Ovarian Cancer Cell Line Panel (OCCP): Clinical Importance of In Vitro Morphological Subtypes

Epithelial ovarian cancer is a highly heterogeneous disease and remains the most lethal gynaecological malignancy in the Western world. Therapeutic approaches need to account for inter-patient and intra-tumoural heterogeneity and detailed characterization of in vitro models representing the different histological and molecular ovarian cancer subtypes is critical to enable reliable preclinical testing. There are approximately 100 publicly available ovarian cancer cell lines but their cellular and molecular characteristics are largely undescribed. We have characterized 39 ovarian cancer cell lines under uniform conditions for growth characteristics, mRNA/microRNA expression, exon sequencing, drug response for clinically-relevant therapeutics and collated all available information on the original clinical features and site of origin. We tested for statistical associations between the cellular and molecular features of the lines and clinical features. Of the 39 ovarian cancer cell lines, 14 were assigned as high-grade serous, four serous-type, one low-grade serous and 20 non-serous type. Three morphological subtypes: Epithelial (n = 21), Round (n = 7) and Spindle (n = 12) were identified that showed distinct biological and molecular characteristics, including overexpression of cell movement and migration-associated genes in the Spindle subtype. Comparison with the original clinical data showed association of the spindle-like tumours with metastasis, advanced stage, suboptimal debulking and poor prognosis. In addition, the expression profiles of Spindle, Round and Epithelial morphologies clustered with the previously described C1-stromal, C5-mesenchymal and C4 ovarian subtype expression profiles respectively. Comprehensive profiling of 39 ovarian cancer cell lines under controlled, uniform conditions demonstrates clinically relevant cellular and genomic characteristics. This data provides a rational basis for selecting models to develop specific treatment approaches for histological and molecular subtypes of ovarian cancer.     

Bioenergetic Analysis of Ovarian Cancer Cell Lines: Profiling of Histological Subtypes and Identification of a Mitochondria-Defective Cell Line

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines'' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O₂ hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs.     

Effect of Ovarian Cancer Ascites on Cell Migration and Gene Expression in an Epithelial Ovarian Cancer In Vitro Model

A third of patients with epithelial ovarian cancer (EOC) present ascites. The cellular fraction of ascites often consists of EOC cells, lymphocytes, and mesothelial cells, whereas the acellular fraction contains cytokines and angiogenic factors. Clinically, the presence of ascites correlates with intraperitoneal and retroperitoneal tumor spread. We have used OV-90, a tumorigenic EOC cell line derived from the malignant ascites of a chemonaive ovarian cancer patient, as a model to assess the effect of ascites on migration potential using an in vitro wound-healing assay. A recent report of an invasion assay described the effect of ascites on the invasion potential of the OV-90 cell line. Ascites sampled from 31 ovarian cancer patients were tested and compared with either 5% fetal bovine serum or no serum for their nonstimulatory or stimulatory effect on the migration potential of the OV-90 cell line. A supervised analysis of data generated by the Affymetrix HG-U133A GeneChip identified differentially expressed genes from OV-90 cells exposed to ascites that had either a nonstimulatory or a stimulatory effect on migration. Ten genes (IRS2, CTSD, NRAS, MLXIP, HMGCR, LAMP1, ETS2, NID1, SMARCD1, and CD44) were upregulated in OV-90 cells exposed to ascites, allowing a nonstimulatory effect on cell migration. These findings were validated by quantitative polymerase chain reaction. In addition, the gene expression of IRS2 and MLXIP each correlated with prognosis when their expression was assessed in an independent set of primary cultures established from ovarian ascites. This study revealed novel candidates that may play a role in ovarian cancer cell migration.     

卵巢上皮性癌细胞系来源肿瘤干细胞的免疫特性研究

目的:探讨卵巢上皮性癌细胞系(SKOV3)来源肿瘤干细胞的免疫特性。方法:通过干细胞特异性标记物乙醛脱氢酶1ALDH1表达鉴定富集肿瘤干细胞(CSCs)的球状细胞(SDC)。将富集CSCs的SDC与相应的贴壁培养的普通卵巢癌细胞(MDC)进行比较。通过Transwell法(细胞侵袭试验)检测SDC和MDC对于T细胞增殖以及分泌功能的影响。RT-PCR检测SDC以及MDC免疫抑制基因表达情况。结果:以球体细胞形成方法获得的CSC富集SDC群比MDC表现出更高比例的ALDH1表达。将T细胞分别与SDC和MDC共培养,SDC组T细胞增殖率显著低于MDC组。MDC共培养的T细胞分泌IFN-γ、IL-2水平明显高于与SDC共培养的T细胞(P0.05)。SDC表达免疫抑制基因ArginaseⅡ、IDO、IL-8、TGF-β水平明显高于MDC,以ArginaseⅡ、IL-8的上调最为明显(P0.05)。结论:球体细胞形成试验是获得卵巢肿瘤干细胞的可靠方法。在卵巢上皮性癌细胞系(SKOV3)中,CSCs比普通肿瘤细胞表现出更强大的免疫抑制性,这可能是妨碍免疫治疗的免疫逃逸机制。     

Morphological Response of Witchweed (Striga asiatica) to In Vitro Culture

Excised sorghum root segments (5–10 mm in length) werecultured for 50 d in four different liquid media containingmineral salts, vitamins, amino acids, glucose, and IAA. Theroots were removed and the remaining medium was solidified withan equal volume of warm 1–6% water agar. Dry unconditionedor conditioned Striga asiatica seeds were transferred to themedium. Some of the seeds germinated and developed into parasitic-typeseedlings. These seedlings had haustoria, tubercles, dense roothairs, branched shoots, and multiple shoot-borne adventitiousroots. The plumule pole developed into a shoot, but the radiclepole displayed only rudimentary development. On the same media,but which had not previously been used to grow sorghum roots,the seedlings displayed a well-developed radicle-derived rootsystem, but the plumule did not grow. Shoots began to appearon the roots only after 35–50 d of culture. These seedlingshad no haustoria, no tubercles, few or scattered root hairs,no shoot-borne adventitious roots and few shoot branches, andappeared to be non-parasitic-type seedlings. Shoots grew ina medium supplemented with IAA and kinetin, but did not in amedium containing NAA plus IBA. On replacement of glucose andIAA with sucrose and 2,4-D, respectively, Striga seeds germinated,and the heart-shaped embryos dedifferentiated into calli. Thecalli have been maintained by subculturing for over 9 months.The results demonstrated that a host signal, in addition tothose for germination and haustorium formation, is requiredfor further development. Moreover, morphogenesis of culturedS. asiatica is influenced by exogenous growth regulators. Key words: Striga asiatica, parasitic weeds, haustoria, Sorghum bicolor, in vitro culture     

人OC-3-VGH卵巢癌细胞裸小鼠肿瘤模型的建立

目的建立人OC-3-VGH细胞株卵巢癌裸小鼠模型并观察该肿瘤生物学生长特性。方法OC-3-VGH细胞株复苏后加入10 mL RPMI-1640培养液,放入培养箱,传2～3代后,取细胞悬液,均以4×106个细胞,每只0.2 mL分别接种至BALB/c雌性裸小鼠皮下,2月后处死取材,观察肿瘤生长特性和转移情况。结果皮下接种一周后,裸鼠长出肿瘤,并随时间而增大,体积呈指数增长,第42天始,明显增大（P〈0.05）。组织学检查发现裸鼠皮下肿瘤细胞均细胞体积较大,细胞核大而染色深,核分裂相较多、异型性明显,接种2个月时,未发生其他组织转移。结论建立了新的卵巢癌动物模型,并初步研究了其生物学特性,为卵巢癌治疗方法的研究拓宽了道路。     

Decorin in Human Colon Cancer: Localization In Vivo and Effect on Cancer Cell Behavior In Vitro

Decorin is generally recognized as a tumor suppressing molecule. Nevertheless, although decorin has been shown to be differentially expressed in malignant tissues, it has often remained unclear whether, in addition to non-malignant stromal cells, cancer cells also express it. Here, we first used two publicly available databases to analyze the current information about decorin expression and immunoreactivity in normal and malignant human colorectal tissue samples. The analyses demonstrated that decorin expression and immunoreactivity may vary in cancer cells of human colorectal tissues. Therefore, we next examined decorin expression in normal, premalignant and malignant human colorectal tissues in more detail using both in situ hybridization and immunohistochemistry for decorin. Our results invariably demonstrate that malignant cells within human colorectal cancer tissues are devoid of both decorin mRNA and immunoreactivity. Identical results were obtained for cells of neuroendocrine tumors of human colon. Using RT-qPCR, we showed that human colon cancer cell lines are also decorin negative, in accordance with the above in vivo results. Finally, we demonstrate that decorin transduction of human colon cancer cell lines causes a significant reduction in their colony forming capability. Thus, strategies to develop decorin-based adjuvant therapies for human colorectal malignancies are highly rational.     

肿瘤干细胞标记分子CD133在胶质瘤细胞U87中的稳定表达

目的：在体外胶质瘤U87细胞中稳定表达肿瘤干细胞标记分子CD133。方法：通过脂质体介导将表达载体质粒CD133-1／pCR3．1-Uni转染U87细胞,G418筛选稳定表达抗性的细胞株;用细胞免疫荧光染色鉴定表达CD133分子的U87细胞。结果：转染CD133表达载体的U87细胞可以被CD133单抗识别,而转染空载体的U87细胞免疫染色结果为阴性,表明CD133分子在U87细胞中稳定表达。结论：U87细胞稳定表达CD133分子,为体内外分析CD133阳性U87细胞特性奠定了基础：U87CD133阳性细胞可以作为免疫组化或流式细胞术等检测其他肿瘤干细胞CD133表达的阳性对照细胞。     

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades ^1,2. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages ^3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren''t specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.     

Attenuation of Cell Mechanosensitivity in Colon Cancer Cells during In Vitro Metastasis

Human colon carcinoma (HCT-8) cells show a stable transition from low to high metastatic state when cultured on appropriately soft substrates (21 kPa). Initially epithelial (E) in nature, the HCT-8 cells become rounded (R) after seven days of culture on soft substrate. R cells show a number of metastatic hallmarks [1]. Here, we use gradient stiffness substrates, a bio-MEMS force sensor, and Coulter counter assays to study mechanosensitivity and adhesion of E and R cells. We find that HCT-8 cells lose mechanosensitivity as they undergo E-to-R transition. HCT-8 R cells'' stiffness, spread area, proliferation and migration become insensitive to substrate stiffness in contrast to their epithelial counterpart. They are softer, proliferative and migratory on all substrates. R cells show negligible cell-cell homotypic adhesion, as well as non-specific cell-substrate adhesion. Consequently they show the same spread area on all substrates in contrast to E cells. Taken together, these results indicate that R cells acquire autonomy and anchorage independence, and are thus potentially more invasive than E cells. To the best of our knowledge, this is the first report of quantitative data relating changes in cancer cell adhesion and stiffness during the expression of an in vitro metastasis-like phenotype.     

88例卵巢上皮性癌行ATP-TCA体外药敏试验结果分析

该研究是探讨三磷酸腺苷生物荧光肿瘤抗癌药物药敏性分析实验（ATP-TCA）在卵巢癌患者化疗中的应用。研究选取88例卵巢上皮性癌新鲜组织行ATP-TCA体外药敏试验,分析结果、计算各种化疗药物敏感性,并与48例对照组患者进行临床近期有效率的比较。结果显示,在体外药敏试验敏感性最强的单药为紫杉醇（51.9%）,敏感性强弱依次为：紫杉醇〉卡铂〉顺铂〉吉西他滨〉拓泊替康〉多西他赛〉依托泊苷〉环磷酰胺〉博来霉素,联合用药方案敏感性较单药增加。药敏组患者临床近期有效率（85.23%）高于对照（68.75%）。ATP-TCA是一种有效的抗癌药物敏感性分析实验,可为卵巢癌患者临床化疗提供个体化的指导方案。     

Anti-Tumor Activity of Eurycoma longifolia Root Extracts against K-562 Cell Line: In Vitro and In Vivo Study

Eurycoma longifolia Jack has been widely used in traditional medicine for its antimalarial, aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer activity has also been recently reported on different solid tumors, however no anti-leukemic activity of this plant has been reported. Thus the present study assesses the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. The K-562 cells (purchased from ATCC) were isolated from patients with chronic myelocytic leukemia (CML) were treated with the various fractions (TAF273, F3 and F4) of E. longifolia root methanolic extract at various concentrations and time intervals and the anti-proliferative activity assessed by MTS assay. Flow cytometry was used to assess the apoptosis and cell cycle arrest. Nude mice injected subcutaneously with 10⁷ K-562 cells were used to study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4 showed various degrees of growth inhibition with IC50 values of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose and time dependent manner. TAF273 arrested cell cycle at G1and S phases. Intraperitoneal administration of TAF273 (50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor in TAF273-treated mice compared with the control mice (P = 0.024). TAF273 shows potent anti-proliferative activity in vitro and in vivo models of CML and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management.     

An Immortalized Human Blood-Nerve Barrier Endothelial Cell Line for In Vitro Permeability Studies

Solute and macromolecular transport studies may elucidate nutritional requirements and drug effects in healthy and diseased peripheral nerves. Endoneurial endothelial cells are specialized microvascular cells that form the restrictive blood-nerve barrier (BNB). Primary human endoneurial endothelial cells (pHEndECs) are difficult to isolate, limiting their widespread availability for biomedical research. We developed a simian virus-40 large T-antigen (SV40-LTA) immortalized human BNB cell line via stable transfection of low passage pHEndECs and observed continuous growth in culture for >45 population doublings. As observed with pHEndECs, the immortalized BNB endothelial cells were Ulex Europaeus agglutinin-1-positive and endocytosed low density lipoprotein, but lost von Willebrand factor expression. Glucose transporter-1, P-glycoprotein (P-gp), γ-glutamyl transpeptidase (γ-GT), large neutral amino acid transporter-1 (LAT-1), creatine transporter (CRT), and monocarboxylate transporter-1 (MCT-1) mRNA expression were retained at all passages with loss of alkaline phosphatase (AP) expression after passages 16–20. Compared with an SV40-LTA immortalized human blood-brain barrier endothelial cell line, there was increased γ-GT protein expression, equivalent expression of organic anion transporting polypeptide-C (OATP-C), organic anion transporter 3 (OAT-3), MCT-1, and LAT-1, and reduced expression of AP, CRT, and P-gp by the BNB cell line at passage 20. Further studies demonstrated lower transendothelial electrical resistance (~181 vs. 191 Ω cm²), equivalent permeability to fluoresceinated sodium (4.84 vs. 4.39 %), and lower permeability to fluoresceinated high molecular weight (70 kDa) dextran (0.39 vs. 0.52 %) by the BNB cell line. This cell line retained essential molecular and biophysical properties suitable for in vitro peripheral nerve permeability studies.     

水蛭素联合阿霉素对人卵巢癌细胞株耐药性的实验观察

目的探讨水蛭素联合阿霉素对人卵巢癌细胞生长的影响。方法通过细胞培养后绘制生长曲线,了解联合用药对抑制癌细胞增殖及诱导凋亡的作用。结果水蛭素联合阿霉素具有杀伤人卵巢癌细胞的效果。结论水蛭素有可能作为临床抗癌和抑癌治疗的潜在辅助用药。     

赫赛汀联合姜黄素对卵巢癌细胞系SKOV3的抑制作用

目的:探讨赫赛汀联合姜黄素对HER2阳性卵巢癌细胞系SKOV3增殖能力的影响。方法:体外培养至对数生长期的人卵巢癌细胞系SKOV3细胞分为对照组、赫赛汀治疗组、姜黄素治疗组以及赫赛汀联合黄素治疗组,利用四唑盐(MTT)比色法和平板集落形成试验比较各组细胞增殖能力,利用AnnexinV-FITC/PI染色比较各组细胞凋亡。结果:赫赛汀联合姜黄素治疗组SKOV3细胞活力明显低于对照组及单一药物处理组,平板集落形成试验结果表明各组细胞集落形成率分别为83.4%、36.8%、59.2%和24.7%,赫赛汀联合姜黄素处理组SKOV3细胞集落形成能力显著低于另外3组(P<0.01),Annexin V-FITC/PI染色并用流式细胞仪分析表明各组SKOV3细胞早期凋亡率分别为1.3%、26.4%、9.3%和39.1%,赫赛汀联合姜黄素处理组细胞早期凋亡率显著低于其余3组(P<0.01)。结论:赫赛汀联合姜黄素能够抑制卵巢癌细胞系SKOV3的增殖,这种抑制作用是通过促进肿瘤细胞凋亡实现的。     

赫赛汀联合姜黄素对卵巢癌细胞系SKOV3的抑制作用

目的：探讨赫赛汀联合姜黄素对HER2阳性卵巢癌细胞系SKOV3增殖能力的影响。方法：体外培养至对数生长期的人卵巢癌细胞系SKOV3细胞分为对照组、赫赛汀治疗组、姜黄素治疗组以及赫赛汀联合黄素治疗组,利用四唑盐（MTT）比色法和平板集落形成试验比较各组细胞增殖能力,利用Annexin V—FITC／PI染色比较各纽细胞凋亡。结果：赫赛汀联合姜黄素治疗组SKOV3细胞活力明显低于对照组及单一药物处理纽,平板集落形成试验结果表明各组细胞集落形成率分别为83．4％、36．8％、59．2％和24．7％。赫赛汀联合姜黄素处理组SKOV3细胞集落形成能力显著低于另外3组（P〈0．01）,Annexin V-FITC／PI染色并用流式细胞仪分析表明各组SKOV3细胞早期凋亡率分别为1．3％、26．4％、9．3％和39．1％,赫赛汀联合姜黄素处理组细胞早期凋亡率显著低于其余3组（P〈0．01）。结论：赫赛汀联合姜黄素能够抑制卵巢癌细胞系SKOV3的增殖,这种抑制作用是通过促进肿瘤细胞凋亡实现的。