Disaggregation and invasion of ovarian carcinoma ascites spheroids

Background

In vivo studies have recently demonstrated that interleukin 21 (IL-21) enhances the anti-tumor function of T-cells and NK cells in murine tumor models, and the combined use of IL-21 and IL-15 has resulted in prolonged tumor regression and survival in mice with previously established tumors. However, the combined anti-tumor effects of IL-21 and low dose IL-2 have not been studied even though IL-2 has been approved for human use, and, at low dose administration, stimulates the proliferation of memory T cells, and does not significantly increase antigen-induced apoptosis or regulatory T cell (Treg) expansion. This study examined whether recombinant IL-21 alone or in combination with low-dose IL-2 could improve the in vivo anti-tumor function of naïve, tumor-antigen specific CD8⁺ T cells in a gp100_25–33 T cell receptor transgenic pmel murine melanoma model.

Methods

Congenic C57BL/6 (Ly5.2) mice bearing subcutaneous B16F10 melanoma tumors were sublethally irradiated to induce lymphopenia. After irradiation naive pmel splenocytes were adoptively transferred, and mice were immunized with bone marrow-derived dendritic cells pulsed with human gp100_25–33 (hgp100_25–33). Seven days after vaccination groups of mice received 5 consecutive days of intraperitoneal administration of IL-2 alone (20 × 10³ IU), IL-21 alone (20 μg) or IL-21 and IL-2. Control animals received no cytokine therapy.

Results

IL-21 alone and IL-2 alone both delayed tumor progression, but only IL-21 significantly augmented long-term survival (20%) compared to the control group. However, combination therapy with IL-21 and IL-2 resulted in the highest long-term (>150 days) tumor-free survival frequency of 46%. Animals that were tumor-free for > 150 days demonstrated tumor-specific protection after rechallenge with B16F10 melanoma cells. At peak expansion (21 days post vaccination), the combination of IL-21 plus IL-2 resulted in a 2- to 3-fold higher absolute number of circulating tumor antigen-specific pmel CD8⁺ T cells than was stimulated by IL-2 or IL-21 alone. Pmel CD8⁺ T cells were predominantly partitioned into central memory (CD62L⁺/CD127⁺) or effector-memory (CD62L^-/CD127⁺) phenotypes by day 28-post vaccination in IL-21 + IL-2 treated mice.

Conclusion

These observations support the potential use of IL-21 and low-dose IL-2 therapy in combination with a tumor-antigen vaccine and lymphopenic conditioning in future cancer clinical trials to maintain high numbers of anti-tumor memory CD8⁺ T cells with the potential to sustain long term tumor regression and survival.     

过表达细胞黏附分子1抑制卵巢癌细胞迁移及侵袭

目的:研究过表达细胞黏附分子1(cell adhesion molecule 1,CADM1)对卵巢癌增殖、迁移和侵袭的影响.方法:qRT-PCR测定CADM1mRNA在卵巢癌细胞系SKOV3及人正常卵巢上皮细胞hose中的表达,将SKOV3细胞分成两组,即CADM1过表达组和对照组,转染48 h后Western印迹测定两组CADM1蛋白表达量,采用lipofectamine 2000分别转染pcDNA3.1-CADM1及pcDNA3.1质粒,采用CCK-8、克隆形成、细胞划痕及Transwell实验分别检测两组细胞增殖、克隆形成、细胞迁移及侵袭能力.结果:CADM1mRNA在SKOV3中表达水平显著低于hose细胞系(1.54±0.34 vs.5.63±0.96,p＜0.05);转染48 h后,CADM1过表达组和对照组CADM1蛋白表达量分别为2.53±0.42,0.37±0.09,差异具有统计学意义(P＜0.05).CADM1过表达组和对照组在0,24,48,72 h 450 nm处的OD值差异无统计学意义(P＞0.05);CADM1过表达组与对照组克隆形成数相比(60.4±7.6 vs.58.3±8.2),差异无统计学意义(P＞0.05);CADM1过表达组细胞迁移率显著低于对照组(20.3％±3.5％vs.60.1％±4.2％,P＜0.05);CADM1过表达组侵袭细胞数显著少于对照组(24.5±5.3 vs.65.1±6.9,P＜0.05).结论:CADM1在卵巢癌细胞系中低表达,过表达CADM1对卵巢癌细胞增殖和克隆形成无影响,但可抑制迁移和侵袭,起抑癌基因的作用.     

Dickkopf-1 is frequently overexpressed in ovarian serous carcinoma and involved in tumor invasion

Dickkopf-1 (DKK1) was known as a negative regulator of the Wnt signaling pathway, which played a crucial role in carcinogenesis. However, its function in human cancers remained elusive. In this study, we aimed to investigate the role of DKK1 in ovarian serous papillary adenocarcinoma (OSC) tumor progression and invasion. Quantitative real-time RT–PCR and Western blot analysis showed that the expression of DKK1 mRNA and protein in 32 OSC tissues were elevated as compared with those in 10 normal ovarian tissues (P = 0.005, P = 0.003, respectively). Immunohistochemical analysis in 178 clinical OSC samples showed that the expression of DKK1 was positively associated with FIGO stage (P = 0.016). Furthermore, the expression of DKK1 protein was positively associated with P-JNK1 protein expression in 178 OSC tissues. DKK1, P-JNK1 and the co-expression of DKK1 and P-JNK1 were all unfavorable prognosis factors for OSC patients (P < 0.0001). DKK1, alone or combined with P-JNK1, was an independent predictor for the 5 year survival (P = 0.015, P < 0.0001, respectively). DKK1 could promote OSC cells invasion and the growth of OSC nude mice xenograft. DKK1 in OSC cells could activate P-JNK1 expression and significantly promote formations of actin filaments and filopodia. Thus, DKK1, alone or combined with P-JNK1, was a novel prognostic predictor for OSC patients and contributed to the invasion of OSC.     

Cell membrane glycosylation mediates the adhesion,migration, and invasion of ovarian carcinoma cells

We have previously shown that ovarian carcinoma cell adhesion to mesothelial cell monolayers and migration toward fibronectin, type IV collagen, and laminin is partially mediated by CD44, a proteoglycan known to affect the functional abilities of tumor cells. The purpose of this study was to determine the role of cell membrane glycosylation in the metastatic abilities of ovarian carcinoma cells. NIH:OVCAR5 cells were treated with glycosidases to remove carbohydrate moieties from molecules on the cells' surface. The ability of the treated cells to adhere to extracellular matrix components or mesothelial cell monolayers, migrate toward extracellular matrix proteins, and invade through Matrigel was assessed. We observed that the loss of different carbohydrate moieties resulted in altered ovarian carcinoma cell adhesion, migration, and/or invasion toward extracellular matrix components or mesothelial cell monolayers. Gene array analysis of NIH:OVCAR5 cells revealed the expression of several proteoglycans, including syndecan 4, decorin, and perlecan. In tissue samples obtained from patients, altered proteoglycan gene expression was observed in primary ovarian carcinoma tumors and secondary metastases, compared to normal ovaries. Taken together, these results suggest that ovarian carcinoma cell proteoglycans affect the cells' ability to adhere, migrate, and invade toward extracellular matrix components and mesothelial cell monolayers. Thus, the carbohydrate modifications of several proteoglycans may mediate the formation and spread of secondary tumor growth in ovarian carcinoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

SIRT2在浆液性卵巢癌细胞系中的表达及其对增殖、迁移和侵袭的影响

目的:研究SIRT2在卵巢表层上皮(ovarian surface epithelium,OSE)及浆液性卵巢癌(serous ovarian carcinoma,SOC)细胞系中的表达情况并从细胞增殖、迁移和侵袭这3个方面探讨SIRT2对SOC恶性生物学行为的影响。方法:运用Western blot技术检测OSE和SOC细胞系中SIRT2蛋白的表达水平;设计靶向SIRT2的siRNA,构建SIRT2过表达载体,分别瞬时转染OSE细胞系HOSEpi C和SOC细胞系HO8910,平板克隆形成实验和CCK-8实验研究SIRT2对细胞生长的影响;细胞划痕实验考察SIRT2在SOC细胞迁移中的作用;Transwell实验研究SIRT2对SOC细胞侵袭能力的影响。结果:5株SOC细胞系中SIRT2的表达水平显著低于OSE细胞系。在HOSEpi C细胞中沉默SIRT2,细胞克隆形成数增多,细胞活力增强。相反,在HO8910细胞中过表达SIRT2,细胞克隆形成数减少,细胞活力降低。沉默SIRT2促进HOSEpi C细胞的迁移,而过表达SIRT2则抑制HO8910细胞的迁移。沉默SIRT2的HOSEpi C细胞侵袭能力明显增加,而过表达SIRT2的HO8910细胞侵袭能力则显著降低。结论:SIRT2在SOC细胞中表达显著下调。SIRT2在OSE细胞中是肿瘤抑制蛋白,抑制细胞增殖、迁移和侵袭。     

肿瘤相关巨噬细胞在晚期卵巢癌组织中的浸润与肿瘤浸润淋巴细胞表型、免疫效能之间的关系

目的:探讨晚期卵巢癌组织中浸润的肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)与肿瘤浸润淋巴细胞(tumor-infiltrating lymphocyte,TIL)表型及免疫效能的关系。方法:免疫组化方法分析175例低分化卵巢癌组织病理切片中TAM分布密度,以中位数为界限将病例分为TAM高密度组和TAM低密度组,对照组为32例良性卵巢病变组织;应用流式细胞术分析TAM高密度组与TAM低密度组中TIL的CD8⁺和CD25⁺表型变化情况;体外扩增培养TIL后取细胞培养上清液,ELISA法分析各组TIL中白细胞介素2(IL-2)、白细胞介素-10(IL-10)、转化生长因子β(TGF-β)和干扰素γ(IFN-γ)细胞因子表达变化。结果:175例低分化卵巢癌组织中TAM平均浸润密度为62.8/高倍镜视野(HP,×400),中位数为53.3/HP,其中TAM高密度组87例,TAM低密度组88例;对照组TAM平均浸润密度10.5/HP(P<0.05)。CD8⁺在TAM高密度组中表达平均值为24%,在TAM低密度组中表达平均值为52%(P<0.05);CD25⁺在TAM高密度组中表达平均值为48%,在TAM低密度组中表达平均值为25%(P<0.05);对照组中CD8⁺和CD25⁺的TIL平均浸润密度为7%, TAM高密度组及TAM低密度组中CD8⁺和CD25⁺的TIL平均浸润密度显著高于对照组(P<0.05)。与TAM低密度组比较,TAM高密度组中TIL的杀伤性细胞因子IL-2和IFN-γ表达明显减少(P<0.05),而抑制性细胞因子IL-10和TGF-β表达明显增加(P<0.05)。结论:高密度TAM浸润的卵巢癌组织中,CD25⁺的TIL表型增多,CD8⁺的TIL表型减少,抑制性细胞因子IL-10和TGF-β表达增加,杀伤性细胞因子IL-2和IFN-γ表达减少,提示TAM浸润密度与TIL表型及免疫效能相关。     

趋化因子受体CCR7的表达与卵巢癌淋巴管入侵及淋巴结转移的关系

目的 观察趋化因子受体CCR7在卵巢癌组织内的表达情况,分析CCR7的表达与肿瘤淋巴管入侵和淋巴结转移之间的关系。方法 取临床资料完整的卵巢癌病例64例,其中,淋巴结转移组40例,无淋巴结转移组24例。应用免疫组化法和Western blot技术观察CCR7在卵巢癌组织内的表达。以D2-40作为淋巴管内皮特异性标记物,观察卵巢癌组织内肿瘤淋巴管入侵的情况。结果 免疫组化法和Western blot检测结果表明,CCR7表达于卵巢癌细胞浆和胞膜内,有淋巴结转移组的表达量明显高于无淋巴结转移组(p<0.05)。D2-40表达于卵巢癌组织内淋巴管内皮细胞,肿瘤淋巴管入侵在CCR7阳性组的发生率明显高于CCR7阴性组的发生率,CCR7表达与肿瘤淋巴管入侵发生率有显著相关性(p<0.05)。结论 CCR7的表达与卵巢癌发生肿瘤淋巴管入侵和淋巴结转移密切相关,提示CCR7在卵巢癌淋巴结转移中可能起重要作用。     

An ovarian steroid cell tumor causing virilization and massive ascites

Steroid cell tumors, not otherwise specified (NOS), are rare ovarian sex cord-stromal tumors with malignant potential. The majority of these tumors produce several steroids, particularly testosterone. Various virilizing symptoms such as hirsutism, temporal balding, and amenorrhea are common in these patients; however massive ascites is an infrequent symptom. A 52-year-old woman with the sudden onset of virilization and massive ascites presented for treatment at Severance Hospital. After clinical evaluation, the patient underwent an exploratory laparotomy and a complete surgical staging procedure. She recovered from the surgery uneventfully and was discharged from the hospital five days after surgery. We present here an unusual case of an ovarian steroid cell tumor, NOS, and a brief review of the literature regarding these types of tumors.     

Familial ovarian carcinoma

Ovarian papillary adenocarcinoma is reported in two families. In one family, this tumor was detected in four females, of whom two had had mammary cancer before the development of the ovarian tumor. In the second family, a mother and daughter died of ovarian adenocarcinoma. The mode of inheritance in both families fits the pattern of autosomal dominant transmission of a single gene. Both are Jewish families from Germany, presently residing in Israel. The two families are presented as examples illustrating the difficult question of genetic counselling in families with recurrent cases of ovarian carcinoma. The difficulty arises because this tumor is relatively frequent but is usually observed in isolated cases without systematic follow-up in the patients' families. Only a few cases, where more than one person in a family is affected, have been selectively reported in the literature.     

Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers

Ovarian carcinoma is the leading cause of gynecological cancer deaths in the United States. Secondary tumor growths form by tumor cell invasion through the mesothelial lining of the peritoneal cavity and peritoneal organs. To study this interaction, we developed a dye-based in vitro model system in which mesothelial cells were grown as confluent monolayers, permeabilized, and then co-cultured with ovarian carcinoma cells for up to seven days. The mesothelial cells were then stained with trypan blue dye, which enabled the visualization of ovarian carcinoma cell invasion through the monolayers of mesothelial cells. Ovarian carcinoma cell invasion was inhibited for up to 7 days by the addition of GRGDSP peptides, a blocking monoclonal antibody against the β1 integrin subunit, or blocking monoclonal antibodies against matrix metalloproteinases 2 and 9. Cell invasion was also inhibited by hyaluronan and GM6001, a chemical inhibitor of matrix metalloproteinases. Differential gene expression of matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases, and disintegrins were observed in primary ovarian carcinoma tumors and secondary metastases, compared to normal ovaries. Taken together, these results suggest that complex interactions between integrins, disintegrins, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinases may mediate ovarian carcinoma cell invasion, and that the dye-based assay described herein is a suitable model system for its study. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of ovarian cancer proliferation and invasion by pachymic acid

To determine the effect of pachymic acid (PA) on proliferation, cell cycle, and invasion in human ovarian carcinoma cell lines HO-8910 and explore some possible mechanisms, HO-8910 cells was treated with different concentrations of PA (0.5, 1, 2 μM). CCK-8 assay, propidium iodide staining, was applied to measuring the growth inhibiting rates of HO-8910 cells. Cell cycle was measured by flow cytometry. In addition, the activity of PA against HO-8910 cells invasion was evaluated in transwell assay. Western blot detected the proteins expression of E-cadherin, β-catenin and COX-2 of different groups treated with PA in different concentrations (0.5, 1, 2 μM) for 48 h. Our results showed that PA could effectively inhibit the in vitro growth of HO-8910 cells in dose-dependent manners in 72 h, suppressed migration and invasion of HO-8910 cells in concentration-dependent manners at 24 h, caused the increased accumulation of G1 phase cells, and caused down-regulation of β-catenin and COX-2 and up-regulation of E-cadherin expression level. Taken together, it could conclude that PA might inhibit proliferation and invasion of ovarian carcinoma cell through decreasing β-catenin and COX-2 expression and increasing E-cadherin exprssion.     

Precursors of endometrial and ovarian carcinoma

This review discusses precursor lesions of endometrial and ovarian carcinoma with an emphasis on the unique molecular alterations that have led to the development of binary classification schemes for tumors of both the endometrium and ovary. While such a system is well established for endometrial carcinoma, only recently has a binary classification scheme been proposed for ovarian carcinoma. For both, the morphologic and molecular genetic-defining characteristics of their respective precursor lesions are described in detail. Furthermore, similarities and differences of the precursor lesions of specific tumors of these two genital tract organs are also addressed with a brief discussion of the clinical implications of their diagnosis.     

Squamous cell carcinoma arising in a mature cystic ovarian teratoma with bladder invasion: a case report

Background

Malignant transformation in a mature cystic ovarian teratoma is rare. Except in cases with high index of suspicion or overt metastasis, oophorectomy is the mainstay of treatment for ovarian teratoma.

Method

A 46-year-old perimenopausal woman who had salpingo-oophorectomy following a clinical diagnosis of benign ovarian tumour that was subsequently reported histologically as mature cystic ovarian teratoma with malignant transformation is presented.

Results

She was referred to our facility based on the histopathology report and haematuria two weeks after surgery. Cystoscopic biopsy done was reported as metastatic squamous cell carcinoma most probably from the ovary. Patient was thereafter referred for radiotherapy but was lost to follow-up after the first course.

Conclusion

Adequate evaluation prior to surgery in suspected ovarian teratoma with malignant transformation is critical to determine extent of surgery and adjuvant therapy. Prognosis in advanced disease condition such as the case presented is generally poor although radical pelvic surgery with resection of the adjacent involved bladder before radiotherapy would probably have improved her prognosis.     

Vagina invasion by cervical carcinoma

Seventy patients with cervical carcinoma who underwent radical hysterectomy and pelvic lymphadenectomy were evaluated to assess spread to the vagina. The overall vaginal invasion rate was 34.2% (24/70), with 36% (21/58) by squamous cell carcinoma, 25% (2/8) by adenocarcinoma and 25% (1/4) by adenosquamous carcinoma. A high vaginal invasion rate (45.7%) was noted in cases in which the cervical lesion was greater than 21 mm (p less than 0.05). Combined parametrial extention (45%) and combined lymph node metastasis (33.3%) were significantly higher in the vaginal invasion cases. The diagnostic accuracy of colposcopy and the Schiller test was 80% and 67% respectively. Histologically, the course of vaginal invasion by squamous cell carcinoma could be divided into : a) continuous invasion (16/21), b) incontinuous invasion via vessel permeation (3/21) and c) combined invasion (2/21). Both cases of vaginal invasion by adenocarcinoma were noted to spread by vessel permeation. Of the 7 cases of vessel permeation, colposcopic examination was positive in only one case. A high percentage of parametrial involvement and lymph node metastasis was noted in the vessel permeation type.     

Purification and characterization of an immunosuppressive factor from ovarian cancer ascites fluid

A nonspecific immunosuppressive factor present in malignant (ovarian carcinoma) ascites fluid has been purified by acid extraction from a high molecular weight (greater than 20000) complex followed by preparative isoelectric focusing on Bio-lyte media. It is an acidic protein (pI = 3.6) of mol. wt. 50000 to 52000 as estimated by gel filtration and composed of subunits of 25000 to 26000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing conditions. It inhibits the phytohemagglutinin-dependent mitogenic response of normal peripheral blood lymphocytes by 50% at 2 microgram/ml concentrations in vitro and suppresses 80% of the plaque-forming cell response to sheep erythrocytes at 100 microgram per mouse in vivo. Its chemical identity with any of the known plasma proteins has not been established. Its failure to stain with periodic acid Schiff's reagent indicates its minimal content of carbohydrates. It is susceptible to tryptic and pronase digestion but insensitive to deoxyribonuclease and ribonuclease digestion. A smaller suppressive factor identified in the same fluid appears to be a lymphotoxin; it differs from the acid-extracted nonspecific suppressive factor in its lack of susceptibility to trypsin.