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PubMed1990

A multicellular tumor spheroid model of cellular immunity against head and neck cancer.

A multicellular tumor spheroid (MTS) model for head and neck cancers has been used to examine the immune function of fresh and 6-day interleukin-2(IL-2)-activated peripheral blood lymphocytes (PBL). MTS are individually cultured in the presence of effector cells, and the spheroids' growth is monitored by sizing them under an inverted microscope. Dose/response studies for IL-2 (0-100 U/ml) alone and for fresh unstimulated PBL (0-10(5) cells/MTS) showed no effects on MTS growth. IL-2-activated PBL (0-10(5) cells/MTS), in contrast, modulated MTS growth in a multiphasic pattern: MTS growth was unperturbed for the first 3 days and then growth inhibition occurred, followed by MTS disintegration. Histological analysis showed that intact MTS histoarchitecture correlated with unperturbed growth, and increasing cell sloughing and MTS dissolution and replacement by activated PBL correlated with growth inhibition and disintegration. Flow-cytometric sorting of lymphocyte subset populations indicated that it was the Leu19+CD3- cells that produced these growth-modulatory effects. In contrast to the initial LAK cell resistance of MTS, single-cell suspensions demonstrated significant lysis in standard 4-h chromium-release assays. Differences between single cells and MTS suggest a potential for tissue-like organization as a factor in lymphokine-activated killing.

Carcinoma, Squamous Cell↗
PubMed1984

Growth and cellular characteristics of multicell spheroids.

The data reviewed here demonstrate that there are many similarities in growth and cellular characteristics for different types of tumor cells grown as multicell spheroids. Furthermore, where comparisons have been made many of the features of spheroids also occur in tumors in vivo. However, as for tumors, there are also many characteristics of individual types of spheroids which are relatively specific and cannot be generalized as properties of all spheroid model systems. The results also demonstrate the marked influence which cellular microenvironments regulated by a supply of oxygen and nutrients may have on the development of cellular heterogeneity. Furthermore, using spheroids it was shown that dynamic cellular and metabolic interactions exist in regulating the development of cellular subpopulations and microenvironments. Spheroids are more sensitive to alterations in culture environment than are monolayer or single-cell suspension cultures. Consequently, researchers who use this model system must characterize, optimize, and standardize the growth conditions for the spheroid cell type being investigated. This information then provides a base from which to undertake detailed studies, which are not possible in experimental tumors, of controlled manipulation of microenvironments in spheroids. The ranges of cellular microenvironments and cellular heterogeneity which exist at different stages of spheroid growth provide a model, at least in part, for coexisting size ranges of microregions in many solid tumors. Thus, spheroids provide a model, which at different stages of growth is readily manipulated and controlled experimentally, to facilitate studies of contributions of individual environmental factors, or concomitant changes in these, on cellular phenotypic expression. It is probable that the cellular changes which can be demonstrated to occur during spheroid growth, also occur in vivo. Modulation of cellular characteristics revealed by research with spheroids requires much more study to determine the mechanisms and effects on tumor cell behavior, as well as response to therapeutic agents and their relevance to tumors in vivo.

Animals↗
PubMed1986

Chemosensitivity testing in V79 spheroids: drug delivery and cellular microenvironment.

Chinese hamster V79 multicell spheroids growing in tissue culture exhibit many of the same properties as solid tumors outgrowing their blood supply, including the spontaneous development of both noncycling and hypoxic cell populations expected to be resistant to many chemotherapeutic agents. Cell-sorting techniques were used to select cells as a function of their position (depth) within the spheroid to test this prediction. "Sensitivity profiles" of cells from various regions within spheroids after treatment with doxorubicin, bleomycin, 5-fluorouracil, carmustine, cisplatin, chlorambucil, and mitomycin are presented. Additionally, exposure of preseparated cells was used to distinguish inherent sensitivity from environmental or locational factors. For these drugs, penetration was a problem only for doxorubicin; in contrast, the microenvironment in the intact spheroid had a much greater influence on cell sensitivity.

Animals↗
PubMed1989

Effect of LAK cells against three-dimensional tumor tissue. In vitro study using multi-cellular human glioma spheroids as targets.

The anti-tumor mechanisms of local LAK cell therapy are difficult to study in vivo. We describe a method to study in vitro the action of LAK cells against three-dimensional tumor tissue. Spherical cell aggregates (spheroids) grown from human glioma cell lines H-2 and U-251 were labeled with 51Cr and then incubated for up to 24 h with LAK cells. After the incubation, most spheroids were still macroscopically identifiable, and the measured reduction of volume did not correlate to the extent of damage. LAK cells infiltrated into spheroid tissue slowly as a frontier which explains why the specific 51Cr release was clearly slower from spheroids than corresponding single cell suspensions. The infiltrated area was at 1 to 2 h very thin but by 8 to 12 h consisted already of several cell layers. Most H-2 spheroids became totally infiltrated by 16 to 24 h whereas in U-251 spheroids the infiltration usually remained peripheral. In accordance with the different extent of infiltration, H-2 spheroids were clearly more sensitive to LAK cells than U-251 spheroids: at E/T ratio 10:1 the mean specific 51Cr release by 24 h was 63 and 36%, respectively. A single exposure to LAK cells released 51Cr from H-2 spheroids approximately 12 h but over 24 h from U-251 spheroids. The spheroid model can be used to study the infiltrative capacity and cytotoxicity of LAK cells against three-dimensional tumor tissue, and the method may help to find an optimal mode of local LAK cell therapy, i.e., proper combination of lymphokines and LAK cells, and proper timing of their administration.

Cell Line↗
PubMed1999

Redox-regulation of intrinsic prion expression in multicellular prostate tumor spheroids.

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.

Antioxidants↗
PubMedDate not supplied

Large-scale expansion of mammary epithelial stem cell aggregates in suspension bioreactors.

Mutations in the pathways regulating mammary epithelial stem cell (MESC) self-renewal and differentiation are currently hypothesized to result in uncontrolled cell division and, in turn, breast tumor formation. Although research is aggressively being pursued to understand how such pathways result in breast cancer formation, current studies have been greatly limited by MESC scarcity. To address this issue, this study has successfully developed large-scale expansion protocols for MESC through the subculture of murine mammary epithelial tissue aggregates, called mammospheres, in suspension bioreactors. Growth kinetics of mammospheres cultured in 125 mL suspension bioreactors and T-flasks were found to be comparable, achieving cell densities of 3.10 x 10(5) and 2.75 x 10(5) cells/mL, respectively. This corresponded to a 4-fold expansion over 8 days. Yields were also found to be strongly affected by liquid shear forces, where high agitation rates reduced overall cell numbers. Bioreactor cultures were scaled up to 1000 mL operating volumes, resulting in the production of 4.21 x 10(8) total cells (5.6-fold expansion) from a single passage. Furthermore, intermittent replacement of culture medium with fresh medium dramatically improved maximum cell densities, resulting in an 11-fold expansion, thereby enabling the generation of stem cells in quantities sufficient for standard biochemical and genetic analyses. After being cultured in suspension bioreactors for several passages, analysis by flow cytometry of Ki-67 revealed that 85% of the population was composed of proliferating cells. The successful development of expansion protocols for MESC aggregates in suspension bioreactors makes available experimental avenues that were not previously accessible for breast cancer research, thereby facilitating future investigations into elucidating the role of MESCs in breast cancer tumorigenesis.

Animals↗
PubMed1987

Multicellular spheroids. A review on cellular aggregates in cancer research.

Cellular aggregates have been used in developmental biology and in experimental cancer research for several decades. Spherical aggregates of malignant cells, i.e. multicellular tumor spheroids, may serve as in vitro models of tumor microregions and of an early, avascular stage of tumor growth. The similarities between the original tumor and the respective spheroids include volume growth kinetics, cellular heterogeneity, e.g. the induction of proliferation gradients and quiescence, as well as differentiation characteristics, such as the development of specific histological structures or the expression of antigens. Research using cell aggregates has been focussed on mechanisms involved in the control of proliferation, invasion and metastasis. Immunological studies with spheroids have resulted in the characterization of defense cells which are responsible for specific host-versus-tumor reactions. The vast majority of investigations on spheroids concerns the simulation of therapy with regard to various treatment modalities, combination treatments and systematic analyses of using various endpoints in predictive assays. Only a few pathophysiological studies on the interrelationship among tumor-specific micromilieu, cellular metabolism, proliferative status, and cellular viability have been undertaken with the spheroid model up to now. Since these studies are indicative of a large influence of the cellular microenvironment on basic biological properties of cancer cells, investigations of these epigenetic mechanisms should be intensified in future research on cell aggregates. Similarly, the molecular basis of the biological peculiarities found in malignant cells grown as three-dimensional aggregates has to be investigated more intensively.

Animals↗
PubMed1993

Characterization of morphology and cellular metabolism during the spheroid formation by fibroblasts.

Normal adult human dermal fibroblasts were cultured at 37 degrees C on a thermoresponsive substratum composed of poly-N-isopropyl acrylamide (PNIPAAm) and type I collagen. The confluent fibroblasts were forced to detach from the substratum as a monolayer cell sheet made of polygonal cells linked together with many microvilli by decreasing an ambient temperature from 37 degrees C to a temperature below the lower critical solution temperature of PNIPAAm (about 30 degrees C). The floating cell sheet shrank and condensed into an aggregate with gap and tight junctions within several hours and finally formed a spheroid by 2 days. Spheroids were covered with squamous cells and contained cuboidal cells inside. Cycloheximide and actinomycin D reversibly inhibited the spheroid formation. Extracellular matrix fibrils deposited among inner cells when the culture period extended over 7 days. A 28-day-old spheroid with a diameter of about 600 microns contained live cells even in the central region. Cellular metabolic activities such as glucose consumption, lactic acid production, and ATP content of spheroids were significantly lower than those of monolayers. The spheroid described in the present study seems to be a useful in vitro model of connective tissues.

Adenosine Triphosphate↗
PubMed1981

Increased radioresistance of cells in cultured multicell spheroids. I. Dependence on cellular interaction.

Monolayers of six different cell lines were investigated with respect to ionic coupling using micro-electrode techniques. In parallel, survival after Co-gamma-irradiation of monolayer- and spheroid cultures of these lines was compared. It was found that spheroids of coupled cell lines were more radioresistant than monolayers ("contact effect"). however, cell coupling did not enhance the survival of monolayers over single cells. This suggests that the contact effect is a "tissue phenomenon" requiring cellular interaction but is expressed only under conditions of three-dimensional growth.

Animals↗
PubMed2004

Inter-patient variation in efficacy of five oncolytic adenovirus candidates for ovarian cancer therapy.

BACKGROUND: Gene therapy offers a new strategy for cancer treatment. Adenoviruses represent the most widely used gene therapy vector and feature an excellent safety record. Conditionally replicative adenoviruses (CRAds) effect solid tumor penetration and tumor selective oncolysis and consequently offer potential efficacy for metastatic disease treatment. We evaluated five CRAds as candidate clinical agents for ovarian cancer therapy: RGDCRADcox-2R, Ad5VEGFE1, Ad5/3VEGFE1, Ad5-Delta24RGD, and Ad5/3-Delta24. METHODS: DNA replication by these five CRAds, wild-type adenovirus, and an E1-deleted control was measured in purified primary ovarian cancer cell spheroids by quantitative PCR. CRAd-mediated oncolysis was quantified in ovarian cancer cell monolayers and three-dimensional spheroids by cellular viability assays. The therapeutic efficacy of each CRAd was tested by intraperitoneal administration in mice with peritoneally disseminated human ovarian cancer. RESULTS: An increase in viral DNA was noted in primary tumor cell spheroids for all replicative viruses tested. Variation was noted in viral DNA replication between patient samples. All five CRAds induced remarkable oncolysis. They also prolonged survival in vivo compared with the wild-type control group. CONCLUSIONS: All five CRAds tested showed robust DNA replication, oncolysis, and in vivo therapeutic efficacy. Each virus has potential for clinical testing, and such further testing will ultimately determine its safety and relative usefulness. Variation of CRAd DNA replication between different patient samples suggests that target tissue features, such as surface receptors and endogenous transcription factors, may affect CRAd infectivity and replicativity. Evaluation of such factors may become important to optimize cancer therapy for individual patients.

Adenocarcinoma↗
PubMed2004

Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids.

Members of the claudin family together with occludin are the major constituents of the tight junction (TJ) complex. The human homologue of the murine CLDN1, previously called SEMP1, was identified by differential expression analysis, and the CLDN1 mRNA was found to be downregulated or completely lost in human breast cancer cells in vitro. Retroviral-induced CLDN1 reexpression in breast cancer cells results in plasma membrane homing of the protein and reconstitution of paracellular flux inhibition, which is not dependent on the presence of occludin protein. In this report, we investigated the physiologic role of CLDN1 in CLDN1-transduced MDA-MB 361 breast tumor cells in adherent 2D and suspension 3D spheroid cell cultures. Retroviral-transduced bulk cultures were FACS-sorted to enrich for 100% CLDN1-positive clonal derivatives with similar expression levels of CLDN1 mRNA and protein. There was no difference in proliferation and cell death characteristics in 2D adherent cell cultures of CLDN1-positive compared to control CLDN1-negative and mock-transduced cell cultures. In contrast, the majority of the CLDN1-transduced derivatives displayed a significant elevation of apoptosis that became evident as early as 2 days after 3D spheroid culture onset. This elevated apoptosis was independent of the volume of established spheroids. The cellular immunofluorescence analysis of CLDN1 protein expression in transduced bulk cultures revealed a CLDN1-positive subfraction with a heterogeneous pattern of membrane and cytosolic immunostaining. In the clonal MDA-MB 361 CLDN1-positive cultures, we found that a more prominent cell membrane localization correlated with a pronounced increase of apoptosis in tumor spheroids. In parallel, inhibition of the paracellular flux rate was observed. These findings support a potential role of the TJ protein CLDN1 in restricting nutrient and growth factor supplies in breast cancer cells, and they indicate that the loss of the cell membrane localization of the TJ protein CLDN1 in carcinomas may be a crucial step during tumor progression.

Apoptosis↗
PubMed1975

Cure, regression and cell survival: a comparison of common radiobiological endpoints using an in vitro tumour model.

Multicell spheroids of Chinese hamster V79-171 cells grown in suspension culture display many of the characteristics of solid tumours in vivo, and can be used as an in vitro tumour model. Two populations of spheroids differing in age and radiosensitivity were exposed to single doses of gamma-radiation and their response assayed by several techniques: (1) spheroids were reduced to single cells by trypsinization at various times post-irradiation, and viability of the single cells determined by colony formation; (2) entire spheroids were placed in petri dishes and observed for cellular outgrowth; (3)spheroid volume and cell content were monitored as a function of time after irradiation. It was found that spheroid volume changes could not be correlated with either the amount of radiation given or with the relative radiosensitivity. In contrast, the number of cells per spheroid, or cellularity, decreased exponentially with exposure dose at sufficiently long times after irradiation. Radiosensitivity was then quantified by calculating the per cent decrease of cellularity per rad. "Cure" of spheroids as defined by lack of outgrowth when entire spheroids were placed in petri dishes, correlated well with single cell survival, and was achieved 50 per cent of the time after 2,630 rads for the smaller spheroids and 3,750 rads for the larger ones. Since these spheroids contained an average of similar 7,600 and similar 30,700 cells respectively, comparison of cure data with single cell survival data showed that cures were achieved only when every cell was killed. This result may have significant therapeutic implications, since cells of the most radioresistant population of the spheroid, the chronically hypoxic internal cells, were capable of proliferation even when the spheroid was not reduced to single cells after irradiation.

Animals↗
PubMed1997

Reconstruction of trabecular tissue from human trabecular cells as a multicellular spheroid.

To understand the development of the trabecular meshwork of the eye, floating cellular aggregates (multicellular spheroids) were formed from human trabecular cells in a non-adherent environment of culture and incubated for up to one month. Dissociated trabecular cells formed multicellular spheroids within one day in the non-adherent environment, and apoptosis continued to occur in the spheroids which had been initially filled with cells. The final structure after one month appeared as a meshwork of cells with large extracellular spaces. Epidermal and basic fibroblast growth factor (EGF and bFGF) protected trabecular cells in the spheroids from apoptosis and, as a result, kept the spheroids filled with cells even after one month. In the absence of excess EGF or bFGF, the multicellular spheroids grown in vitro from human trabecular cells mimicked the mesh-like structure of normal trabecular tissue. In contrast, under an excess of these growth factors, spheroids of high cellularity, resembling the abnormal trabecular tissues of patients with congenital glaucoma, were formed.

Humans↗
PubMed1997

Transfection of reaggregating embryonic chicken retinal cells with an antisense 5'-DNA butyrylcholinesterase expression vector inhibits proliferation and alters morphogenesis.

The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5' upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate-mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina-like spheres, so-called rosetted or stratified retinospheroids. We show that antisense 5'-BChE gene expression decreased the steady-state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5'-BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation.

Animals↗
PubMed2003

Accumulation and photocytotoxicity of hypericin and analogs in two- and three-dimensional cultures of transitional cell carcinoma cells.

The aim of this study was to investigate the in vitro cellular accumulation, distribution and photocytotoxic effect of hypericin in two-dimensional (2-D) and three-dimensional (3-D) cultured RT-112 transitional cell carcinoma cells of the bladder. In addition, two iodinated derivatives of hypericin were incorporated to investigate whether these analogs, with their increased lipophilicity and heavy-atom effect, display a different biological behavior and optimized photodynamic effect. The results indicate that hypericin and mono-iodohypericin behave similarly in terms of cellular accumulation, spheroidal distribution and photocytotoxic effect. In contrast, di-iodohypericin concentrated to a higher extent in monolayers and spheroids, but the accumulation was restricted to the outermost part of the spheroid. An inverse correlation therefore seems to exist between the extent of cellular uptake under 2-D conditions and the penetration of the compounds in multicellular systems. Moreover, a less pronounced photocytotoxic effect was observed for di-iodohypericin in both 2-D and 3-D cell culture systems. It can be concluded that iodinated derivatives of hypericin do not show an increased cytotoxic effect upon irradiation in either monolayers or spheroids. Moreover, this study shows that when new photosensitizers are preclinically developed, the use of 3-D cell aggregates is critical for a correct evaluation of their efficacy.

Anthracenes↗
PubMed1995

Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.

Carcinoma↗
PubMed2006

A multicellular spheroid array to realize spheroid formation, culture, and viability assay on a chip.

We describe a novel multicellular spheroid culture system that facilitates the easy preparation and culture of a spheroid microarray for the long-term monitoring of cellular activity. A spheroid culture device with an array of pyramid-like microholes was constructed in a silicon chip that was equipped with elastomeric microchannels. A cell suspension was introduced via the microfluidic channel into the microstructure that comprised silicon microholes and elastomeric microwells. A single spheroid can be formed and localized precisely within each microstructure. Since the culture medium could be replaced via the microchannels, a long-term culture (of approximately 2 weeks) is available on the chip. Measurement of albumin production in the hepatoma cell line (HepG2) showed that the liver-specific functions were maintained for 2 weeks. Based on the cellular respiratory activity, the cellular viability of the spheroid array on the chip was evaluated using scanning electrochemical microscopy. Responses to four different chemical stimulations were simultaneously detected on the same chip, thus demonstrating that each channel could be evaluated independently under various stimulation conditions. Our spheroid culture system facilitated the understanding of spheroid formation, culture, and viability assay on a single chip, thus functioning as a useful drug-screening device for cancer and liver cells.

Albumins↗