Rapid Growth and Spontaneous Metastasis of Human Germinal Tumors Ectopically Transplanted into Scid (Severe Combined Immunodeficiency) and Scid-nudestreaker Mice

Xenograft acceptance, growth and spontaneous metastasis of ectopically transplanted human germinal tumors were compared among scid mice, athymic nude mice and F₂ hybrids constructed from scid and nude mice, in relation to the impairments of T and B cell functions in these mice. In scid mice which are deficient in T and B cell functions, human yolk sac tumor (YST-2) that originated from the ovary grew to enormous sizes in 100% of the animals after both subcutaneous and intraperitoneal transplantation, while only half (59.1% and 51.9%) of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (BALB/c- nu/nu and CDl- nu/nu ). The YST-2 grew rapidly in scid mice, developing 3 to 10 times larger tumors compared to nude-streaker (AKR/J- nu^str/nu^str ) and usual nude mice, respectively. Furthermore, ectopically transplanted tumors spontaneously metastasized to distant organs (mostly to the lung) in scid mice (but less frequently in leaky scid mice), while metastases have never been found in nude mice. Although a xenograft of human classic (typical or pure) seminoma of the testis has never been established in nude mice, it grows slowly in one-third (36.4%) of scid mice and very rapidly in all of scid-nu ( scid/scid; nu^str/nu^str ) double mutant mice. Spontaneous metastases of xenografted seminomas were also observed in distant organs (lymph node, lung, liver, spleen, and kidney). The metastastic distribution of the two human germinal tumors in scid and scid- nu^str mice mimics that found in human. These results (xenograft acceptance, growth of transplanted tumors and degree of metastatic spread) were compatible with the level of T and B cell impairments indicated by FACS analysis, as well as mitogen responses, serum IgG and morphological features of the thymus.     

Inhibition of in vitro Tumor Cell Invasion by Ginsenoside Rg3

The effect of plant glycosides on tumor cell invasion was examined. Among the glycosides tested, ginsenoside Rgs was found to be a potent inhibitor of invasion by rat ascites hepatoma cells (MM1), B16FE7 melanoma cells, human small cell lung carcinoma (OC10), and human pancreatic adenocarcinoma (PSN-1) cells, when examined in a cell monolayer invasion model. Structurally analogous ginsenosides, Rb₂, 20(R)-ginsenoside Rg₂ and 20(S)-ginsenoside Rg₃ (a stereoisomer of Rg₃), showed little inhibitory activity. Neither Rh₁, Rh₂, 20(R)-ginsenosides Rh₁ Rb₁, Rc nor Re had any effect. The effective ginsenoside, Rg₃, tended to inhibit experimental pulmonary metastasis by highly metastatic mouse melanoma B16FE7 cells as well. Taking account of our previous finding that 1-oleoyl-lysophosphatidic acid (LPA) induced invasion by MM1 cells in the monolayer invasion model, the effect of Rg₃ on molecular events associated with the invasion induced by LPA was analyzed in order to understand the mechanism of the inhibition. Rg₃, which suppressed the invasion induced by LPA, dose-dependently inhibited the LPA-triggered rise of intracellular Ca²⁺. Protein tyrosine phosphorylation triggered by LPA was not inhibited by Rg₃.     

Expression of Tumor-associated Glycoantigen, Sialyl Lewisa, in Human Head and Neck Squamous Cell Carcinoma and Its Application to Tumor Immunotherapy

The glycoantigen sialyl Lewis^a (sLe^a) is widely expressed on a variety of gastrointestinal tumor cells. Here, we immunohistochemically demonstrated the expression of sLe^a antigen in 54% (7 out of 13) of human head and neck squamous cell carcinoma (H-NSCG) samples. Frequent expression of sLe^a antigen was also demonstrated on a variety of H-NSCC cell lines using flow cytometry. Both CD4⁺ and CD8⁺ T cells, which were activated with immobilized OKT3 monoclonal antibody plus inter-leukin-2, showed augmented cytotoxicity against sLe^a-positive H-NSCC, including autologous tumor cells, on targeting with anti-CD3 x anti-sLe^a bispccific antibody, suggesting that sLe^a antigen is a good target molecule for bispecific antibody-dependent adoptive tumor immunotherapy of human head and neck cancer.     

Significance of Expression of Human METCAM/MUC18 in Nasopharyngeal Carcinomas and Metastatic Lesions

Human METCAM/MUC18, a cell adhesion molecule (CAM) in the immunoglobulin-like gene super family,plays a dual role in the progression of several epithelium cancers; however, its role in the nasopharyngealcarcinoma (NPC) remains unclear. To initiate the study we determined human METCAM/MUC18 expression intissue samples of normal nasopharynx (NP), NPCs, and metastatic lesions, and in two established NPC cell lines.Immunoblotting analysis was used for the determination in lysates of frozen tissues, and immunohistochemistry(IHC) for expression in formalin-fixed, paraffin-embedded tissue sections of 7 normal nasopharynx specimens, 94NPC tissue specimens, and 3 metastatic lesions. Human METCAM/MUC18 was expressed in 100% of the normalNP, not expressed in 73% of NPC specimens (or expressed at very low levels in only about 27% of NPC specimens),and expressed again in all of the metastatic lesions. The level of human METCAM/MUC18 expression in NPCtissues was about one fifth of that in the normal NP and metastatic lesions. The low level of human METCAM/MUC18 expression in NPC specimens was confirmed by a weak signal of RT-PCR amplification of the mRNA.Low expression levels of human METCAM/MUC18 in NPC tissues were also reflected in the seven establishedNPC cell lines. These findings provided the first evidence that diminished expression of human METCAM/MUC18is an indicator for the emergence of NPC, but increased expression then occurs with metastatic progression,suggesting that huMETCAM/MUC18, perhaps similar to TGF-β, may be a tumor suppressor, but a metastasispromoter for NPC.     

Alkaline phosphatase isozymes in human testicular germ cell tumors, their precancerous stage, and three related cell lines

The four known isozymes of the human alkaline phosphatase (ALP) were detected by isoelectric focusing in extracts of various types of germ cell tumors, three related cell lines, and their precancerous elements (atypical germ cells). In seminoma, placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (PLAP-like) could be separated by isoelectric focusing following isolation by immunoaffinity. The occurrence of both isozymes in seminoma could explain partial heat sensitivity and variation in electrophoretic patterns of the seminoma isozyme frequently observed upon starch gels, in comparison to the normal placental phenotype. The four ALP isozymes are produced not only in germ cell tumors, but already in precancerous tissues. Quantitative analysis showed that the amount of the four isozymes varies in parallel in the tumors tested. Maximal expression was found in seminoma. The relation between ALP gene overexpression and gene amplification by polyploidy of chromosomes 1 and 2 in these lesions is discussed. On the other hand, the ectopic expression of intestinal alkaline phosphatase and PLAP associated with overexpression of PLAP-like in tumor cells as well as in their precancerous stage indicates gene activation by some unknown mechanisms, probably a regulatory process affecting the three tissue-specific ALP genes simultaneously.     

Human epithelial ovarian carcinoma cell-derived cytokines cooperatively induce activated CD4+CD25−CD45RA+ naïve T cells to express forkhead box protein 3 and exhibit suppressive ability in vitro

Regulatory T cells play an important role in tumor escape from host antitumor immunity. Increased frequencies of CD4⁺CD25⁺ regulatory T cells have been documented in the tumor sites, malignant effusions, and peripheral blood of patients with ovarian carcinoma. However, the mechanism involved remains unclear. In the present study, we collected high-purity human CD4⁺CD25⁻CD45RA⁺ naïve T cells by microbead cell separation. These cells did not express FOXP3 by single-cell analysis, and few cells expressed FOXP3 when they were activated with anti-CD3/CD28 dual signal. However, more cells expressed FOXP3 when the supernatant of human epithelial ovarian carcinoma cell culture was added, yet not the supernatant of normal human ovarian surface epithelia cell culture. Neutralization assays revealed that neutralizing antibody against transforming growth factor β (TGF-β), interleukin-10, and interleukin-4 did not abrogate elevated FOXP3 expression induced by carcinoma cell culture supernatant, whereas neutralizing leukemia inhibitory factor (LIF) partially abrogated FOXP3 expression, but LIF alone could not increase FOXP3 expression in activated naïve T cells. Further, an in vitro coculture suppression assay showed that these cells could suppress the proliferation of autologous CD4⁺CD25⁻CD45RA⁻ T cells. In summary, our findings show that ovarian carcinoma cells are able to induce expression of FOXP3 and exhibit suppressive ability in activated naïve T cells by producing soluble substances, and multiple cytokines involve in the induction of FOXP3 expression. ( Cancer Sci 2009)     

Regulatory (FOXP3+) T cells as target for immune therapy of cervical intraepithelial neoplasia and cervical cancer

Regulatory (FOXP3⁺) T cells (T_regs) comprise a subpopulation of CD4⁺ T cells that suppress autoreactive immune cells, thereby protecting organs and tissues from autoimmunity. T_regs have also been detected in human malignancies and their depletion or inactivation substantially improves cellular antitumor immunity in preclinical studies. Novel therapeutic strategies for cervical cancer and precancerous cervical intraepithelial neoplasia (CIN) focus on immune-modulatory and cancer vaccination approaches. In this context, the frequency of T_regs in cervical cancer and precancerous CIN could influence therapeutic strategies. We determined the frequency of infiltrating CD4⁺ and CD8⁺ T cells as well as FOXP3⁺ T_regs in high-grade CIN lesions (CIN III) and cervical carcinoma compared to colon carcinoma, skin melanoma, and bronchial carcinoma. We show that human papilloma virus-derived lesions have a significantly higher number of infiltrating lymphocytes and FOXP3⁺ T_regs compared to three other common tumor entities. In addition we explored the therapeutic effect of agonistic anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein antibodies that, by single systemic application, inactivate T_regs and induce strong intratumoral invasion of CD8⁺ T cells and complete tumor eradication in 70% of treated animals. The large number of T_regs in human papilloma virus-derived lesions suggests a pivotal role of T_regs for counteracting the host immune response. We therefore regard CIN and cervical cancer as prime targets for new immune-based non-invasive therapies. ( Cancer Sci 2009; 100: 1112–1117)     

Expression of Mucin-associated Sulfo-Lea Carbohydrate Epitopes on Human Colon Carcinoma Cells

The level of sulfo-Le^a (SO₃-3Galβ1-3(Fucα1-4)GlcNAc) epitope recognized by monoclonal antibody (mAb) 91.9H in hepatic metastasis of colon carcinoma is known to be lower than at the primary sites. We examined 19 human colon carcinoma cell lines for their production of this epitope. Sixteen cell lines were found to produce high M _r components that metabolically incorporated [³⁵S]sulfate and were resistant to heparitinase I and chondroitinase ABC, and 8 of them were reactive with mAb 91.9H as shown by western blotting analysis. These were all of the 4 cell lines derived from well differentiated primary tumors (HCCP-2998, LS174T, GEO, and CBS), 2 of 10 cell lines (DLD-1 and HCT116) from moderately to poorly differentiated primary tumors, and 2 of 5 cell lines (SW480 and HCC-M1544) from metastases. Incubation of LS174T cells with benzyl- N -acetyl-α- d -galactosaminide abrogated the incorporation of [³⁵S]sulfate and the reactivity of mAb 91.9H with high M _r components in the cell lysates. Sodium chlorate, which inhibits the formation of 3'-phosphoadenosine 5'-phosphosulfate, also inhibited the [³⁵S]sulfate incorporation and reactivity with mAb 91.9H. These treatments did not change the incorporation of [¹⁴C]threonine into high M _r components. These results indicated that sulfo-Le^a epitopes were expressed on O -linked carbohydrate chains in sulfomucins. Immunohistochemical studies of tumor tissues in nude mice indicated that sulfo-Le^a was expressed at the site of orthotopic transplantation in the cecum. The expression appeared to be suppressed in liver metastatic foci in nude mice.     

精原细胞瘤的细胞病理学特征（附10例报告及文献复习）

目的总结原发及转移性精原细胞瘤的细胞学特征,提高细胞病理医生对其认识。方法回顾性分析10例男性精原细胞瘤患者[4例原发纵隔,3例原发睾丸（隐睾）,1例原发腹膜后,2例转移瘤]的11个针吸标本,从涂片背景,肿瘤细胞排列和特征等方面总结,并与相应的组织病理学结果 （6例）进行对照。结果精原细胞瘤穿刺涂片显示主要由分散的大细胞组成,核圆形到轻度不规则,有一个位于中心的明显的核仁或是2~3个小核仁,胞浆少到中等量,有多个小空泡。背景中有数量不等的淋巴细胞,特征性的＂虎皮样＂物质以及固缩的核,只有少数病例有上皮样组织细胞。结论针吸细胞学可以为原发及转移部位的精原细胞瘤提供初步诊断,对后续检查和治疗非常有帮助。     

Serum neuron-specific enolase. A marker for responses to therapy in seminoma

Neuron-specific enolase (NSE) was evaluated as a serum marker in 105 patients with testicular cancer and compared with the established tumor markers alphafetoprotein (AFP) and human chorionic gonadotropin (HCG). Increased serum NSE activity was measured in eight of 11 (73%) patients with metastatic seminoma. Serum NSE concentrations fell to within the normal range following chemotherapy. Localization of NSE in seminoma cells was demonstrated immunohistochemically. Only six of 40 (15%) patients with metastatic nonseminomatous germ cell tumors showed elevated serum NSE levels. AFP and HCG were both positive in 70% of patients in this group, and NSE determination gave no additional information. Serum NSE concentrations were normal in 53 of 54 testicular cancer patients after orchiectomy and there was no evidence of metastatic disease; only one had borderline NSE levels, indicating the specificity of serum NSE determination. NSE is a new marker of seminoma and its measurement may be of clinical value in monitoring chemotherapy in patients with metastatic seminoma.     

Metastatic cancer cells with macrophage properties: evidence from a new murine tumor model

Metastasis is the process by which cancer cells disseminate from the primary neoplasm and invade surrounding tissue and distant organs, and is the primary cause of morbidity and mortality for cancer patients. Most conventional cancer therapies are ineffective in managing tumor metastasis. This has been due in large part to the absence of in vivo metastatic models that represent the full spectrum of metastatic disease. Here we identify 3 new spontaneously arising tumors in the inbred VM mouse strain, which has a relatively high incidence of CNS tumors. Two of the tumors (VM-M2 and VM-M3) reliably expressed all of the major biological processes of metastasis to include local invasion, intravasation, immune system survival, extravasation and secondary tumor formation involving liver, kidney, spleen, lung and brain. Metastasis was assessed through visual organ inspection, histology, immunohistochemistry and bioluminescence imaging. The metastatic VM tumor cells also expressed multiple properties of macrophages including morphological appearance, surface adhesion, phagocytosis, total lipid composition (glycosphingolipids and phospholipids) and gene expression (CD11b, Iba1, F4/80, CD68, CD45 and CXCR4). The third tumor (VM-NM1) grew rapidly and expressed properties of neural stem/progenitor cells, but was neither invasive nor metastatic. Our data indicate that spontaneous brain tumors can arise from different cell types in VM mice and that metastatic cancer can represent a disease of macrophage-like cells similar to those described in several human metastatic cancers. The new VM tumor model will be useful for defining the biological processes of cancer metastasis and for evaluating potential therapies for tumor management.     

Enhanced Liver Metastatic Potential of Alpha-fetoprotein-producing Human Gastric Carcinoma after Carbon Tetrachloride-induced Liver Damage in Nude Mice

The liver metastatic potential of alpha-fetoprotein (AFP)-producing human gastric carcinoma (NSC-3) was examined in male, BALB/c, nude mice. Metastatic nodules in the liver were produced by intrasplenic (IS) injection of tumor cell suspension prepared by trypsinization from subcutaneous NSC-3 tumor. The serum AFP level increased exponentially after IS injection along with the growth of metastatic nodules in the liver, and a positive correlation was observed between the estimated weight of metastatic nodules and serum AFP level. To investigate the effect of liver damage by carbon tetrachloride (CCI₄) on the metastatic potential of NSC-3 cells injected intrasplenlcally, the mice were divided into 4 groups: Group 1 received IS injection of 1×10⁶ of NSC-3 cells without CCI₄, treatment; Groups 2, 3 and 4 received IS injection 7 days, 2 days and 1 day after CCI₄ treatment, respectively. All mice were killed 64 days after IS injection. The incidence of liver metastasis was 80% in Group 1, but 100% in Groups 2, 3 and 4. The mean numbers of metastatic nodules per liver were 4.2 in Group 1, 16.8 in Group 2, 18.0 in Group 3 and 44.5 in Group 4. Significant differences in the mean numbers of metastatic nodules were observed between Group 4 and the other groups. It was clearly demonstrated that the metastatic potential of AFP-producing human gastric carcinoma cells (NSC-3) is enhanced in the situation prevailing after liver parenchymal cells are damaged by CCI₄.     

Insulin and Insulin-like Growth Factor 1 Stimulate Proliferation of Metastatic Variants of Colon Carcinoma 26

The proliferation rate of malignant cells in vivo is one of the important factors which affect the formation of tumor metastasis. A highly metastatic variant of mouse colon adenocarcinoma 26 (NL- 17) grew more rapidly than a low-metastatic variant (NL-44) both in vitro and in vivo. The effect of growth factors on the proliferation of NL-17 and NL-44 cells was examined in serum-free medium. Among growth factors examined, human insulin and insulin-like growth factor 1 (IGF-1), which were produced by gene engineering techniques, stimulated the growth of metastatic NL-17 and NL-44 cells as determined by thymidine incorporation and cell counts. DNA synthesis and cell proliferation of the high-metastatic NL-17 was stimulated to a greater extent by insulin and IGF-1 than those of the low-metastatic NL-44. These findings suggest that circulating growth factors could enhance the formation of tumor metastasis. Scatchard analysis of [¹²⁵I]IGF-1 binding to NL-17 and NL-44 showed that each cell line had an almost equal number of IGF-1 receptors (1.37 × 10⁵/cell and 1.26 × 10⁵/cell, respectively), which had similar dissociation constants (8.94×10⁻¹⁰ M and 9.54×10⁻¹⁰ M , respectively). Since the number and affinity of IGF-1 receptors are equivalent between low- and high-metastatic cells, the intracellular events which result in the cell growth after binding of IGF-1 may differ between NL-17 and NL-44 cells.     

Endosialin/CD248 may be a potential therapeutic target to prevent the invasion and metastasis in osteosarcoma

Endosialin/CD248/tumor endothelial marker 1 is classified as a C-type lectin-like transmembrane receptor, found on the plasma membrane of activated mesenchymal cells, which binds to fibronectin. Although endosialin is expressed at high levels in stem-like cells of sarcomas, its role has not been fully uncovered. The present study aimed to determine whether endosialin expression is associated with tumor progression and metastasis, and whether endosialin has the potential to act as a novel therapeutic target in osteosarcoma (OS) using MORAb-004/ontuxizumab, a humanized monoclonal antibody, which targets the type C lectin domain of endosialin. The results demonstrated that endosialin was highly expressed in OSs with metastatic disease. Furthermore, MORAb-004 had no cytostatic effect on OS cells in vitro and did not change the expression of stem cells and differentiation markers; however, it inhibited migration of OS cells. Taken together, these results suggest that endosialin may play a role in migration, and may be involved in the metastatic process of OSs. Furthermore, MORAb-004 reduces the motility of OS cells, and suppresses invasion and the development of metastatic lesions.     

2-18fluoro-deoxy-D-glucose positron emission tomography is a reliable predictor for viable tumor in postchemotherapy seminoma: an update of the prospective multicentric SEMPET trial.

PURPOSE: To define the clinical value of 2-18fluoro-deoxy-D-glucose positron emission tomography (FDG PET) as a predictor for viable residual tumor in postchemotherapy seminoma residuals in a prospective multicentric trial. PATIENTS AND METHODS: FDG PET studies in patients with metastatic pure seminoma who had radiographically defined postchemotherapy residual masses were correlated with either the histology of the resected lesion or the clinical outcome documented by computer tomography (CT), tumor markers, and/or physical examination during follow-up. The size of the residual lesions on CT, either >3 cm or < or =3 cm, was correlated with the presence or absence of viable residual tumor. RESULTS: Fifty-six FDG PET scans of 51 patients were assessable. All 19 cases with residual lesions >3 cm and 35 (95%) of 37 with residual lesions < or =3 cm were correctly predicted by FDG PET. The specificity, sensitivity, positive predictive value, and negative predictive value of FDG PET were 100% (95% CI, 92% to 100%), 80% (95% CI, 44% to 95%), 100%, and 96%, respectively, versus 74% (95% CI, 58% to 85%), 70% (95% CI, 34% to 90%), 37%, and 92%, respectively, for CT discrimination of the residual tumor by size (>3 cm/< or =3 cm). CONCLUSION: This investigation confirms that FDG PET is the best predictor of viable residual tumor in postchemotherapy seminoma residuals and should be used as a standard tool for clinical decision making in this patient group.     

Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas

Sixteen monoclonal antibodies that were obtained after immunization of BALB/c mice with intact melanoma cells or extracts of melanoma cells were tested for reactivity with normal and malignant melanocytic cells in situ, using an immunoperoxidase technique on frozen tissue sections. Sections representing six histopathologically defined stages of tumor progression, ranging from normal melanocytes to highly malignant metastatic lesions, were used. Thirteen monoclonal antibodies (MAbs) did not stain normal melanocytes in situ, whereas three MAbs weakly stained between 1 and 12.5% of melanocytes in 6-22% of the skin sections examined. MAb B 73.1, which was produced by immunization of mice with human natural killer cells and which binds to the Fc receptor of natural killer cells and granulocytes, reacted exclusively with malignant cells that represent the last two stages of tumor progression, vertical growth phase (VGP) primary melanoma and metastatic melanoma. All other antibodies showed variable reactivity with benign proliferative lesions or radial growth phase (RGP), an early stage of primary melanoma. Staining by MAbs that were reactive with gangliosides, unknown antigens, receptors, and two proteins (120/94 kDa protein and 250 kDa glycoprotein) showed a gradual increase in subsequent stages of tumor progression. Two steps in tumor progression were characterized by significant quantitative changes in the expression of antigens detected by the MAbs used in this study. First, mature nevus cells showed significantly higher reactivity with a panel of six MAbs, when compared to normal melanocytes. Second, a separate panel of six MAbs discriminated between RGP and VGP primary melanoma cells. No significant differences in antigen expression were found between dysplastic nevus cells and RGP melanoma, except that some antigens (nerve growth factor receptor and GD2/GD3 gangliosides) appear to be expressed at lower levels in RGP lesions, nor did VGP primary and metastatic melanomas show significant differences in antigen expression. These results suggest that (a) tumor progression of melanocytic cells in vivo is accompanied by significant quantitative differences in the expression of antigens, (b) some of the antigens examined here are associated with biologically aggressive malignant lesions but not normal or premalignant melanocytic cells, and (c) RGP primary melanoma cells are antigenically more similar to nevus cells than to VGP primary melanoma cells.     

Cytotoxicity of Histocompatibility Leukocyte Antigen–DR8–restricted CD4+ Killer T Cells against Human Autologous Squamous Cell Carcinoma

Although CD8⁺ killer T cells reacting against human autologous tumor cells have recently been studied in detail, little is known about the cytotoxic mechanism of CD4⁺ T cells against such tumor cells. In order to investigate this, we have established CD4⁺ cytotoxic T lymphocyte TcOSC–20 lines. TcOSC–20 showed selective cytotoxic activity against autologous OSC–20 cells, derived from a cancer of the tongue, in an HLA–DR–restricted fashion. HLA–DR8 (DRB1* 08032) is the only DR molecule expressed on OSC–20 cells, and anti–DRS monoclonal antibody could inhibit the Cytotoxicity, suggesting that HLA–DRB1^★ 08032 is the tumor rejection antigen–presenting moleculeto TcOSC–20. The Fas ligand was expressed on TcOSC–20 lines, and its expression was induced upon mixed lymphocyte–tumor cell culture of autologous peripheral blood lymphocytes. Furthermore, the Cytotoxicity of TcOSC–20 was inhibited by anti–Fas ligand antibody.These data imply that TcOSC–20 lines recognize the tumor antigenic peptide presented by HLA–DR8, and exert Cytotoxicity against autologous tumor cells via a Fas–mediated cytotoxic pathway.