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Purpose: Based on previous findings in SCC of the human uterine cervix and of the head and neck, metastasis- and survival-related metabolic classification was performed in human rectal adenocarcinomas. Such a characterization is suggested as basis of a novel clinical guideline for an appropriate therapy of epithelium-derived malignancies., Methods: Tissue concentrations of ATP, glucose, and lactate in viable tumor regions of 33 pretherapeutically taken cryobiopsies from 24 patients were measured at a microscopic level using the technique of imaging bioluminescence., Results: In metastatic carcinomas, lactate levels were significantly higher (mean+/-SD: 13.4+/-3.8 vs 6.9+/-2.5 micro mol/g, P = 0.0046) and glucose levels significantly lower (0.3+/-0.2 vs 1.0+/-0.4 micro mol/g, P = 0.0020) than in non-metastatic carcinomas. No patients had distant metastasis with tumor lactate levels below the population median of 8.0 micro mol/g or glucose levels above 0.9 micro mol/g., Conclusions: These results support the hypothesis that lactate and glucose levels measured in human primary carcinomas may serve as an early prognostic tool in the clinic with regard to formation of metastasis and patient survival. The present data encourage further studies on possible strategies for a metabolic classification of tumors as a prognostic tool.

Tumor-associated fibroblasts (TAF) and tumor-associated macrophages are the main stromal components in desmoplastic breast tumors. These host cell types were extensively studied individually with regard to tumor development and progression but little is known about their reciprocal interactions. To elucidate the role of TAF in the recruitment of monocytes (MO) we designed a 3D co-culture system of multicellular fibroblast spheroids of different origin, co-cultured with MO suspensions from healthy donors. Spheroids of tumor-derived but not of normal fibroblasts were extensively infiltrated by MO. A linear correlation between number of infiltrated MO and number of MO applied per spheroid was shown, indicating a distinct migratory MO subpopulation (approximately 15%) within the peripheral blood MO pool. Our data imply that MO migration into fibroblastic tumor areas may partially result from high expression of CCL2 (monocyte chemotactic protein-1), which was regulated by endogenous IL-6 as shown by neutralization experiments. The effect of CCL2 on MO migration was inhibited by CCL2 neutralizing antibody in tumor-derived fibroblast conditioned media in a Boyden chamber migration assay but not in spheroid culture. While this phenomenon needs further evaluation, our data clearly support the concept of fibroblasts as "sentinel cells" relevant for tumor progression.

Transforming growth factor-beta (TGF-beta) is known to induce alpha-smooth muscle actin (alpha-SMA) in fibroblasts and is supposed to play a role in myofibroblast differentiation and tumor desmoplasia. Our objective was to elucidate the impact of TGF-beta1 on alpha-SMA expression in fibroblasts in a three-dimensional (3-D) vs. two-dimensional (2-D) environment. In monolayer culture, all fibroblast cultures responded in a similar fashion to TGF-beta1 with regard to alpha-SMA expression. In fibroblast spheroids, alpha-SMA expression was reduced and induction by TGF-beta1 was highly variable. This difference correlated with a differential regulation in the TGF-beta receptor (TGFbetaR) expression, in particular with a reduction in TGF-betaRII in part of the fibroblast types. Our data indicate that 1) sensitivity to TGF-beta1-induced alpha-SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast type-independent regulatory mechanisms, such as a general reduction/loss in TGF-betaRIII, contribute to an altered TGFbetaR expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGFbetaR types I and II determine the sensitivity to TGF-beta1-induced alpha-SMA expression in the 3-D setting. We suggest that fibroblasts that can be induced by TGF-beta1 to produce alpha-SMA in spheroid culture reflect a "premyofibroblastic" phenotype.

To develop effective therapeutic strategies aimed at treating tumor metastasis, critical steps in this process must be better understood. For this purpose we have established a new model to visualize and quantify early metastasis. Murine CT-26 colon adenocarcinoma cells were stably transfected with green fluorescent protein (GFP). Tumor cells were intraportally delivered to the liver of Balb/c mice and subsequently tracked by intravital fluorescence microscopy. Coinjection of fluorescent beads and in vivo propidium iodide staining allowed examination of initial tumor cell arrest, extravasation, viability and proliferation. Results showed that GFP-transfection compared to conventional labeling procedures (Calcein, cytoplasmic microspheres) did not alter early metastatic properties. However, the long-term development of liver metastases expressing GFP was markedly reduced compared to wild type CT-26 tumor cells. An increase in the size and the number of liver metastases in T- and B-cell-deficient SCID mice suggested an immune response to the GFP transfected cells responsible for the reduced metastatic growth in wild-type mice. Based on our findings, this model can be used to examine the early steps of metastasis in vivo. However, in immunocompetent mice, the use of GFP-labeled tumor cells should be limited to tracking cell arrest and extravasation, whereas evaluations of long-term metastatic growth should be performed in immunodeficient mice.

Most malignant tumors contain so-called tumor-associated macrophages (TAM) as a major component of their leukocytic infiltrate. To investigate the impact of the tumor microenvironment on activation and differentiation of macrophages, we established a 3-dimensional model system by culturing human monocytes within multicellular tumor spheroids. After 7 days, monocyte-derived TAM were isolated and analyzed for phenotypic alterations as compared to macrophages cultured without tumor cell contact. We found the known macrophage differentiation marker Carboxypeptidase M to be suppressed while CD14, HLA-DR, and CD16 were up-regulated. Using Differential Display, we identified several genes that were differentially expressed between TAM and control macrophages. Prolidase, a peptidase known to influence the chemoattraction of neutrophils and macrophage activity, was down-regulated in TAM. In contrast, the Toll-like receptor family-related molecules MD-1 and RP105 were up-regulated by tumor cell contact, both at the RNA and protein level. From our data we conclude that TAM represent a distict macrophage population characterized by low expression of differentiation-associated macrophage antigens but also by a constitutive state of activation.

Aim: Monoclonal antibodies against the human homologue of mouse macrosialin, CD68, are generally commercialized as markers for human monocytes and macrophages. Indeed, CD68 is considered as a selective marker for human myeloid cells, although several previous immunohistochemical studies indicate that some antibody clones also react with other hematopoietic and non-hematopoietic cell types. The aim of our study was to verify these observations and to evaluate the reliability of CD68 as a macrophage marker., Methods: We investigated protein and RNA expression of CD68 in various fibroblast types and carcinoma cell lines as compared to monocytes and macrophages using immunohistochemistry, flow cytometry, and specific RT-PCR. Different monoclonal antibody clones against CD68 were applied including KP-1 and EBM11., Results: As expected, the intensity of immunohistochemical and flow cytometric CD68 staining was dependent on both the antibody clone and the fixation procedure. However, fibroblasts isolated from normal skin, normal breast, breast tumor tissue, and osteoarthritis synovia clearly expressed CD68 protein at levels comparable to macrophages. The specificity of CD68 expression in fibroblasts was verified by RT-PCR which also showed some tumor cell types to express CD68 mRNA., Conclusion: Our findings clearly demonstrate that the expression of CD68 is not restricted to the macrophage lineage. This is highly relevant for experimental and diagnostic purposes, since anti CD68 antibodies cannot be accepted without reservations for the discrimination of myeloid cells and fibroblasts even in paraffin sections after formalin fixation.

Fibroblasts are the most abundant stromal cell type in desmoplastic ductal invasive breast tumors, are a substrate of tumor cell invasion and modify tumor cell behavior. However, the complex interaction between tumor cells and fibroblasts remains incompletely understood. Our aim was to identify candidate genes in tumor cells that are affected by fibroblasts and to reveal new molecules involved in this heterologous interaction. To reflect the in vivo situation, a 3-D multicellular spheroid coculture model of breast tumor cells and fibroblasts was combined with cell separation technology (MACS and FACS) and molecular analyses, including RAP-PCR, semiquantitative RT-PCR and reverse Northern blotting. Three tumor cell lines (BT474, T47D and MCF-7) with different invasive potential were applied in coculture with normal skin and/or breast tumor-derived fibroblasts. With the techniques mentioned, 18 different bands in the tumor cDNA band pattern of mono- as opposed to cocultures were identified. One of the mRNAs is considered worth further analysis: rack1. Rack1 mRNA was reproducibly suppressed in tumor cells following interaction with fibroblasts. Downregulation was recorded in all tumor cell lines and for cocultures with both types of fibroblast. This indicates a general regulatory mechanism between epithelial tumor cells and fibroblasts, which may be an interesting tumor cell differentiation-independent target for therapy., (Copyright 2002 Wiley-Liss, Inc.)

Seeding of hematopoietic progenitor cells (HPC) into the bone marrow requires a complex interaction between cell membrane and adhesion systems and cell signaling pathways. We established a multicellular, spheroid coculture model to study HPC migration in a three-dimensional stromal environment. Here, entry of primary CD34(+) cells into stroma cell spheroids was independent of the integrins very late antigen (VLA)-4, VLA-5, lymphocyte function-associated antigen-1, and the chemokine receptor CXCR4. Experiments using a panel of bacterial toxins selectively targeting key regulators of cellular locomotion, the Rho family small GTPases Rho, Rac, and Cdc42, revealed a considerable reduction or even abrogation of TF-1 cell migration without an increase of apoptosis or impairment of proliferation. Pertussis toxin, an inhibitor of Galpha(i) proteins, showed a similar effect. In some in vitro invasion assays, phosphatidylinositol-3 kinase (PI-3K) was shown to mediate Rac- and Cdc42-induced cell motility and invasion. However, inhibition of the PI-3K pathway by LY294002 did not impair TF-1 cell migration in our three-dimensional model system.

Rat1-T1 and MR1 spheroids represent separate transformed phenotypes originated from the same rat fibroblasts that differ in three-dimensional (3D) growth kinetics, histological structure, and oxygenation status. In the present study, (31)P-NMR spectroscopy of perfused spheroid suspensions was used to investigate cellular energetics relative to 3D growth, development of necrosis, and cell cycle distribution. Both spheroid types were characterized by a remarkably low amount of free (inorganic) phosphate (P(i)) and a low phosphocreatine peak. The ratio of nucleoside triphosphate (NTP) to P(i) ranged between 1.5 and 2.0. Intracellular pH, NTP-to-P(i) ratio, and NTP/cell remained constant throughout spheroid growth, being unaffected by the emergence of oxygen deficiency, cell quiescence, and necrosis. However, a 50% decrease in the ratio of the lipid precursors phosphorylcholine and phosphorylethanolamine (PC/PE) was observed with increasing spheroid size and was correlated with an increased G(1)/G(0) phase cell fraction. In addition, the ratio of the phospholipid degradation products glycerophosphorylcholine and glycerophosphorylethanolamine (GPC/GPE) increased with spheroid diameter in Rat1-T1 aggregates. We conclude that changes in phospholipid metabolism, rather than alterations in energy-rich phosphates, reflect cell quiescence in spheroid cultures, because cells in the inner oxygen-deficient zones seem to adapt their energy metabolism to the environmental conditions before necrotic cell destruction.

Phenotypic and functional characteristics of tumor associated fibroblasts (TAF) in contrast to normal fibroblasts are reviewed in this first synopsis (part I). Terms as tumor stroma, desmo-plasia, TAF, myofibroblast, and fetal-type fibroblast are defined, and experimental systems to study heterologous cell interactions are presented. While we only start to gather information on the genotype of TAF, a broad range of data deals with the expression profile of these cells, covering e.g. ECM and ECM-modulating molecules, growth factors and cytokines. Summarizing the recent state of knowledge indicates that TAF provide sources for tumor diagnosis and therapy, that have to be further defined in an organ-specific approach in terms of the functional impact on the tumor cell and its environment (see part II).

The article focuses on the functional impact of tumor-associated fibroblasts (TAF) on its surrounding cells. It intends to cover the recent knowledge on TAF, the phenotype, and expression profile of which have been described in the first part of the review series (Kunz-Schughart and Knuechel, 2002). The present review is subdivided into two main chapters: (1) functional impact of TAF on tumor cells and (2) fibroblast-host cell interactions in tumor tissue. In the first paragraph of chapter (1) about the role of fibroblasts in tumor cell growth and differentiation it is revealed, how strongly cellular interaction is dependent on fibroblast and tumor cell type as well as the spatial ratio between the cells. The variation of cellular behavior depending on quantity of molecules holds also true for the group of ECM molecules, e.g. the balance between MMPs and TIMPs, which provide an interesting therapeutic target in tumor tissue. This is one of the topics addressed in the second paragraph which focuses on tumor cell dissemination. Chapter (2) addresses the relation of TAF to other intra- or peritumoral host cells. The hypoxia-related angiogenesis induction of fibroblasts via growth factor secretion (e.g. VEGF) is considered as important as the immune modulatory properties of fibroblasts on immune cells, such as monocytes/macrophages. These cellular properties can be tested under controlled conditions in three-dimensional heterologous cultures of human cells, providing the chance for systematic modification to assess therapeutic effects in an in vivo like environment.

Invasion and dissemination of well-differentiated carcinomas are often associated with loss of epithelial differentiation and gain of mesenchyme-like capabilities of the tumor cells at the invasive front. However, when comparing central areas of primary colorectal carcinomas and corresponding metastases, we again found the same differentiated epithelial growth patterns. These characteristic phenotypic changes were associated with distinct expression patterns of beta-catenin, the main oncogenic protein in colorectal carcinomas, and E-cadherin. Nuclear beta-catenin was found in dedifferentiated mesenchyme-like tumor cells at the invasive front, but strikingly, as in central areas of the primary tumors, was localized to the membrane and cytoplasm in polarized epithelial tumor cells in the metastases. This expression pattern was accompanied by changes in E-cadherin expression and proliferative activity. On the basis of these data, we postulate that an important driving force for progression of well-differentiated colorectal carcinomas is the specific environment, initiating two transient phenotypic transition processes by modulating intracellular beta-catenin distribution in tumor cells.

The objective of our study was to establish spheroid cocultures as a valid 3-D in vitro model mimicking tumor-fibroblast interactions in scirrhous breast tumors. The experimental setup was designed to verify if in cocultures (a) adherence and migration reflect the invasive potential of breast tumor cells, (b) breast tumor cells induce tumor-associated fibroblast differentiation, and (c) tumor-derived fibroblasts better reflect the in vivo situation than normal skin fibroblasts. Only one (SK-BR-3) out of five tumor cell types showed extensive fibroblast infiltration, MCF-7 cells frequently invaded fibroblast spheroids; BT474, T47D, and ZR-75-1 were noninvasive. While tumor cell invasion was independent of fibroblast origin, tumor-associated myofibroblast differentiation defined by alpha-SMA expression was demonstrated for tumor-derived but not normal skin fibroblasts in coculture indicating that (a) tumor cell invasion and myofibroblast differentiation are autonomous processes and (b) cocultures with tumor-derived fibroblasts resemble advanced stages of desmoplastic carcinomas while cocultures with normal skin fibroblasts rather reflect the early tumor development. The latter is also implied by fibroblast-associated alterations in tumor cell morphology and ECM distribution in the system. By using RNA arbitrarily primed PCR and cells isolated from cocultures by fluorescence-activated and magnetic cell separation, peripheral myelin protein PMP22/SR13 has been identified as a novel candidate with potential relevance in the interaction between tumor cell and normal fibroblast since PMP22 mRNA was significantly reduced in normal skin fibroblasts in coculture with BT474 cells., (Copyright 2001 Academic Press.)

The purpose of the present study was to examine mitochondrial function in differently transformed cells relative to their tumorigenic state and proliferative activity in vitro. An established two-step carcinogenesis model consisting of immortal and tumorigenic rat embryo fibroblasts that can be cultured as monolayers and multicellular spheroids was investigated. Flow cytometric measurements were carried out using the two mitochondrial-specific fluorochromes rhodamine 123 (Rh123) and 10-N-nonyl acridine orange (NAO), in combination with the DNA dye Hoechst 33342 for simultaneous cell cycle analysis. Since the accumulation of Rh123 depends on mitochondrial membrane potential, Rh123 fluorescence intensity gives an estimate of mitochondrial activity per cell, as determined by both overall mitochondrial function and mass. In contrast, NAO uptake reflects mitochondrial mass only, as it binds to cardiolipin in the inner mitochondrial membrane independently of membrane potential. Aliquots of cell suspensions derived from exponential monolayer, confluent monolayer, and a range of sizes of multicellular spheroids were stained with either Rh123 or NAO and Hoechst 33342, then mitochondrial mass and activity per unit cell volume and cellular DNA content were measured by flow cytometry. Differences in the average mitochondrial activity per cell in different cell lines and culture conditions were primarily due to alterations in cell volume. Importantly, tumorigenic conversion by ras-transfection did not consistently change mitochondrial activity per unit cell volume. The mitochondrial mass per unit cell volume increased for all cells when cellular quiescence was induced, either in monolayers or spheroids. However, mitochondrial function (activity/mass) decreased when cells became quiescent, resulting in a positive correlation between mitochondrial function and S-phase fraction, independent of transformation status or culture condition. We conclude that mitochondrial function reflects proliferative activity rather than tumorigenic conversion., (Copyright 2001 Academic Press.)

Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 (myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha-ras-transfected Rat1) and MR1 (myc/T24Ha-ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 micrometer. In contrast, the histomorphological emergence of cell death at a diameter of around 500 micrometer in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture.

Oxygen consumption, glucose, lactate, and ATP concentrations, as well as glucose and lactate turnover rates, have been studied in a three-dimensional carcinogenesis model of differently transformed rat embryo fibroblasts (spontaneously immortalized Rat1 and myc-transfected M1, and the ras-transfected, tumorigenic descendants Rat1-T1 and MR1) to determine metabolic alterations that accompany tumorigenic conversion. Various bioluminescence techniques, thymidine labeling, measurement of PO(2) distributions with microelectrodes, and determination of cellular oxygen uptake rates (Qc(O(2))) have been applied. In the ras-transfected, tumorigenic spheroid types, the size dependencies of some of the measured parameters exhibited sharp breaks at diameters of approximately 830 microm for Rat1-T1 and approximately 970 microm for MR1 spheroids, respectively, suggesting that some fundamental change in cell metabolism occurred at these characteristic diameters (denoted as "metabolic switch"). Qc(O(2)) decreased and lactate concentration increased as functions of size below the characteristic diameters. Concomitantly, glucose and lactate turnover rates decreased in MR1 spheroids and increased in Rat1-T1. Spheroids larger than the characteristic diameters (exhibiting cell quiescence and lactate accumulation) showed an enhancement of Qc(O(2)) with size. Systematic variations in the ATP and glucose levels in the viable cell rim were observed for Rat1-T1 spheroids only. Proliferative activity, Qc(O(2)), and ATP levels in small, nontumorigenic Rat1 and M1 aggregates did not differ systematically from those recorded in the largest spheroids of the corresponding ras transfectants. Unexpectedly, respiratory activity was present not only in viable but also in the morphologically disintegrated core regions of M1 aggregates. Our data suggest that myc but not ras transfection exerts major impacts on cell metabolism. Moreover, some kind of switch has been detected that triggers profound readjustment of tumor cell metabolism when proliferative activity begins to stagnate, and that is likely to initiate some other, yet unidentified energy-consuming process.

Background: Over the past decade, cell separation technology has become an important tool in various fields of cell biology allowing for the analysis or subsequent cultivation of specific cell subsets. The objective of the present study was to evaluate if the established sorting techniques fluorescence-activated (FACS) and magnetic cell separation (MACS) affect cell membrane physiology in order to define the most non-perturbing application for the separation of tumor and stromal cells., Materials and Methods: Membrane physiology was monitored in single cell suspensions of adherently grown BT474 breast tumor cells and N1 normal skin fibroblasts using flow cytometry. Cell membrane integrity was evaluated by propidium iodide (PI) staining. Microviscosity within the lipophilic membrane layer was determined by a monomer/excimer method utilizing pyrene decanoic acid, membrane potential measurements were carried out using the fluorescence indicator DiBAC4(3), and Annexin-V-staining reflected transversal membrane asymmetry, and an altered phospholipid distribution., Results: Not only the number of preparative cycles prior to cell separation but also the sort conditions during FACS resulted in loss of membrane integrity of a certain cell fraction. If these PI-positive cells were excluded from further analysis, neither MACS nor FACS affected membrane microviscosity while a clear hyperpolarization in both cell types after MACS resulting from exposure to the ferromagnetic matrix of the depletion column and the inhomogeneous magnetic field was shown. In addition, cell sorting of BT474 tumor cells by MACS and FACS was accompanied by the generation of an Annexin-V-positive/PI-negative cell fraction with altered phospholipid distribution. Data were discussed with regard to the sort-induced "stress" conditions such as exposure to hydrodynamic forces or magnetic fields., Conclusions: Both separation procedures modify cell membrane with neither technique being physiologically preferable for subsequent analysis or recultivation of the sorted cells.

In every latently Epstein-Barr virus-infected cell the viral genes EBER-1 and EBER-2 are transcribed by polymerase III. In lytically infected cells in vivo the EBER genes could not be detected. However, in cell culture downregulation could not be confirmed, and hence the relevance of this shutdown to the replication of the virus was not clear. We assayed the transcriptional activity of the EBER genes by nuclear run-on assays with enriched lytically infected cells and demonstrated that EBER-1 and EBER-2 are differentially downregulated on the transcriptional level during the switch to lytic viral replication. This downregulation was an early event during the lytic replication of the virus.

The growth of tumour cells as three-dimensional multicellular spheroids in vitro has led to important insights in tumour biology, since properties of the in vivo-tumour such as proliferation or nutrient gradients, can be studied under controlled conditions. While this review starts with an update of recent data on spheroid monocultures, especially concerning tumour microenvironment and therapeutic modalities, the main emphasis is put on the spectrum of heterologous cultures which have evolved in previous years. This type of culture includes tumour cell interaction with endothelial, fibroblast or immunocompetent cells. The relation of the spheroid culture model to other types of three-dimensional culture and our critical evaluation and presentation of the technical aspects of growing and analysing spheroids are included in the text. These topics are chosen to help the experimental pathologist design experiments with tumour spheroids and to stimulate discussion.

Two mitochondrion-specific fluorochromes, 10-N-nonyl acridine orange (NAO) and rhodamine 123 (Rh123), were used to determine the mechanism responsible for alterations in energy metabolism of transformed rat embryo fibroblast cells isolated from different locations within multicellular spheroids. Accumulation of Rh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thus represents mitochondrial distribution only. A reproducible selective dissociation procedure was used to isolate cells from different locations within the spheroids. After isolation, cells were simultaneously stained with one mitochondrial stain and the DNA dye Hoechst 33342, and several parameters, including cell volume, were monitored via multilaser-multiparameter flow cytometry. Our data clearly show a decrease in the uptake of Rh123 in cells from the periphery to the inner regions of the tumor spheroids, reflecting a persistent alteration in mitochondrial function. However, NAO staining experiments showed no reduction in the total mitochondrial mass per unit cell volume. Because cells were exposed to stain under uniform conditions after isolation from the spheroid, these data indicate the downregulation of mitochondrial function is associated with cell quiescence rather than a transient effect of reduced nutrient availability. This result, which is in accordance with data from two other cell lines (EMT6 and 9L), might reflect a general phenomenon in multicellular spheroids, supporting the hypothesis that quiescent cells in the innermost viable spheroid layer stably reduce their mitochondrial function, presumably to compensate for lower nutrient supply and/or decreased energy demand.

To date, cell biological characteristics of oncogene-transfected cells have been investigated either in relatively homogeneous monolayer cultures or in heterogeneous tumors in vivo. To evaluate the emergence of cellular heterogeneity during tumor formation, we have established a multicellular spheroid system from an oncogene-dependent, genetically determined 2-stage carcinogenesis model for 3-dimensional growth under well-defined conditions. The effect of T24Ha-ras transfection on cellular growth, proliferation, cell viability and oxygenation was investigated using spontaneously immortalized (Rat1) and c-myc-transfected (M1) Fisher 344 rat embryo fibroblasts and a tumorigenic T24Ha-ras-transfected clone of each (Rat1-T1 and MR1). Spheroid volume growth curves and [3H]thymidine autoradiographs clearly demonstrated that spheroids better reflect the degree of tumorigenicity in vivo as opposed to monolayer cultures. Studies on Rat1 and M1 aggregates showed that the potential for tumor formation of Rat1 cells might be manifested in vitro as an increased capability of the cells to survive in 3D culture. pO2 measurements confirmed that neither cell quiescence nor cell death in the pseudo-normal cell aggregate types is due to an oxygen deficiency. In contrast, depletion of oxygen coincided with necrotic cell death in Rat1-T1 spheroids and proliferation arrest in MR1 cultures. Cell-line-specific attributes in 3D culture that were not specifically related to ras transfection of the cells included histological structure, development of necrosis and thickness of viable cell rim. However, growth behavior, proliferation characteristics and their association with the oxygen supply might be correlated with the extent of transformation.