced arthritis monkey model

To understand the contribution of IL-6/IL-6R to subchondral bone and bone marrow abnormality in RA patients and the effects of tocilizumab on those abnormalities, we evaluated early change in a collagen-induced arthritis (CIA) monkey model with or without a single administration of tocilizumab. Six CIA cynomolgus monkeys received tocilizumab and 3 CIA monkeys received vehicle only. Their interphalangeal joints were analyzed using HE, silver impregnation (SI), or immunohistochemistry (RANKL) staining. The number of osteoclasts increased in the arthritis control but was suppressed in the tocilizumab-treated animals. Osteoblast/stromal cells of the arthritis control monkeys were of monolayer, while in the tocilizumab-treated monkeys, the cells were multi-layer or differentiated osteoblasts, and the meshwork of the reticulum fibers showed recovery in the SI. Hematopoietic marrow was replaced by interstitial fluid and reticulum fibers were eliminated in the arthritic model but showed recovery in the tocilizumab-treated animals. RANKL showed overproduction with arthritis and suppressed with tocilizumab treatment. The evidence indicates that IL-6/IL-6R is involved in subchondral bone and bone marrow change in RA patients. Tocilizumab treatment recovered changes in the CIA monkeys as a result of the co-differentiation between the osteoclasts and the osteoblast/stramal cells, at least partially through the suppression of RANKL overproduction.

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.

Histopathological analyses of the antitumor activity of anti-glypican-3 antibody (GC33) in human liver cancer xenograft models: The essential role of macrophages

Previously, we demonstrated the antitumor efficacy of the anti-glypican-3 (GPC3) antibody GC33 in several human liver cancer xenograft models and the important role of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor mechanism of GC33. Involvement of other mechanisms such as modulation of the functions of GPC3 in antitumor activity remains to be elucidated. In this study, we investigated histopathologically time-course changes in xenografts in mice following a single administration of GC33 to clarify the morphological changes contributing to the tumor growth inhibition of GC33, including the changes in GPC3-related factors/components (proliferation, extracelluar matrices (ECMs) and macrophage). Histopathological changes peaked 3–5 days after GC33 administration and included increased tumor cell death, tumor cells with round morphology, multinucleated tumor cells and small spindle/round-like cells (mostly F4/80-positive macrophages). No direct effects of GC33 on proliferation activity of tumor cells were observed. Meanwhile, alteration of ECM structures and a remarkable increase in macrophages was noted in the GC33-treated group. Increase in macrophages was observed mainly in the outer layer of tumor nodules; the area of the increase approximately included the area where the change in tumor cells and ECMs were observed. Interestingly, depletion of macrophages in the xenograft models resulted in a marked reduction of the antitumor activity of GC33. In the in vitro ADCC assay, ADCC was only slightly induced by mouse peritoneal macrophages. These data suggest that macrophages play an essential role in the antitumor activity of GC33 and the possible involvement of macrophage-mediated non-ADCC action.

Novel genetically-humanized mouse model established to evaluate efficacy of therapeutic agents to human interleukin-6 receptor

For clinical trials of therapeutic monoclonal antibodies (mAbs) to be successful, their efficacy needs to be adequately evaluated in preclinical experiments. However, in many cases it is difficult to evaluate the candidate mAbs using animal disease models because of lower cross-reactivity to the orthologous target molecules. In this study we have established a novel humanized Castlemans disease mouse model, in which the endogenous interleukin-6 receptor gene is successfully replaced by human IL6R, and human IL6 is overexpressed. We have also demonstrated the therapeutic effects of an antibody that neutralizes human IL6R, tocilizumab, on the symptoms in this mouse model. Plasma levels of human soluble IL6R and human IL6 were elevated after 4-week treatment of tocilizumab in this mouse model similarly to the result previously reported in patients treated with tocilizumab. Our mouse model provides us with a novel means of evaluating the in vivo efficacy of human IL6R-specific therapeutic agents.

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.