FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by WARTENBERG, M.
Right arrow Articles by SAUER, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by WARTENBERG, M.
Right arrow Articles by SAUER, H.
(The FASEB Journal. 2001;15:995-1005.)
© 2001 FASEB

Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells

MARIA WARTENBERG, FATMA DÖNMEZ, FREDERIKE C. LING, HELMUT ACKER*, JÜRGEN HESCHELER and HEINRICH SAUER1

Department of Neurophysiology, University of Cologne, D-50931 Cologne, Germany; and
* Max-Planck-Institute of Molecular Physiology, D-44227 Dortmund, Germany

1Correspondence: Department of Neurophysiology, Robert-Koch-Str. 39, D-50931 Cologne, Germany. E-mail: hs{at}physiologie.uni-koeln.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor vascularization is the rate-limiting step for the progression of cancer. Differential steps of tumor-induced angiogenesis were studied by a novel in vitro confrontation culture of avascular multicellular prostate tumor spheroids and embryoid bodies grown from pluripotent embryonic stem (ES) cells. Vascularization in embryoid bodies started on day 5 of cell culture and was paralleled by down-regulation of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) and vascular endothelial growth factor (VEGF). In parallel, a dissipation of gradients in the pericellular oxygen pressure was observed as measured by O2-sensitive microelectrodes. After 24–48 h of confrontation culture, cells positive for platelet endothelial cell adhesion molecule (PECAM-1) became visible in the contact region between the embryoid body and the tumor spheroid and sprouted within the confrontation cultures during subsequent days. Tumor-induced angiogenesis resulted in growth stimulation of tumor spheroids, disappearance of central necrosis and a reduction of the pericellular oxygen pressure. Furthermore, tumor vascularization resulted in elevated levels of HIF-1{alpha}, VEGF, heat shock protein 27 (HSP27), and P-glycoprotein. Tumor-induced angiogenesis may augment the oxygen consumption in tumors resulting in an increased expression of hypoxia-related, proangiogenic genes as well as of HSP27 and P-glycoprotein, which are involved in a multidrug resistance phenotype.—Wartenberg, M., Dönmez, F., Ling, F. C., Acker, H., Hescheler, J., Sauer, H. Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells.


Key Words: VEGF • hypoxia • multidrug resistance • P-glycoprotein • HSP27


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IT IS WELL KNOWN that avascular tumors in vivo or tumor spheroids in vitro will grow until they reach a size at which passive diffusion can no longer provide nutrients for the cells in the depth of the tissue nor can waste products adequately diffuse out of the tumor tissue. The gradients in oxygen, pH, nutrients, and the absence of sufficient detoxification then result in growth arrest, development of central necrosis as well as induction of a multidrug resistance (MDR) phenotype, which is presumably correlated with the increased expression of the MDR transporter P-glycoprotein. Such tumors usually remain in this state of equilibrium for decades unless they are vascularized by the host vasculature in the process of tumor-induced angiogenesis. Compelling evidence shows that tumor-associated neovascularization is a prerequisite for rapid tumor growth and metastasis (1) . However, the transition from a nonangiogenic to an angiogenic phenotype and subsequent tumor vascularization is not well understood, although it is generally accepted that tumor angiogenesis is regulated by the production of angiogenic stimulators including members of the fibroblast growth factor and vascular endothelial growth factor (VEGF) families (1) . Expression of these angiogenic factors is likely to be induced by the hypoxic microenvironment present in the avascular tumor tissue (2) . Significant evidence indicates that the VEGF gene is under the control of HIF-1{alpha}, which itself is strongly induced by hypoxia (3 4 5) . Furthermore, vascular growth may be regulated by complex interactions of extracellular matrix molecules, proteolytic enzymes, and cell adhesion molecules. The inhibition of angiogenesis is considered one of the most promising strategies leading to the development of novel antineoplastic therapies (6) . Each signaling and differentiation step in the angiogenic process represents a potential target for therapeutic anticancer strategies. Several antiangiogenic agents, including naturally occurring inhibitors of angiogenesis, i.e., angiostatin (7) , endostatin (8) , platelet factor-4 (9) , and others, have been described in recent years. However, intense efforts are still needed to evaluate novel antiangiogenic agents that could be exploited in anticancer therapies (6) .

The present study compares angiogenesis in the nonneoplastic tissue of embryoid bodies with tumor-induced angiogenesis by using a novel confrontation culture system consisting of embryoid bodies and multicellular tumor spheroids. This confrontation culture system was developed with reference to the cocultures of glioma tumor spheroids and brain cell aggregates, which have been used before to study tumor cell invasion (10 , 11) . Embryoid bodies are grown from pluripotent murine ES cells that differentiate to cell types of all three germ layers. Previous studies have demonstrated that vasculogenesis and angiogenesis occur in embryoid bodies, making this in vitro model suitable for studies of tumor-induced angiogenesis (12 13 14) . We establish that tumor spheroids grown in confrontation culture with embryoid bodies are efficiently vascularized via tumor-induced angiogenesis. Our data show that vasculogenesis and angiogenesis in the host tissue of embryoid bodies result in improved oxygen supply and down-regulation of HIF-1{alpha} and VEGF. In contrast, upon vascularization of the tumor spheroid increased oxygen consumption leads to severe hypoxia with the consequence of up-regulation of HIF-1{alpha}-, VEGF-, and MDR-related proteins. The hypoxic conditions in the vascularized tumor may be responsible for the maintenance of the proangiogenic phenotype, which is a prerequisite for the further sprouting of vessel structures into the tumor tissue as well as uncontrolled progression of tumor growth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Spinner culture technique for cultivation of embryoid bodies
The ES cell line D3 (15) was grown on mitotically inactivated feeder layers of primary murine embryonic fibroblasts for a maximum of eight passages in Iscove’s medium (Gibco-Life Technologies, Helgerman Court, Md.) supplemented with 20% heat-inactivated (56°C, 30 min) fetal calf serum (FCS) (Boehringer Mannheim, Mannheim, Germany), 2 mM glutamax (Gibco), 100 µM ß-mercaptoethanol (Sigma, Deisenhofen, Germany), 1% minimum essential medium (MEM) nonessential amino acids stock solution (Gibco), 100 IU/ml penicillin, and 100 µg/ml streptomycin (both Gibco) in a humidified environment containing 5% CO2 at 37°C and passaged every 2 or 3 days. At day 0 of differentiation, adherent cells were enzymatically dissociated using 0.2% trypsin and 0.05% EDTA in phosphate-buffered saline (PBS) (Gibco) and seeded at a density of 1·107 cells ml-1 in 250 ml siliconized spinner flasks (Integra Biosciences, Fernwald, Germany) containing 100 ml Iscove’s medium supplemented with the same additive as described above. After 24 h, 150 ml medium was added to give a final volume of 250 ml. The spinner flask medium was stirred at 20 r.p.m. using a stirrer system (Integra Biosciences) and 150 ml cell culture medium were exchanged every day.

Spinner culture technique for cultivation of multicellular tumor spheroids
The human prostate cancer cell line DU-145 was used throughout the study. The cell line was grown routinely in 5% CO2/humidified air at 37°C with Ham’s F10 medium (Gibco) supplemented with 10% FCS (Boehringer), 2 mM glutamine, 100 µM ß-mercaptoethanol, 2 mM MEM, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Gibco). Spheroids were grown from single cells. Cell monolayers were trypsinized with 0.2% trypsin, 0.05% EDTA (Gibco) and seeded in 250 ml spinner flasks as described previously (12) . For the experiments, tumor spheroids with mean diameters of 440 ± 80 µm were used. Central necrosis was obvious in these tumor spheroids.

Generation of confrontation cultures
A schematic depiction of the experimental procedure of confrontation culture generation from embryoid bodies and multicellular tumor spheroids is given in Fig. 1 . At the indicated times embryoid bodies and multicellular tumor spheroids were removed from the spinner flasks. One embryoid body (5- to 6-day-old) together with one tumor spheroid (18- to 30-day-old) was added to a 40 µl drop of Iscove’s cell culture medium placed onto the lid of a 10 cm petri culture dish. After adding 20–30 drops to the lid of the petri dish, the lid was turned around and placed on a petri dish filled with 10 ml sterile PBS. Within 24–48 h, embryoid bodies and tumor spheroids closely attached within the hanging drops and were transferred to 10 cm bacteriological petri dishes filled with 10 ml Iscove’s cell culture medium. In the experiments with the VEGF antagonist SU5614 (Calbiochem, Bad Soden, Germany), the cell culture medium was supplemented with 1 µM of the compound. Confrontation cultures were used for the experiments at times as indicated.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 1. Schematic diagram of the experimental protocol used for the generation of confrontation culture objects of multicellular tumor spheroids and embryoid bodies. Tumor spheroids are derived from prostate cancer cells of the DU-145 cell line grown in monolayer culture. Embryoid bodies are derived from ES cells of the D3 cell line grown on feeder layers of embryonic fibroblasts. After enzymatic dissociation, ES cells and DU-145 cells were inoculated into spinner flasks and tumor spheroids and embryoid bodies were cultivated. Confrontation objects were generated by coincubation of single tumor spheroids with single embryoid bodies in a hanging drop.

Confocal laser scanning microscopy (CLSM)
Fluorescence recordings were performed by means of a confocal laser scanning setup (LSM 410, Zeiss, Jena, Oberkochen, Germany) connected to an inverted microscope. (Axiovert 135, Zeiss). The confocal setup was equipped with a 5 mW helium/neon laser, single excitation 633 nm (excitation of Cy5), and an argon laser, single excitation 488 nm (excitation of Lucifer yellow/VS and CMF). Emission was recorded using the long-pass filter sets RG665 and LP515, respectively. A 16x, numerical aperture (N.A.) 0.5, oil immersion-corrected objective (Neofluar, Zeiss) and a 10x, N.A. 0.3 objective (Neofluar, Zeiss) were used.

Long-term labeling of multicellular tumor spheroids
To discriminate tumor spheroids grown in confrontation culture from embryoid bodies, tumor spheroids were labeled with the long-term cell tracker dye 5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes, Eugene, Oreg.). In brief, tumor spheroids were incubated for 60 min in F10 cell culture medium that contained 10 µM CMFDA (stock solution 10 mM, dissolved in DMSO). Subsequently they were washed and incubated for further 24 h in bacteriological petri dishes. The penetration of CMFDA into the tumor tissue was assessed in diffusion experiments as described previously (12) . Tumor spheroids were incubated with the dye; the CMFDA fluorescence was determined after 30 min by performing optical sections with a thickness of 5 µm from the periphery toward the center of the tumor spheroids and evaluated in selected regions of interest (600 µm2, 40 x 40 pixel). The diffusion coefficient D was then calculated according to the Einstein-Smoluchovski equation D = x2/2t, where D is the diffusion coefficient, x is the maximal diffusion distance of CMFDA from the tumor spheroid periphery, and t is the diffusion time. From this equation, the diffusion coefficient D for CMFDA in the tissue of multicellular tumor spheroids was calculated to 3.4 · 10-8 cm2 · s-1. Accordingly, a penetration depth 156 µm for CMFDA was achieved within 1 h of incubation. Stable CMF fluorescence was observed for more than 5 days of tumor spheroid culture. Fluorescence excitation was performed by the 488 nm line of an argon-ion laser of the confocal setup. Emission was recorded using a long-pass LP515 nm filter set.

Immunohistochemistry
Immunohistochemistry was performed with whole-mount embryoid bodies, tumor spheroids, and confrontation cultures. For primary antibodies, we used monoclonal anti-mouse platelet endothelial cell adhesion molecule (PECAM-1; CD31) (Endogen, Woburn, Mass.; concentration of 5 µg/ml), the monoclonal anti-HIF-1{alpha} (clone mgc3) (Alexis Biochemicals, Grünberg, Germany) (dilution 1:200), the monoclonal anti-VEGF (clone JH121) (Upstate Biotechnology, Lake Placid, N.Y.; concentration 20 µg/ml), the monoclonal anti-HSP27 (Chemicon, Temecula, Calif.; concentration 2 µg/ml), and the polyclonal anti-P-glycoprotein antibody (Ab-1) (Calbiochem-Novabiochem) (concentration 5 µg/ml. For PECAM-1, HIF-1{alpha}, HSP27, and P-glycoprotein staining, the respective tissues were fixed in ice-cold methanol/acetone (7:3) for 60 min at -20°C, and washed with PBS containing 0.1% Triton X-100 (PBST) (Sigma). For VEGF staining, the tissues were fixed for 1 h at 4°C in 4% formaldehyde in PBS. Blocking against unspecific binding was performed for 60 min with 10% fat-free milk powder (Heirler, Radolfzell, Germany) dissolved in PBS. The tissues were subsequently incubated for 90 min (for VEGF staining overnight at 8°C) at room temperature with primary antibodies dissolved in PBS supplemented with 10% milk powder. The tissues were thereafter washed three times with PBST (0.01% Triton) and reincubated with either a Cy5-conjugated rabbit anti-Syrian hamster IgG (H+L) (PECAM-1), a Cy5-conjugated goat anti-mouse IgG (H+L) (HIF-1{alpha}, HSP27, VEGF), or a Cy5-conjugated goat anti-rabbit IgG (P-glycoprotein) (all from Dianova, Hamburg, Germany) at a concentration of 3.8 µg/ml in PBS containing 10% milk powder. After washing three times in PBST (0.01% Triton) the tissues were stored in PBS until inspection. For excitation of the Cy5 fluorochrome the 633 nm band of a helium/neon laser of the confocal setup was used. Emission was recorded using a 655 nm long-pass filter set. The pinhole settings of the confocal setup were adjusted to give a full-width half maximum (FWHM) of 9 µm. The photomultiplier tube (PMT) sensitivity was set to 898 V, 794 V, and 834 V to evaluate VEGF, HIF-1{alpha}, P-glycoprotein, and HSP27 immunofluorescence, respectively. Fluorescence was recorded in a depth of 80–120 µm in the depth of the tissue and the fluorescence values in the respective optical section were evaluated by the image analysis software of the confocal setup.

The immunofluorescence values obtained in the experiments were corrected for fluorescence arising from unspecific binding of the primary and secondary antibodies within the tissue. To assess the magnitude of unspecific fluorescence, the tissues were incubated with nonsense antibodies directed against antigens that are not expressed in the respective tissue, i.e., an antibody against prostate-specific acid phosphatase (Sigma) used in embryoid bodies and the erythroid cell specific antibody TER-119 (PharMingen, Hamburg, Germany) used in tumor spheroids. Labeling with these primary antibodies and a Cy-5 labeled secondary antibody resulted in a weak unspecific fluorescence, which was subtracted from the fluorescence values achieved in the immunofluorescence experiments with PECAM-1, HIF-1{alpha}, VEGF, P-glycoprotein, and HSP27.

Quantitative immunohistochemistry
The immunofluorescence grey level values are not necessarily proportional to the amounts of expressed antigen protein. To correlate the immunofluorescence values to the protein levels of antigen, calibration measurements were performed with solutions of the fluorochrome-labeled secondary Cy-5-labeled antibody in a concentration range of 0.175 µM to 5.8 µM (Fig. 2 ). The number of emitted photons and the concentration of the antibody follow a linear relation up to a maximal antibody concentration Cmax, which is defined by Cmax = 0.05/2.303 · x · {varepsilon}, where x is the thickness of the optical section penetrated by the laser beam and {varepsilon} represents the molar extinction coefficient (250,000 M-1 cm-1 for Cy5). Under the experimental conditions used in the present study, Cmax was calculated to be 8.7 µM. The relation between the emitted photons and the concentration of the fluorochrome can be estimated according to Parker’s law: IF/I0 = 2.303 · {Phi}r · x · C · {varepsilon}, where IF is the emitted fluorescence, I0 is the excitation light intensity, {Phi}r is the fluorochrome-specific fluorescence yield, i.e., {Phi}r = 0.28 for Cy5, and C is the concentration of the secondary antibody. The slope of this linear relation is dependent on the settings of the confocal microscope: the excitation light intensity, the pinhole settings, the settings of the PMT, as well as the specific properties of the fluorochrome, i.e., the quantum yield and the molar extinction coefficient. Since tumor spheroids, embryoid bodies, and confrontation objects are incubated during the immunolabeling in a surplus of antibody, the amount of bound primary and secondary antibody directly correlates to the amount of expressed antigen. The calibration measurements were performed using the CLSM settings, which were applied in the respective experiments with either confrontation objects or embryoid bodies and tumor spheroids alone.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Emitted Cy5 (secondary antibody) fluorescence in relation to the concentration of the secondary antibody. The number of emitted photons and the concentration of the fluorochrome follows a linear relation, which can be described by Parker’s law: IF/I0 = 2.303 · {Phi}r · x · C · {varepsilon}, where IF is the emitted fluorescence, I0 is the excitation light intensity, {Phi}r is the fluorochrome-specific fluorescence yield, i.e., {Phi}r = 0.28 for Cy5, x is the thickness of the optical section penetrated by the laser beam, C is the concentration of the secondary antibody, and {varepsilon} represents the molar extinction coefficient. The data were recorded in solutions of secondary antibody using the settings of the confocal laser scanning microscope described in Materials and Methods. The sensitivity of the PMT tube amounted to 898 V. The data were fitted by linear regression analysis.

Necrosis staining with Lucifer yellow/VS
Lucifer yellow/VS (Sigma) was used in a final concentration of 100 µM as lethal cell stain for necrotic areas (16) . Lucifer yellow/VS was loaded by a 40 min incubation at room temperature. The samples were washed three times and resuspended in 2 ml cell culture medium. After 8 h in cell culture, central necrosis in embryoid bodies was examined by CLSM using the argon ion laser of the confocal setup, and a 10 x objective (Plan-Neofluar, Zeiss, Jena). Excitation was performed at 488 nm. Emission was recorded using a long-pass 515 nm filter set.

PO2 measurements
PO2 distribution profiles in embryoid bodies and multicellular tumor spheroids were measured in a perfusion chamber as described previously (17) . To stabilize the objects for perfusion with isotonic salt solution (Locke’s solution) and insertion of the microelectrode, they were positioned onto a small plastic plate containing bore holes. Single channel microelectrodes with a tip diameter of 2–4 µm were used for polarographic Po2 measurements. Two-point calibration was performed as described previously (17) .

Statistical analysis
Data are given as mean values ± SD, with n denoting the number of experiments unless otherwise indicated. In each experiment at least 15 culture objects were analyzed. Student’s t test for unpaired data was applied as appropriate. A value of P<0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of HIF-1{alpha} and VEGF during the time course of vasculogenesis and angiogenesis in embryoid bodies
Embryoid bodies derived from pluripotent ES cells were used as a vascularized host tissue from which sprouting of capillary-like structures into the tumor tissue should occur during tumor-induced angiogenesis. Endothelial precursor cells as detected by PECAM-1 immunohistochemistry were obtained in 5-day-old embryoid bodies (Fig. 3A ). At day 8 of embryoid body culture, a branched network of capillary-like structures was observed that traversed the whole tissue of the embryoid bodies and did not further augment during subsequent days (Fig. 3B ). These steps are in line with previous investigations on the differential steps of vasculogenesis and angiogenesis in embryoid bodies of the CCE ES cell line (12 , 13) .



View larger version (39K):
[in this window]
[in a new window]
  		Figure 3. Vasculogenesis and angiogenesis in embryoid bodies. Embryoid bodies efficiently differentiate vascular structures from day 5 (A) to day 8 (B). Shown are representative embryoid bodies stained with a monoclonal antibody directed against PECAM-1 (bar represents 100 µm). In embryoid bodies, a transient up-regulation of HIF-1{alpha} (C) and VEGF (D) was observed (the maximal immunofluorescence in each experiment was set to 100%). The steep oxygen gradients observed in avascular embryoid bodies were dissipated on vascularization (E). The pericellular oxygen pressure was determined by oxygen-sensitive microelectrodes that were stepwise punctured into the tissue. The first point in the curve represents the oxygen pressure in the incubation medium.

Vasculogenesis and angiogenesis are known to require the expression of VEGF. The VEGF gene has previously been shown to be under the control of HIF-1{alpha} (19) . We therefore investigated the expression of HIF-1{alpha} as well as of VEGF during the time course of vasculogenesis and angiogenesis in embryoid bodies. Since HIF-1{alpha} is induced under hypoxic conditions (20) , an elevated expression was expected in embryoid bodies before the onset of vasculogenesis. As assumed, HIF-1{alpha} (Fig. 3C ) as well as VEGF (Fig. 3D ) were up-regulated in avascular embryoid bodies during the first 4 days of cell culture. In contrast, during the period of vasculogenesis (from day 5 to day 8) a significant down-regulation of HIF-1{alpha} as well as VEGF was observed (n=4). The changes in expression of HIF-1{alpha} correlated well with the limited oxygen supply in the avascular tissue of 3-day-old embryoid bodies as compared to the vascularized tissue in 8-day-old embryoid bodies. By the use of O2-sensitive microelectrodes, steep oxygen gradients were observed in avascular 3-day-old embryoid bodies resulting in PO2 values of 5.2 ± 0.1 mm Hg in a depth of 300 µm in the tissue. In contrast, in 8-day-old vascularized embryoid bodies O2 gradients were almost completely dissipated and amounted to 123 ± 6 mm Hg (Fig. 3E ) (n=10 embryoid bodies for each experimental condition).

Time course of tumor-induced angiogenesis in the confrontation culture of embryoid body and multicellular tumor spheroid
To investigate tumor-induced angiogenesis in the confrontation culture model, we selected avascular embryoid bodies before the onset of angiogenesis, which allowed us to study differential steps of tumor-induced angiogenesis. Embryoid bodies were confronted on days 5 and 6 of development with 18- to 30-day-old tumor spheroids containing central necrosis. The tumor spheroids were stained with the long-term cell tracker dye CMFDA to distinguish between tumor tissue and the tissue of the embryoid body. Subsequently, the confrontation objects were investigated for the formation of capillary-like structures using PECAM-1 immunohistochemistry and confocal laser scanning microscopy. We observed that after 24–48 h in confrontation culture, PECAM-1-positive cells appeared predominantly within the contact region between the embryoid body and the tumor spheroid (Fig. 4A ) (n=10 confrontation objects). During the days following, sprouting of capillary-like structures occurred from the contact region toward more central parts of the embryoid body and the tumor spheroid (Fig. 4B ) (n=10 confrontation objects). Within 5 days of confrontation culture, extended areas of capillary networks were observed in embryoid bodies, as well as in tumor spheroids (Fig. 4C ) (n=10 confrontation objects). After this time the tissue of the embryoid body and the tumor spheroid could still be clearly distinguished, as demonstrated by the distribution of the CMF staining in tumor spheroids. Prolonged culture of confrontation objects, i.e., longer than 8 days, resulted in a complete amalgamation of tumor spheroids and embryoid bodies (data not shown).



View larger version (42K):
[in this window]
[in a new window]
  		Figure 4. Time course of tumor-induced angiogenesis in confrontation culture objects of multicellular tumor spheroids and embryoid bodies. After 24–48 h in confrontation culture, the first PECAM-1-positive cells appeared in the contact region between the embryoid body and the tumor spheroids (A). Within 5 days of confrontation cultures, endothelial cells migrated toward the tumor tissue, which resulted in vascularization of the tumor spheroid (B, C). Shown are false color overlays of confocal fluorescence images (red, green) with a nonconfocal transmission image (blue). The tumor tissue was labeled with the long-term cell tracker dye CMFDA (green). Vascular structures were labeled with a monoclonal antibody directed against PECAM-1 (red). PECAM-1-positive vascular structures were recorded in a depth of 100 µm in the tissue. The bar represents 100 µm.

Expression of VEGF and HIF-1{alpha} in multicellular tumor spheroids in confrontation culture
In tumor spheroids, increased VEGF expression has recently been attributed to the development of hypoxic cell areas in the depth of the tissue (20 , 21) . Increased levels of HIF-1{alpha} are present in several human cancers and their metastases and have been suggested to confer the proangiogenic phenotype of solid tumors (22) . HIF-1{alpha} has recently been reported to be expressed in prostate cancer cells of the DU-145 cell line (23) . We therefore investigated the expression levels of VEGF (n=4) as well as HIF-1{alpha} (n=3) in avascular tumor spheroids as compared to vascularized tumor spheroids cultivated for 5–6 days in confrontation culture. HIF-1{alpha} immunofluorescence was significantly increased from 100 ± 31% in avascular control spheroids to 165 ± 21% in vascularized tumor spheroids cultivated in confrontation culture, which corresponds to an ~threefold increase HIF-1{alpha} protein levels (Fig. 5A ). In embryoid bodies cultivated in confrontation culture, HIF-1{alpha} levels were not significantly different from control embryoid bodies. VEGF expression apparently paralleled the expression of HIF-1{alpha}. VEGF immunofluorescence was significantly increased from 100 ± 17% in avascular control spheroids to 200 ± 45% in vascularized tumor spheroids cultivated in confrontation culture, which corresponds to an ~fivefold increase in VEGF protein levels (Fig. 5B ). The highest expression level of VEGF was observed in the contact region between the embryoid body and the tumor spheroid (Fig. 5C ), indicating that the observed onset of tumor-induced angiogenesis in this region may be owing to the increased VEGF levels. To investigate whether VEGF is biologically active in confrontation cultures of embryoid bodies with tumor spheroids, the culture objects were incubated in cell culture medium supplemented with 1 µM SU5614, which is an inhibitor of VEGF receptor tyrosine kinases. Under these conditions, only rudimentary angiogenesis occurred in embryoid bodies and no invasion of vascular structures from the embryoid body toward the tumor spheroid was observed (n=10 culture objects) (Fig. 6 ).



View larger version (65K):
[in this window]
[in a new window]
  		Figure 5. Changes in the expression of HIF-1{alpha} (A) and VEGF (B, C) after tumor-induced angiogenesis. HIF-1{alpha} and VEGF expression were evaluated by quantitative immunohistochemistry in whole-mount avascular control tumor spheroids and vascularized tumor spheroids in confrontation culture as well as in vascularized control embryoid bodies and vascularized embryoid bodies in confrontation culture. The immunofluorescence of control tumor spheroids was set to 100%. Fluorescence was recorded in a depth of 80–120 µm within the tissue. Tumor-induced angiogenesis resulted in an up-regulation of HIF-1{alpha} and VEGF protein in the tumor spheroid. A pronounced elevation of VEGF was observed in the contact region between the tumor spheroid and the embryoid body (C). The bar represents 50 µm. *P < 0.05, significantly different from control tumor spheroids.



View larger version (100K):
[in this window]
[in a new window]
  		Figure 6. Effects of the VEGF antagonist SU5614 on angiogenesis in confrontation cultures of embryoid bodies and tumor spheroids. During the entire period of cultivation (6 days), 1 µM of the compound was present in the incubation medium. Shown is a representative overlay image between a transmission image (green) and a fluorescence image (red) showing the PECAM-1 immunofluorescence. The tumor spheroid is on the right side of the image, whereas the embryoid body is on the left side. Note that treatment of the confrontation object with SU5614 resulted in significant inhibition of angiogenesis in the embryoid body. Tumor-induced angiogenesis in the tumor spheroid was completely absent. The bar represents 100 µm.

PO2 profiles in vascularized tumor spheroids cultivated in confrontation culture
The expression of HIF-1{alpha} is supposed to correlate with the pericellular oxygen tension in vascularized tumor spheroids cultivated in confrontation culture. Measurements of pericellular oxygen tension in avascular tumor spheroids (n=5 tumor spheroids) revealed steep oxygen gradients resulting in a PO2 of 40 ± 5 mm Hg in a depth of 300 µm in the tissue of the tumor spheroids (Fig. 7 ). Vascularization of tumor spheroids apparently did not dissipate the oxygen gradients. The PO2 at a depth of 300 µm amounted to 8 ± 4 mm Hg, which was significantly lower than in the avascular control (n=5 tumor spheroids) and may indicate increased oxygen consumption on vascularization.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 7. Pericellular oxygen pressure in avascular tumor spheroids and vascularized tumor spheroids cultivated in confrontation culture. Tumor spheroids were punctured with oxygen-sensitive microelectrodes. The first point in the curve represents the oxygen pressure in the bath solution. Note that vascularization of tumor spheroids resulted in a decreased pericellular oxygen pressure.

Growth stimulation and disappearance of central necrosis in multicellular tumor spheroids on vascularization
Vascularization of tumors is the limiting step for cancer progression. In avascular tumors hypoxia, gradients of nutrients, accumulation of waste products, and low pH occur. This results in the development of a necrotic core in the center of tumor spheroids surrounded by a rim of quiescent, cell cycle inactive cells and—at the spheroid periphery—a rim of proliferating cells (24) . On the other hand, vascularization of the tumor tissue with the concomitant improvement in the supply with nutrients results in growth stimulation of the tumor cells and expansion of the solid tumor. To investigate whether comparable effects occurred in tumor spheroids cultivated in confrontation culture, the growth of vascularized tumor spheroids in confrontation culture was compared to the growth of avascular control spheroids. It was observed that vascularization of tumor spheroids resulted in significant growth stimulation compared with the growth kinetics of control tumor spheroids (Fig. 8A ). After 6 days of confrontation culture the size of the tumor spheroids was increased to 557 ± 64%, whereas only a minor increase to 156 ± 18% was observed in control spheroids (n=3). The size of the vascularized embryoid bodies did not increase during the time course of confrontation culture. Since the observed growth stimulation of tumor spheroids grown in confrontation culture might be caused by the secretion of mitogens from the embryoid bodies rather than by an improved supply with nutrients via the vasculature of the host tissue, we conducted control experiments with tumor spheroids cultivated together with embryoid bodies (but not in confrontation culture). The growth of the tumor spheroids in the presence of embryoid bodies was then compared with the growth of control tumor spheroids cultivated alone. If the embryoid bodies would secrete mitogens, an increased growth of the tumor spheroids should be expected. We observed that coincubation of tumor spheroids with embryoid bodies did not result in a growth stimulation of the tumor spheroids (data not shown). Hence, we conclude that the growth stimulation of tumor spheroids observed in our confrontation culture system was not caused by the secretion of mitogens by the embryoid bodies.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 8. Growth stimulation (A) and disappearance of central necrosis (B) in tumor spheroids on tumor-induced angiogenesis. In tumor spheroids cultivated in confrontation culture, a marked growth stimulation was observed, indicating an improved supply with nutrients. In vascularized embryoid bodies, central necrosis was absent (Ba) whereas in avascular tumor spheroids prominent central necrosis was evident (Bb). Tumor-induced angiogenesis resulted in a disappearance of the central necrosis (Bc). The bar represents 100 µm.

Since vascularization should dissipate gradients of nutrients and allow the export of catabolic waste products, we assumed that central necrosis in multicellular tumors should disappear on vascularization. To verify this, we stained control vascularized embryoid bodies and avascular multicellular spheroids as well as vascularized confrontation cultures (n=15 objects for each experimental condition) with Lucifer yellow/VS, which has been demonstrated to accumulate in necrotic areas of tumor spheroids (18) . As shown previously (12) , necrosis was absent in vascularized embryoid bodies (Fig. 8Ba ), whereas prominent necrosis was observed in avascular tumor spheroids (Fig. 8Bb ). In vascularized tumor spheroids grown in confrontation culture, only a few Lucifer yellow/VS-positive cells were observed (Fig. 8Bc ), indicating that upon vascularization the central necrotic area had been recolonized by cell cycle active cells from the more peripheral parts of the tumor spheroid.

Expression of P-glycoprotein and HSP27 in multicellular tumor spheroids on vascularization
We previously reported on the development of an intrinsic P-glycoprotein-meditated MDR in quiescent cell areas of large, non-necrotic tumor spheroids of the DU-145 prostate cancer cell line (25 , 26) . Occurrence of drug resistance has been attributed to hypoxic conditions in the depth of the tumor tissue (27) . Furthermore, the low molecular weight HSP27 has been reported to be involved in resistance to chemotherapy and to be coexpressed with P-glycoprotein (28 , 29) . To investigate changes in the expression of drug resistance-related proteins after tumor-induced angiogenesis, immunohistochemical analysis of P-glycoprotein and HSP27 expression was performed in vascularized tumor spheroids as well as in embryoid bodies cultivated in confrontation culture. After confrontation culture, P-glycoprotein (n=8) and HSP27 (n=3) immunofluorescence was significantly up-regulated in tumor spheroids from 100 ± 9% and 100 ± 6% to 171 ± 6% and 151 ± 19%, respectively, corresponding to an ~four- and twofold increase in P-glycoprotein and HSP27 protein levels (Fig. 9A , B ). In embryoid bodies, confrontation culture resulted in a moderate but significant up-regulation of P-glycoprotein immunofluorescence to 139 ± 12%, whereas the expression of HSP27 remained unchanged.



View larger version (52K):
[in this window]
[in a new window]
  		Figure 9. Up-regulation of P-glycoprotein (A) and HSP27 (B) in vascularized tumor spheroids cultivated in confrontation culture. P-glycoprotein and HSP27 were evaluated by quantitative immunohistochemistry in control embryoid bodies and control tumor spheroids as well as in embryoid bodies and tumor spheroids cultivated in confrontation culture. The immunofluorescence of control tumor spheroids was set to 100%. Vascularization resulted in a pronounced increase in P-glycoprotein as well as in HSP27 levels. Note that a significant increase in P-glycoprotein levels was likewise observed in embryoid bodies grown in confrontation culture. *P < 0.05, significantly different from control tumor spheroids.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study reports on the differential steps of tumor-induced angiogenesis and the concomitant changes in the expression of HIF-1{alpha} and VEGF as well as of HSP27 and P-glycoprotein. We introduce a novel in vitro cell culture system based on embryoid bodies derived from pluripotent embryonic stem cells and multicellular tumor spheroids, which are well characterized as model systems of avascular micrometastases or avascular regions of solid tumors (24 , 30) . Within the multicellular tissue of embryoid bodies, ES cells differentiate to cell types of all three germ layers, including the endothelial (13) and hematopoietic (31 , 32) cell lineage. We have previously demonstrated that the capillary-like structures differentiated within embryoid bodies are functional and improve the diffusion properties of the tissue (12) . Furthermore, this and earlier studies (17) demonstrate that the vascularization in embryoid bodies results in increased oxygen supply in the depth of the tissue

The in vitro confrontation culture system used in the present study is superior to previously published confrontation cultures of multicellular tumor spheroids based on endothelial cells (33 , 34) , which appeared to be unsuitable for studying the process of tumor induced-angiogenesis since no invasion of endothelial cells into the tumor tissue was observed (35) . In contrast, tumor spheroids cultivated in our novel confrontation culture with embryoid bodies were efficiently vascularized within few days. This may be because the endothelial cells of embryoid bodies are embedded in a normal tissue microenvironment with defined cytoarchitecture and formation of basement membranes, which may represent an inevitable condition for the directed capillary growth. The induction of PECAM-1, VEGF, HIF-1{alpha}, and P-glycoprotein apparently was selective for the tumor tissue. Angiogenesis occurred in confrontation cultures of embryoid bodies with embryoid bodies; however, the area covered by vascular structures was not significantly different from control embryoid bodies. Likewise, the expression of VEGF, HIF-1{alpha}, and P-glycoprotein did not change when embryoid bodies were cultivated with embryoid bodies in confrontation culture as compared to control embryoid bodies (data not shown).

In the confrontation culture, the first endothelial cells were found in the direct contact region between the tumor spheroid and the embryoid body, suggesting that growth factors, which induce endothelial cell growth and sprouting, are expressed predominantly in this tissue zone. This assumption was validated by immunohistochemical stainings of VEGF expression, which demonstrated that this growth factor was indeed increased in the contact region between the tumor spheroid and the embryoid body. Vascularization of the tumor also resulted in an increased level of HIF-1{alpha} and VEGF expression within the tumor tissue. This may be a consequence of the observed reduction of the pericellular oxygen pressure in the vascularized tumor spheroids vs. control avascular tumors. In contrast, in vascularized embryoid bodies, a marked decrease in HIF-1{alpha} as and VEGF as compared to avascular embryoid bodies was observed and correlated well with the dissipation of oxygen gradients on vascularization. Hypoxia is a well-known feature of several aggressive tumors and correlates with a poor therapeutic outcome and a high vascular density (36 , 37) . Tumor oxygenation depends on the cellular oxygen consumption and on the oxygen supply to the respiring cells. This implies that the pericellular oxygen pressure in tumors is critically determined by the proliferation status of the tumor cells and not simply by diffusion gradients of oxygen. It has been demonstrated for several solid malignancies with highly proliferating cells that the oxygen status of tissue is poorer than in normal tissue at the site of tumor growth (38) .

In the present study, vascularization of tumor spheroids resulted in a pronounced growth stimulation. In contrast, the growth of embryoid bodies was unaffected during confrontation culture. Vascularization of the tumor may have, as a consequence, the presence of an increased oxygenation status in the close vicinity of the invading microvessels. This should result in cell cycle activation and increased oxygen consumption of the tumor cells. The enhanced oxygen consumption by proliferating cells may be the cause of the reduced pericellular oxygen pressure in the depth of the vascularized tumor spheroids as well as for the increased expression of the proangiogenic genes coding for HIF-1{alpha} and VEGF. The elevated oxygen consumption may lead to more severe hypoxic conditions in deeper avascular regions of the tumor tissue and may add to the maintenance of a proangiogenic phenotype, which is inevitable for further vascularization of the expanding tumor and the progression of the cancer disease. The increased cell proliferation after tumor vascularization furthermore resulted in a disappearance of the central necrosis despite low oxygen pressures present in the vascularized tumor tissue. From this observation, we concluded that hypoxia may not be the sole prerequisite for the development of central necrosis.

Another well-known feature of hypoxia in solid tumors is the development of drug resistance (39) and the concomitant expression of stress-induced genes (40) , including heat shock proteins. The molecular mechanism of hypoxia-induced drug resistance and the involvement of the MDR transporter P-glycoprotein are still matters of debate (41) . In tumor spheroids of the EMT6/Ro cell line, hypoxia failed to induce P-glycoprotein mRNA levels (42) . However, another study demonstrated that the ability and efficiency of hypoxia to induce P-glycoprotein expression and drug resistance was cell line dependent (43) . The data of the present study demonstrate elevated levels of P-glycoprotein in vascularized tumor spheroids concomitant with increased levels of HSP27. Coexpression of HSP27 and P-glycoprotein has been evidenced before, indicating that HSP27 may be involved in the MDR phenotype (28) . The increased expression of MDR-related proteins on tumor vascularization may be of therapeutic significance, since it has been demonstrated that antiangiogenic therapy of experimental cancer did not induce acquired drug resistance (44) . Inhibitors of angiogenesis coadministered with conventional antineoplastic agents have been proved to inhibit the growth of drug resistant tumors and to circumvent the MDR phenotype (45) . In view of the results of our study, it appears feasible to avoid acquired and/or intrinsic MDR by antiangiogenic treatment of tumors. Prevention of the development of a MDR phenotype by inhibition of tumor vascularization may uncover novel benefits of the well-established antiangiogenic therapy in the treatment of cancer.

Received for publication June 16, 2000. Revision received September 18, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Grunstein, J., Roberts, W. G., Mathieu-Costello, O., Hanahan, D., Johnson, R. S. (1999) Tumor-derived expression of vascular endothelial growth factor is a critical factor in tumor expansion and vascular function. Cancer Res 59,1592-1598[Abstract/Free Full Text]
  2. Richard, D. E., Berra, E., Pouyssegur, J. (1999) Angiogenesis: how a tumor adapts to hypoxia. Biochem. Biophys. Res. Commun. 266,718-722[Medline]
  3. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., Semenza, G. L. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16,4604-4613[Abstract]
  4. Ratcliffe, P. J., O’Rourke, J. F., Maxwell, P. H., Pugh, C. W. (1998) Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression. J. Exp. Biol. 201,1153-1162[Abstract]
  5. Zhu, H., Bunn, H. F. (1999) Oxygen sensing and signaling: impact on the regulation of physiologically important genes. Resp. Physiol. 115,239-247[Medline]
  6. Folkman, J., Ingber, D. (1992) Inhibition of angiogenesis. Semin. Cancer Biol. 3,89-96[Medline]
  7. O’Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., Folkman, J. (1994) Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79,315-328[Medline]
  8. O’Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., Folkman, J. (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88,277-285[Medline]
  9. Jouan, V., Canron, X., Alemany, M., Caen, J. P., Quentin, G., Plouet, J., Bikfalvi, A. (1999) Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action. Blood 94,984-993[Abstract/Free Full Text]
  10. Nygaard, S. J., Pedersen, P. H., Mikkelsen, T., Terzis, A. J., Tysnes, O. B., Bjerkvig, R. (1995) Glioma cell invasion visualized by scanning confocal laser microscopy in an in vitro co-culture system. Invasion Metastasis 15,179-188[Medline]
  11. Bjerkvig, R., Laerum, O. D., Mella, O. (1986) Glioma cell interactions with fetal rat brain aggregates in vitro and with brain tissue in vivo. Cancer Res 46,4071-4079[Abstract/Free Full Text]
  12. Wartenberg, M., Gunther, J., Hescheler, J., Sauer, H. (1998) The embryoid body as a novel in vitro assay system for antiangiogenic agents. Lab. Invest. 78,1301-1314[Medline]
  13. Vittet, D., Prandini, M. H., Berthier, R., Schweitzer, A., Martin-Sisteron, H., Uzan, G., Dejana, E. (1996) Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88,3424-3431[Abstract/Free Full Text]
  14. Risau, W., Sariola, H., Zerwes, H. G., Sasse, J., Ekblom, P., Kemler, R., Doetschman, T. (1988) Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development 102,471-478[Abstract]
  15. Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., Kemler, R. (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87,27-45[Medline]
  16. Wartenberg, M., Acker, H. (1995) Quantitative recording of vitality patterns in living multicellular spheroids by confocal microscopy. Micron 26,395-404
  17. Gassmann, M., Fandrey, J., Bichet, S., Wartenberg, M., Marti, H. H., Bauer, C., Wenger, R. H., Acker, H. (1996) Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells. Proc. Natl. Acad. Sci. USA 93,2867-2872[Abstract/Free Full Text]
  18. Wartenberg, M., Acker, H. (1996) Induction of cell death by Doxorubicin in multicellular spheroids as studied by confocal laser scanning microscopy. Anticancer Res 16,573-579[Medline]
  19. Liu, Y., Cox, S. R., Morita, T., Kourembanas, S. (1995) Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5' enhancer. Circ. Res. 77,638-643[Abstract/Free Full Text]
  20. Waleh, N. S., Brody, M. D., Knapp, M. A., Mendonca, H. L., Lord, E. M., Koch, C. J., Laderoute, K. R., Sutherland, R. M. (1995) Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker. Cancer Res 55,6222-6226[Abstract/Free Full Text]
  21. Shweiki, D., Neeman, M., Itin, A., Keshet, E. (1995) Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis. Proc. Natl. Acad. Sci. USA 92,768-772[Abstract/Free Full Text]
  22. Zhong, H., De Marzo, A. M., Laughner, E., Lim, M., Hilton, D. A., Zagzag, D., Buechler, P., Isaacs, W. B., Semenza, G. L., Simons, J. W. (1999) Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res 59,5830-5835[Abstract/Free Full Text]
  23. Zhong, H., Agani, F., Baccala, A. A., Laughner, E., Rioseco-Camacho, N., Isaacs, W. B., Simons, J. W., Semenza, G. L. (1998) Increased expression of hypoxia inducible factor-1alpha in rat and human prostate cancer. Cancer Res 58,5280-5284[Abstract/Free Full Text]
  24. Sutherland, R. M. (1988) Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240,177-184[Abstract/Free Full Text]
  25. Wartenberg, M., Frey, C., Diedershagen, H., Ritgen, J., Hescheler, J., Sauer, H. (1998) Development of an intrinsic P-glycoprotein-mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids. Int. J. Cancer 75,855-863[Medline]
  26. Wartenberg, M., Hescheler, J., Acker, H., Diedershagen, H., Sauer, H. (1998) Doxorubicin distribution in multicellular prostate cancer spheroids evaluated by confocal laser scanning microscopy and the ’optical probe technique‘. Cytometry 31,137-145[Medline]
  27. Sanna, K., Rofstad, E. K. (1994) Hypoxia-induced resistance to doxorubicin and methotrexate in human melanoma cell lines in vitro. Int. J. Cancer 58,258-262[Medline]
  28. Schneider, J., Jimenez, E., Marenbach, K., Marx, D., Meden, H. (1998) Co-expression of the MDR1 gene and HSP27 in human ovarian cancer. Anticancer Res 18,2967-2971[Medline]
  29. Kasimir-Bauer, S., Ottinger, H., Meusers, P., Beelen, D. W., Brittinger, G., Seeber, S., Scheulen, M. E. (1998) In acute myeloid leukemia, coexpression of at least two proteins, including P-glycoprotein, the multidrug resistance-related protein, bcl-2, mutant p53, and heat-shock protein 27, is predictive of the response to induction chemotherapy. Exp. Hematol. 26,1111-1117[Medline]
  30. Knuechel, R., Keng, P., Hofstaedter, F., Langmuir, V., Sutherland, R. M., Penney, D. P. (1990) Differentiation patterns in two- and three-dimensional culture systems of human squamous carcinoma cell lines. Am. J. Pathol. 137,725-736[Abstract]
  31. Keller, G., Kennedy, M., Papayannopoulou, T., Wiles, M. V. (1993) Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol. Cell. Biol. 13,473-486[Abstract/Free Full Text]
  32. Wiles, M. V., Johansson, B. M. (1997) Analysis of factors controlling primary germ layer formation and early hematopoiesis using embryonic stem cell in vitro differentiation. Leukemia 11,454-456
  33. Knuchel, R., Feichtinger, J., Recktenwald, A., Hollweg, H. G., Franke, P., Jakse, G., Rammal, E., Hofstadter, F. (1988) Interactions between bladder tumor cells as tumor spheroids from the cell line J82 and human endothelial cells in vitro. J. Urol. 139,640-645[Medline]
  34. Offner, F. A., Bigalke, I., Schiefer, J., Wirtz, H. C., Klosterhalfen, B., Feichtinger, H., Kirkpatrick, C. J. (1993) Interaction of human malignant melanoma tumor spheroids with endothelium and reconstituted basement membrane: modulation by RGDS. Int. J. Cancer 54,506-512[Medline]
  35. Kunz-Schughart, L. A., Kreutz, M., Knuechel, R. (1998) Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology. Int. J. Exp. Pathol. 79,1-23[Medline]
  36. Hockel, M., Schlenger, K., Mitze, M., Schaffer, U., Vaupel, P. (1996) Hypoxia and radiation response in human tumors. Semin. Radiat. Oncol. 6,3-9[Medline]
  37. Hockel, M., Knoop, C., Schlenger, K., Vorndran, B., Baussmann, E., Mitze, M., Knapstein, P. G., Vaupel, P. (1993) Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix. Radiother. Oncol. 26,45-50[Medline]
  38. Vaupel, P., Kallinowski, F., Okunieff, P. (1990) Blood flow, oxygen consumption and tissue oxygenation of human tumors. Adv. Exp. Med. Biol. 277,895-905[Medline]
  39. Koomagi, R., Mattern, J., Volm, M. (1999) Glucose-related protein (GRP78) and its relationship to the drug-resistance proteins P170, GST-pi, LRP56 and angiogenesis in non-small cell lung carcinomas. Anticancer Res 19,4333-4336[Medline]
  40. Heacock, C. S., Sutherland, R. M. (1990) Enhanced synthesis of stress proteins caused by hypoxia and relation to altered cell growth and metabolism. Br. J. Cancer 62,217-225[Medline]
  41. Tomida, A., Tsuruo, T. (1999) Drug resistance mediated by cellular stress response to the microenvironment of solid tumors. Anticancer Drug Des 14,169-177[Medline]
  42. Sakata, K., Kwok, T. T., Murphy, B. J., Laderoute, K. R., Gordon, G. R., Sutherland, R. M. (1991) Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance. Br. J. Cancer 64,809-814[Medline]
  43. Luk, C. K., Veinot-Drebot, L., Tjan, E., Tannock, I. F. (1990) Effect of transient hypoxia on sensitivity to doxorubicin in human and murine cell lines. J. Natl. Cancer Inst. 82,684-692[Abstract/Free Full Text]
  44. Boehm, T., Folkman, J., Browder, T., O’Reilly, M. S. (1997) Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature (London) 390,404-407[Medline]
  45. Browder, T., Butterfield, C. E., Kraling, B. M., Shi, B., Marshall, B., O’Reilly, M. S., Folkman, J. (2000) Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60,1878-1886[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 FASEB J.Home page
M. Schmelter, B. Ateghang, S. Helmig, M. Wartenberg, and H. Sauer
Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation
FASEB J, June 1, 2006; 20(8): 1182 - 1184.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
B. Ateghang, M. Wartenberg, M. Gassmann, and H. Sauer
Regulation of cardiotrophin-1 expression in mouse embryonic stem cells by HIF-1{alpha} and intracellular reactive oxygen species
J. Cell Sci., March 15, 2006; 119(6): 1043 - 1052.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
K. Baar
New dimensions in tissue engineering: possible models for human physiology
Exp Physiol, November 1, 2005; 90(6): 799 - 806.
[Abstract] [Full Text] [PDF]

Home page
 J Biomol ScreenHome page
L. A. Kunz-Schughart, J. P. Freyer, F. Hofstaedter, and R. Ebner
The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model
J Biomol Screen, June 1, 2004; 9(4): 273 - 285.
[Abstract] [PDF]

Home page
 J. Cell Sci.Home page
W. E. G. Muller, N. L. Thakur, H. Ushijima, A. N. Thakur, A. Krasko, G. Le Pennec, M. M. Indap, S. Perovic-Ottstadt, H. C. Schroder, G. Lang, et al.
Matrix-mediated canal formation in primmorphs from the sponge Suberites domuncula involves the expression of a CD36 receptor-ligand system
J. Cell Sci., May 15, 2004; 117(12): 2579 - 2590.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P. Wachsberger, R. Burd, and A. P. Dicker
Tumor Response to Ionizing Radiation Combined with Antiangiogenesis or Vascular Targeting Agents: Exploring Mechanisms of Interaction
Clin. Cancer Res., June 1, 2003; 9(6): 1957 - 1971.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. M. Comerford, T. J. Wallace, J. Karhausen, N. A. Louis, M. C. Montalto, and S. P. Colgan
Hypoxia-inducible Factor-1-dependent Regulation of the Multidrug Resistance (MDR1) Gene
Cancer Res., June 1, 2002; 62(12): 3387 - 3394.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow