D. Kumagai

Establishment and characterization of cell lines derived from a transplantable rat malignant meningioma: morphological heterogeneity and production of nerve growth factor

Abstract A cell line (KMY-J) was established from a transplantable tumor (MM-KMY) derived from a spontaneous malignant meningioma arising in an aged F344 rat, and three cloned cell lines (KMY-1, KMY-2 and KMY-3) were induced from the parent KMY-J. Morphologically, KMY-J and tumors induced in syngeneic rats by KMY-J showed cell pleomorphism. All neoplastic cells in KMY-J and its tumors were immunoreactive to vimentin; occasional cells reacted to ED1 (rat macrophage/histiocyte-specific antibody) and α-smooth muscle actin (α-SMA), indicating expression of histiocytic or myofibroblastic immunophenotypes of meningioma cells. In contrast, KMY-1, KMY-2 and KMY-3 consisted of a uniform cell population differing from each other. KMY-1-induced tumors were similar histologically to meningeal fibrosarcomas. Dendritic cells seen in KMY-2 cultures gave an appearance of arachnoid trabecular cells. In KMY-3 and its tumors, large round cells and multinucleated giant cells were predominant. Cells of these cloned cell lines also reacted to vimentin, but were negative for ED1 and α-SMA. By the bioassay using PC12 cells and reverse transcription-polymerase chain reaction for nerve growth factor (NGF) mRNA, production of NGF was demonstrated in the parent and cloned cell lines. The present cell lines may prove useful for studying the histological features of meningeal tumors and the bioactive factors produced by meningeal cells.

Characteristics of a rat fibrosarcoma-derived transplantable tumour line (SS) and cultured cell lines (SS-P and SS-A3-1) showing myofibroblastic and histiocytic phenotypes.

Abstract A transplantable tumour line (SS) was established in syngeneic rats from a spontaneous fibrosarcoma that had arisen in the submandibular salivary gland of a 24-month-old male F344 rat. A cell line (SS-P) was induced from SS, and a cloned cell line (SS-A3-1) was isolated from SS-P. The primary tumour consisted of oval to spindle-shaped cells arranged in bundles with abundant collagen fibres; ultrastructurally, neoplastic cells exhibited fusiform morphology with prominent rough endoplasmic reticulum. SS tumours showed marked interlacing fascicle and herring-bone growth patterns. SS-P and SS-A3-1 were simmilar morphologically to each other, consisting of oval, spindle or polygonal cells and occasional multinucleated giant cells. Tumours induced by SS-P and SS-A3-1 were histologically similar to SS tumours. Immunohistochemically, all cells in the primary tumour, SS tumours and tumours induced both by SS-P and SS-A3-1 and by SS-P and SS-A3-1 cultures gave a positive reaction to vimentin. Interestingly, neoplastic cells reacting to ED1 (rat macrophage/histiocyte-specific antibody) and α-smooth muscle actin (α-SMA) appeared in SS tumours and tumours induced by SS-P and SS-A3-1 and by SS-P and SS-A3-1 cultures. Cells with histiocytic fine structures and myofibroblastic cells with cytoplasmic actin-like microfilaments were also observed by electron microscopy. The present rat fibrosarcoma-derived transplantable tumour line (SS) and cell lines (SS-P and SS-A3-1) might express myofibroblastic and histiocytic phenotypes, probably depending on the surrounding conditions. These cell lines may prove useful for studying the mechanisms of phenotypic plasticity in neoplastic fibroblasts.

Morphological characteristics of a transplantable histiocytic sarcoma (HS-J) in F344 rats and appearance of renal tubular hyaline droplets in HS-J-bearing rats

A transplantable tumour (HS-J) was established from a spontaneous histiocytic sarcoma found in a 24-month-old male F344 rat. Serial transplantations (seven generations) were made in syngeneic male and female rats by means of intraperitoneal or subcutaneous implants, with a 100% take rate. Rats given HS-J implants developed large nodules locally, with metastasis to distant organs. HS-J tumours consisted mainly of round to oval cells with abundant cytoplasm, arranged in a compact sheet. Enzyme- and immuno-histochemical examination showed that neoplastic cells reacted with ED1 (rat monocyte/macrophage-specific antibody), lysozyme, alpha 1-antitrypsin and lysosomal enzymes (acid phosphatase and non-specific esterase), indicating derivation from cells of the monocyte/macrophage lineage. The majority of neoplastic cells were negative for ED2 (rat tissue macrophage-specific antibody). Abnormal accumulations of hyaline droplets in the proximal renal tubular epithelial cells were seen in HS-J-bearing rats. The droplets were faintly immunopositive for lysozyme, but negative for alpha-2u globulin and albumin. It was considered that excessive production of the protein by tumour cells might lead to subsequent overload in renal tubules. HS-J may prove beneficial for studying the biological behaviour of monocyte/macrophage-derived tumours in the rat.

Characterization of newly established tumor lines from a spontaneous malignant schwannoma in F344 rats: nerve growth factor production, growth inhibition by transforming growth factor-β1, and macrophage-like phenotype expression

Transplantable tumor (KE) and clone cell (KE-F11) lines were established from a spontaneous malignant schwannoma found in an aged F344 rat. The primary tumor and KE tumors consisted of oval or spindle cells arranged in ill-defined bundles. Cultured KE-F11 cells exhibited polygonal or spindle configurations. Immunohistochemically, neoplastic cells in KE and KE-F11 reacted to vimentin, S-100 protein, neuron-specific enolase, myelin basic protein, and glial fibrillary acidic protein in varying degrees, indicating neurogenic features; occasional cells reacted to α-smooth muscle actin. Cells positive for lysosomal enzymes (acid phosphatase and non-specific esterase), and ED1 (rat macrophage specific) were observed in KE-F11, and electron microscopically, cells with many lysosomes were frequently present, indicating expression of macrophage-like phenotypes. Bioassay analysis revealed that KE-F11 cells produced high levels of nerve growth factor. DNA synthesis was inhibited by addition of transforming growth factor-β1 (TGF-β1), and Northern blot analysis revealed that expression of c-myc, a cell cycle-related immediate early gene, was depressed by TGF-β1. Likely, TGF-β1 is a factor capable of inhibiting cellular growth of Schwann cells. mRNA expression of the low-density lipoprotein receptor-related protein (LRP) was seen in KE-F11 cells by Northern blot analysis, and the level was decreased by lipopolysaccharide (LPS) treatment. LRP may be attributable to regulation of Schwann cell functions. KE-F11 cells seeded on laminin-coated dishes exhibited more extended cytoplasmic projections than on collagen type I-coated dishes. The present study provides evidence that biological properties of malignant schwannoma-derived cells might be affected by exogenous factors such as TGF-β1, LPS and laminin. These tumor lines may be useful for studies on pathobiological characteristics of Schwann cells.

INFLUENCE OF PROGESTERONE AND OESTROGEN ON GROWTH AND MORPHOLOGY OF A TRANSPLANTABLE RAT UTERINE SMOOTH MUSCLE TUMOUR (SMT-Y)

Tumours of uterine smooth muscle are poorly understood neoplasms in which the effects of steroid sex hormones are complex. The influence of progesterone and oestrogen on a transplantable rat uterine smooth muscle tumour line (SMT-Y) was investigated. Female F344 rats given subcutaneous transplants of tumour fragments developed tumours, 1.5-2 cm in diameter, and were then treated with progesterone (10 mg/rat) or 17 beta-oestradiol (50 mg/rat). Tumours in treated groups were compared with those in untreated controls. During a 9-week observation period after treatment, progesterone promoted tumour growth from 4 weeks, with increased numbers of proliferating cells. In contrast, oestradiol inhibited tumour growth from 6 weeks; the degraded tumours, consisting mainly of vacuolated neoplastic cells, had decreased numbers of proliferating cells and increased numbers of apoptotic cells, demonstrable by in-situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick labelling. Immunohistochemically, tumours in control and progesterone groups were labelled positively for alpha-smooth muscle actin (SMA) and desmin but not for vimentin, whereas the degraded tumours in the oestradiol group had reduced reactivity for SMA and desmin but an increased reactivity for vimentin. These results indicate that progesterone may act as a promoter for uterine smooth muscle tumour growth by stimulating mitotic activity, whereas oestrogen may have suppressive effects on tumour growth, accompanied by morphological changes.

Cutaneous Rhabdoid Tumor in a Cat

Rhabdoid tumor is a highly aggressive neoplasm of unknown cellular origin in humans, usually occurring in the kidney and central nervous system of infants or children. In older patients, it occurs rarely in other organs, including the skin and soft tissues. A subcutaneous mass in a 13-year-old male mixed-breed cat was composed of nests or sheets of round to polygonal cells with glassy eosinophilic cytoplasmic inclusions. Immunohistochemically, many neoplastic cells expressed vimentin, localized to the cytoplasmic inclusions, whereas the cytoplasm of some neoplastic cells was diffusely positive for neuron-specific enolase, neurofilament, or S-100 protein. By electron microscopy, the cytoplasmic inclusions were found to be composed of aggregates of intermediate filaments. These findings are quite similar to the histologic, immunohistochemical, and ultrastructural features of human rhabdoid tumors and the few rhabdoid tumors reported in animals.