To investigate the effect of manganese superoxide dismutase (MnSOD) silence on the in vitro tumorigenicity in human small cell lung cancer NCI-H446 cells and the underlying mechanisms.
 Methods: Sphere formation cells from NCI-H446 cells were obtained by suspension culture, while the expression of MnSOD and urokinase type plasminogen activator (uPAR) was analyzed by Western blot. Silence of MnSOD was performed by adenovirus infection in the second passage formation cells, and the effect of MnSOD silence on tumorigenicity in NCI-H446 cells was evaluated by sphere formation assay and soft-agar colony formation assay, while the expression of uPAR was analyzed by Western blot.
 Results: Compared with NCI-H446 cells, the sphere formation rate, colony formation rate, and the expression of MnSOD and uPAR were significantly increased in the second passage sphere formation cells in NCI-H446 cells (P<0.05). Silence of MnSOD inhibited the sphere formation rate, colony formation rate, and the expression level of uPAR in the second passage sphere formation cells in NCI-H446 cells.
 Conclusion: MnSOD may promote tumorigenicity in NCI-H446 cells by up-regulation of uPAR expression in vitro.
Adenoviridae ; Carcinogenesis ; Cell Line, Tumor ; Humans ; In Vitro Techniques ; Lung Neoplasms ; etiology ; metabolism ; RNA Interference ; Receptors, Urokinase Plasminogen Activator ; genetics ; metabolism ; Small Cell Lung Carcinoma ; etiology ; metabolism ; Spheroids, Cellular ; pathology ; Superoxide Dismutase ; genetics ; metabolism ; Tumor Stem Cell Assay ; Up-Regulation

To investigate the effect of apigenin on self-renewal for sphere-forming cells in human small cell lung cancer cell line NCI-H446 and the underlying mechanisms.
 Methods: Sphere-forming cells from NCI-H446 cell line were cultured in stem cell-conditioned culture medium with ultra-low attachment surface plates. The rate of sphere-forming cells in the second passage sphere-forming cells was used to evaluate the inhibitory effects of apigenin on the self-renewal for sphere-forming cells. The protein level of urokinase-type plasminogen activator receptor (uPAR) in spheroids was analyzed by Western blot.
 Results: Apigenin signifcantly inhibited the self-renewal of the second passage sphere-forming cells [0, 5.0, 10.0, 20.0 μmol/L apigenin: (18.2±1.9)%, (13.6±1.7)%, (10.6±1.6)%, (6.9±1.3)%, respectively] and down-regulated uPAR expression in a concentration-dependent manner (P<0.05).
 Conclusion: Apigenin inhibits the self-renewal capacity of sphere-forming cells in NCI-H446 cells, which may be associated with down-regulation of uPAR expression.
Apigenin ; pharmacology ; Cell Line, Tumor ; Down-Regulation ; drug effects ; genetics ; Humans ; Lung Neoplasms ; Neoplastic Stem Cells ; drug effects ; pathology ; physiology ; Receptors, Cell Surface ; Receptors, Urokinase Plasminogen Activator ; drug effects ; genetics ; metabolism ; Signal Transduction ; Small Cell Lung Carcinoma ; drug therapy ; pathology ; Spheroids, Cellular ; drug effects ; physiology ; Stem Cells

OBJECTIVETo fabricate an innovative scaffold for lung cancer cell culture and establish a three-dimensional lung cancer model in vitro, and to reveal the differences in biological functions of lung cancer cells under the two-dimensional and three-dimensional culture conditions.

METHODSWe chose agarose and alginate as the scaffold materials, and 3D printing technique was applied to construct cell culture scaffold. 95D cells were co-cultured with this scaffold. The differences of cell morphology, proliferation ability, protein expression, etc. in the cells cultured under 2D and 3D cultural conditions were evaluated by light microscopy using HE staining, MTT assay, scanning electron microscopy, and Western blot analysis.

RESULTSCells cultured in 2D wells displayed a spindle and polygonal morphology, whereas those grown in the 3D culture aggregated into spheroids, which invaded, migrated and disseminated into the surrounding scaffold. MTT assay showed that the proliferation rates of the 3D-cultured cells for 2-6 days were significantly lower than, but those cultured for 8-9 days were significantly higher than that of the 2D-cultured cells, indicating that proliferative activity of the cells grown in 2D cultures for 8-9 days was inhibited. In contrast, cells grown on 3D scaffolds still maintained a higher proliferation. The Western blot assay showed that the expression of Cdc42, p53, mTOR were significantly down-regulated in 3D scaffold-cultured group (0.529±0.103, 0.820±0.038 vs. 1.967±0.066), compared with that of the 2D-cultured group (3.063±0.139, 1.738±0.122 vs. 2.472±0.151) (P<0.05 for all), while the expression of MMP-2 was up-regulated in the 3D-cultured cells (1.110±0.029), significantly higher than that of the 2D-cultured cells (0.017±0.001) (P<0.05).

CONCLUSIONSThe cell morphology, proliferation and associated protein expression of lung cancer cells in 3D-culture systems are distinctively different as compared to those of the 2D-cultural cells. 3D-bioprinted agarose-alginate scaffold can better mimic the growth microenvironment of lung cancer in vivo and may provide a promising model for lung cancer research in vitro.

Alginates ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; physiopathology ; Cell Culture Techniques ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Glucuronic Acid ; Hexuronic Acids ; Humans ; Lung Neoplasms ; metabolism ; pathology ; physiopathology ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; Printing, Three-Dimensional ; Sepharose ; Spheroids, Cellular ; pathology ; Time Factors ; Tissue Scaffolds ; Tumor Microenvironment

OBJECTIVETo observe the histological features of tumor-bearing tissues formed by human fibroblasts after reprograming by spermatogonial stem cell self-renewal key regulating gene Piwil2 (Piwil2-iCSC).

METHODSPiwil2-iCSC tumor spheroids-like colonies were selected for tumor formation assay in four nude mice. Pathological features of Piwil2-iCSC tumors were observed by histology. Stem cell markers and common triploblastic markers were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) assay and immunohistochemistry. Germ cell tumor markers were detected by immunohistochemical examination.

RESULTSTwo weeks after inoculation, subcutaneous tumors were formed in all the four nude mice with a tumor formation rate of 100%. In the Piwil2-iCSC tumor tissues, Piwil2-GFP(+) cells showed high-density nuclear expression and were widely observed in DAPI-stained sections. Numerous mitotic figure of the neoplastic cells were seen (>10 cells/field of vision under high magnification) in HE-stained sections. Enlarged abnormal cell nuclei were observed. RT-PCR assay showed that Piwil2-iCSC tumors still expressed Piwil2 and some self-renewal and pluripotent markers of stem cells and some markers of triploblastic differentiation. Immunohistochemical staining showed that the tumors expressed stem cell markers, triploblastic markers and germ cell tumor markers AFP and HCG.

CONCLUSIONSPiwil2-iCSC tumors are probably undifferentiated embryonic small cell carcinoma, most likely to be immature teratoma, mixed with yolk sac tumor and choriocarcinoma components. It can be used as a useful model for the research of origin or genesis mechanism of cancer stem cells and the treatment of relevant tumors.

Adult Stem Cells ; Animals ; Argonaute Proteins ; genetics ; Cellular Reprogramming Techniques ; Choriocarcinoma ; pathology ; Endodermal Sinus Tumor ; pathology ; Fibroblasts ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Mice ; Mice, Nude ; Neoplasms, Germ Cell and Embryonal ; chemistry ; genetics ; pathology ; Neoplastic Stem Cells ; chemistry ; pathology ; Real-Time Polymerase Chain Reaction ; Spheroids, Cellular ; Teratoma ; pathology ; Time Factors

Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell (CSC) traits in a number of cancers, but this phenomenon has not yet been reported in the VX2 rabbit oral cancer model. Hence, this study aimed to study the spheroid cells from VX2 rabbit buccal squamous cell carcinomas (SCCs) and assess their CSC characteristics. Five adult male New Zealand white outbred rabbits were used to generate VX2 rabbit buccal SCC. Sphere-forming cell culture was performed for the VX2 rabbit buccal SCC specimens. The self-renewal capability; cluster of designation (CD) 44, CD133, acetaldehyde dehydrogenase 1 (ALDH1), B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), Nestin, octamer-binding transcription factor 4 (Oct4) and reduced expression protein-1 (Rex-1) expression with reverse transcription-polymerase chain reaction (RT-PCR); chemoresistance to cisplatin and 5-fluorouracil; and in vivo tumorigenicity of spheroid cell transplantation in nude mice were evaluated to determine the CSC characteristics of the resulting spheroid cells. We successfully obtained spheroid cells from the VX2 rabbit OSCC tissues. The spheroid cells exhibited CSC traits, including the expression of CSC and stem cell markers (CD44, Bmi-1, Nestin, Oct4 and Rex-1), capacity to generate new spheroid colonies within 1 week of reseeding from single-dissociated spheroid cells, chemoresistance capacity and generation of tumour xenografts (with histological features resembling those of the original VX2 rabbit buccal SCC) from the transplantation of 10(3) undifferentiated spheroid cells into nude mice. In summary, we demonstrated that spheroid cells with CSC cell traits can be derived from VX2 rabbit buccal SCCs, indicating that this animal cancer model is applicable for studying CSCs in human oral cancers.
AC133 Antigen ; Animals ; Antigens, CD ; analysis ; Antineoplastic Agents ; pharmacology ; Carcinoma, Squamous Cell ; pathology ; Cell Culture Techniques ; Cisplatin ; pharmacology ; DNA-Binding Proteins ; analysis ; Disease Models, Animal ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Glycoproteins ; analysis ; Heterografts ; transplantation ; Hyaluronan Receptors ; analysis ; Isoenzymes ; analysis ; Male ; Mice ; Mice, Nude ; Mouth Neoplasms ; pathology ; Neoplasm Transplantation ; Neoplastic Stem Cells ; classification ; Nestin ; analysis ; Octamer Transcription Factor-3 ; analysis ; Peptides ; analysis ; Polycomb Repressive Complex 1 ; analysis ; Proto-Oncogene Proteins ; analysis ; Rabbits ; Retinal Dehydrogenase ; analysis ; Spheroids, Cellular ; classification

Hereditary diffuse leukoencephalopathy with neuroaxonal spheroids (HDLS) is a rare autosomal dominant leukoencephalopathy disease, and colony stimulating factor 1 receptor (CSF1R) is the only gene in which mutations are known to cause HDLS. HDLS should be suspected in individuals with progressive neurological decline, characteristic MR imaging findings, and positive family history. This article reviews recent advance in imaging findings, clinical manifestations, genetic counseling and management in HDLS.
Brain ; diagnostic imaging ; Humans ; Leukoencephalopathies ; diagnosis ; diagnostic imaging ; genetics ; physiopathology ; Radiography ; Spheroids, Cellular ; cytology

OBJECTIVETo imitate tumor microenvironment in vivo through construction of two-layer cell spheroid as a three-dimensional tumor model, and to validate its application in the study of Cx43 expression in colorectal cancer cell-fibroblast interactions and colorectal cancer progression.

METHODSThe two-layer cell spheroid was constructed from SW620 colorectal cancer cells and HELF fibroblasts. The expression of Cx43 in the spheroid was detected by immunocytochemistry. The expression of Cx43 in cultured SW620 cells with or without co-cultured fibroblasts was detected by immunocytochemistry and immunofluorescence. The expression of Cx43 in colorectal cancer tissue was detected by immunohistochemistry.

RESULTSThe spheroid showed well-defined cellular morphology and clear boundary between two cell lines.Significant expression of Cx43 was found along the boundary.SW620 cells had no expression of Cx43 when cultured alone, while the expression of Cx43 was induced upon co-culturing with fibroblasts.In the colorectal cancer tissue, expression of Cx43 was minimal in the centre of tumor in contrast to an upregulated expression at invasive front.

CONCLUSIONSThe two-layer cell spheroid is an observable and sensitive model for cell-cell interaction for studies of tumor microenvironment.It can simulate colorectal cancer cell-fibroblast interactions through up-regulation of Cx43 expression.

Cell Communication ; Cell Line, Tumor ; Coculture Techniques ; Colorectal Neoplasms ; metabolism ; pathology ; Connexin 43 ; metabolism ; Fibroblasts ; cytology ; Humans ; Spheroids, Cellular ; cytology ; metabolism ; Tumor Microenvironment ; Up-Regulation

OBJECTIVE: To prepare Arg-Gly-Asp (RGD) and cell penetrating peptide TAT co-modified paclitaxel loaded liposome (RGD/TAT-LP-PTX) for MCF-7 cell inhibition. METHODS: The co-modified liposome was prepared by film-ultrasonic method. The appearance, particle size and zeta potential were evaluated. The cellular uptake by MCF-7 cells in vitro was used to evaluate the targeting efficiency. The anti-proliferation efficiency of RGD/TAT-LP-PTX was evaluated by MTT assay. Tumor spheroids were used to evaluate anti-tumor ability of RGD/TAT-LP-PTX in vitro. RESULTS: The particle diameter of the co-modified liposome was (138.8 ± 12.4) nm with the Zeta potential of (25.85 ± 2.75) mV. The entrapment efficiency of PTX was 88.3%. The RGD/TAT-LP uptaken by MCF-7 cells at 4 h was 1.79 times higher than that at 2 h. The co-modified liposome uptaken by MCF-7 cells was 2.25 and 2.72 times higher than that of TAT-LP and RGD-LP, respectively. The anti-proliferation rate of RGD/TAT-LP-PTX increased with time. The inhibition rate of RGD/TAT-LP-PTX for MCF-7 cells at 48 h was 1.78 times higher than that at 24 h. The MTT assay demonstrated the cell viability of RGD/TAT-LP-PTX was 1.65, 1.82 and 2.55 times higher than that of TAT-LP-PTX, RGD-LP-PTX and LP-PTX, respectively. CONCLUSION Co-modified liposome may serve as a promising breast cancer delivery system for antitumor drugs.
Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; Cell Survival ; Humans ; Liposomes ; MCF-7 Cells ; drug effects ; Oligopeptides ; chemistry ; Paclitaxel ; pharmacology ; Particle Size ; Peptide Fragments ; chemistry ; Spheroids, Cellular ; drug effects

BACKGROUNDMonolayer cell culture models are the traditional culture models used for in vitro research of pancreatic carcinoma chemosensitivity. However, these models neglect the interactions between tumor cells and the impact of the tumor microenvironment. Such tumor cell monolayers poorly mimic the solid tumor microenvironment. The present study aimed to investigate the chemosensitivity characteristics of pancreatic cancer cells in a three-dimensional culture system by analyzing the differences in drug sensitivity between a scattered cell culture model and a multicellular spheroid culture model.

METHODSThree pancreatic cancer cell lines (SW1990, ASPC-1 and PCT-3) were cultured in three-dimensional collagen gels as well as in traditional two-dimensional monolayers. The chemosensitivities of the pancreatic carcinoma cells to 5-fluorouracil (5-FU), gemcitabine, and oxaliplatin in vitro were detected by both the Cell Counting Kit-8 test and the collagen gel droplet-embedded culture drug-sensitivity test.

RESULTSIn the two-dimensional culture model, differences in the chemosensitivities of the cloned pancreatic carcinoma cells and scattered cells existed for some concentrations of 5-FU, gemcitabine and oxaliplatin. In the three-dimensional culture model, there were significant differences in the chemosensitivities of the pancreatic cancer cells between the scattered cells and multicellular spheroids (P < 0.05).

CONCLUSIONPancreatic carcinoma cells exhibit multicellular resistance in three-dimensional cultures.

Antineoplastic Agents ; pharmacology ; Cell Culture Techniques ; methods ; Cell Line, Tumor ; Cell Survival ; drug effects ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Fluorouracil ; pharmacology ; Humans ; Organoplatinum Compounds ; pharmacology ; Pancreatic Neoplasms ; drug therapy ; Spheroids, Cellular ; drug effects

BACKGROUND AND OBJECTIVESince the proposal of the tumor stem cell hypothesis, considerable interest has been devoted to the isolation and purification of tumor stem cells. Tumor stem cell enrichment from primary tumor derived cell spheres has been demonstrated in specific, serum-free media. This goal of this study is to establish a method of cultivating floating tumor spheres from neuroblastoma cells and to confirm that neuroblastoma spheres are rich in tumor stem cells.

METHODSBone marrow aspirates were obtained from pediatric patients diagnosed with stage IV neuroblastoma. Primary tumor cells were isolated and cultivated in serum-free, stem cell-selective medium. Single sphere-forming cells were cultivated under serum-free conditions; their cloning efficiency and monoclonal tumor sphere formation rates were calculated. The expression of stem cell marker genes Oct-4 and Bmi-1 was detected by RT-PCR in sphere-forming cells and parental neurolastoma cells. Sphere-forming cells were injected into the armpit of nude mice with subsequent assessment for tumor growth. Sphere-forming cells were cultivated in differentiation medium containing 5 μmol/L 13-cis retinoic acid; changes in cell morphology were observed.

RESULTSNeuroblastoma cells formed non-adherent neurospheres under serum-free, stem cell-selective conditions after a period of 4 to 6 days. A single cell dissociated from a neurosphere could reform a monoclonal sphere; cloning efficiency and monoclonal sphere formation rates were 55.3% and 26.3%, respectively. RT-PCR results revealed heightened tumor sphere expression of Oct-4 and Bmi-1 as compared with parental tumor cells. Fourteen days after injection of 10(4) sphere-forming cells into nude mice, a neuroblastoma xenograft formed. Treatment of sphere-forming cells with 13-cis retinoic acid induced a gradual differentiation to neuronal cell morphology.

CONCLUSIONSNeuroblastoma derived tumor spheres enrich tumor stem cells and the cultivation of primary neuroblastoma cells in serum-free, stem cell-selective medium is an effective method to dissociate and purify tumor stem cells in vitro.

Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Child ; Culture Media, Serum-Free ; Humans ; Isotretinoin ; pharmacology ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplastic Stem Cells ; metabolism ; pathology ; Neuroblastoma ; metabolism ; pathology ; Nuclear Proteins ; metabolism ; Octamer Transcription Factor-3 ; metabolism ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; metabolism ; Repressor Proteins ; metabolism ; Spheroids, Cellular ; pathology ; Xenograft Model Antitumor Assays