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PubMed2005

A twelve-step program for evolving multicellularity and a division of labor.

The volvocine algae provide an unrivalled opportunity to explore details of an evolutionary pathway leading from a unicellular ancestor to multicellular organisms with a division of labor between different cell types. Members of this monophyletic group of green flagellates range in complexity from unicellular Chlamydomonas through a series of extant organisms of intermediate size and complexity to Volvox, a genus of spherical organisms that have thousands of cells and a germ-soma division of labor. It is estimated that these organisms all shared a common ancestor about 50 +/- 20 MYA. Here we outline twelve important ways in which the developmental repertoire of an ancestral unicell similar to modern C. reinhardtii was modified to produce first a small colonial organism like Gonium that was capable of swimming directionally, then a sequence of larger organisms (such as Pandorina, Eudorina and Pleodorina) in which there was an increasing tendency to differentiate two cell types, and eventually Volvox carteri with its complete germ-soma division of labor.

Animals↗
PubMed2006

Multicellular redox regulation: integrating organismal biology and redox chemistry.

Early in the 20th century, Charles Manning Child attributed organismal gradients in metabolism to interactions among groups of cells. Metabolic gradients are now firmly grounded in redox chemistry, yet modern work on metabolic signaling has consistently focused on the cellular level. Multicellular redox regulation, however, may occur when redox state is determined by the behavior of a group of cells. For instance, typically an abundance of substrate will shift the redox state of mitochondria in the direction of reduction, leading to increased reactive oxygen species (ROS). These ROS, in turn, may modify the conformation and activity of proteins involved in signaling pathways, resulting in phenotypic changes. In contrast, if substrate triggers the contractions of a muscular structure comprising mitochondrion-rich cells, the resulting metabolic demand may shift the redox state in the direction of oxidation, with a corresponding decrease of ROS and different phenotypic effects. Indeed, colonial hydroids exemplify this process. Parallel examples may occur whenever mitochondria are concentrated in cells of structures that can respond to environmental perturbations with increased metabolic demand. In these circumstances, predicting the direction of metabolic signaling may require an understanding of events at the organismal level.

Animals↗
PubMed2003

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.

Breast Neoplasms↗
PubMed1992

In vitro modulation of haematoporphyrin derivative photodynamic therapy on colorectal carcinoma multicellular spheroids by verapamil.

Photodynamic therapy has possible applications in the treatment of colorectal carcinoma. The photosensitizer haematoporphyrin derivative (HpD) is selectively retained by tumours. Agents which block p-glycoprotein, an export protein expressed to increased levels in a high proportion of colorectal carcinomas, may modulate photodynamic therapy by reducing HpD efflux from cells. Multicellular spheroids derived from the colorectal cell lines HRT18 and HT29 were incubated for 24 h with 1 microgram ml-1 HpD and 0, 1, 2 and 4 microM verapamil. Bioactivity demonstrated a dose-dependent potentiation of HpD-photodynamic therapy growth retardation. Clonogenic survival of cells disaggregated from spheroids treated with HpD-photodynamic therapy was reduced when spheroids were coincubated with verapamil. The mean(s.e.m.) efflux of HpD from spheroids into fresh medium assessed by fluorimetry was greater in spheroids treated with HpD alone (93.2(18.8) arbitrary units ml-1) than in those treated with verapamil (18.1(2.8) arbitrary units ml-1), P = 0.003. Flow cytometry demonstrated increased HpD fluorescence in cells derived from spheroids coincubated with verapamil over a range of HpD incubation concentrations. Verapamil can potentiate the bioactivity of HpD-photodynamic therapy and HpD may be a substrate for p-glycoprotein.

Colorectal Neoplasms↗
PubMed1980

Invited review: guidance cue patterns and cell migration in multicellular organisms.

In multicellular organisms, guidance cues are either diffusible molecules or cellular or extracellular surfaces that are found in reproducible locations and that orient migrating cells and cell processes. The pattern of the guidance cues usually determines the complex in vivo migration routes of motile cells and cell processes. Within organisms, guidance cues are found to be organized in two general patterns: a) broad gradients--such as diffuse chemotactic gradients; b) discrete routes (substrate pathways)--such as chemotactic gradients confined to long channels, and such as the axon surface which represents a long specific highway for migrating Schwann cells.

Amphibians↗
PubMed1980

Correlation of the growth of Chinese hamster V279-17lb multicellular spheroids with cytokinetic parameters.

The volumes of Chinese hamster V279-17lb multicellular spheroids grown in spinner culture were measured and correlated to DNA distribution and cell volume distribution information. Spheroid growth curves were obtained over a 21-day period using both electronic cell volume measurements and microscope micrometer sizing. The spheroids were collected from the aperture tube after electronic sizing, and single cell suspensions were prepared via trypsinization. Saline-washed cells were then stained with mithramycin and analyzed as unfixed or ethanol-fixed cells for both cell volume and DNA fluorescence measurements. Spheroid growth was determined to have an initial 8- to 9-day rapid growth phase, and a slow phase from 10 to 21 days. The single cell volume distributions of cells obtained from spheroids were dependent on spheroid volume and the relative proportions of small volume noncycling cells to large volume cycling cells comprising the spheroids were also dependent on spheroid volume. DNA distribution data and dual parameter DNA distribution cell volume contour patterns obtained for single cells dissociated from spheroids were closely related to the measured spheroid volume and revealed that the fraction of small, hypoxic G0-like cells was greatest in old, large spheroids.

Animals↗
PubMed1987

Viable sorting of intact multicellular spheroids by flow cytometry.

A flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly-sized populations with diameters in the range of 50-100 microns. A FACS II instrument was modified for this purpose by installing a 200-microns-diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 microns diameter) and 41-96-microns-diameter spheroids could be sorted and recovered with 70-100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small-angle forward light scatter, 90 degree light scatter, and autofluorescence using a 488-nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90 degrees light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90 degree light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5-9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as investigations of the contact effect or in the initiation of growth curve studies.

Animals↗
PubMedDate not supplied

The Dictyostelium discoideum proteome--the SWISS-2DPAGE database of the multicellular aggregate (slug).

The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2-D PAGE with immobilised pH gradients (pH 3.5-10) in the first dimension and sodium dodecyl sulfate (SDS)-PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (M(r)) against the SWISS-PROT database with the ExPASy AAcompID tool (http:// expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by cross-species matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS-2DPAGE database.

Animals↗
PubMedDate not supplied

Retinoic acid inhibition of a head and neck multicellular tumor spheroid model.

The effects of retinoic acid (RA) on multicellular tumor spheroids (MTS) derived from a head and neck squamous carcinoma cell line, MDA 886Ln, were studied. Growth of MTSs was shown to be inhibited by 10(-5) to 10(-7) M RA; dose response studies demonstrated that by 10(-10) M RA, MTS growth was inhibited by less than 20%. MTSs treated with RA for 10 days had a decreased labeling index (15% compared with 23% for controls). Histologic studies at 10 days showed both an alteration in tissue-like architecture and an inhibition of squamous differentiation in RA-treated spheroids. Morphologic inhibition of differentiation was confirmed by immunohistochemical staining for involucrin. Histologic sections were also probed with a series of biotinylated lectins to search for RA-induced changes in glycosylation. Changes in staining occurred with two lectins, soybean agglutinin and peanut agglutinin. This study showed that RA induced perturbations in biological processes such as growth and differentiation in a new model system for squamous carcinomas of the head and neck, MDA 886Ln MTS.

Carcinoma, Squamous Cell↗
PubMed2003

Reactive oxygen species-mediated regulation of eNOS and iNOS expression in multicellular prostate tumor spheroids.

Nitric oxide (NO) generated by either endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) may be involved in prostate tumorigenesis through the inhibition of reactive oxygen species (ROS)-induced apoptosis. Multicellular DU-145 prostate tumor spheroids endogenously generated NO that paralleled the production of ROS. With increasing spheroid size, eNOS expression was downregulated, whereas an upregulation of iNOS expression was observed. In parallel, NO generation declined, as evaluated by the NO indicator diaminofluorescein-2 diacetate (DAF-2DA), suggesting that NO generation in DU-145 tumor spheroids is mainly mediated by eNOS. Elevation of ROS by treatment of tumor spheroids with either buthionine sulfoximine (BSO) or hydrogen peroxide resulted in upregulation of eNOS, whereas iNOS was downregulated. Furthermore, eNOS expression was increased by epidermal growth factor (EGF) in a redox-sensitive manner. Upregulation of eNOS after treatment with hydrogen peroxide was apparently transduced through receptor tyrosine kinase signaling pathways since it was abolished by the protein kinase C (PKC) inhibitor bisindolylmaleimide-1 (BIM-1), the p21(ras) inhibitor S-trans-trans-farnesylthiosalicylic acid (FTS), the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1/2 activation. Endogenous NO may serve to escape from oxidative stress-induced apoptosis since treatment of tumor spheroids with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO) as well as the NO synthase inhibitor N-omega-amino-L-arginine (L-NAA) increased cleaved caspase-3. Consequently, lowering intracellular NO levels with either L-NAA or PTIO significantly raised ROS levels, indicating that endogenously generated NO may play a role as a ROS scavenger, thereby protecting exponentially growing tumor spheroids from ROS-induced apoptosis.

Antioxidants↗
PubMed2005

Regulation of the multidrug resistance transporter P-glycoprotein in multicellular prostate tumor spheroids by hyperthermia and reactive oxygen species.

Hyperthermia is an important component of many cancer treatment protocols. In our study the regulation of the multidrug resistance (MDR) transporter P-glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids. Hyperthermia treatment of small (50-100 microm) tumor spheroids significantly increased P-glycoprotein and mdr-1 mRNA expression with a maximum effect at 42 degrees C, whereas only moderate elevation of P-glycoprotein was found in large (350-450 microm) tumor spheroids. Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS). Inhibition of ROS generation with NADPH-oxidase inhibitors diphenylen iodonium (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P-glycoprotein expression but did not affect its transcript levels following heat treatment. This indicates that P-glycoprotein levels are controlled by regulating its translation rate or stability. Hyperthermia incubation resulted in a differential activation of p38 mitogen-activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c-jun N-terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment. Furthermore, upregulation of hypoxia-inducible factor 1alpha (HIF-1alpha) was observed. Elevation of HIF-1alpha and P-glycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580. In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF-1alpha and P-glycoprotein in the control as well as the hyperthermia-treated samples, indicating negative regulation of intrinsic HIF-1alpha and P-glycoprotein expression by ERK1,2 and JNK signaling cascades. In summary our data demonstrate that hyperthermia-induced upregulation of P-glycoprotein and HIF-1alpha is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P-glycoprotein and HIF-1alpha under control conditions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1979

Identification of murine endogenous xenotropic retrovirus in cultured multicellular tumour spheroids from nude-mouse-passaged nasopharyngeal carcinoma.

After nude-mouse-passage, a carcinoma of the nasopharynx was found to contain a few scattered C-type retrovirus particles. Culture of this nude-mouse-grown material over solid agar allowed the human tumour cells to grow as multicellular spheroids which increased in number by budding and could be subcultured for up to 6 months. Within the spheroids the human tumour cells expressed increased retrovirus replication and large numbers of C-type particles were observed. Treatment with a halogenated pyrimidine further enhanced the virus production. This tissue culture system provided sufficient virus production for the identification of the retrovirus as an endogenous xenotropic murine leukaemia virus and not a human agent.

Animals↗
PubMed1984

Multicellular spheroids grown directly from human tumour material.

Human tumour cells from surgical material were grown as multicellular spheroids. In 16 out of 20 cultures spheroids with a diameter of more than 250 microns could be observed. In 6 out of 20 cultures more than 30 spheroids with a diameter of at least 300 microns were obtained, i.e. 30% of the cultures fulfilled the criterion for a possible chemosensitivity test on primary cell spheroids. A study of stained sections from the spheroids and the respective tumours, showed that the morphology of the spheroid was very similar that of the tumour from which the cells were derived. Samples from 9 malignant melanomas, 4 bladder carcinomas, 2 renal-cell carcinomas, 2 ovarian carcinomas, 1 lymphoma, 1 colon carcinoma, and 1 schwannoma were tested for spheroid growth. Spheroids were obtained from at least one representative of all these tumour types. However, investigations involving larger numbers of tumours are needed to find out which tumour types are most suitable for further biological characterization of primary cell spheroids and tests for therapy response.

Adult↗
PubMed1985

The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs--an in vitro study using EMT6 multicellular spheroids.

Multicellular spheroids of the EMT6/Ca/VJAC mouse mammary tumour cell line have been used in an investigation of the effect of tumour geometry on the response of tumour cells to 3 cytotoxic drugs, adriamycin (ADM), nitrogen mustard (HN2) and CCNU. In addition to the inherent cellular drug response, factors related to spheroid structure, namely cell-cycle distribution, intercellular contact, drug penetration and microenvironment (pH, oxygen, glucose, etc.) are believed to influence the response of cells within spheroids to cytotoxic drugs. Selective enzymatic dissociation (with bacterial neutral protease) has been used to separate the cells within large (approximately 800 micron in diameter) spheroids into 4 distinct subpopulations. The cells within the subpopulations have been characterized by their DNA content, RNA content, tritiated thymidine labelling index, cell size and clonogenic capacity. It was found that cells at the surface of spheroids are relatively larger and more proliferative than cells towards the centre while their clonogenic capacity is similar. Studies on the responses of EMT6/Ca/VJAC log and plateau-phase monolayer cells have been carried out in parallel and have shown that cycling cells are more sensitive to ADM and HN2 than are non-cycling cells but somewhat less sensitive for the response to CCNU. Since the response patterns of cells from different regions of spheroids to HN2, treated either before disaggregation (intact spheroid) or after disaggregation (isolated spheroid cells), are similar and the surviving fraction increases from the surface towards the centre of the spheroid, cell cycle distribution is thought to be the only factor involved in the cytotoxicity of HN2 towards cells within the spheroids. Although the patterns of response to ADM of cells within intact spheroids and isolated spheroid cells are similar to those for HN2, the initial slope of the curve for intact spheroids is much steeper than that of the isolated spheroid cells. Therefore, in addition to the factor of cell-cycle distribution, drug penetration also appears to be involved in the action of ADM on spheroids, while the factors of intercellular contact and microenvironment appear to be relatively less important. The reverse pattern was found for the response of cells within different regions of spheroids to CCNU, treated as intact spheroids or as isolated spheroid cells (i.e., greater killing of inner compared with outer cells).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Modulation of growth, differentiation and glycoprotein synthesis by beta-all-trans retinoic acid in a multicellular tumor spheroid model for squamous carcinoma of the head and neck.

Cell line MDA 886Ln was established from a laryngeal lymph node metastasis. When grown as a multicellular tumor spheroid (MTS), it exhibits squamous differentiation. We studied the effects of beta-all-trans retinoic acid (RA) on the growth, differentiation and glycoprotein content of this MTS model for squamous carcinomas of the head and neck. The growth of MTSs was inhibited in a dose-dependent manner by 10(-6) to 10(-10) M RA. Growth inhibition occurred between 3 and 5 days of RA treatment (10(-6)M). Immunohistochemical and electrophoretic analyses revealed that RA suppressed the morphological markers of squamous differentiation (squames), involucrin expression, and keratin expression. Gly-coprotein expression was examined by metabolic labelling using 3H-glucosamine, in situ labelling of polyacrylamide gels with 125I-labelled wheat-germ agglutinin (WGA), localization of fluorescein isothionate-WGA in frozen sections, and determination of sialyltransferase activity. Treatment using 10(-6) M RA altered glycoprotein expression both biochemically and morphologically, and WGA was shown to bind preferentially to sialic acid residues. The sensitivity of this MTS model to RA treatment and its ability to be analyzed through morphological, immunohistochemical and biochemical techniques suggest that it will prove useful in studying the relationships between growth, differentiation and RA-induced alterations in squamous carcinomas.

Carcinoma, Squamous Cell↗
PubMed1990

Characterization of a new model of human prostatic cancer: the multicellular tumor spheroid.

Multicellular tumor spheroids (MTS) provide a closer in vitro correlate to in vivo malignancy than do conventional monolayer cultures; while simulating many parameters of in vivo growth, MTS systems provide those perquisites (i.e., experimental control, economy, expediency) associated with in vitro evaluation of preclinical therapeutic strategies. For these reasons, we exploited the proclivity of the highly metastatic human prostatic carcinoma subline I-LN-PC3-IA to spontaneously assume a spheroid morphology under routine culture conditions. I-LN spheroids demonstrate salient features described in other spheroid systems and exhibit histologic characteristics of human prostate carcinoma. Cells encompassed in the I-LN spheroid format demonstrated functional divergence from their monolayer counterparts with respect to immunoreactivity for prostatic acid phosphatase, positional dependence of prostate-restricted p40 antigen expression, and chemotherapeutic drug response. This new in vitro-in vivo transition model of human prostatic carcinoma should provide a valuable in vitro context to expediently evaluate in vivo correlates of oncolytic protocols on a malignancy that remains refractive to therapy.

Antineoplastic Agents↗
PubMed1991

Biological response of multicellular EMT6 spheroids to exogenous lactate.

The influence of elevated lactate concentrations, as found in tumor microregions, on cellular growth, viability, and metabolic state was studied employing the multicellular spheroid model. Spheroids of EMT6/Ro cells were cultured at 37 degrees C in 5% or 20% (v/v) oxygen, using stirred media with various concentrations of exogenous lactate ranging from 0.0 mM (standard conditions) to 20.0 mM. Elevated concentrations of exogenous lactate led to a considerable decrease of the maximum spheroid diameter at growth saturation, e.g., for 20% O2 from around 1700 microns to 700 microns in 0.0 and 20.0 mM lactate respectively. Histological investigations showed that the thickness of the viable cell rim was increased by elevated lactate concentrations in 20% O2, whereas this correlation was reversed in 5% O2. Cultivation of spheroids in increasing lactate concentrations was associated with a shift of metabolic pathways from net production to increased utilization of lactate in both 20% and 5% O2, as determined by standard enzymatic assays. Oxygen tension (PO2) values measured with micro-electrodes were less in spheroids cultured in high lactate (9.0 and 20.0 mM) than under standard conditions, irrespective of the external oxygen concentration. This finding reflected a substantial increase in the cellular O2 consumption with elevated external lactate levels. At given lactate concentrations, respiration rates that were derived from measured PO2 distributions by theoretical considerations were significantly lower in 5% O2 than in 20% O2.

Animals↗
PubMed1991

Efficacy and selectivity of monoclonal-antibody-targeted drugs and free methotrexate in fluorescence-labelled mixed tumour-cell monolayer cultures and multicellular spheroids.

Free methotrexate (MTX) and 2 monoclonal antibody (MAb)-MTX conjugates were tested against mixed human tumour-cell cultures, in which 2 cell lines of differing antigenicity or drug sensitivity, pre-labelled with fluorescent dyes, were added together in microtitre wells. Conjugates were selectively cytotoxic for cells bearing high concentrations of the relevant antigen, and MTX was preferentially cytotoxic for wild-type cells rather than MTX-resistant variants when tested on separate cultures. In mixed cultures these selectivities were substantially retained, although there was a varying tendency towards intermediate cytotoxicity for each cell line of the pair. MTX was cytotoxic for cell line 79 IT grown as multicellular spheroids, but a MAb-MTX conjugate and a MAb-RTA immunotoxin showed little cytotoxicity against spheroids at the highest concentrations tested, although they were highly effective against monolayer cells. Mixed spheroids could be formed efficiently from most cell lines, although in some cases cell distribution was non-random. In mixed spheroids prepared between wild-type and MTX-resistant 79 IT variants, relative MTX sensitivities conformed broadly to those seen in separate monolayer cultures. Fluorescence-labelling was a reliable method for determining cell behaviour under the above conditions. We conclude that (a) selectivity of therapeutic agents in mixed cultures was partially, but not completely, impaired compared to that observed in separate cultures, and (b) that low Mr drugs are effective against 3-dimensional tumour-cell structures but antibody-targeted conjugates are not.

Antibodies, Monoclonal↗