Identification and analysis of CXCR4-positive synovial sarcoma-initiating cells.

Synovial sarcoma accounts for almost 10% of all soft tissue sarcomas, and its prognosis is poor with 5-year survival rates at 36%. Thus, new treatments and therapeutic targets for synovial sarcoma are required. Tumor-initiating cells have been defined by the ability for self-renewal and multipotent differentiation, and they exhibit higher tumorigenic capacity, chemoresistance and radiation resistance, expecting to be a new therapeutic target. In synovial sarcoma, the presence of such stemness remains largely unclear; thus, we analyzed whether synovial sarcoma possessed tumor-initiating cells and explored specific markers, and we discovered that synovial sarcoma cell lines possessed heterogeneity by way of containing a sphere-forming subpopulation highly expressing NANOG, OCT4 and SOX2. By expression microarray analysis, CXCR4 was identified to be highly expressed in the sphere subpopulation and correlated with stem-cell-associated markers. Inhibition of CXCR4 suppressed the cell proliferation of synovial sarcoma cell lines in vitro. The tumor-initiating ability of CXCR4-positive cells was demonstrated by xenograft propagation assay. CXCR4-positive cells showed higher tumorigenicity than negative ones and possessed both self-renewal and multipotent differentiation ability. Immunohistochemical analysis of 39 specimens of synovial sarcoma patients revealed that CXCR4 strongly correlated with poor prognosis of synovial sarcoma. Thus, we conclude that CXCR4 is the marker of synovial sarcoma-initiating cells, a new biomarker for prognosis and a new potential therapeutic target.

Regression of gastric de novo diffuse large B-cell lymphoma following Helicobacter pylori eradication: a case report.

We report a case of primary gastric diffuse large B-cell lymphoma (DLBCL), de novo DLBCL without the features of mucosa-associated lymphoid tissue (MALT) lymphoma, which regressed after Helicobacter pylori (HP) eradication. A 27-year-old Japanese female with epigastralgia was revealed to have ulcerated lesions in the angle and antral regions on gastroscopy. Biopsy specimen was consistent with a diagnosis of DLBCL without MALT lymphoma component, indicating de novo development. Her clinical staging on the Lugano system was Stage I. HP was positive on a rapid urease test, and she received HP eradication therapy twice, because the first therapy was not successful. On gastroscopy performed 1 month after the second HP eradication therapy, no ulcerated lesion was noted, and the lymphoma cells had regressed histopathologically. (Acta gastro-enterol. belg., 2016, 79, 367-369A).

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells.

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.

Isolation and characterization of a low metastatic variant from EL-4 mouse T-lymphoma.

A variant cell line (EL-4ad) which adhered to a tissue culture dish was isolated from highly metastatic EL-4 murine T-lymphoma. The experimental and spontaneous metastatic ability of EL-4ad was lower than that of the EL-4 parent cell line. The cell surface phenotypes of both cell lines were CD2+3+4-8-45+TCR alpha beta+TCR gamma delta-, but the level of CD2 expression of EL-4ad was much lower than that of EL-4. Furthermore, EL-4ad had higher binding ability to fibronectin and expressed more PNA receptors on the cell surface than EL-4. These differences indicated that either the maturation stage of the less metastatic variant was lower than that of the parent cell line or the activation state of the two cell lines differed. EL-4ad showed higher in vitro invasiveness and adhesiveness to liver cells, and these characters were not consistent with the reduced metastatic ability of this variant. Neuraminidase-releasable cell surface sialic acid levels did not differ significantly between the cell lines. Neither cell line was adhesive to laminin, type IV collagen or reconstituted basement membrane. These metastasis-related properties could not explain the decreased metastatic ability of EL-4ad. On the other hand, EL-4ad was more sensitive to NK activity than EL-4 in vivo, and this was thought to be a major cause of its decreased metastatic ability. The molecules or mechanisms involved in the differentiation or activation of T-cells may be responsible for the sensitivity of tumor cells to NK activity.

Inhibition of metastasis by a dialysable factor in fetal bovine serum in B16 melanoma cells.

Fetal bovine serum (FBS) supplemented to the culture medium inhibited the metastasis of B16BL6 melanoma cells in a dose-dependent manner. This metastasis-inhibiting activity was accompanied by growth-promoting activity, up to the concentration of 10% FBS. However, among the clones isolated from B16BL6 cells, there were clones in which FBS significantly inhibited the metastasis without promoting their growth. Dialysis (cutoff M.W. 8000 Da) removed the metastasis-inhibiting activity but not the growth-promoting activity from FBS. These results indicate that FBS has metastasis-inhibiting activity which is independent of growth-promoting activity, and that the metastasis-inhibiting activity is carried by molecules removed by dialysis.

Decrease of metastatic ability after selection for intravasating ability in Lewis lung carcinoma (3LL) cell line.

The release of cancer cells from the primary site and penetration into blood vessels are obligatory preliminary steps for metastasis. To investigate the mechanism of such steps we isolated variant cells (designated as Int-3LL) possessing enhanced intravasating ability from Lewis lung carcinoma (3LL) cells by in vivo selection. In spite of the enhanced intravasating ability of Int-3LL, the spontaneous and experimental metastatic abilities of Int-3LL decreased significantly compared to parent cells. Such a cell line has never been reported so far. The matched pair of cell lines described in this report provides a useful system for investigating the primary steps of metastasis.

Secretory function of adrenal chromaffin cells cultured on polypyrrole films.

Polypyrrole (PPy) is a conducting polymer and is obtained electrochemically on an electrode such as indium-tin oxide (ITO). In this study, in order to develop a novel cell-culture system which makes it possible to communicate with cultured mammalian cells, bovine adrenal chromaffin cells were cultured on PPy-coated ITO plates for 7 days and the influence of PPy-coating on the cell functions was investigated. Since the chromaffin cells synthesize and secrete catecholamines such as adrenaline and noradrenaline, the amount of synthesized and released catecholamines from the chromaffin cells cultured on PPy-coating and ITO itself were measured. The cells on the PPy-coated ITO plate could be kept in culture, without any significant changes in morphology and in the secretory responsiveness to acetylcholine as compared with those of the cells cultured on collagen. On the contrary, the cells on the ITO plate lost the responsiveness, while the amount of catecholamines synthesized was affected little by both PPy and ITO surfaces. It is suggested that PPy supports the secretory function of the chromaffin cells when they are cultured on it. This paper describes that PPy films are applicable as a polymer-modified electrode which support the cell function without collagen.

Involvement of peanut agglutinin-binding sugar chains in experimental metastasis of B16 melanoma cells.

Involvement of PNA (peanut agglutinin)-binding sugar chains in experimental metastasis of B16M4 cells and their metastatic variants (B16F1, B16F10, and B16BL6) was investigated by using ginsenoside Rh2 (Rh2), which increased PNA binding to the cell surface without increasing concanavalin A or wheat germ agglutinin binding. The PNA binding to cell surface increased in parallel with the experimental metastatic ability in Rh2-treated B16M4 cells, and this increase in metastatic ability was suppressed when the cells were treated with PNA before i.v. injection of cells. Concanavalin A and wheat germ agglutinin did not have such inhibitory effects. Increased PNA binding was observed on glycoproteins of M(r) 75,000-85,000, and was suggested to be due to reduced sialylation of PNA-binding sugar chains on these glycoproteins. In Rh2-treated B16F10 and B16BL6 cells, the increase in experimental metastatic ability was also associated with increased PNA binding to cell surface and M(r) 75,000-85,000 glycoproteins. Although the mechanisms of these effects of Rh2 are unknown, these results suggested that the reduced sialylation of PNA-binding sugar chains on M(r) 75,000-85,000 is related to the increased experimental metastatic ability of Rh2-treated B16 melanoma cells.

Isolation of a highly metastatic cell line to lymph node in human oral squamous cell carcinoma by orthotopic implantation in nude mice.

To isolate a variant cell line with increased lymph node metastasising potential from human oral squamous cell carcinoma (SCC) we performed in vivo selection using orthotopic implantation in nude mice. Human oral SCC cells (HSC-3) were injected orthotopically into the tongue of nude mice. After 3 weeks their cervical lymph nodes were excised and the tumour cells metastasised to the lymph nodes were isolated and injected into the tongue of nude mice again, this procedure was performed three times. The resultant cells, designated as HSC-3-M3, metastasised to cervical lymph nodes in 90% of mice, while parental HSC-3 cells metastasised in only 30% of mice after injection into the tongue. HSC-3 and HSC-3-M3 cell lines which have the same origin but different lymphatic metastatic capacities could be a useful model system for studying mechanisms involved in lymph node metastasis of human oral SCC.

Functional suppression of integrin beta 4-mediated adhesion caused by in vivo sequential selection for cancer cell intravasation.

Intravasation is essential for hematogenous metastasis in cancer cells, but its cellular determinants have not been well elucidated because of a lack of suitable experimental cell systems. Int-3LL was originally developed by in vivo sequential selection for intravasation from Lewis lung carcinoma (3LL) cells. Here, we found that these variant cells showed a highly penetrating ability in vitro as well as an augmented intravasating potential in vivo. In three-dimensional collagen-gel, Int-3LL cells formed diffusive colonies with less plating efficiency than their parental cells. Despite these properties, Int-3LL cells showed an ability of invasive migration in vitro similar to parental cells. On the other hand, a reduced adhesiveness and less spreading on extracellular matrices were revealed in Int-3LL cells. Analyses using anti-integrin antibodies indicated that the dysadhesion phenotype in Int-3LL cells was associated with integrin beta 4 dysfunction, which is known to produce epithelial detachment. Also, the types and the levels of integrins were not indistinguishable between Int-3LL and parental 3LL cells. Thus, the impaired function of integrin beta 4-mediated adhesion is considered to be an important factor in intravasation during metastasis.

CD44 participates in tumor cell adhesion to endothelial cells in the experimental metastatic process in B16BL6 melanoma cells.

There are several pieces of evidence suggesting a relationship between CD44 expression and tumor metastasis, but the role of CD44 in the metastatic processes is unclear. We analysed the role of CD44 in the experimental metastatic processes of B16BL6 melanoma cells using anti-CD44 monoclonal antibody (clone IM7). B16BL6 melanoma cells expressed CD44 and these cells treated with anti-CD44 monoclonal antibody increased in the experimental metastatic ability, indicating that CD44 participates in metastatic processes in B16BL6 cells. Furthermore, the adhesiveness of B16BL6 cells to endothelial cells and the retention of these cells in the lung increased by treatment with anti-CD44 monoclonal antibody. These results suggested that the CD44-mediated adhesion of tumor cells to endothelial cells was involved in the experimental metastatic process of B 16BL6 cells.

Citrus limonoids obacunone and limonin inhibit azoxymethane-induced colon carcinogenesis in rats.

Obacunone and limonin are bitter limonoids in citrus. Their modifying effects on the development of aberrant crypt foci (ACF), the activity of detoxification enzymes, glutathione S-transferase (GST) and quinone reductase (QR), and cell proliferation activity were investigated in male F344 rats treated with azoxymethane (AOM). Obacunone and limonin were administered in the diet, during the initiation (for 4 weeks) or postinitiation phase (for 4 weeks) of AOM-induced tumorigenesis. Feeding of obacunone and limonin (0.02% or 0.05%) caused significant reduction (55-65% by "initiation" feeding and 28-42% by "postinitiation" feeding) in the yield of ACF. The ability to reduce the proliferating cell nuclear antigen-labeling index in crypts and correlated well with the prevention of ACF. In a subsequent long-term experiment (38 weeks), in which rats were initiated with AOM and fed 0.05% obacunone or 0.05% limonin during the initiation or post-initiation phase, both compounds in diet caused significant reduction (65%-92% inhibition) in the incidence of colonic adenocarcinoma. Thus, citrus bitter limonoids obacunone and limonin possess chemopreventive effects on chemically induced rat colon carcinogenesis.

Hepatocellular carcinoma complicating biliary cirrhosis caused by biliary atresia: report of a case.

A case of hepatocellular carcinoma complicating biliary cirrhosis caused by biliary atresia is reported. The patient had persistent severe jaundice with hepatosplenomegaly. A liver tumor was suspected because of the elevated serum alpha-fetoprotein and was shown by ultrasonography at 6 years of age. The tumor was treated with percutaneous ethanol injection therapy (PEIT). Nine months after initiation of PEIT, the patient died of massive bleeding from a metastatic tumor.

[A pathologic review of thymic neoplasms].

Pathology, particularly histology and histogenesis, of thymic neoplasms is reviewed. In this report, thymic neoplasms include thymoma, lymphoma, carcinoid and germ cell tumors (teratoma group) originating from the thymus. Ultrastructures, enzymohistochemistry and immunohistochemistry have increased the knowledge on the relation between the histogenesis, morphology and function of each neoplasm. Recently, various kinds of monoclonal antibodies have easily been available, and the use of them in immunohistochemistry is useful for differentiating the subpopulation of lymphocytes in thymomas. Some interpretations referring to the microenvironment by thymoma cells and developing T lymphocytes are described. We stressed that peanut lectin was useful for identifying cortical thymocytes in paraffin sections, and functions of S-100 protein positive cells in thymomas should be investigated for elucidating one of the questions concerning the microenvironment by thymoma cells.

[A long-term survival after partial nephrectomy in a case of pelvic tumor arising in a solitary kidney].

A 44-year-old man was admitted on January 21, 1975 because of asymptomatic hematuria. The patient had nephrectomy of his left kidney due to nephritis at the age of three. Cystoscopy revealed no abnormalities, and excretory urography showed an irregular filling defect and slight ectasia in right upper calyx. A clinical diagnosis of pelvic tumor of the right kidney was made and partial nephrectomy was performed on April 18, 1975. The resected kidney was 4.5 X 5.0 X 6.5 cm in greatest dimension and the tumor was well localized in the upper calyx. Pathological diagnosis was transitional cell carcinoma, papillary, grade 11, stage 1. About 2 years after the operation, the patient developed a rice-sized tumor in the bladder neck followed by transurethral resection. Otherwise he is in good condition to date, 7 years and 4 months after the partial nephrectomy.

[Acute respiratory failure caused by inhalation of waterproofing spray fumes].

A 25-year-old woman was admitted to our hospital because of dry cough, slight fever, and severe dyspnea 5 hrs after inhalation of waterproofing spray. She had used the spray indoors near an oil heater, and then had smoked with spray-contaminated fingers. Chest roentgenograms revealed diffuse interstitial shadows mixed with patchy alveolar infiltration, and computed tomographic (CT) scans confirmed diffuse infiltration in both lungs. Marked leukocytosis and severe hypoxemia were noted. A transbronchial lung biopsy performed 4 days later demonstrated extensive alveolitis characterized by edema in alveolar septa and marked neutrophil migration into alveoli as well as alveolar septa. Oxygen therapy and the administration of methylprednisolone (1 g/day) achieved a complete recovery in about 1 week. We speculated that acute lung injury in this patient may have been induced by direct inhalation not only of the waterproofing spray itself, but also of spray by-products resulting from heat-decomposition. When using waterproofing spray, precautions should be taken to avoid inhaling the spray fumes or the more toxic by-products of thermal degradation.

[A comparative study of computed tomographic techniques for the detection of emphysema in middle-aged and older patient populations].

Helical-scan computed tomography (CT) is now widely utilized as a mass screening procedure for lung cancer. By adding 3 slices of high-resolution CT (HRCT) to the standard screening procedure, we were able to compare the efficacy of helical-scan CT and HRCT in detecting pulmonary emphysema. Additionally, the prevalence of emphysema detected by HRCT was examined as a function of patient age and smoking history. The subjects (106 men and 28 women) were all community-based middle-aged and older volunteers who participated in a mass lung cancer screening program. Based on visual assessments of the CT films, emphysema was detected in 29 subjects (22%) by HRCT, but in only 4 (3%) by helical-scan CT. Although the prevalence of emphysema was higher among subjects with a higher smoking index, no correlations with age were observed. We concluded that the efficacy of helical scan CT in detecting pulmonary emphysema can be significantly improved with the inclusion of 3 slices of HRCT, and confirmed that cigarette smoking is linked to the development of pulmonary emphysema.

[Experimental chemotherapy of human testicular carcinoma transplanted in nude mice].

The testicular carcinoma serially transplanted in nude mice with BALB/c genetic background was used for experimental chemotherapy. A stable growth and a high production of alpha-fetoprotein (AFP) were observed in this tumor line. The effect and side effect of Cis-platinum (CDDP) and other anticancer agents on this tumor line in nude mice were studied by the chemotherapy with single administration of CDDP 2 mg/kg, 4 mg/kg, 6 mg/kg and 8 mg/kg, and the combination chemotherapy with CDDP, Vinblastine (VBL) 0.1 mg/kg, Bleomycin (BLM) 0.5 mg/kg and Cyclophosphamide (CTX) 2 mg/kg. The body weight of the tumor bearing nude mice, the tumor size (length X breadth) and serum AFP level were measured every week up to 10 weeks after inoculation of the tumor mass. Six weeks after administration of these anticancer agents, the tumor mass was removed out and examined histologicaly. The effects of CDDP and other anticancer agents were observed as inhibition of the tumor growth and regression of the tumor mass. In the groups treated by the combination chemotherapy with either CDDP + VBL + BLM or CDDP + VBL + CTX, the most remarkable inhibition and regression were observed. The AFP levels were remarkably decreased in contrast with those of the control group. The changes of serum AFP levels were reflected in the tumor growth. The serum AFP levels fell down to normal level, however, the tumor mass was clearly recognized. The tumor tissue was damaged histologicaly by the single administration of CDDP. The most remarkable change was shown in the group treated by the combination chemotherapy CDDP 4mg/kg + VBL + BLM. The tumor cells were arranged one or two layers like the epithelium. This histological findings suggested that the malignant tumor could be differentiated to benign tumor. The side effect of CDDP and other anticancer agents was observed as a loss of weight. All of mice treated by the single administration at a dose of CDDP 6 mg/kg and 8 mg/kg died of the side effect of CDDP.

[Chronic pulmonary thromboembolism diagnosed on the basis of characteristic mosaic patterns on lung computed tomograms].

We report on a 59-year-old woman who presented with characteristic findings on lung computed tomographic (CT) scans and was therefore suspected to have chronic pulmonary thromboembolism. She visited our hospital because of worsening exertional dyspnea over the preceding year, and because she was dissatisfied with an earlier diagnosis made by another institution. Chest roentgenograms disclosed nonhomogeneous hyperlucency in both lungs associated with linear and bundle shadows, dullness of the right costophrenic angle, and dilatation of the descending branch of the right pulmonary artery. The patient experienced moderate hypoxemia even at rest. Pulmonary function tests demonstrated a restrictive ventilatory pattern associated with reduced diffusion capacity. The lung CT scans disclosed a mosaic pattern of attenuation in the lung parenchyma, which probably reflected scattered areas of low perfusion. The unique characteristics of such CT findings drew our attention to chronic pulmonary thromboembolism as a possible diagnosis. We eventually confirmed the diagnosis on the basis of enhanced CT scans, pulmonary perfusion and ventilation scintigrams, and digital subtraction angiography. In our view, chronic pulmonary thromboembolism should be kept in mind as a possible differential diagnosis of the mosaic patterns of attenuation on lung CT scans.