Cells In Spheroid Culture Research Articles

Maintenance Therapy for Pancreatic Cancer, a New Approach Based on the Synergy between the Novel Agent GP-2250 (Misetionamide) and Gemcitabine.

The novel Oxathiazinane derivative GP-2250 (Misetionamide) displays antineoplastic activity in vitro and in vivo, as previously shown in pancreatic cancer cells and in patient-derived mouse xenografts (PDX). Currently, GP 2250 is under phase I clinical trial in pancreatic ductal adenocarcinoma (PDAC). GP-2250 in combination with Gemcitabine displays a high synergistic capacity in various primary and established pancreatic cancer cell lines. Additionally, in the eight PDX models tested, the drug combination was superior in reducing tumor volume with an aggregate tumor regression (ATR) of 74% compared to Gemcitabine alone (ATR: 10%). Similarly, in a PDX maintenance setting following two weeks of treatment with nab-Paclitaxel plus Gemcitabine, the combination of GP-2250 plus Gemcitabine resulted in outstanding tumor control (ATR: 79%) compared to treatment with Gemcitabine alone (ATR: 60%). Furthermore, GP-2250 reduced the ratio of tumor-initiating CD133+ markers on the surface of PDAC cells in spheroid cultures, indicating a possible mechanism for the synergistic effect of both substances. Considering the high tolerability of GP 2250, these results may open up a new approach to maintenance therapy with GP-2250/Gemcitabine combination following nab-Paclitaxel plus Gemcitabine as first-line treatment.

Monolayer culture alters EGFR inhibitor response through abrogation of microRNA-mediated feedback regulation

Ex vivo drug screening is a potentially powerful tool for the future of cancer care, but the accuracy of results is contingent on the culture model. Both monolayer (2D) and spheroid (3D) culture systems offer advantages, but given the differences in mechanical environment, we hypothesized that that the suitability of one system over another would be critical for screening drugs with mechanical targets in mechanical tissues. HCC827 lung adenocarcinoma cells were challenged with EGFR tyrosine kinase inhibitors in monolayer and spheroid culture. RNA sequencing was performed on cells in both conditions to assess culture-induced transcriptional changes that could account for differences in drug response and differences in EGFR expression detected by immunostain. A microRNA microarray was performed to assess culture-induced differences in regulation of microRNA, and the impact of miR-146a-5p on drug response was verified by inhibition. Results were confirmed in human lung adenocarcinoma tissue. HCC827 spheroids were resistant to erlotinib and gefitinib, but significantly more sensitive in 2D culture. RNA-seq and immunostaining show a discrepancy in EGFR transcript and protein expression between the two conditions, which we attribute to miR-146a-5p. This microRNA targets EGFR and is differentially expressed between 2D and 3D culture. Inhibition of miR-146a-5p significantly increased erlotinib cytotoxicity, but validation in patient-derived spheroids suggests that the effect may be mutation-specific. Analysis of RNA-seq data suggests that cells in 2D culture become highly dependent on EGFR signaling to drive proliferation and cell spreading, resulting in a misleading level of sensitivity to EGFR TKIs, while the same cells in spheroid culture retain microRNA-driven EGFR feedback regulation that leaves them less vulnerable to EGFR inhibition. These findings underscore the need for close scrutiny of culture-induced effects on drug target regulation in model design for ex vivo drug screening.

Characterization and Study of Gene Expression Profiles of Human Periodontal Mesenchymal Stem Cells in Spheroid Cultures by Transcriptome Analysis.

A spheroid is known as a three-dimensional culture model, which better simulates the physiological conditions of stem cells. This study is aimed at identifying genes specifically expressed in spheroid-cultured human periodontal ligament mesenchymal stem cells (hPDLMSCs) using RNA-seq analysis to evaluate their functions. Transcriptome analysis was performed using spheroid and monolayer cultures of hPDLMSCs from four patients. Cluster and Gene Ontology analyses revealed that genes involved in cell-cell adhesion as well as the G2/M and G1/S transitions of mitotic cell cycles were strongly expressed in the monolayer culture group. However, genes involved in the negative regulation of cell proliferation, histone deacetylation, and bone morphogenetic protein signaling were strongly expressed in the spheroid culture group. We focused on the transcription factor nuclear receptor subfamily 4 group A member 2 (NR4A2) among the genes that were strongly expressed in the spheroid culture group and analyzed its function. To confirm the results of the transcriptome analysis, we performed real-time polymerase chain reaction and western blotting analyses. Interestingly, we found that the mRNA and protein expressions of NR4A2 were strongly expressed in the spheroid-cultured hPDLMSCs. Under osteogenic differentiation conditions, we used siRNA to knock down NR4A2 in spheroid-cultured hPDLMSCs to verify its role in osteogenesis. We found that NR4A2 knockdown significantly increased the levels of mRNA expression for osteogenesis-related genes alkaline phosphatase (ALP), Osteopontin (OPN), and type 1 collagen (COL1) (Student's paired t-test, p < 0.05). ALP activity was also significantly increased when compared to the negative control group (Student's paired t-test, p < 0.05). Additionally, spheroid-cultured hPDLMSCs transfected with siNR4A2 were cultured for 12 days, resulting in the formation of significantly larger calcified nodules compared to the negative control group (Student's paired t-test, p < 0.05). On the other hand, NR4A2 knockdown in hPDLMSC spheroid did not affect the levels of chondrogenesis and adipogenesis-related genes under chondrogenic and adipogenic conditions. These results suggest that NR4A2 negatively regulates osteogenesis in the spheroid culture of hPDLMSCs.

Modeling adaptive drug resistance of colorectal cancer and therapeutic interventions with tumor spheroids.

Drug resistance is a major barrier against successful treatments of cancer patients. Various intrinsic mechanisms and adaptive responses of tumor cells to cancer drugs often lead to failure of treatments and tumor relapse. Understanding mechanisms of cancer drug resistance is critical to develop effective treatments with sustained anti-tumor effects. Three-dimensional cultures of cancer cells known as spheroids present a biologically relevant model of avascular tumors and have been increasingly incorporated in tumor biology and cancer drug discovery studies. In this review, we discuss several recent studies from our group that utilized colorectal tumor spheroids to investigate responses of cancer cells to cytotoxic and molecularly targeted drugs and uncover mechanisms of drug resistance. We highlight our findings from both short-term, one-time treatments and long-term, cyclic treatments of tumor spheroids and discuss mechanisms of adaptation of cancer cells to the treatments. Guided by mechanisms of resistance, we demonstrate the feasibility of designing specific drug combinations to effectively block growth and resistance of cancer cells in spheroid cultures. Finally, we conclude with our perspectives on the utility of three-dimensional tumor models and their shortcomings and advantages for phenotypic and mechanistic studies of cancer drug resistance.

Upregulation of Peroxiredoxin-2 in Well-Differentiated Pancreatic Neuroendocrine Tumors and Its Utility as a Biomarker for Predicting the Response to Everolimus.

Pancreatic neuroendocrine neoplasms (pNENs) account for 2–3% of pancreatic malignancies. Peroxiredoxins (Prdxs), which are major cellular antioxidants, are involved in multiple oncogenic signaling pathways. We investigated the role of peroxiredoxin-2 in QGP-1 human pNEN cell line and patient-derived pNEN tissue. To validate the cancer stem cell-like cell characteristics of QGP-1 cells in spheroid culture, in vitro analyses and xenografting were performed. Furthermore, immunohistochemical staining was conducted to verify the overexpression of Prdx2 in pNEN tissue. Prdx2 expression was high at the mRNA and protein levels in QGP-1 cells. Prdx2 was also overexpressed in patient-derived pNEN tissue. Silencing of Prdx2 using siRNA induced overexpression and phosphorylation of ERK and AKT in QGP-1. Cell proliferation was increased by treating QGP-1 cells with siPrdx2, and the IC50 of everolimus increased suggesting resistance to everolimus. Interestingly, QGP-1 spheroid cells, which exhibited cancer stem cell-like features, exhibited lower expression of Prdx2 and mTOR. The results suggest that Prdx2 expression level and its activity may be a potential predictive biomarker for therapeutic response or resistance to everolimus in pNEN.

Mevalonate Pathway Provides Ubiquinone to Maintain Pyrimidine Synthesis and Survival in p53-Deficient Cancer Cells Exposed to Metabolic Stress.

Oncogene activation and loss of tumor suppressor function changes the metabolic activity of cancer cells to drive unrestricted proliferation. Moreover, cancer cells adapt their metabolism to sustain growth and survival when access to oxygen and nutrients is restricted, such as in poorly vascularized tumor areas. We show here that p53-deficient colon cancer cells exposed to tumor-like metabolic stress in spheroid culture activated the mevalonate pathway to promote the synthesis of ubiquinone. This was essential to maintain mitochondrial electron transport for respiration and pyrimidine synthesis in metabolically compromised environments. Induction of mevalonate pathway enzyme expression in the absence of p53 was mediated by accumulation and stabilization of mature SREBP2. Mevalonate pathway inhibition by statins blocked pyrimidine nucleotide biosynthesis and induced oxidative stress and apoptosis in p53-deficient cancer cells in spheroid culture. Moreover, ubiquinone produced by the mevalonate pathway was essential for the growth of p53-deficient tumor organoids. In contrast, inhibition of intestinal hyperproliferation by statins in an Apc/KrasG12D-mutant mouse model was independent of de novo pyrimidine synthesis. Our results highlight the importance of the mevalonate pathway for maintaining mitochondrial electron transfer and biosynthetic activity in cancer cells exposed to metabolic stress. They also demonstrate that the metabolic output of this pathway depends on both genetic and environmental context. SIGNIFICANCE: These findings suggest that p53-deficient cancer cells activate the mevalonate pathway via SREBP2 and promote the synthesis of ubiquinone that plays an essential role in reducing oxidative stress and supports the synthesis of pyrimidine nucleotide.

Changes in characteristics of periodontal ligament stem cells in spheroid culture.

The periodontal ligament (PDL) has important roles in maintaining homeostasis, wound healing, and regeneration of periodontal tissues by supplying stem/progenitor cells. Periodontal ligament stem cells (PDLSCs) have mesenchymal stem cell (MSC)-like characteristics and can be isolated from periodontal tissues. The aim of this study was to examine the effect of three-dimensional spheroid culture on the characteristics of PDLSCs. Periodontal ligament stem cells were isolated and cultured from healthy teeth, and PDLSC spheroids were formed by pellet culture in polypropylene tubes. The proliferation of PDLSCs in spheroids and conventional two-dimensional (2D) cultures were examined by immunostaining for Ki67. Cell death and cell size were analyzed using flow cytometry. Gene expression changes were investigated by quantitative real time PCR. Periodontal ligament stem cells spontaneously formed spheroid masses in pellet culture. The size of PDLSC spheroids was inversely proportional to the culture period. Fewer Ki67-positive cells were detected in PDLSC spheroids compared to those in 2D culture. Flow cytometry revealed an increase in dead cells and a decrease in cell size in PDLSC spheroids. The expression levels of genes related to anti-inflammation (TSG6, COX2, MnSOD) and angiogenesis (VEGF, bFGF, HGF) were drastically increased by spheroid culture compared to 2D culture. TSG6 gene expression was inhibited in PDLSC spheroids in the presence of the apoptosis signal inhibitor, Z-VAD-FMK. Additionally, PDLSC spheroid transplantation into rat periodontal defects did not induce the regeneration of periodontal tissues. We found that spheroid culture of PDLSCs affected several characteristics of PDLSCs, including the expression of genes related to anti-inflammation and angiogenesis; apoptosis signaling may be involved in these changes. Our results revealed the characteristics of PDLSCs in spheroid culture and have provided new information to the field of stem cell research.

C-Kit+ Cells in Adult Salivary Glands do not Function as Tissue Stem Cells

A rare population of salivary gland cells isolated based on c-Kit immunoreactivity are thought to represent tissue stem cells since they exhibit the most robust proliferative and differentiation capacity ex vivo. Despite their high promise for cell-based therapies aimed at restoring salivary function, the precise location and in vivo function of c-Kit+ stem cells remain unclear. Here, by combining immunostaining with c-KitCreERT2-based genetic labeling and lineage tracing in the adult mouse salivary glands, we show that c-Kit is expressed in a relatively large and heterogeneous cell population that consists mostly of differentiated cells. Moreover, c-Kit does not mark ductal stem cells that are known to express cytokeratin K14. Tracking the fate of in vivo-labeled c-Kit+ or that of K14+ cells in spheroid cultures reveals a limited proliferative potential for c-Kit+ cells and identifies K14+ cells as the major source of salispheres in these cultures. Long-term in vivo lineage tracing studies indicate that although c-Kit marks at least two discrete ductal cell lineages, c-Kit+ cells do not contribute to the normal maintenance of any other cell lineages. Our results indicate that c-Kit is not a reliable marker for salivary gland stem cells, which has important implications for salivary gland regenerative therapies.

CRISPR-Cas9 targeting of MMP13 in human chondrocytes leads to significantly reduced levels of the metalloproteinase and enhanced type II collagen accumulation

To investigate the efficacy of CRISPR-Cas9 mediated editing in human chondrocytes, and to develop a genome editing approach relevant to cell-based repair. Transfection of human articular chondrocytes (both healthy and osteoarthritic) with ribonucleoprotein complexes (RNP) containing Cas9 and a crisprRNA targeting exon2 of MMP13 was performed to assess editing efficiency and effects on MMP13 protein levels and enzymatic activity. Using spheroid cultures, protein levels of a major target of MMP13, type II collagen, were assessed by western blot and immunofluorescence. With an editing efficiency of 63-74%, secreted MMP13 protein levels and activity were significantly reduced (percentage decrease 34.14% without and 67.97% with IL-1β based on median values of MMP13 enzymatic activity, N=7) comparing non-edited with edited cell populations using an exon-targeting gRNA resulting in premature stop codons through non-homologous end joining (NHEJ). Accumulation of cartilage matrix protein type II collagen was enhanced in edited cells in spheroid culture, compared to non-edited controls. CRISPR-Cas9 mediated genome editing can be used to efficiently and reproducibly establish populations of human chondrocytes with stably reduced expression of key genes of interest without the need for clonal selection. Such an editing approach has the potential to greatly enhance current cell-based therapies for cartilage repair.

Integrin α2β1 decelerates proliferation, but promotes survival and invasion of prostate cancer cells.

High expression level of integrin α2β1 is a hallmark of prostate cancer stem cell like cells. The role of this collagen receptor is controversial since it is down regulated in poorly differentiated carcinomas, but concomitantly proposed to promote metastasis. Here, we show that docetaxel resistant DU145 prostate cancer cells express high levels of α2β1 and that α2β1High subpopulation of DU145 cells proliferates slower than the cells representing α2β1Low subpopulation. To further study this initial observation we used Crispr/Cas9 technology to create an α2β1 negative DU145 cell line. Furthermore, we performed rescue experiment by transfecting α2 knockout cells with vector carrying α2 cDNA or with an empty vector for appropriate control. When these two cell lines were compared, α2β1 positive cells proliferated slower, were more resistant to docetaxel and also migrated more effectively on collagen and invaded faster through matrigel or collagen. Integrin α2β1 was demonstrated to be a positive regulator of p38 MAPK phosphorylation and a selective p38 inhibitor (SB203580) promoted proliferation and inhibited invasion. Effects of α2β1 integrin on the global gene expression pattern of DU145 cells in spheroid cultures were studied by RNA sequencing. Integrin α2β1 was shown to regulate several cancer progression related genes, most notably matrix metalloproteinase-1 (MMP-1), a recognized invasion promoting protein. To conclude, the fact that α2β1 decelerates cell proliferation may explain the dominance of α2β1 negative/low cells in primary sites of poorly differentiated carcinomas, while the critical role of α2β1 integrin in invasion stresses the importance of this adhesion receptor in cancer dissemination.

Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells

The aim of this study was to investigate the effect of hyperthermia, 6MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133+/CD44+ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133+/CD44+ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6Gy) and hyperthermia (60 and 90min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.

Proton NMR characterization of intact primary and metastatic melanoma cells in 2D & 3D cultures

ObjectiveTo characterize the differences between the primary and metastatic melanoma cell lines grown in 2D cultures and 3D cultures.MethodsPrimary melanoma cells (WM115) and metastatic melanoma cells (WM266) extracted from a single donor was cultured in 2D as well as 3D cultures. These cells were characterized using proton NMR spectrometry, and the qualitative chemical shifts markers were identified and discussed.ResultsIn monolayer culture (2D), we observed one qualitative chemical shift marker for primary melanoma cells. In spheroid cultures (3D), we observed nine significant chemical shifts, of which eight markers were specific for primary melanoma spheroids, whereas the other one marker was specific to metastatic melanoma spheroids. This study suggests that the glucose accumulation and phospholipid composition vary significantly between the primary and metastatic cells lines that are obtained from a single donor and also with the cell culturing methods. 14 qualitative chemical shift markers were obtained in the comparison between monolayer culture and spheroids cultures irrespective of the differences in the cell lines. Among which 4 were unique to monolayer cultures whereas 10 chemical shifts were unique to the spheroid cultures. This study also shows that the method of cell culture would drastically affect the phospholipid composition of the cells and also depicts that the cells in spheroid culture closely resembles the cells in vivo.ConclusionThis study shows the high specificity of proton NMR spectrometry in characterizing cancer cell lines and also shows the variations in the glucose accumulation and phospholipid composition between the primary and metastatic melanoma cell lines from the same donor. Differences in the cell culture method does plays an important role in phospholipid composition of the cells.

Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes

PurposeCorneal stromal cells transform to precursor cells in spheroid culture. We determined whether keratocytes derived from spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes.MethodsSpheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes.ResultsStromal cells formed spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in spheroids as compared to adherent cells whereas alpha-smooth muscle actin (α-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and α-SMA in spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the spheroids.ConclusionStemness transcription factor genes are upregulated in spheroids. Keratocytes derived from spheroids resemble tissue resident keratocytes, thus increasing manifolds the quantity of these cells for in-vitro experiments.

In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture

It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell-enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to genetic modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell-enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.

Expression of CD133 in SW620 colorectal cancer cells is modulated by the microenvironment

The use of CD133 as a marker for identifying and isolating colon cancer stem cells (CSCs) is controversial. In particular, it is not clear whether the pattern of expression is influenced by the tumor microenvironment. We analysed the expression of CD133 in the SW620 colon cancer cell line, which produces subpopulations with different levels of CD133. CD133(neg) and CD133(hi) SW620 cells were isolated and compared in hanging drop spheroid cultures. The expression of CD133 was examined under different culture conditions and following nutrient withdrawal. The results showed that SW620 cells express the CD133 antigen at variable levels. CD133(neg) and CD133(hi) SW620 cells can be separated by fluorescence-activated cell sorting and, when grown as adherent cells in liquid culture, are serially passaged without changes to the CD133 levels. However, the CD133 antigen is re-expressed when the CD133(neg) cells are deprived of nutrients or grown as non-adherent cells in spheroid cultures. Our observations indicate that the presence of the CD133 antigen does not identify a stem cell population of SW620 colorectal cancer cells. The expression of CD133 is dynamic and is modulated during certain cell culture conditions, suggesting that changes in expression may be mediated by the energy supply available to the cells.

Dienogest inhibits aromatase and cyclooxygenase-2 expression and prostaglandin E 2 production in human endometriotic stromal cells in spheroid culture

To determine the effect of dienogest (DNG) on the expression of aromatase and cyclooxygenase-2 (COX-2) and the production of prostaglandin E(2) (PGE(2)) in human endometriotic stromal cells (ESCs). Experimental study in vitro. University hospital. Seventeen patients with ovarian endometrioma. ESCs from chocolate cyst linings of ovaries were treated with DNG. Expression of aromatase and COX-2 evaluated in spheroid cultures of human ESCs by real-time quantitative polymerase chain-reaction and immunocytochemistry, production of PGE(2) quantified by enzyme-linked immunosorbent assay (ELISA), and nuclear factor kappa B (NF-κB) DNA-binding examined by ELISA and immunocytochemistry. The pharmaceutical actions of DNG on the expression of aromatase and COX-2 and the production of PGE(2) were examined using spheroid cultures of human ESCs. More aromatase, COX-2, and PGE(2) were expressed in spheroid cultures than in conventional ESCs monolayers. In the spheroid cultures, DNG (10(-7) M) and progesterone (10(-7) M) inhibited the expression of aromatase, COX-2, and PGE(2). DNG also inhibited NF-κB DNA-binding activity and reduced the immunocytochemical protein expression of aromatase, COX-2, and NF-κB p50 nuclear localization. Because DNG inhibits aromatase and COX-2 expression as well as PGE(2) production in ESCs, these pharmacologic features might contribute to a therapeutic effect of DNG on endometriosis.

Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells

Human hepatic cancer cells, HepG2, formed spheroids on a poly-L-glutamic acid-coated dish. Doxorubicin (DOX) efflux activity of the cells in spheroid culture was higher than that in monolayer culture due to the higher expression of MDR1 protein of the cells in spheroids compared with those in monolayer. The amount of MDR1 per cell in spheroids was similar to that of hepatic tumor tissue in vivo. Consequently, it was suggested that the drug efflux activity of cells in spheroid culture reflected the activity of hepatic cancer cells. Furthermore, the IC(50) of DOX and epirubicin (EPI) in HepG2 cells, both of which are known to be exported by MDR1, were higher in spheroid compared with monolayer cells, while IC(50) of 5-fluorouracil (5-FU), which is not exported by MDR1 protein, was almost the same in both types of culture. The higher IC(50) of DOX and EPI in HepG2 cells in spheroid culture was associated with a higher efflux activity of the drugs in the spheroid-cultured cells, which appeared to reflect the IC(50) of DOX and EPI in cancer cells in vivo. Therefore, a spheroid culture of hepatic cancer cells seems to provide a promising cell-based in vitro assay system for examining the proper IC(50) values of anticancer agents that would reflect the drug resistance of cancer cells in vivo. In addition, the system would be useful in screening for inhibitors of MDR1 activity, which will help to overcome the multidrug resistance of cancer cells.

Nitric oxide attenuates resistance to doxorubicin in three-dimensional aggregates of human breast carcinoma cells

Compared with monolayer culture, tumour cells cultured as multicellular aggregates (spheroids) exhibit much higher levels of resistance to chemotherapeutic agents, a phenomenon known as multicellular resistance (MCR). Associated with multicellular aggregates is a heterogeneous microenvironment characterised by gradients in oxygen, pH, and nutrients. We previously showed that nitric oxide (NO) signalling plays an important role in the regulation of chemosensitivity in cancer cells cultured as monolayer, and that hypoxia increases resistance to anti-cancer agents largely through a mechanism involving the inhibition of NO signalling. The goal of the present study was to determine whether NO mimetics chemosensitize breast cancer cells in spheroid cultures. Survival of MDA-MB-231 breast carcinoma cells was determined by clonogenic assay following spheroid culture, doxorubicin exposure, and NO mimetic administration. When spheroids were incubated for 24 h with the NO mimetics diethylenetriamine/nitric oxide adduct (DETA/NO) and glyceryl trinitrate (GTN), cell survival after doxorubicin (200 microM) exposure was decreased by 33% (p<0.006) and by up to 47% (p<0.02), respectively. Nitric oxide-mediated signalling involves the generation of the second messenger cyclic guanosine monophosphate (cGMP). Administration of a non-hydrolysable cGMP analogue, 8-Bromo-cGMP, significantly decreased MCR (p<0.04). The effect of NO mimetic exposure on tumour cell chemosensitivity was not due to increased penetration of doxorubicin into spheroids, nor was it associated with an increase in cell proliferation. These results suggest that NO mimetics attenuate MCR to doxorubicin through a mechanism involving cGMP-dependent signalling. Therefore, NO-mimetics may potentially be used as chemosensitizers in cancer therapy.

Spheroid formation and functional restoration of human fetal hepatocytes on poly–amino acid-coated dishes after serial proliferation

Primary human fetal hepatocytes proliferated in monolayer culture up to the 9th passage. During proliferation, the cells changed their morphology from a fibroblast-like shape after inoculation to an epithelia-like polygonal shape after they reached confluence. The proliferation was associated with the loss of ammonia detoxification capacity, which is essential for the function of bioartificial liver. The cells formed spheroids on a poly-glutamic acid- or poly-aspartic acid-coated polystyrene dish that had a negatively charged surface at neutral pH. However, the cells did not form spheroids on a poly-lysine- or poly-arginine-coated dish that had a positively charged surface, which is reportedly suitable to form spheroids for adult hepatocytes. The activity of cytochrome P450 (CYP 1A1, CYP1A2) of the cells in spheroid culture was about twice as high as that of the cells in monolayer culture. The ammonia detoxification activity of the cells was restored in spheroid culture by treatment with 2% dimethylsulfoxide. These results suggest that the conditions for human fetal hepatocytes to form spheroids are different from that for adult hepatocytes, and the use of poly-glutamic acid or poly-aspartic acid coating may improve spheroid culture of proliferative human fetal hepatocytes.