Fundamental studies of targeting cancer chemotherapy against choriocarcinoma--application of liposome

For the fundamental study of the targeting cancer chemotherapy against choriocarcinoma using the anti-human chorionic gonadotropin beta subunit (hCG beta) antibody-conjugated-liposome containing anticancer drugs, we evaluated the specific accumulation of the antibody to the choriocarcinoma tissue and the uptake of liposome by choriocarcinoma cells. In vivo, the mice bearing human choriocarcinoma were injected anti-hCG beta antibody (30 micrograms/body) and the localization of the antibody in the tumor and other mouse organs were investigated by the PAP method. The antibody specifically accumulated to the choriocarcinoma tissue within 4 hours after the injection of the antibody, and gradually increased. In vitro, cultured BeWo cells were exposed to the liposome containing adriamycin, the antibody conjugated liposome containing adriamycin or the adriamycin alone, respectively, for 2 hours. Then the uptake of the adriamycin by the tumor cells was studied under fluorescent microscopy. The nuclear accumulation of adriamycin was clearly observed in the tumor cells exposed to the liposome containing adriamycin or the antibody conjugated liposome containing adriamycin, whereas only faint cytoplasmic accumulation of the adriamycin without any nuclear accumulation was observed in the cells exposed to the adriamycin alone. These results indicate that the liposome is a good carrier of anticancer drugs for immunotargeting chemotherapy.     

Biology of testicular tumors.

Testicular tumors arise from the germ cell line and therefore exhibit characteristics of both neoplastic and normal growth and differentiation. Experimental model systems of animal and human tumors have been reviewed with emphasis on the biologic characteristics of these tumors. The embryonal carcinoma cell is the totipotential stem cell that resembles normal germ cells in many ways and is capable of differentiating along the somatic pathways to form endoderm, mesoderm and ectoderm cell types (teratoma) or along extraembryonic pathways to form trophoblast (choriocarcinoma) or yolk sac (endodermal sinus tumor). Markers of the extraembryonic cell types have been defined, and the cell surface characteristics of embryonal carcinoma cells are being intensively studied. Clarification of the biology of testicular tumors will provide the basis for future rational therapy.     

Testicular seminoma occurring 8 years after treatment of a metastatic extragonadal germ cell tumor

A 30-year-old man was admitted with a chief complaint of left-sided scrotal enlargement, and was diagnosed as having testicular seminoma after orchiectomy. Eight years earlier, he had been treated with chemotherapy for an extragonadal germ cell tumor, without orchiectomy, leading to complete remission. His histological diagnosis at that time was a germ cell tumor, composed of choriocarcinoma and embryonal carcinoma. He was followed up without testicular biopsy. Routine pretreatment testicular biopsy in patients with extragonadal germ cell tumor is controversial, but regular long-term follow up and information on the risk of developing a metachronous testicular tumor are needed after treatment of extragonadal germ cell tumors, even when there seems to be a partial or complete clinical response.     

Radioimmunotherapy of xenografted human bladder cancer using monoclonal antibody against blood group A-related antigen

An IgG3 murine monoclonal antibody 1G3.10 was generated against a human urinary bladder carcinoma cell line TSGH-8301. The tumor-associated antigen recognized by the monoclonal antibody is a blood group A-related substance. From fluorescence-activated cell sorter analysis, a direct cytolytic effect of tumor cells by the antibody at concentrations of more than 10 micrograms/ml was seen. The antibody could target iodine-131 to established subcutaneous human bladder carcinoma xenografts in BALB/c nude mice. Monoclonal antibody alone also showed some inhibitory effect on xenografted tumor growth. In this study, radiolabeled monoclonal antibody 1G3.10 presented more effective cytotoxicity than antibody alone from in vitro and in vivo experiments.     

Treatment of nonseminomatous testicular tumor with liver metastases

Therapeutic course of 2 cases of nonseminomatous testicular tumor with liver metastasis is reported. One case had mixed tumors including embryonal carcinoma, choriocarcinoma and yolk sac carcinoma, and was positive pulmonary metastasis already at the initial examination. In the other case having mixed tumors of embryonal carcinoma and choriocarcinoma, metastasis to the supraclavicular lymph node was detected at the initial examination. In both cases liver metastasis occurred after complete response could be obtained by treatment chiefly consisting of PVB therapy. For liver metastasis four-drug combination treatment using cisplatin, vinblastine, adriamycin and actinomycin D was performed with partial response. However, this patient eventually died. The other case received VAB-6 therapy with complete response for liver metastasis. It is advisable to consider other modalities therapy in addition to conventional chemotherapy in cases of testicular tumor with liver metastasis since the prognosis is poor in these cases.     

Testicular germ cell tumor with pineal metastases

A patient with a mixed testicular germ cell tumor (choriocarcinoma, teratocarcinoma and embryonal carcinoma) that had metastasized to the lungs, cerebrum, and pineal gland is presented. The metastases had resulted in localized neurological signs and initially, on clinical grounds, a primary intracranial lesion could not be excluded. The occurrence of tumor metastases to the pineal gland is discussed and the literature is reviewed.     

Characterization of a human chorionic gonadotropin-producing testicular choriocarcinoma cell line

A testicular choriocarcinoma cell line designated JHTK-1 was established in vitro from a mixed germ cell tumor xenografted into nude mice, and may prove to be the first human choriocarcinoma cell line of testicular origin in long-term culture. The cells have been kept in culture for three years and produce human chorionic gonadotropin persistently. The chromosome number distribution ranged from tetraploidy to septaploidy, and trypsin G-band karyotyping showed that all the chromosomes were of human origin. Dibutyryl cyclic adenosine monophosphate at a concentration of 1 mM caused more than 15-fold stimulation of human chorionic gonadotropin secretion with morphological alteration in JHTK-1 cells. This suggests that dibutyryl cyclic adenosine monophosphate induces the differentiation of cytotrophoblastic cells into syncytiotrophoblastic cells among JHTK-1 cells. When JHTK-1 cells were heterotransplanted into the subcutis of BALB/c nude mice, large cystic tumors were produced with histological characteristics similar to choriocarcinoma.     

Significance of DNA quantification in testicular germ cell tumors

A cytophotometric quantification of DNA in tumor cells was performed in histological sections of orchidectomy specimens from 36 men with testicular germ cell tumors (TGCT), 7 of them showing more than one tumor type. Among the variants of seminoma (classic and spermatocytic) the lowest DNA content were in spermatocytic seminoma. With respect to non-seminomatous tumors (yolk sac tumor, embryonal carcinoma, teratoma, and choriocarcinoma), choriocarcinomas showed the highest DNA content, and the lowest value was found in teratomas. No significant differences were found between the average DNA content of seminomas (all types) and non-seminomatous tumors (all types). Both embryonal carcinoma and yolk sac tumor showed similar DNA content when they were the sole tumor and when they were found associated with other tumors. In this study, except for the 4 cases of teratoma and the case of spermatocytic seminoma, all TGCT examined did not show modal values of DNA content in the diploid range. Such an elevated frequency of aneuploidism in these tumors may be helpful for their diagnosis.     

The synchronous appearance of multiple testicular epidermoid cysts and of a malignant mixed germinal tumor in the contralateral testis]

We describe the simultaneous appearance of multiple epidermoid cysts in the right and an adult teratoma containing embryonal carcinoma and choriocarcinoma in the left testis. No similar case has previously been described. Epidermoid cysts of the testis are rare, accounting for about 1% of all testicular tumors. Epidermoid cysts are now regarded as monoepidermally developed teratomas of germ cell origin. Testicular teratomas in adults, however, are always malignant. Because epidermoid cysts are rare tumors, primary therapy often consists in ablation of the testis. In the case described, excision of the epidermoid cyst protected the patient from complete castration. This case demonstrates the simultaneous appearance of a malignant and a benign testicular germ cell tumor.     

Expression of embryonic stem cell markers in cultured JKT-1, a cell line derived from a human seminoma

Testicular germ cell tumours (TGCTs), the most frequent solid tumour of the young men, originate from the primitive germ cells. They share some pluripotency stem-cell markers which may help to distinguish between seminoma, the most frequent TGCTs and non-seminoma tumours, such as embryonal carcinoma, teratocarcinoma or choriocarcinoma. Due probably to the propensity of seminoma to apoptosis, only two cell lines originated from pure testicular seminoma, TCam-2 and JKT-1 have been up to now, established, maintained and proposed as representative models of human testicular seminoma. However, both seem, following recent reports, to be able to drift. Thus, the molecular signature of embryonic stem-cell markers of the JKT-1 cells cultured in our laboratory, were studied by RT-PCR, Western blot and immunofluorescence (IF). JKT-1 cells analysed after 30 passages, expressed placenta alkaline phosphatase but not alphafoetoprotein (αFP) nor beta-human chorionic gonadotropin. JKT-1 cells also expressed markers of pluripotency such as NANOG and OCT3/4 and more specific seminoma markers, such as AP2γ and HIWI. However, protein expression of OCT3/4 and AP2y was weak and these JKT-1 cells expressed SOX2, a marker of embryonal carcinoma and did not express c-KIT usually expressed in most seminoma. Possible derivation through in vitro culture conditions was supported by looking at later passages (61) which showed a decrease of NANOG and HIWI protein expression. JKT-1 cells express a signature of markers which is still near from the one express by seminoma cells, allowing carcinogenetic studies. However, because of their great ability to drift as shown for TCam-2, it is recommended to verify and to precise this molecular signature before reporting functional results.     

Prognostic factors in nonseminomatous testicular cancer

The clinical, pathological and laboratory data for 60 patients with pathological stage I, 50 with stage II and 55 with stage III nonseminomatous testicular cancer were reviewed to identify prognostic factors that may be used to guide therapeutic approaches. In patients with stage I disease the presence of embryonal carcinoma (p less than 0.001) and vascular invasion in primary tumors (p less than 0.01) are significant predictors of metastases and/or recurrence after retroperitoneal lymphadenectomy. In patients with clinical stage II disease, lymph nodes greater than 2 cm. in diameter or more than 5 in number, vascular and/or lymphatic invasion and the presence of embryonal carcinoma with or without choriocarcinoma indicated an increased risk for recurrent tumor after retroperitoneal lymphadenectomy. In patients with stage III disease the involvement of 3 or more organ systems was associated with decreased survival. Patients in this group with intrathoracic metastases smaller than 2 cm., retroperitoneal metastases less than 4 cm., normal kidneys and liver, and no evidence of yolk sac tumor or choriocarcinoma had improved survival. These observations can be used to guide the type and duration of therapy selectively in patients with nonseminomatous testicular cancer.     

Glypican 3: a novel marker in testicular germ cell tumors

Glypican 3 (GPC3), a membrane-bound heparin sulfate proteoglycan, may play a role in promoting embryonic cell growth and differentiation. GPC3 is mutated in Simpson-Golabi-Behmel syndrome, characterized by tissue overgrowth and an increased risk of embryonal malignancies. Recently, GPC3 was reported to be one of the over-expressed genes in testicular yolk sac tumors by gene expression microarray analysis. However, the presence of the GPC3 protein in germ cell tumors has never been investigated. The purpose of the study was to investigate the GPC3 expression in various histologic components of testicular germ cell tumors using immunohistochemistry and to assess its possible utility as a diagnostic marker. Tumors from 71 patients were examined, including components of 42 seminomas, 37 embryonal carcinomas, 24 yolk sac tumors, 20 teratomas with mature elements, 16 teratomas with immature elements, and 7 choriocarcinomas. Cytoplasmic and membranous immunoreactivity was semiquantitatively evaluated. All yolk sac tumor (24/24) and choriocarcinoma (7/7) components were immunoreactive for GPC3, whereas only 38% of teratomas with immature elements and 8% of embryonal carcinomas expressed GPC3. There was no immunoreactivity in benign testicular tissue, intratubular germ cell neoplasia, seminomas (0/42), or teratomas with mature elements (0/20). We conclude that the oncofetal protein GPC3 is a novel immunohistochemical marker in testicular germ cell tumors with differential expression in defined histologic subtypes. Our findings suggest a possible role of GPC3 in tumor cell differentiation. Furthermore, GPC immunostaining may be useful in the pathologic diagnosis of nonseminomatous germ cell tumors, particularly yolk sac tumor, and choriocarcinoma.     

Localization of alpha-fetoprotein and human chorionic gonadotropin to specific histologic types of nonseminomatous testicular cancer

We used an indirect immunoperoxidase technique to localize alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) to specific histologic types of testicular germ cell cancers. Among 20 nonseminomatous tumors studied, yolk sac tumor reacted for AFP in 13 of 15 cases, teratoma in 3 of 11 cases, and embryonal carcinoma in 3 of 14 cases. Syncytiotrophoblasts alone reacted for HCG in 14 of 15 cases, and syncytiotrophoblasts associated with choriocarcinoma reacted for HCG in 2 of 2 cases. There was a close correlation between the tissue demonstration of AFP and HCG and elevated serum levels of AFP and HCG, respectively. We conclude that in nonseminomatous testicular cancer yolk sac tumor is the primary site of synthesis of AFP, and syncytiotrophoblasts are the only site of synthesis of HCG.     

Sonographically-Detected Impalpable Testicular Cancer with Retroperitoneal Bulky Metastases: A Case Report

We report a case of a sonographically-detected impalpable embryonal cell carcinoma with bulky retroperitoneal metastases. A 34-year-old man, who presented with left flank pain, was presumed to have an extragonadal retroperitoneal germ cell tumor. Scrotal sonography revealed a hypoechoic lesion, 7 mm in diameter, which was histologically diagnosed as a primary embryonal cell carcinoma. Evidence suggested that the primary tumor had grown slowly, as the tumor was well encapsulated. This case suggests that some extragonadal germ cell tumors arise from a primary testicular cancers, and that successful treatment of these tumors should include consideration that they may have arisen as a primary testicular mass.     

乳腺癌小鼠动物模型中白细胞介素-17的表达及其意义

目的 探讨小鼠乳腺癌动物模型中白细胞介素-17(IL-17)的表达及意义.方法 以MA782/5S28102及4T1细胞株建立两种乳腺癌动物模型.分别于接种后1周和4周采用Western blot检测肿瘤组织中IL-17的表达.使用PMA+CD3单抗+CD28单抗刺激后,采用酶联免疫吸附试验(ELISA)检测肿瘤细胞和淋巴细胞培养体系上清中IL-17的含量.同时观察IL-17对培养体系及4T1荷瘤小鼠中肿瘤细胞生长的影响.结果 乳腺癌模型中晚期肿瘤组织中IL-17的表达水平均明显较早期有所升高.分离的肿瘤细胞接受刺激后,几乎不分泌IL-17,淋巴细胞分泌大量IL-17(P＜0.01).IL-17不能促进4T1细胞的生长(增长率分别为1.11±0.11和1.28 ±0.21,P＞0.05);将IL-17输注至4T1荷瘤小鼠的体内,可见肿瘤生长速度明显加快(P＜0.05).输注IL-17的荷瘤小鼠肿瘤组织中微血管密度(MVD)明显增加(MVD分别为:35.79±9.49,13.52±3.55,P＜0.01).结论 IL-17在晚期肿瘤组织中明显升高,IL-17可能通过促进肿瘤组织内微血管形成加快肿瘤的生长.

Abstract：

Objective To explore the impact of interleukin (IL) -17 expression on tumor growth in experimental models of murine mammary carcinoma and potential mechanisms. Methods Two murine cell lines, MA782/5S28102 and 4T1 were used to establish experimental models of murine mammary carcinoma. The IL-17 expression in tumor tissues derived from MA782-bearing mice or 4T1-bearing mice was detected in early and late stages of the tumor by Western blotting. The tumor cells and tumor-infiltrated-lymphocytes were separated from tumor tissues and cultured for 5 days with stimulation of PMA, anti-CD3 antibody and anti-CD28 antibody. The supernatants of culture media of stimulated tumor cells or tumor-infiltrated-lymphocytes were harvested and tested for IL-17 concentration by enzyme linked immunosorbent assay (ELISA). To evaluate the effect of IL-17 on the proliferation of tumor cells, 4T1 cells were culture in media with or without IL-17 and the cell number was counted on the day 5. For ire vivo assay, 4T1-bearing mice were injected with IL-17 or culture media via tail vein, and the tumor volume was measured. To assay the angiogenesis, the tumor tissues from 4T1-bearing mice with or without injection of IL-17 were stained with anti-CD31 antibody by immunohistochemistry. Results The IL-17 expression was significantly higher in late stage than in early stage of tumor in two experimental models. The tumor expression of IL-17 was secreted by tumor infiltrated lymphocytes (P ＜0.01). IL-17 could not increase the generation of tumor cells in vitro (1. 11 ±0. 11, 1. 28 ±0. 21 ,P ＞0. 05). But IL-17, injected into 4T1 -bearing mice, markedly enhanced in vivo tumor growth and significantly increased tumor vascularity (35. 79 ±9. 49, 13. 52 ±3. 55,P ＜0.01). Conclusion IL-17 in tumor tissue probably promotes tumor growth through enhancing angiogenesis.     

Retroperitoneal lymph node dissection for testicular tumor with renal autotransplantation: a case report

A case of testicular tumor (embryonal cell carcinoma with choriocarcinoma) in a 42-year-old man treated with renal autotransplantation is presented. After four courses of chemotherapy (carboplatin, vinblastine and bleomycin), we performed retroperitoneal lymph node dissection. At the operation, one of the swelling lymph nodes involved left renal artery completely, so we performed left renal autotransplantation and promoted more effective lymph node dissection. After the operation, renal function was impaired but returned completely in a short time. Autotransplantation provides excellent bench visualization of renal lesions and stable graft function. Therefore, this procedure should be taken into consideration when nephrectomy cannot be avoided.     

An assessment of prolonged reactivity of seven monoclonal antibodies against CX-1 tumor xenografts using a hand-held gamma-detecting probe

The biodistribution and kinetics of 7 monoclonal antibodies (MAb) with known reactivity against CX-1 tumor were examined over 21 days using a hand-held gamma-detecting probe (Neoprobe system). Twenty-eight immuno-deprived (athymic) nude mice implanted with human colon adenocarcinoma CX-1 xenografts were injected intraperitoneally with 50 microCi of 125I-labeled antibodies (4 mice/antibody). Of the 7 monoclonal antibodies, 4 were anti-CEA (MA, MB, MC, and MD), 2 were anti-TAG 72 (B72.3 NCI and B72.3 fermented) and one was anti-colorectal cancer (17-1A). Daily probe counts were recorded in duplicate over the tumor site and the contralateral nontumor site (background), and tumor-to-background (Tu/Bkg) ratios were calculated. Animals were sacrificed on day 21, and blood, heart, liver, spleen, lungs, kidneys, intestine, muscle, and the tumor were removed for gamma well counting. All antibodies identified the tumor as early as 24 h postinjection and specific tumor localization improved over time. Patterns of prolonged tumor binding varied considerably from one antibody to another, although all but one (MB) showed continuously increasing Tu/Bkg ratios. These data indicate progressive clearance of the antibodies from the background tissue and a persistence of labeled MAb activity in tumor resulting in improved tumor localization with increasing postinjection time.     

Anti-PD-L1/TGFβR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines,rendering them more susceptible to immune-mediated recognition and lysis

Background

Avelumab has recently been approved by the Food and Drug Administration for the therapy of Merkel cell carcinoma and urothelial carcinoma. M7824 is a novel first-in-class bifunctional fusion protein comprising a monoclonal antibody against programmed death-ligand 1 (PD-L1, avelumab), fused to the extracellular domain of human transforming growth factor beta (TGFβ) receptor 2, which functions as a TGFβ “trap.” Advanced urothelial tumors have been shown to express TGFβ, which possesses immunosuppressive properties that promote cancer progression and metastasis. The rationale for a combined molecule is to block the PD-1/PD-L1 interaction between tumor cells and immune cell infiltrate and simultaneously reduce or eliminate TGFβ from the tumor microenvironment. In this study, we explored the effect of M7824 on invasive urothelial carcinoma cell lines.