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Cultures of human tumor-derived
capillary endothelial cells (TdCEC) were obtained from neoplastic tissue
specimens. After collagenase digestion of tumor fragments, the cell suspension
was plated on culture dishes previously coated with cellular human SV-40-transformed
fibroblast-derived fibronectin (WI38VA-FN). This step allowed us to obtain
an enrichment of the endothelial cell population after 5 to 7 days of incubation.
The TdCEC were purified from the nonendothelial cells by selective binding
to the lectin ulex europaeus or to the monoclonal antibody anti-cd31 bound
to magnetic Dynabeads. TdCEC proliferate poorly and age rapidly in comparison
to cultures of normal capillary endothelial cells isolated from human adrenal
glands or from angioma tissue samples. However, when TdCEC were cultured
on dishes coated with WI38VA-FN, which contains a high percentage oncofetal
fibronectin (FN) isoform (> 70% of the total FN purified), and in the
presence of vascular endothelial growth factor, a significant increase of
the TdCEC proliferation was obtained. Our findings indicate that the oncofetal
FN isoform is the most promising candidate for maintaining, in culture,
the microvascular cells derived from human tumors. TdCEC may represent a
unique tool for analyzing the influence of the tumor microenvironment on
microvascular endothelium.
We have extensively studied the extra domain (ED-B) of FN, which is also
known as oncofetal FN because of its high expression on fetal and tumor
blood vessels and its absence in normal adult tissue microvessels (Carnemolla
et al, 1992; Castellani et al, 1987; Zardi et al, 1987). However, despite
strong implications of its involvement in tumor blood vessel development,
the biologic function of oncofetal FN has yet to be determined because of
the difficulty in obtaining microvascular endothelial cells from tumor tissue.
In a previous work, we observed an increase in the expression of oncofetal
FN in a SV-40-transformed human cell line fibroblast (WI38VA), with nearly
70% of the total FN purified from the culture medium containing the oncofetal
isoform (Borsi et al, 1992). Using this FN, we describe herein the purification
and culture of capillary endothelial cells isolated from 13 human malignant
tissue samples.
Human tissue specimens were obtained during surgical procedures. After several
washes, tissue fragments were finely minced with scissors and then incubated
for 2 hours at 37[degree]C in a M199 medium (GIBCO, Grand Island, New York)
containing 0.25% of collagenase D (Boehringer-Mannheim, Mannheim, Germany)
+ 0.25% bovine serum albumin (BSA). The cell suspensions were washed in
M199 medium + 10% fetal bovine serum (FBS) and passed through a 20-[mu]m
pore-size filter (ConsulTS, Turin, Italy). This procedure allows the removal
of all macroaggregates containing the most undesired cells. After centrifugation
at 1000g, the pellets were resuspended in a growth medium of endothelial
basal medium (EBM) supplemented with 10% FBS, 100 ug/ml heparin, 1 ug/ml
hydrocortisone, 10 ng/ml epidermal growth factor, and 10 ug/ml bovine brain
extract (all purchased from Clonetics, San Diego, California). The cells
were seeded in Petri dishes coated with 1 ug/cm2 collagen type I (Coll type
I) (Boehringer-Mannheim) and 1 ug/cm2 of WI38VA-FN. As described above,
nearly 70% of this FN is oncofetal FN isoform and was purified from the
WI39VA (American Type Culture Collection, Bethesda Maryland) cell line culture
supernatant following the method of Engvall and Rouslahti (1977). The dishes
were incubated for 5 to 7 days, and the colonies of endothelial cells arising
in the culture dishes as well as the contaminating nonendothelial cells
(tumor cells and fibroblasts) were detached by trypsin (0.1% w/v), centrifuged,
and resuspended in PBS + 1% BSA at the concentration of 106/ml. The purification
of capillary endothelial cells was obtained following the procedure described
by Jackson et al (1990). Briefly, approximately 5 x 106 magnetic beads (Dynal,
Oslo, Norway) covalently bound with ulex europaeus 1 (UEA-1) lectin (Sigma,
St. Louis, Missouri) or cd31 antibodies (Dako, Carpenteria, California)
were added to 106 cells (ratio of cells/beads = 1:5) and incubated for 30
minutes at 4[degree]C. After incubation, the endothelial cells bound to
the beads were separated from the unbound cells using the magnetic particles
concentrator (Dynal). Most TdCEC were released from the beads by pipetting
the cells against the wall of the tube. After counting, the TdCEC were plated
onto Coll type I+WI38VA-FN-coated dishes at a density of no less than 2
x 104/cm2. TdCEC primary cultures were fed with EBM growth medium and reached
confluence after 10 to 14 days of incubation. TdCEC cultures were then analyzed
for endothelial cell origin. Strong perinuclear immunostaining for factor
VIII was observed in all TdCEC cultures. Immunofluorescence for cd31 antigens
and Ve-cadherins, which are specific markers of endothelium (Lampugnani
et al, 1992; Newmann et al, 1990), was positive and typically found along
the cell borders in culture. Furthermore, TdCEC were able to form capillary-like
structure when seeded on Matrigel (Becton Dickinson, Bedford, Massachusetts)
(data not shown). The use of WI38VA-FN was essential to enrichment of the
endothelial cell population after collagenase digestion of tumor samples.
To test whether WI38VA-FN may be a useful growth substrata for TdCEC, tumor-derived
cells were seeded on dishes coated with Coll type I alone or Coll type I+plasma
FN (plFN) or with Coll type I+WI38VA-FN and left to grow for 7 days. At
the end of incubation, cells were harvested, and TdCEC were measured quantitatively
by counting the number of cells bound to UEA-1-coated magnetic beads. The
percentage of endothelial cells obtained in the different tumor types (meningioma,
neuroblastoma, and mammary carcinoma) was 30 to 50 times higher on Coll
type I+WI38VA-FN than on Coll type I alone or 3 to 5 times than on Coll
type I+plFN. Thus, the oncofetal FN present in WI38VA-FN, but absent in
plFN, was able to enrich the amount of TdCEC obtained from different tumor
samples tested. However, on both FN substrates, the numbers of endothelial
cells isolated from normal adrenal tissue or benign angioma were almost
identical. On the basis of this observation, the WI38VA-FN was used in a
routine manner in the purification of endothelial cells from different malignant
tumors (for a summary of these results, see Table 1). The percentages obtained
of endothelial cells binding the UEA-1 or the monoclonal antibody anti-cd31
were highly variable according to the tumor specimen. Although the amount
of capillary endothelial cells isolated from tumor tissues was relatively
low, we were able to obtain 10 of 13 TdCEC primary cultures. However, not
all endothelial cell preparations successfully produced primary cultures.
A number of factors---namely, differences in the weight of tumor samples,
the grade of tumor vascolarization, and, most likely, the type of tumor
tested--may have contributed to this result. Costello and Del Mastro (1990)
reported the isolation of TdCEC in 50% of brain tumor samples. In our study,
the best results were obtained with brain tumor (4 of 5) and ovary carcinoma
(3 of 3) specimens, whereas the worst were seen with mammary carcinoma (0
of 2) samples. It is interesting to note that the percentage of endothelial
cells isolated from tumor samples was generally lower than from a benign
angioma or normal adrenal tissues (Table 1). Although this finding is an
indirect evaluation of tumor vascularization, it is in keeping with previous
observations that the vasculature of tumors might be less extensive than
that of their normal counterparts (Coomber et al, 1987; Tannock and Stell,
1969). TdCEC growth requirements were studied to determine the optimal growth
culture conditions.
In a proliferation assay, we observed that the addition of 5 ng/ml of human
recombinant vascular endothelial growth factor (VEGF) to EBM growth medium
was essential to the sustenance of TdCEC proliferation. The rate of growth
increase in the three different TdCEC cultures of meningioma, neuroblastoma,
and glioblastoma were 38%, 37%, and 33%, respectively, in the absence of
VEGF, and significantly increased to 79%, 72%, and 85% in presence of the
growth factor. Capillary endothelial cells isolated from normal adrenal
glands grew equally well in EBM growth medium with or without VEGF. The
finding that VEGF raised the proliferation of TdCEC is not surprising given
that this factor is a potent endothelial cell mitogen (Connolly et al, 1989;
Ferrara et al, 1991). However, the observation that VEGF was significantly
effective on TdCEC confirms its role in tumor angiogenesis (Plate et al,
1992).
Although TdCEC have a much lower rate of proliferation and age more rapidly
than endothelial cells isolated from normal adrenal tissues, subcultures
were achieved in 60% of the cases (Table 1). Examination by light microscopy
of living cultures of normal and TdCEC did not seem to differ a few days
after isolation of cells. In contrast, substantial morphologic differences
were noted when cultures reached respective confluences: whereas normal
adrenal capillary endothelial cells typically formed a very homogeneous
monolayer, tumor-derived endothelium appeared very heterogeneous in cell
shape, and giant cells were particularly evident because cultures were at
their initial stage (data not shown). The explanation for this phenomenon
is complex, and two hypotheses are suggested: (a) tumor microvessels in
vivo may undergo a number of replications during tumor growth which may
limit further mitotic events in vitro; or (b) the lack of ``specific'' growth
factors present in the tumor microenvironment may contribute to the acceleration
of in vitro aging. In support of the second hypothesis are findings that
tumor microvessels differ from pre-existing capillaries in many respects
(Hory et al, 1990). For example, there are some promising molecules found
in tumors that are lacking in normal vessels (Thorpe and Burrows, 1995;
Dvorak et al, 1991; Rettig et al, 1992), which could be useful for tumor
vascular targeting; oncofetal FN isoform is the most interesting candidate
among these. Moreover, our findings showing that oncofetal FN and VEGF are
important culture condition requirements for TdCEC also support, at least
in part, this second hypothesis. In conclusion, we have developed a reproducible
method for the isolation and culture of TdCEC. The use of the FN/VEGF combination
described herein could contribute to revealing specific differences between
tumor and normal vessels, which may in turn lead to new strategies for the
therapy of solid tumors.
References
Borsi L, Balza E, Alemanni G, and Zardi l (1992). Differential expression
of the fibronectin isoform containing ED-B oncofetal domain in normal human
cell line originating from different tissues. Exp Cell Res 199:98-105.
Carnemolla B, Leprini A, Allemanni G, Saginati M, and Zardi L (1992). The
inclusion of the type-III repeat ED-B in the fibronectin molecule generates
conformational modifications that unmask a cryptic sequence. J Biol Chem
267:24689-24692.
Castellani P, Viale G, Dorcatto A, Nicolo G, Kaczharek J, Querze G, and
Zardi L (1987). The fibronectin isoform containing ED-B oncofetal domain:
A marker of angiogenesis. Int J Cancer 59:612-618.
Connelly DT, Hewelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, Siegel
RN, Liengruber RS, and Feder J (1989). Tumor vascular permeability factor
stimulates endothelial cell growth and angiogenesis. J Clin Invest 84:1470-1478.
Coomber BL, Stewart PA, and Hayakawa EM (1987). A quantitative morphology
of human glioblastoma multiform microvessels: Structural basis of blood-brain
barrier defect. J Neurol Oncol 5:299-307.
Costello P and Del Mastro (1990). Human cerebral endothelium: Isolation
and characterization of cells derived from microvessels of non-neoplastic
and malignant glial tissue. J Neurol Oncol 8:231-243.
Dvorak HF, Siussat TM, Brown LF, Berse B, Nagy JA, Sotrel A, Manseau EJ,
van de Water L, and Senger DR (1991). Distribution of vascular permeability
factor (vascular endothelial growth factor) in tumors-concentration in tumor
blood vessels. J Exp Med 174:1275-1278.
Envall E and Rouslathi E (1977). Binding of soluble form of fibroblast surface
protein fibronectin to collagen. Int J Cancer 20:1-5.
Ferrara N, Houck KA, Jakeman LB, Winer J, and Leung DW (1991). The vascular
endothelial growth factor family of polypeptides. J Cell Biochem 47:211-218.
Hory K, Suzuky M, Tanda S, and Saito S (1990). In vivo analysis of tumor
vascularization in the rat. Jpn J Cancer Res 81:279-288.
Jackson CJ, Garbett PK, Nissen B, and Schrieber S (1990). Binding of human
endothelium to Ulex Europaeus coated Dynabeads. J Cell Science 96:257-262.
Lampugnani MG, Resnati M, Raiteri M, Pigott R, Pisacane A, Hoven G, Ruco
LP and Dejana E (1992). A novel endothelial specific membrane protein is
a marker of cell-cell contacts. J Cell Biol 118:1511-1522.
Newmann PJ, Berndt MC, Gorsky J, White GC, Lyman S, Paddock C and Muller
WA (1990). Pecam-1 (cd31) cloning and relation to adhesion molecules of
the immunoglobulin gene superfamily. Science 247:1919-1922.
Plate KH, Breier G, Weich HA, and Risau W (1992). Vascular endothelial growth
factor is a potential tumor angiogenic factor in human glioma in vivo. Nature
359:845-848.
Rettig WJ, Garin-Chesa P, Healey JH, Su SL, Jaffe EA, and Old LJ (1992).
Identification of endosialin, a cell surface glycoprotein of vascular endothelial
cells in human cancer. Proc Natl Acad Sci USA 89:10832-10836.
Tannock IF, and Stell GG (1969). Quantitative technique for study of the
anatomy and function of small blood vessels in tumors. J Natl Cancer Inst
42:771-782.
Thorpe PF and Burrows FJ (1995). Antibody-directed targeting of the vasculature
of solid tumors. Breast Cancer Res Treat 36:237-251.
Zardi L, Carnemolla B, Siri A, Petersen TE, Paolella G, and Baralle FE (1987).
Transformed human cells produced a new fibronectin isoform by preferential
alternative splicing of a previously unobserved exon. EMBO J 6:2337-2342.AU:
Who do the numbers in parentheses (first column) indicate?
Received August 5, 1997.
Affiliations: Laboratories of Cell Biology (GA, PC, LZ) and of Pathology
(GN), Istituto Nazionale per la Ricerca sul Cancro, Genova, and Laboratory
of Biology and Therapy of Metastasis (GGSC, RG), Mario Negri Institute for
Farmacological Research, Bergamo, Italy.
This study was supported by funds of the Associazione Italiana per la Ricerca
sul Cancro and Consiglio Nazionale delle Ricerche (Progetto Finalizzato:
Applicazioni Cliniche Della Ricerca Oncologica).
Address reprint requests to: Dr. G. Alessandri, Laboratory of Cell Biology,
Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16132
Genova, Italy. Fax: 39 10 352855. |
