First published online 23 January 2003
doi: 10.1242/jcs.00281
Tumour-endothelium interactions in co-culture: coordinated changes of gene expression profiles and phenotypic properties of endothelial cells
Nikolai N. Khodarev1,
Jianqing Yu1,
Edwardine Labay1,
Thomas Darga1,
Charles K. Brown1,
Helena J. Mauceri1,
Reza Yassari2,
Nalin Gupta3 and
Ralph R. Weichselbaum1,*
1 Department of Radiation and Cellular Oncology, University of Chicago, Chicago,
IL 60637, USA
2 Section of Neurosurgery, University of Chicago, Chicago, IL 60637, USA
3 Department of Neurosurgery, University of California San Francisco, San
Francisco, CA 94143, USA
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Fig. 6. Tumour cells stimulate release of self-activating factors in endothelial
cells. (A) Schematics of transfer experiments. HUVECs were cultivated in the
inserts of Transwell chambers in EBM-2 medium alone as acceptor cells and were
also used as negative controls. HUVECs were also co-cultivated with HUVECs
(naive HUVECs) in both compartments or with U87 cells in inserts (activated
HUVECs). Inserts were discarded and cells in the bottom compartment were
intensively washed and co-incubated with acceptor cells for 24 hours. (B)
Increase in formation of net-like structures by acceptor cells co-cultivated
with naive or activated HUVECs. Net-like structures were quantified as
described in Materials and Methods and shown in
Fig. 2. Significance was
estimated by unpaired Student's t-test. (C) Stimulation of RANTES and
FGF7 production by HUVECs activated by tumour-conditioned medium. U87 cells
and HUVECs were set up in T150 flasks and treated as described in Materials
and Methods. HUVEC-conditioned medium is indicated as HCM and
tumour-conditioned medium as TCM. The concentrations are shown in pg
ml–1 of FGF7 and RANTES in HUVEC+HCM culture medium (open
bars, –FGF7; dashed bars, –RANTES) and in HUVEC+TCM culture medium
(grey bars, –FGF7; black bars, –RANTES). Error bars are standard
deviations.
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Fig. 2. Migration and net-like formation. (A) The number of migrated cells counted
from 4 fields each on duplicate membranes (see Materials and Methods). (B)
Number of net-like structures in monoculture (M) and co-culture (C). (C,D)
Images of monoculture and co-culture, used for quantification (magnification
x100). (E,F) Morphology of HUVECs in monoculture and co-culture
respectively (magnification x400). (G) The appearance of net-like
structures is abrogated by anti-angiogenic agents. The figure shows average
numbers of enclosed spaces per field for HUVEC monoculture (M), HUVECs in
co-culture (C) and HUVECs in co-culture with added angiostatin (C/angio) or
endostatin (C/endo) at 100 ng ml–1. Error bars are standard
deviations.
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Fig. 3. Genes in HUVECs related to the angiogenic phenotype undergo transcriptional
reprogramming when exposed to GFP-U87. Endothelial cells and tumour cells were
co-cultured in EBM-2 medium for 0, 5, 12 and 24 hours. RNA was purified from
endothelial cells and cDNA array analysis was performed. After normalization
and filtration of data, we obtained 290 genes. Ratios of expression were
normalized to a +1/–1 scale and data were clustered as described in
Materials and Methods. (A) Cell structure/motility/extracellular matrix genes.
(B) Receptors for growth factors/cytokines/chemokines. (C) Growth response
genes (cell proliferation/cell cycle/DNA repair and recombination). (D)
Receptors for secreted ligands. Red corresponds to upregulated genes and green
to downregulated genes. Data are from a representative experiment with GF211
arrays as ratios of expression in co-culture to monoculture at 5, 12 and 24
hours of co-cultivation.
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Fig. 4. Tie-2 staining is increased when tumour cells are present. (A) HUVECs were
cultured on Transwell inserts as for net-like formation and stained with CD31
or with Tie-2. Staining is comparable in cells in monoculture and in
co-culture for CD-31, but Tie-2 staining is visible only in HUVECs cultured
with U87 in the well. (B) Control brain tissue is compared with tissue from
U87 xenografts. CD31 staining is not significantly different in control and
tumour sections, with only endothelial cells in vessel lumina staining
positive. For Tie-2, staining is visible in vessels in control tissue. In U87
xenografts, more structures are Tie-2 positive, possibly caused by an
increased number of microvessels.
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© The Company of Biologists Ltd 2003
, HUVEC monoculture; 
,
HUVEC co-culture; 
, U87 monoculture; 
, U87 co-culture. (B) Growth
after 48 hours with cells physically separated on Transwell inserts (0.4 µm
pores). HUVECs established on inserts with either HUVECs (M) or GFP-U87 cells
(C) added to wells in starvation medium. (C) Development of angiogenic
phenotype in HUVECs cocultured with GFP-U87 cells. 0 hours is HUVEC
monoculture at the start of the experiment, with 5 hour, 12 hour and 24 hour
time points showing elongation (insert, 5 hours) and migration of cells to
form a morphologically distinct phenotype.
) both interact with FGFRII (
) and CFR-1 (