Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion.     

A colonic tissue architecture assay applied to human colon carcinoma cells

Summary A two-component tissue architecture assay system has been devised that tests the ability of human colon carcinoma cells to conform to the specific three-dimensional cell-cell and cell-substratum interactions characteristic of normal colonic tissues. Dissociated fetal rat colonic cells (FRCC) were allowed to reaggregate in suspension with or without the addition of different proportions (0.1%, 1%, and 10% of the total cells) of the human colon carcinoma cell lines, SW-1222 and LS-174T. Cellular aggregates obtained after 36 hours’ incubation exhibited cell sorting by the formation of recognizable epithelial colonic crypt-like structures with glandular lumens in a mesenchyme-like background. Carcinoembryonic antigen (CEA)-positive SW-1222 cells in 10% mixed aggregates were organized into numerous well-formed glandular structures with a polarized apical distribution of CEA. LS-174T cells, on the other hand, were self-sorted but structurally disorganized with a continuous cell surface CEA distribution. Pure FRCC and mixed aggregates were implanted under the kidney capsules of Swiss nu/nu (nude) or CD-1 nu/nu mice and allowed to grow for a period of 7–10 days. Whereas the normal FRCC readily formed colonic tissue, the SW-1222 cells exhibited a capacity for differentiation into colonic crypts which became progressively less normal and more tumor-like as the proportion of carcinoma cells in the aggregates was increased. The LS-174T cells demonstrated poor differentiation at all concentrations. Cell surface levels of CEA and the CEA family member nonspecific crossreacting antigen (NCA), both overexpressed in colon cancer, were higher in LS-174T than in SW-1222 cells, whereas family member biliary glycoprotein (BGP), downregulated in colon carcinoma was higher in the SW-1222 cells. These results thus support the suggestion that deregulated expression of CEA family members can be involved in the ability of colonocytes to differentiate and conform to normal tissue architecture as assessed by the assay. The assay is therefore amenable to genetic analysis of normal and perturbed architectural phenotypes. This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. C. I. is a recipient of a studentship from “le Fonds pour la Formation de Chercheurs et l’Aide à la Recherche.”     

Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions

Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO^® MSC SFM) or conventional serum-containing medium (α-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105⁺⁺ and CD146^dim. After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium.     

分子细胞遗传学在软组织肿瘤中的应用

软组织肿瘤是发生于纤维组织、脂肪组织、平滑肌、骨骼肌、滑膜等间叶组织的一组病因复杂,组织形态各异,临床表现多样的肿瘤.软组织肉瘤是此类肿瘤中的一小部分,以独特的临床袁现和特异性的遗传学改变为特点,虽然占人类所有恶性肿瘤的比例不到1%[1],但它们同样严重威胁着人类的生命并具有重要的诊断和治疗意义.随着组织化学染色、电镜技术和免疫组化技术等辅助手段的广泛应用,人们对软组织肿瘤的发生、发展和分类、诊断等方面有了更深刻的认识,特别是对其分子水平的研究表明除癌基因和抑癌基因的点突变之外,染色体易位和缺失所致的基因重排和丢失也是软组织肿瘤发生的重要分子机制.这使得分子细胞遗传学在软组织肿瘤研究中具有了重要作用和特殊意义.本综述通过分析分子细胞遗传学技术在软组织肿瘤的分类、诊断及预后等方面的应用,客观评价了此技术的作用、优缺点及未来的发展方向.     

Rapid chemosensitivity assay with human normal and tumor cells in vitro

Summary Neutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes and fibroblasts were incubated for 2, 24, and 48 h with graded concentrations of cis-platinum (0.1 to 80 μM), doxorubicin (0.01 to 100 μM), and 5-fluorouracil (1 to 1000 μM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2–4 times more sensitive than the normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference in cytotoxic response. Methotrexate (1 to 10 μM) toxicity could be reversed in the presence of 100 μM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development. This work has been supported, in part, by funds from Schering Corporation, New Jersey, and Chevron Environmental Health Center, Inc., California.     

Contribution of bone marrow-derived endothelial cells to human tumor vasculature

It has been shown that bone marrow-derived stem cells can form a major fraction of the tumor endothelium in mouse tumors. To determine the role of such cells in human tumor angiogenesis, we studied six individuals who developed cancers after bone marrow transplantation with donor cells derived from individuals of the opposite sex. By performing fluorescence in situ hybridization (FISH) with sex chromosome-specific probes in conjunction with fluorescent antibody staining, we found that such stem cells indeed contributed to tumor endothelium, but at low levels, averaging only 4.9% of the total. These results illustrate substantial differences between human tumors and many mouse models with respect to angiogenesis and have important implications for the translation of experimental antiangiogenic therapies to the clinic.     

An electron microscopic study of the behavioral characteristics of human lung tumor cells cultured on soft agar]

The ultrastructure of cells from seven human lung cancers and from the colonies formed by these cells in soft agar was investigated. Tumor cells developed to display the morphofunctional potentials of the initial tumors. Cultured cells of squamous-cell carcinomas contained numerous tonofilaments, those of adenocarcinomas developed microvilli on their apical surfaces and intracellular lumens. On the other hand, cells of squamous-cell carcinomas showed features specific of adenoma epithelium, i.e. well developed microvilli and intracellular lumens. Besides, cells of adenocarcinoma often contained large quantities of tonofilaments considered to be characteristic of epidermoid epithelium. The results obtained suggest a possibility of metaplastic transformation of the lung epithelium.     

Feasibility of using gene expression analysis to study canine soft tissue sarcomas

The prognosis given for canine soft tissue sarcomas (STSs) is based primarily on histopathologic grade. The decision to administer adjuvant chemotherapy is difficult since less than half of patients with high-grade STSs develop metastatic disease. We hypothesize that there is a gene signature that will improve our ability to predict development of metastatic disease in STS patients. The objective of this study was to determine the feasibility of using cDNA microarray and quantitative real-time PCR (qRT-PCR) analysis to determine gene expression patterns in metastatic versus nonmetastatic canine STSs, given the inherent heterogeneity of this group of tumors. Five STSs from dogs with metastatic disease were evaluated in comparison to eight STSs from dogs without metastasis. Tumor RNA was extracted, processed, and labeled for application to the Affymetrix Canine Genechip 2.0 Array. Array fluorescence was normalized using D-Chip software and data analysis was performed with JMP/Genomics. Differential gene expression was validated using qRT-PCR. Over 200 genes were differentially expressed at a false discovery rate of 5%. Differential gene expression was validated for five genes upregulated in metastatic tumors. Quantitative RT-PCR confirmed increased relative expression of all five genes of interest in the metastatic STSs. Our results demonstrate that microarray and qRT-PCR are feasible methods for comparing gene signatures in canine STSs. Further evaluation of the differences between gene expression in metastatic STSs and in nonmetastatic STSs is likely to identify genes that are important in the development of metastatic disease and improve our ability to prognosticate for individual patients.     

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.     

Gemcitabine-mediated radiosensitization of human soft tissue sarcoma

Background/Purpose

Local and systemic control of soft tissue sarcoma (STS) remains a clinical challenge, particularly for retroperitoneal, deep truncal, or advanced extremity disease. 2′,2′-Difluoro-2′-deoxycytidine (gemcitabine) is a potent radiosensitizer in many tumor types, but it has not been studied in human STS. The purpose of this study was to determine the radiosensitizing potential of gemcitabine in preclinical models of human STS.

Materials and Methods

The in vitro radiosensitizing activity of gemcitabine was assessed with clonogenic survival assay on three human STS cell lines: SK-LMS-1 (leiomyosarcoma), SW-872 (liposarcoma), and HT-1080 (fibrosarcoma). Cell cycle distribution was determined using dual-channel flow cytometry. The in vivo radiosensitizing activity of gemcitabine was assessed with subcutaneous SK-LMS-1 nude mice xenografts. Tumor-bearing mice were treated with concurrent weekly gemcitabine and fractionated daily radiotherapy (RT) (2 Gy daily) for 3 weeks (a total dose of 30 Gy).

Results

The 50% inhibitory concentration (IC₅₀) of gemcitabine for the human STS cell lines ranged from 10 to 1000 nM. Significant in vitro radiosensitization was demonstrated in all three human STS cell lines using gemcitabine concentrations at and below the IC₅₀. Maximal radiosensitization was associated with accumulation of cells in early S-phase. SK-LMS-1 xenografts displayed significant tumor growth delay with combined gemcitabine and RT compared to either treatment alone. Treatment related toxicity was greatest in the gemcitabine plus RT arm, but remained at an acceptable level.

Conclusions

Gemcitabine is a potent radiosensitizer in preclinical models of human STS. Clinical trials combining gemcitabine and RT in human STS are warranted.     

Host-supplied connective tissue as a guide for proliferating tumor cells in human tumor xenografts

The morphologic alterations of 7 human tumors of different origin and various histologic types, heterotransplanted to athymic nude mice, were investigated in the present study. Constant patterns of histologic and ultrastructural changes were observed in all 7 tumors. Following the initial dying of most inoculated tumor cells, host-supplied inflammatory cells invaded the xenografts and phagocytosed necrotic tumor cells. Fibroblasts which vivaciously produced collagenous material invaded the xenografts and built up solid strands of connective tissue which tightly contacted surviving tumor cells. These stands were used as guide-rails for ingrowing blood vessels. Immediately after their immigration, the first mitoses of tumor cells occurred in close proximity to capillary-conducting strands of connective tissue resulting in a revival of tumor cell proliferation near to the fibrous cords and a spreading of newly formed tumor cells along the strands of connective tissue. These results point to the host-supplied connective tissue as playing an important role for tumor proliferation and local tumor expansion.     

A radioligand binding assay for antitubulin activity in tumor cells

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.     

Characteristics of human mesenchymal stem cells isolated from bone marrow and adipose tissue

Populations of human mesenchymal stem cells were derived from bone marrow and adipose tissue. Here analysis of six individuals is represented. Cells were isolated, expanded and evaluated by the expression of surface antigens using flow cytometry. These cells displayed similar characteristics for many markers. Cells isolated from bone marrow and adipose tissue were found to be homogeneously positive for CD13, CD44, CD90, CD105, and negative for CD45, CD34, CD31 and CD117. Besides, differences in surface antigene CD10 expression between narrow and adipose tissue-derived cells were detected. All these findings indicate that both bone marrow and adipose tissue are important sources of mesenchymal stem cells, which could be used in cell therapy protocols.     

A bioinformatic assay for pluripotency in human cells

Pluripotent stem cells (PSCs) are defined by their potential to generate all cell types of an organism. The standard assay for pluripotency of mouse PSCs is cell transmission through the germline, but for human PSCs researchers depend on indirect methods such as differentiation into teratomas in immunodeficient mice. Here we report PluriTest, a robust open-access bioinformatic assay of pluripotency in human cells based on their gene expression profiles.     

A hypo-osmotic medium to disaggregate tumor cell clumps into viable and clonogenic single cells for the human tumor stem cell clonogenic assay

A hypo-osmolar medium and tissue processing technique is described which is useful for disaggregation of residual human tumor cell clumps persisting after mechanical or enzymatic treatment of solid tumors and malignant effusions. The addition of the hypo-osmolar procedure to the standard methods for disaggregation increased the viable single cell yield in solid tumors by 47% and in malignant effusions by 67%. In 5 of the 26 solid tumor specimens tested in the human tumor stem cell assay, clonogenic single cells were obtained with the hypo-osmolar procedure, whereas no growth was observed using standard methods. Overall, the success rate for clonogenicity increased from 46% to 65% for the 26 solid tumors, with the major improvement occurring in ovarian cancer. Clonogenicity was obtained in 80% of malignant effusions both by standard methods and the hypo-osmolar techniques. The increased total yield of clonogenic cells obtained with this procedure enhances the opportunity for experimental versatility and in vitro drug testing.     

A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue

To evaluate the potential of three stem cells for cell therapy and tissue engineering applications, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). There was no significant difference between the rates of proliferation of the three stem cells. During osteogenic differentiation, alkaline phosphatase (ALP) activity peaked on day 7 in USSC compared to BM-MSC which showed the maximum value of ALP activity on day 14. However, BM-MSC had the highest ALP activity and mineralization during osteogenic induction. In addition, AT-MSC showed the lowest capacity for mineralization during differentiation and had the lowest ALP activity on days 7 and 14. Although AT-MSC expressed higher levels of collagen type I, osteonectin and BMP-2 in undifferentiated state, but these genes were expressed higher in BM-MSC during differentiation. BM-MSC also expressed higher levels of ALP, osteocalcin and Runx2 during induction. Taking together, BM-MSC showed the highest capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.