Kozo Ohkusu-Tsukada

Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines.

There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44+CD24- population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10(4)sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.

Targeted inhibition of IL-10-secreting CD25- Treg via p38 MAPK suppression in cancer immunotherapy.

Cancer‐induced immunotolerance mediated by inducible Treg (iTreg) is a major obstacle to cancer immunotherapy. In a basic study of immunotolerance, injection of an endogenous superantigen, i.e. the minor lymphocyte stimulatory (Mls)‐1a, into specific TCR Vβ8.1‐Tg mice enabled generation of anergic CD25− iTreg, the immunosuppressive function of which was maintained by IL‐10 production via p38‐MAPK activation. Interestingly, although p38‐chemical inhibitor (p38‐inhibitor) is capable of breaking CD25− iTreg‐induced immunotolerance, the p38‐inhibitor had hardly any immunotolerance breaking effect when CD25+ Treg were present, suggesting that depletion of CD25+ Treg is necessary for p38‐inhibitor to be effective. Peptide OVA323–339 iv.‐injection into its specific TCR‐Tg (OT‐II) mice also induced adaptive tolerance by iTreg. Peptide immunotherapy with p38‐inhibitor after CD25+ Treg‐depletion was performed in an OVA‐expressing lymphoma E.G7‐bearing tolerant model established by adoptive transfer of OT‐II CD25− iTreg, which resulted in suppression of tumor growth. Similarly, the antitumor immunity induced by peptide immunotherapy in colon carcinoma CT26‐bearing mice, in which the number of IL‐10‐secreting iTreg is increased, was augmented by treatment with p38‐inhibitor after CD25+ Treg‐depletion and resulted in inhibition of tumor progression. These results suggest that simultaneous inhibition of two distinct Treg‐functions may be important to the success of cancer immunotherapy.

Increased Presence of Stromal Myofibroblasts and Tenascin-C With Malignant Progression in Canine Mammary Tumors

The aims of this study were to determine whether the appearance of stromal myofibroblasts and the expression of tenascin-C (Tn-C) correlate with the grade of malignancy in canine mammary tumors and to determine the main cellular source of Tn-C in these tumors. Single or double immunostaining using antibodies against α-smooth muscle actin (α-SMA) and Tn-C was performed on serial sections of normal canine mammary glands as well as those with lobular hyperplasia, simple adenoma, and simple carcinoma. Thirty-nine of 42 simple carcinomas (93%) exhibited stromal α-SMA–positive myofibroblasts and Tn-C expression. Only 6 of 11 cases of simple adenoma (55%) showed these changes, whereas no changes were observed in normal mammary gland tissue or cases of lobular hyperplasia. The distribution of stromal Tn-C correlated with the presence of myofibroblasts. However, Tn-C immunoreactivity was also occasionally observed in the basement membrane zone surrounding the myoepithelial layer in normal tissue, benign lesions, and tubulopapillary carcinomas. This pattern of staining was not related to the presence of myofibroblasts. The appearance of stromal myofibroblasts and expression of Tn-C were significantly correlated with higher histological grades of malignancy and vascular/lymphatic invasion in simple carcinomas. Stromal myofibroblasts appear to be a major cellular source of Tn-C and play an important role in the development of canine mammary tumors. The Tn-C expressed in the basement membrane zone of normal, hyperplastic, and neoplastic mammary tissue, which is likely produced by neighboring myoepithelial cells, may differ functionally from the Tn-C produced by myofibroblasts.

Differences in Indicators of Malignancy Between Luminal Epithelial Cell Type and Myoepithelial Cell Type of Simple Solid Carcinoma in the Canine Mammary Gland

Routinely diagnosed simple solid carcinoma (SSC) of the canine mammary gland comprises a heterogeneous group of tumors. Seventy-two cases that had been diagnosed as SSC based on hematoxylin and eosin–stained tissue sections were reclassified immunohistochemically on the basis of myoepithelial markers p63 and α-smooth muscle actin, as well as a luminal epithelial marker cytokeratin 8. Only 23 cases (32%) were true SSC, composed only of luminal epithelial cells, whereas 11 cases (15%) were malignant myoepithelioma (MM), composed predominantly of myoepithelial cells, and 38 cases (53%) were biphasic carcinoma (BC), characterized by biphasic proliferation of luminal epithelial and basal/myoepithelial components. As the pathological parameters were compared between the reclassified tumor types, infiltrative potential, vascular/lymphatic invasion, lymph node metastasis, and Ki-67 labeling index were higher in true SSC compared with MM and BC, suggesting that the former may exhibit a poorer prognosis compared with the latter two.

Acinar Cell Cystadenoma of the Pancreas in a Cat

Cystic tumours of the pancreas are heterogeneous lesions with a spectrum of morphology and biological behaviour in people. These are poorly characterized in animals. A multicystic tumour of the pancreas was identified in an 11-year-old, female, mixed breed cat. The tumour was 5.5 cm in diameter and the largest cysts were 1.5 cm in diameter. Microscopically, the cysts were lined by single layered or pseudostratified, flat, cuboidal or columnar epithelial cells that occasionally formed papillary structures with a thin fibrous core. The tumour cells had eosinophilic granules in the apical cytoplasm, similar to zymogen granules, and the nuclei were uniform in size and shape. Mitotic figures were not observed. Immunohistochemically, the tumour cells expressed trypsin, but not cytokeratin 7. A diagnosis of acinar cell cystadenoma of the pancreas was made and this is the first report of this tumour in a cat.

Cellular Sources of Tenascin-C in Canine Mammary Carcinomas

Tenascin-C (Tn-C) is an extracellular matrix glycoprotein implicated in the progression of several human cancers. In canine mammary carcinomas, accumulation of Tn-C has been recognized in 3 different areas: regions of proliferating myoepithelial cells in complex carcinoma, basement membrane zone in low-grade simple carcinoma, and reactive stroma in high-grade simple carcinoma. To identify the Tn-C synthesizing cells in these areas, we utilized double-labeling immunohistochemistry, branched DNA in situ hybridization, and in situ hybridization–immunohistochemistry double-labeling techniques. In complex carcinomas, Tn-C was generated by proliferating myoepithelial cells. Tn-C in low-grade simple carcinomas was also derived from myoepithelial cells existing as a basal monolayer. However, stromal Tn-C in high-grade carcinomas was mainly synthesized by fibroblasts/myofibroblasts, similar to human breast cancer. Thus, the origin of Tn-C in canine mammary carcinomas differs between low- and high-grade malignancies. The role of myoepithelial cell-generated Tn-C is not yet understood.

Erratum: Regulation of the maintenance of peripheral T-cell anergy by TAB1-mediated p38α activation (Molecular and Cellular Biology (2004) 24, 16 (6957-6966))

Volume 24, no. 16, p. 6957–6966, 2004. In this paper we identified an 56-kDa protein expressed in anergic CD4 T cells by Western blotting using anti-TAB1 peptide antibody (N-19; Santa Cruz Biotechnology). This molecule was not present in naı̈ve CD4 T cells, and we concluded that TAB1 is expressed in anergic and not in naı̈ve CD4 T cells. However, our recent work indicated that this is not correct. After publication of the paper, we generated monoclonal antibodies (MAbs) specific for recombinant human TAB1 protein to confirm the expression of TAB1 in naı̈ve and anergic CD4 T cells. Contrary to our expectations, these MAbs identified an 70-kDa molecule in naı̈ve CD4 T cells at levels similar to those in anergic CD4 T cells. Subsequent experiments have shown that the 70-kDa protein is the TAB1 molecule, because these antibodies detect a molecule of the same size in COS7 cells transfected with the expression vector containing human TAB1 cDNA and not with the control vector and, furthermore, this band is specifically absent in cells lacking the TAB1 gene. Therefore, the 56-kDa molecule that we identified in anergic CD4 T cells by anti-TAB1 antibody (N-19) was not TAB1. The expression of TAB1 was shown in Fig. 2 of the paper, and we retract these data. We apologize for releasing and misinterpreting the immature data. While we are unable to conclude that the activation of p38 in anergic CD4 T cells is regulated by up-regulation of TAB1 protein, the main conclusions that anergic CD4 T cells show enhanced p38 mitogen-activated protein kinase activity and that signals involving TAB1 could be a critical regulatory point in maintaining CD4 T-cell anergy by activating p38 remain unchanged.

Clonal deletion of T cell repertoires with specific T cell receptor Vβ chains by two endogenous superantigens in NC/Nga mice

Superantigens (SAgs) are powerful T-cell stimulatory proteins. Because an atopic dermatitis (AD) model NC/Nga mice had two endogenous SAgs, namely minor lymphocyte-stimulating locus-1a (Mls-1a) and mouse mammary tumor virus (MMTV)(SHN), SAg-responsive T-cells bearing Vβ5.1, Vβ6, Vβ8.1, Vβ8.2, Vβ8.3, Vβ9, and Vβ11 should be endogenously deleted. Here, we discuss that the endogenous SAgs-expression may be involved in AD-sensitivity in NC/Nga mice.

Lipid-rich carcinoma in the mammary gland of a Djungarian hamster (phodopus sungorus).

A lipid-rich carcinoma of the mammary gland was diagnosed in a female Djungarian hamster (Phodopus sungorus), which was kept as an indoor pet. The animal underwent surgery for a primary tumor arising in the mammary gland at the age of 16 months, and also for a recurrent tumor 6 months later. Histologically, the primary neoplasm was composed of 2 different cell populations: nonvacuolated glandular neoplastic cells with moderate atypia, and vacuolated neoplastic cells with marked atypia. Transition from the nonvacuolated glandular cells to the vacuolated cells was frequently seen. The recurrent neoplasm was composed predominantly of vacuolated neoplastic cells that often invaded the surrounding soft tissue. The cytoplasmic vacuoles contained neutral lipids, as confirmed by oil red O and Nile blue staining. The vacuolated neoplastic cells were immunopositive for cytokeratin and negative for vimentin, α-smooth muscle actin, p63, estrogen receptor α, and androgen receptor. Presumably, this high-grade, lipid-rich mammary carcinoma had developed from a low-grade mammary adenocarcinoma.

Low expression of a D dm7 /L dm7 -hybrid mutant (D/L dm7 ) in the novel haplotype H-2 nc identified in atopic dermatitis model NC/Nga mice

Environmental factors and the major histocompatibility complex (MHC) are involved in the pathogenesis of atopic dermatitis (AD). However, MHC type (H2 haplotype) of AD model mice NC/Nga is poorly understood. Alloreactive CD8+ or CD4+ T cells in NC/Nga strongly responded to each antigen-presenting cells (A/J: H-2a, C57BL/6: H-2b, BALB/c: H-2d, or C3H/HeJ: H-2k), suggesting that NC/Nga has other H2 haplotype. Polymorphic microsatellite (CA)n repeats in TNF-α gene differ based on the H2 haplotype at present. NC/Nga’s (CA)n repeats (n = 19) were different from other examined strains, A/J (n = 14), BALB/c (n = 14), C3H/HeJ (n = 16), and C57BL/6 (n = 20). Using flow cytometry and genotyping, we demonstrated the NC/Nga H2 haplotype had a unique phenotype (Kd, I-Ak, and I-Ek) in which Dd and Ld lacked as protein despite sensitive mRNA detection. The loss of Dd and Ld was caused by forming a unique Ddm7/Ldm7-hybrid mutant (D/Ldm7). We propose to call this novel H2 haplotype the “H-2nc,” and provide the important information regarding the AD research using NC/Nga mice.