Drug Targeting Organ-Specific Strategies

240 9 Tumour Vasculature Targeting
dothelial cells are cultured on top of a matrix gel (i.e. collagen [50], fibrin [51], or matrigel
[52]) and are induced to form sprouts into the matrix by stimulation with either growth factors,
tumour biopsies or tumour cell lines grown as spheroids. An essential feature in all of
these assays is that a lumen is formed in these sprouts and that not merely migration and cell
rearrangement has taken place.
Endothelial cells grown on gelatin-coated beads embedded in a matrix can be induced to
form sprouts into the matrix [53]. The sprouting can be quantified either by measuring the
distance over which vessels were formed or by computer-aided measurement of total vessel
length. Other methods reflect the in vivo situation even more closely. These methods are
based on endothelial cell sprouting from freshly isolated tissues embedded in matrix gels, including
the rat aortic ring [54] and human placenta tissue [55]. This procedure is not applicable
for all tissues.Tumour biopsies, for example, often produce an excess of proteases.As a result,
the matrix is digested and endothelial cell sprouting prevented [50]. A recently published
in vitro angiogenesis assay that mimics the in vivo situation quite well, exploits the use
of embryoid bodies [56]. In vitro cultured mouse blastocyst cells [57] recapitulate several
steps of murine embryogenesis, including vasculogenesis and angiogenesis [58].There is complete
blood vessel development in these embryoid bodies [59] which makes this system suitable
for studying the effects of a wide spectrum of angiogenesis modulators.
The advantages of in vitro assays are (1) the ability to control the assay variables, (2) the
potential opportunity to study the various steps within the complete process, (3) cellular and
molecular events can be more carefully monitored and (4) the costs and the duration of the
experiments are often lower than those of in vivo assays. The disadvantages of in vitro assays
are that the cells, reagents and conditions used in different laboratories are not standardized
and that the in vitro effects seen, do not always match the activities observed in vivo.This has
been demonstrated for e.g.TNFα, which inhibits angiogenesis in vitro, but induces angiogenesis
in vivo [6]. Particularly in the light of the possible influence of various cells of the immune
system on angiogenesis, extrapolation of data from in vitro to in vivo needs to be carefully
addressed.
9.2.4 In Vivo Assays to Study Angiogenesis and Targeting of Angiogenic
Blood Vessels
It is well recognized that in vitro angiogenesis assays can have clear advantages. However, the
major drawback of all of these assays is that they require the endothelial cells to be removed
from their natural microenvironment, which alters their physiological properties. To study
angiogenesis in vivo, the most frequently used assay systems exploit chicken chorio-allantoic
membrane (CAM) [28,60], the corneal pocket [61], transparent chamber preparations such
as the dorsal skin fold chamber [62,63], the cheek pouch window [64] and polymer matrix implants
[65,66].
The CAM assay is based on the developmental angiogenesis occurring in the CAM during
chick embryo development. The developing vasculature can easily be observed and regulators
of angiogenesis can be tested in this model by intra-vessel injection or by addition of soluble
compounds, either by release from within a silicone ring placed onto the membrane or
by release from a methylcellulose or alginate pellet. This assay is relatively inexpensive and