Characterization of Autocrine Growth Factors, Their Receptors and Extracellular Matrix Present in Three-Dimensional Cultures of DU 145 Human Prostate Carcinoma Cells Grown in Simulated Microgravity

J Cellular Molecular Medi

David

et al. 2003

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Neoplastic cells self-assemble into multicellular spheroids that resemble micrometastases and avascular regions of larger tumors [1]. The applications of these tissue constructs include basic oncological research on invasion [2] and angiogenesis [3], and applied research on drug development [4] and delivery [5]. Culture conditions are critical to spheroid composition for these applications. For instance, mixing culture medium enhances collagen content of regenerated cartilage and calcium deposition in bone constructs for implantation [6,7]. These examples are illustrative of an existing paradigm that normal tissue becomes more differentiated upon mixing. During malignant transformation, however, differentiation mechanisms are disrupted [8]. The present study tests the validity of AbstractNeoplastic multicellular spheroids are in vitro models of solid tumors employed in drug testing and basic research. This study compares differentiation in static and mixed prostate cancer spheroids. Staining intensity of prostate specific antigen (PSA) was down-regulated upon mixing, from 0.21 ± 0.03 to 0.13 ± 0.03 in LNCaP multicellular spheroids, and from 0.13 ± 0.04 to 0.03 ± 0.02 in DU 145 multicellular spheroids. This was accompanied by 65% increase in the expression of cytokeratins 8 and 18 in DU 145 spheroids. PSA expression extended 60 µm within static spheroids and was disrupted in mixed culture. Diminished PSA expression and spatial organization suggests a more aggressive cancer. Higher cytokeratin expression could result from either differentiation towards a luminal phenotype or activation of the Ras pathway during dedifferentiation. Thus, the existing paradigm of differentiation established for normal tissue does not apply for our neoplastic spheroids. Cell dedifferentiation is attributed to improved interstitial transport and synthesis of extracellular matrix.

Section: Significance and Applicationsmentioning

confidence: 99%

Immunohistochemical analysis of differentiation in static and mixed prostate cancer spheroids

Song

J Cellular Molecular Medi

David

et al. 2003

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“…The spheroids behave as well-differentiated solid tumors, while the bilayer behaves as an aggressive invading tumor margin. The spheroids are more differentiated, are slower growing, have greater cytoskeletal and extracellular organization, and have far different staining intensities for autocrine growth factors and their receptors [4]. In this study, we are using the spheroid/bilayer system to investigate during physiological stimulation, the signal transduction associated with the lipid second messengers AA, DAG, PA, ceramide and phospholipase A 2 (PLA 2 ), phospholipase C (PLC), phospholipase D(PLD), PI 3 kinase (PI 3 K), SMase and cAMP.…”

Section: Discussionmentioning

confidence: 99%

“…Our aim was to determine the early signal transduction pathways without supplementation of the culture medium with additional hormones or growth factors. This was based on our previous studies which implied at least EGF and TGF-β autocrine growth factor regulation mediated HARV-induce changes [4]. The expression of TGF receptor and ligand in both HARV and T. Well, the increased activity of TGF in HARV and EGF in T. Well culture, made us try to map the early signal transduction pathways without additional supplementation of what the fetal calf serum contained.…”

Section: Discussionmentioning

confidence: 99%

“…The HARV was mixed at 15 rpm. For all chambers, cells were fed to maintain the glucose concentration between 50 and 130 mg/dl, and pH between 7.0 and 7.4 [4].…”

Section: Methodsmentioning

confidence: 99%

“…By day 17, the ratio of staining intensity for the HARV to that for the T. Well was 0.15 for the epidermal growth factor (EGF), 0.52 for EGF receptor, 1.2 for transforming growth factor (TGFβ), 0.52 for EGF receptor, and 0.17 for TGF-β receptor, also 3.7 for collagen and 2.4 for laminin. These differences were mediated in part through EGF and TGF-β, autocrine loops and cell-cell interactions [4].…”

Section: Introduction Abstractmentioning

confidence: 99%

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Tri‐dimensional prostate cell cultures in simulated microgravity and induced changes in lipid second messengers and signal transduction

Clejan

J Cellular Molecular Medi

Rosensweig

2001

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The high aspect rotating‐wall vessel (HARV) was designed to cultivate cells in an environment that simulate microgravity. We studied previously the effects of HARV cultivation on DU‐145 human prostate carcinoma cells. We determined that HARV cultivation produced a less aggressive, slower growing, less proliferative, more differentiated and less pliant cell than other cell cultivation methods. The result was a 3‐dimensional (3D) growth model of prostate cancer which mimics in vivo tissue growth. This work examines the signal transduction‐second messenger pathways existing temporarily in these HARV cells and correlates these features with the special properties in growth and 3D spheroid formation. We found an initial very active ceramide, a diacylglycerol increase together with increases in PI‐PLC and PLA2 a central defect in PLD (no phosphatic acid or phosphatidylethanol at any time during 15 days of HARV cultivation). There is a cross‐talk between ceramide and PI3K pathways with activation of PI3K, after 6 days of HARV growth concomitant with down‐regulation of ceramide. At this time, there is also an increase of cAMP (seen by increases in arachidonic acid). Taken together these results can explain the 3D organoidlike growth. We therefore developed a model for growth in HARV prostate cancer cells which involve temporal “switches” between second messengers, activation and cross‐talk between multiplicity of signaling pathways and a central defect in PLD pathways. Essential to the late slow growth, and 3D organotypic formation are the apoptotic, anti‐survival, anti‐proliferation and differentiation pathways in the first days of HARV, with growth of “new” different types of prostate cancer cells which set‐up for later “switch” in ceramide‐PI3K to survival and proliferation.

Characterization of bimodal cell death of insect cells in a rotating-wall vessel and shaker flask

Cowger

Hammond

et al. 1999

Biotechnol. Bioeng.

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In previous publications, we reported the benefits of a high-aspect rotating-wall vessel (HARV) over conventional bioreactors for insect-cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker-flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first-order term. Cellcycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell-cell interactions, waste accumulation, and mass transport. These studies may benefit insect-cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity.