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Experimental Therapeutics |
Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex, HA6 2JR, United Kingdom [G. M. T., V. E. P., J. W., M. C., P. R. B., B. V., D. J. C.], and Department of Radiation Oncology, Duke University Medical Center, Duke University, Durham, North Carolina [S. S., M. W. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-nitro-L-arginine (L-NNA) was also
assessed, because this combination has been shown previously to have a
potentiating effect. The early effect of CA-4-P on tumor vascular
permeability to albumin was determined to assess whether this could be
involved in the mechanism of action of the drug. Tumor blood flow
reduction was extremely rapid after CA-4-P treatment, with red cell
velocity decreasing throughout the observation period and dropping to
<5% of the starting value by 1 h. NOS inhibition alone caused a
50% decrease in red cell velocity, and the combined treatment of
CA-4-P and NOS inhibition was approximately additive. The mechanism of
blood flow reduction was very different for NOS inhibition and CA-4-P.
That of NOS inhibition could be explained by a decrease in vessel
diameter, which was most profound on the arteriolar side of the tumor
circulation. In contrast, the effects of CA-4-P resembled an acute
inflammatory reaction resulting in a visible loss of a large proportion
of the smallest blood vessels. There was some return of visible
vasculature at 1 h after treatment, but the blood in these vessels
was static or nearly so, and many of the vessels were distended. The
hematocrit within larger draining tumor venules tended to increase at
early times after CA-4-P, suggesting fluid loss from the blood. The
stacking of red cells to form rouleaux was also a common feature,
coincident with slowing of blood flow; and these two factors would lead
to an increase in viscous resistance to blood flow. Tumor vascular
permeability to albumin was increased to 
160% of control values at
1 and 10 min after treatment. This could lead to an early decrease in
tumor blood flow via an imbalance between intravascular and tissue
pressures and/or an increase in blood viscosity as a result of
increased hematocrit. These results suggest a mechanism of action of
CA-4-P in vivo. Combination of CA-4-P with a NOS
inhibitor has an additive effect, which it may be possible to exploit
therapeutically. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The tubulin-binding agents, vincristine and vinblastine, are
well-established cytotoxic drugs that have also been found to cause
extensive vascular damage in animal tumors at close to their maximum
tolerated doses (5)
. The tubulin-binding agent,
colchicine, was found to produce hemorrhagic necrosis in human tumors,
but toxicity prevented its further development (6)
.
CA-4-P,3
which is cleaved to its active form, CA-4, by endogenous, nonspecific
phosphatases, is one of a number of compounds isolated from the South
African bush willow tree, Combretum caffrum (7
, 8)
. CA-4 has a high affinity for tubulin at or near the
colchicine binding site (9)
and, like colchicine, causes
the destabilization of the tubulin cytoskeleton. This results in
extensive tumor vascular damage and necrosis in transplanted (ectopic
and orthotopic) and spontaneous tumors (10, 11, 12, 13)
. However,
unlike colchicine, it can have these effects at relatively nontoxic
doses (14)
. It was shown recently that blood flow rate to
a s.c. transplanted rat tumor decreased rapidly, to almost undetectable
levels, by 6 h after i.p. treatment with 100 mg/kg CA-4-P, whereas
the blood-flow rate to normal tissues was much less affected
(15)
. In addition, it was found that systemic NOS
inhibition using N-nitro-L-arginine methyl
ester potentiated the effect in tumor tissue but not in normal
tissues (15)
. This finding links the mechanism of action
of CA-4-P to that of other vascular-damaging strategies, such as
photodynamic therapy and ischemia-reperfusion injury, the effects of
which have also been enhanced by NOS inhibition (16
, 17)
.
A significant increase in vascular resistance was also obtained in
tumors perfused ex vivo with a cell-free perfusate, although
the effect was much less than that observed in vivo
(15)
. In vitro studies have shown that
endothelial cells are particularly sensitive to the effects of CA-4-P
compared with various other cell types (18)
. Proliferating
human umbilical vein endothelial cells round up when exposed to CA-4-P
(13
, 19) , and this is associated with condensation of the
tubulin and reorganization of the actin cytoskeletons and increased
permeability of an endothelial cell monolayer to macromolecules
(18)
. The time course of these changes is very similar to
the CA-4-P-induced vascular effects in ex vivo perfused
tumors (15)
, suggesting that endothelial cell shape change
and an increase in vascular permeability may be related to the vascular
effects of CA-4-P observed in vivo.
CA-4-P has the potential for combination with conventional cytotoxic agents and radiotherapy (14 , 20 , 21) , and Phase I clinical trials of CA-4-P as a single agent are near completion in the United Kingdom and the United States. However, more mechanistic information is needed to optimize treatment with CA-4-P and to develop more effective analogues of the drug. The aims of the current study were: (a) to extend our understanding of the vascular effects of CA-4-P by direct microscopic observation of tumor blood vessels, subsequent to treatment, in a rat window chamber model; (b) to administer CA-4-P with and without systemic NOS inhibition to clarify the role of nitric NO production in the CA-4-P-induced vascular effects; and (c) to determine whether CA-4-P treatment causes a change in tumor vascular permeability in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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200 µm apart, were surgically implanted
into the dorsal skin of male BD9 rats weighing 200 g. Surgery
involved removing the epidermal and dermal layers of both skin layers
of a dorsal skin flap, except for the deepest fascia layer on each
side, and then securing the two sides of the chamber to the skin using
stainless steel screws and sutures (22
, 23)
. After this
procedure, the two fascia layers moved freely between the two glass
windows. Early-generation s.c. transplants of the P22 rat
carcinosarcoma (24)
were used as donor tumors. Tumor
fragments (0.5 mm in diameter) were placed onto one of the fascia
layers, within the window chamber, immediately following surgery.
Animals were given a s.c. injection of a few ml of
dextrose-saline immediately after surgery and kept on a warmed pad
until recovery from anesthesia. Subsequently, animals were kept in a
warm room, 32–34°C, until the day of the experiment.
Marker Red Cells.
Donor red cells were obtained by cardiac puncture from anesthetized
male BD9 rats into a heparinized syringe and fluorescently labeled with
DiI (D-282 Molecular Probes; Cambridge Bioscience) for the measurement
of red cell velocity and flux in recipient animals. This method
has been published previously (25
, 26)
. Briefly, labeling
consisted of separating red cells from plasma and other cellular
constituents by centrifugation and incubating the washed red cells with
DiI for 30 min at room temperature. Approximately 25 µg of DiI were
used per 50 µl of packed red cells. After incubation, red cells were
washed twice and resuspended in PBS to a concentration of 50 µl of
DiI-labeled red cells/ml. Labeled cells were kept for up to 3 days at
4°C before use. Viability and uniformity of labeling were checked by
microscopy.
Intravital Microscopy.
Intravital microscopy was carried out 7–14 days after surgery, when
tumors measured 3.4 ± 0.2 mm in diameter
(mean ± 1 SE for the whole group). An inverted Nikon
Diaphot 200 fluorescence microscope with a stage modified in-house for
taking rats, was used. Animals were anesthetized with Hypnorm and
midazolam and placed on the stage such that the window chamber
was located centrally above the objectives using locating screws.
Rectal temperature was maintained between 35–37°C throughout the
experiment using a thermostatically controlled heating pad beneath the
rat and an infrared overhead lamp. MABP was monitored continuously via
a pressure transducer connected to a cannulated tail artery.
CA-4-P (provided by OxiGene, Inc., Lund, Sweden) was administered i.p. at 30 or 100 mg/kg in a volume of 3 ml/kg, made up in 0.9% saline with a few drops of 5% Na2CO3. Thirty mg/kg CA-4-P (or the vehicle for the drug) was administered 15 min after a bolus i.v. injection of the NOS inhibitor, L-NNA, or the vehicle for L-NNA (water acidified with a few drops of 1 N HCl). NOS inhibition was continued throughout the experiment by supplementing the i.v. bolus dose of L-NNA (10 mg/kg in a volume of 3 ml/kg) with a continuous i.v. infusion of L-NNA (10 µg/kg/h in a volume of 100 µl/kg/h) starting immediately after the bolus dose.
Either the upper or lower surface of the tumors was investigated. The
upper surface enables a large number of venules and capillaries to be
monitored. The lower surface is adjacent to the fascia layer from which
the tumor microcirculation develops and allows monitoring of the
supplying arteriolar vessels and larger draining venules. Intravital
microscopy of tumors was carried out before and after administration of
CA-4-P, with and without systemic NOS inhibition. Preceding the start
of each experiment, an aliquot of fluorescently labeled red cells
(0.2 ml) was injected i.v. Tumor preparations were alternately
viewed under transmitted visible light for the subsequent measurement
of vessel numbers and diameters and under fluorescence
-illumination using a 100-W mercury arc lamp for the
subsequent measurement of red cell flux and velocity under fluorescence
conditions. Fluorescence was set up to excite and detect the emissions
from the red cells labeled with DiI (550 and 565 nm, respectively).
Before the administration of drugs, a tumor ROI, approximately half-way
between the center and the periphery of the tumor, was selected, using
the x20 objective, to include a range of different-sized vessels (the
high-power ROI). At each time point, this ROI was monitored for 30 s using transmitted light and then for 60 s using
-illumination. In addition, the tumor was monitored on transmitted
light at each time point using the x4, x10, and, where possible, x40
objective. Most of the tumor was visible using the x4 objective. In
addition, at the beginning and end of each experiment, the whole tumor
was examined under high-power transmitted light (x20 objective) and
under very low power (x1.5 objective). Exposure to transmitted light
and -illumination was limited to 90 s and 60 s,
respectively, at each time point. XY stage micrometers could be
used to return to a specific ROI (to within <10 µm) when required.
Return to a specific focal plane within the ROI (Z direction) could be
ensured by reference to specific vascular features within the field of
view.
Data Analysis for Intravital Microscopy.
Observations were recorded in a digital format, using a Sony DSR-30P
digital videocassette recorder, for off-line analysis. Multiple frames
(typically 10) were captured onto computer, and the images were
averaged for the analysis of vessel numbers. Vessel numbers, rather
than vessel density or length, were analyzed in order that each vessel
could be described as "large" or "small" (see below) and so
that the fate of individual vessels could be monitored. In all of the
analyses, a single vessel was defined as a vessel length with no
visible branches. No attempt was made to discriminate between small
venules and true capillaries, where red cells proceed in single file,
because of intermittent flow in some tumor vessels, which makes
discrimination difficult. Therefore, venules were defined as being
small or large if their diameters were 
10 µm or >10 µm,
respectively. Vessels on the lower surface of tumors were categorized
as arterioles if they were narrow, fast-flowing, straight, with few
branches, and with divergent flow afferent to the tumor. For analysis
of vessel numbers, vessels were identified in images acquired before
treatment, and these vessels were scored as being present or absent in
subsequent images. Numbers of vessels, which were invisible in the
images acquired before treatment but which appeared in subsequent
images, were noted separately.
Image analysis software was developed in-house for the analysis of
vessel numbers, combining automatic and manual vessel identification
and counting. The software was written in the LabWindowsCVI 5.0
(National Instruments, Redmond, TX) environment to run under
WindowsNT 4.0 (Microsoft Corporation). Working on the 10-frame-averaged
images from a digital video cassette, the software performed
image-processing functions to delineate the visible vessels and produce
a skeleton "map" of vessels for each field from which the number of
vessels was automatically counted. The algorithm performed image
enhancement using spatial filters matched to vessel-like intensity
profiles in two orthogonal orientations. These ridge-shaped spatial
filters had a Gaussian profile with a width appropriate to approximate
the transverse intensity profile of a vessel of known approximate
diameter. They caused vessels to appear bright in the image, while
suppressing all other structures. Hysteresis thresholding and
skeletonization (27)
were used to obtain a binary vessel
map of connected 1-pixel-width lines automatically from the enhanced
image. At this point, the operator could correct the map, if necessary,
by observation. To determine vessel numbers, the points where vessels
branch, cross, and "end" have to be identified within the vessel
map. The end of a vessel is normally where it progresses out of the
plane of image focus. Although this was largely achieved automatically,
the operator was required to intervene in cases where two very close
branches and two vessels that cross but are not joined cannot be
automatically distinguished. This often required reference to the taped
video sequences. On average, the automated procedure correctly
identified 60% of the visible vessels, leaving the user to draw the
remainder on screen using the computer mouse and custom user interface.
Additional analysis was carried out manually on all of the vessels in the higher power ROI (x20 objective) to identify functioning versus nonfunctioning vessels. Nonfunctioning vessels were defined as those vessels which had disappeared from the field of view (assessed by comparison of each image with the starting image) plus those vessels that contained stationary red cells for at least 30 s (the monitoring period for each time point).
Quantitative data on flow characteristics and diameter measurements were obtained from selected vessels within the high power ROI (x20 objective). Selection of four venules from the upper surface and up to four venules from the lower surface of tumors was based on examination of the pretreatment tapes. Vessels were chosen for their length (>30 µm, typically 100 µm), uniformity of flow, and a range of diameters within each tumor. Measurements were made with the aid of an image overlay hardware system (DAVID; Brian Reece Scientific Ltd., Reading, United Kingdom). This was calibrated using microscope distance standards (1, 10, and 100 µm) so that the distance between any pair of selected pixels could be directly read out, on the image, in microns. Diameters of selected venules on the upper and lower surfaces of tumors and of selected arterioles on the lower surface were measured. For these, pairs of points were chosen on opposite sides of the red cell column and perpendicular to it in each vessel (10 pairs/vessel) under transmitted visible light. Small vessels (<10 µm diameter) were not used for the diameter analysis. Measurement of the width of the red cell column may slightly underestimate the true vessel diameters. However, we were more interested in detecting changes in vessel diameter after treatment than in measuring absolute values, and we felt that the method chosen would be a reasonable reflection of this. In addition, alternative methods would have involved the use of exogenous fluorescent markers for identifying the vessel wall, and these may be vasoactive in themselves and can extravasate from hyperpermeable vessels leading to the obfuscation of vessel margins.
Red cell velocity and flux in all selected vessels were calculated from
recorded data of the high-power ROI on fluorescence -illumination.
At least 10 fluorescent red cells were monitored for velocity
measurements over 60 s of recorded images. Velocity was calculated
in µm/s from the number of video frames taken for each red cell to
travel between two points of measured distance, marked on the
vessel image. If, after treatment, no red cells were observed within
the observation period, a value of 0.85 µm/s was assumed. This is a
very small fraction of the average pretreatment velocity observed (see
"Results") and represents the maximum possible velocity under this
condition, assuming equal spacing of fluorescently labeled red cells.
Using this method, there is a tendency to underestimate very slightly
the degree of vascular shut-down observed. Changes in red cell flux
were calculated from the number of fluorescent red cells crossing a
single point marked on the vessel within a measured time (1 min). On
average, 57 fluorescent red cells/vessel were counted at the initial
time point for the calculation of changes in red cell flux.
Flow rates through individual vessels on the upper surface were
calculated from the red cell velocity and diameter measurements
described above (flow rate = red cell
velocity * 
/4 *
2), where
d is vessel diameter. This assumes that the red cells are
traveling with the bulk plasma flow. A few red cells, which were
observed to be traveling slower than the majority within a particular
vessel, were not used for velocity calculations. Relative changes in
vascular resistance with time after treatment were estimated from the
relative change in MABP divided by flow rate. Relative changes in
hematocrit within individual vessels were calculated from changes in
red cell flux relative to changes in the flow rate. That is, hematocrit
[red cell flux/(
2 * red cell
velocity)], where r is vessel
radius. This approximates that treatment does not affect the mean
volume of red cells, and that red cells travel with the bulk plasma
flow.
Tumor Vascular Permeability to Albumin.
For the measurement of changes in tumor vascular permeability to
albumin at various times subsequent to CA-4-P treatment, the P22
carcinosarcoma was grown in the right inguinal fat pad of BD9 rats such
that the tumor developed with only a single supplying artery and a
single draining vein. This method has been published previously
(28)
. Tumors were treated 2 weeks after implantation
(mean tumor weight ± SE was 0.59 ± 0.07 g for the whole group) and assayed after treatment with drug
vehicle only (control) or 1, 10, 20, or 30 min after 30 mg/kg CA-4-P
given i.p., as described above. Access to the tumor-supplying artery
allowed changes in tumor vascular permeability to be determined via a
slightly modified version of the classical Oldendorf method
(29)
, originally used to determine changes in vascular
permeability to various plasma solutes in the cerebral circulation.
Briefly, at the assay time, an admixture of 0.019 MBq (0.5 µCi) of
the test compound (in this case, 125I-labeled
BSA; ICN Pharmaceuticals Ltd., Basingstoke, United Kingdom) and 0.008
MBq (0.2 µCi) of a low-molecular weight reference compound (in this
case, 86RbCl; Amersham Pharmacia Biotech UK Ltd.,
Little Chalfont, United Kingdom) was injected, in saline (0.1 ml), into
the retrograde-cannulated saphenous artery. This meant that the
admixture was washed directly into the tumor-supplying artery via
arterial blood flow. Only a short time (10 s) was allowed to elapse, to
minimize backflux into the vasculature and to ensure minimal
recirculation, before the tumor was excised, weighed, and counted
together with an injectate sample. Samples were counted on a Wallac
Autogamma counter with suitable gating for the simultaneous assessment
of counts from 125I and
86Rb. Because the admixture of tracers was
administered directly to the tumor, the ratio of
125I counts:86Rb counts in
the injectate was assumed to be equivalent to the ratio in the
supplying blood. Therefore, any change in tumor vascular permeability
to albumin subsequent to treatment was assessed by calculating the
uptake of 125I albumin into tumor tissue
(125I counts in tumor ÷ 125I counts
in injectate) as a percentage of the uptake of 86RbCl
(86Rb counts in tumor ÷
86Rb counts in injectate). This tumor
uptake index was considered to be a good index of changes in
vascular permeability to BSA because the uptake of both tracers would
be equally affected by blood flow changes after treatment, and the
uptake of 86RbCl is unlikely to be permeability-limited in
tumors.
Statistics.
Statistical analysis was carried out using JMP Statistics Version 3 for
the Apple Macintosh (SAS Institute, Inc., Cary, NC). Intravital
microscopy data were fitted to a multivariate model (MANOVA) with
repeated measures to determine the effects of the different treatments
on vessel numbers, diameters, MABP, red cell velocity, vascular
resistance, and hematocrit over time. Responses were fitted to effects
using least squares. The effect of variation between individual vessels
was accounted for by using a nested design, and any variation between
rats was described as random. Differences in responses caused by
treatment or time were tested for significance using an approximate
F test, based on comparison of the matrix for the hypothesis
sum of squares and cross products with the matrix for the residual.
Differences in response were described as significant if the
probability corresponding to the F value was <0.05. The
Student t test for unpaired data was used to test for
significant differences in tumor uptake indices for different treatment
groups. A P < 0.05 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the effect being more profound for the
100 mg/kg dose than for the 30 mg/kg dose. After 30 mg/kg CA-4-P (Fig. 1a)
, total visible vessel number decreased to 65% of the
pretreatment number by 10 min after treatment but subsequently
recovered to around 80% by 45 min. For 100 mg/kg CA-4-P (Fig. 1b)
, there was a similar initial reduction in total vessel
number, but with no indication of recovery at the later times.
Reduction in small venule number was more pronounced than reduction in
large venule number, especially for the 100 mg/kg group, the difference
becoming statistically significant at early times after treatment in
this group (Fig. 1b)
. Interestingly, a slight decrease in
the total number of vessels over time was observed for untreated tumors
(shaded regions in Fig. 1, a and b
).
However, this was not the case if vessels that were invisible in the
pretreatment images, but which appeared in subsequent images, were
included in the analysis (data not shown). This is indicative of some
temporal fluctuations in the flow in the untreated tumors. A small
number of "new" vessels also appeared in the treated tumors after
the start of treatment, but inclusion of these did not make a
significant difference to the data shown in Fig. 1
.
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shows that, for venules on the upper surface of tumors, the number of
visible vessels, as shown in Fig. 1a
, is a poor reflection
of the functionality of vessels that reappear at later times.
Approximately 4% of vessels in untreated tumors were nonfunctioning.
The number of nonfunctioning vessels increased with time for rats
treated with 30 mg/kg CA-4-P or with the combination of 30 mg/kg CA-4-P
and NOS inhibition, with no indication of recovery at later times.
There were significantly more nonfunctioning vessels in the combination
group than in the CA-4-P alone group at 45 and 60 min post-CA-4-P.
However, because L-NNA alone caused some vessels to stop functioning,
this effect may be no more than additive (Fig. 2)
. Nonfunctioning
vessels were defined as those which disappeared from the field of view
plus those which were visible but contained nonflowing red cells.
Therefore, the recovery in total vessel number, as shown in Fig. 1a
, predominantly consists of this latter group of
nonflowing vessels. Presumably, red cells began to reflow in some of
the vessels, which had disappeared from view, accounting for the
recovery in numbers; but this returned flow was only temporary. By
1 h after the start of CA-4-P, there were two to three times more
vessels in the visible, nonflowing category than in the disappeared
category for both CA-4-P treatment alone and for the combination with
NOS inhibition (data not shown). In the L-NNA group, most of the
nonfunctioning vessels were in the disappeared category, for the whole
time course. There is no obvious explanation for the difference between
the L-NNA group and the combination group before time 0, except that
the data are very heterogeneous for the L-NNA group, principally
because of a large number of nonfunctioning vessels in one tumor of
this group. Venules on the lower surface of tumors responded very
similarly to those on the upper surface. Arterioles tended to be more
resistant, but this did not reach statistical significance with the
number of vessels analyzed (data not shown).
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shows the changes in the MABP observed for rats treated
with 30 mg/kg CA-4-P alone, L-NNA alone (10 mg/kg plus constant
infusion), or the combination of the two agents. MABP was significantly
increased for both L-NNA alone and CA-4-P alone; however, the pattern
of change was different for the two agents. MABP was significantly
raised after L-NNA throughout the whole time course of the experiment,
reaching a maximum (150% of the starting value) at 10 min after the
bolus administration. After 10 min at this pressure, MABP decreased
to around 120% of the pretreatment value despite a continuous infusion
of L-NNA throughout this period. After CA-4-P treatment, MABP gradually
increased to around 125% of the starting value, which was maintained
throughout the time course of the experiment. These results are
consistent with previously reported values for NOS inhibition and
CA-4-P (15
, 30
, 31)
. The effect of the combination of
CA-4-P and L-NNA on MABP was not significantly different from that of
L-NNA alone.
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shows the changes in red cell velocity within
selected tumor blood vessels of the upper surface of tumors within
high-power ROIs. The effect of CA-4-P on red cell velocity is extremely
rapid, with velocity falling throughout the time course of the
experiment and reaching <5% of its starting value by 1 h. This
blood flow shut-down is impressive in light of the increase in MABP
over the same time course (Fig. 3a)
, which will tend to
increase the perfusion pressure of individual vessels and therefore
maintain flow. L-NNA caused a significant reduction in red cell
velocity almost immediately, reaching a maintained value of 50% by 15
min after bolus administration. The combination treatment is more
effective than CA-4-P alone but, as for the data shown in Fig. 2
for
the whole tumor field, there is little indication of anything more than
an additive effect of the two agents. Similar results were obtained for
venules on the lower surface of tumors [preliminary data published
previously (19)
].
Blood flow rate was calculated from the velocity data shown in Fig. 3b
and the diameter of the individual vessels. Results were
almost identical to those shown for velocity, except that the increase
in flow for the control group was smaller than the increase in velocity
(data not shown). This similarity reflects the fact that average
changes in venule diameter were very minor for all treated groups (Fig. 4)
, and that blood flow rate in the treated groups was determined
predominantly by changes in local perfusion pressures and/or blood
viscosity rather than by venular diameters.
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were calculated from the MABP data shown in Fig. 3a
and the blood flow rate data. This calculation assumes
that a change in MABP produces an identical change in perfusion
pressure in all of the individual vessels analyzed. Although this is
unlikely to be the case, and the resulting values are very variable for
the treated groups (see legend to Fig. 3
), the data do indicate
that vascular resistance is greatly increased after treatment with
CA-4-P or the combination of CA-4-P with L-NNA. In addition, the
calculated vascular resistance for the control group was almost
constant with time (Fig. 3c)
, in contrast to velocity and
flow, which significantly increased. This is attributable to a
balancing effect of the increase in MABP and the increase in flow rate
for individual vessels in this group.
There was a tendency for hematocrit to increase in the larger venules
analyzed on the lower surface of the tumors after treatment with
CA-4-P, which reached significance at 10 min posttreatment but not at
20 min (data not shown). Fig. 4
shows the effects of the different
treatments on vessel diameter. There is a small but significant
reduction in arteriole diameter at 45 and 60 min after CA-4-P
treatment, but there is no significant effect on the venules (Fig. 4, a
and b, respectively). L-NNA caused a
significant reduction in arteriolar diameter to 80% of control, but
this was lost at later times (Fig. 4c)
. No significant
effects were found for venules (Fig. 4d)
. This result is
similar to previously published data for this agent (32)
.
For the combination treatment, there was a progressive and profound
reduction in arteriolar diameter, which reached 60% of control by
45 min (Fig. 4e)
. There was also an early significant but
smaller reduction in the diameter of venules to 85% of control, but
these vessels recovered at later times, with some vessels dilating
beyond their starting values (Fig. 4f)
. A similar response
pattern was seen for both CA-4-P and the combination treatment in the
venules of the upper surface (Fig. 5)
. It is apparent that the pattern of response to CA-4-P or the
combination treatment is different for arterioles and venules, with
arterioles tending to show a sustained reduction in diameter, and
venules tending to recover from any decrease, at later times.
Some venules, but not arterioles, seemed to be dilated compared with
their starting values (Fig. 4
, a, b, e, and
f; and Fig. 5
).
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shows example images from video records of tumors from rats at
different times after treatment. These images illustrate the decrease
in number of visible vessels with time after treatment with CA-4-P or
CA-4-P combined with L-NNA and then a partial recovery at later times,
as quantified in Fig. 1a
. It also shows that most of the
vessel loss is in the center of tumors. An increase in diameter of some
of the larger venules, by the end of the observation time, is also
apparent, especially for the combination treatment. Other observations,
which were not quantified, are illustrated in Fig. 5
. First, a general
darkening at the tumor periphery was apparent for the CA-4-P- and
CA-4-P-plus-L-NNA-treated tumors at the later times. This is most
likely attributable to hemorrhage from the peripheral vessels.
Difficulty in focusing the x40 objective was noted in a majority of
treated tumors, starting at early times after treatment; and this may
also be related to an increased permeability of tumor venules,
resulting in edema. Red cell stacking (formation of rouleaux), as blood
flow slowed, was very apparent and is illustrated in Fig. 5
. Recordings
were made of the granulation tissue surrounding tumors at the start and
finish of the observation period. It was obvious that blood flow was
reduced in this tissue by CA-4-P and the combination treatment,
although the effect was not as profound as in the tumor tissue. On a
few occasions, the visible tumor vasculature in the high-power ROI or
in a larger area completely disappeared for a few seconds.
Presumably, this is attributable to a temporary shut-down of a
supplying vessel or vessels upstream from the ROI.
Fig. 6
shows the effect of CA-4-P on the uptake of BSA into the P22 tumor. The
tumor uptake index for 125I
albumin/86RbCl was significantly increased by
60% at 1 and 10 min after CA-4-P treatment compared with control
values. This indicates a significant increase in tumor vascular
permeability to albumin at early times after treatment.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the difference between the two doses may be predictive of a
later recovery in flow. Indeed, using a radiotracer method, there was
no recovery of blood flow rate in subcutaneously transplanted P22
tumors at 6 h after 100 mg/kg CA-4-P (15)
. At
24 h, there was a partial recovery for 30 mg/kg CA-4-P but not for
100
mg/kg.4
Very small venules accounted for most of the disappeared tumor blood
vessels after CA-4-P treatment. It is not clear whether these vessels
collapsed completely or whether they collapsed/constricted or became
blocked with red cells locally, which allowed the down-stream passage
of plasma but not red cells. Because vessels could only be
identified under visible light by the presence of red cells, we could
not distinguish between these events. Attempts to label the plasma with
TRITC-albumin for identification of vessels under fluorescence
conditions were inconclusive because of leakage from the vessels (data
not shown). A very small decrease in diameter of a small venule would
close it off completely. This could occur via various means. First, the
observed increase in tumor vascular permeability to plasma proteins
could result in a net transport of water from the plasma into the
interstitial space (edema), resulting in an imbalance between the
intravascular and tissue hydrostatic pressures, causing vascular
collapse. Second, a decrease in diameter could be caused by the
rounding-up of endothelial cells, an effect consistently observed after
exposure of endothelial cells in culture to low concentrations of
CA-4-P (13
, 18
, 19)
. A third possibility, that leukocyte
adherence to the vascular wall could account for changes in venular
diameter (33)
, is unlikely because, in an in
vitro flow system, neutrophil adherence to an endothelial cell
monolayer was only observed after several hours’ exposure to CA-4-P
(34)
. In addition, a very rapid increase in vascular
resistance of P22 tumors was observed after exposure to CA-4-P in an
isolated perfusion system where leukocytes were absent
(15)
. Some venules were obviously widened and engorged
with red cells by the end of treatment, an effect which was most
profound in tumors from rats treated with the combination treatment
(Fig. 5)
. This suggests some recovery from the initial effects of
CA-4-P combined with local increases in intravascular pressures,
perhaps caused by continued down-stream vessel narrowing, which caused
the vessels to distend. Using conventional histology, the widening and
engorgement of venules in the P22 tumor were not commonly associated
with coagulation at those
times.5
Diameter changes in venules are unlikely to be caused by active
vasoconstriction/vasodilation, especially as the pattern of change in
venule diameter after treatment was very different from those observed
in arterioles (Fig. 4)
. The reduction in arteriolar diameter to 80%
of the starting value by 15 min after administration of L-NNA alone is
consistent with reported values for other tumors (32)
.
Arteriolar vasoconstriction (or, strictly, reversal of vasodilation)
could account for all of the flow reduction observed for L-NNA alone,
and this is consistent with the absence of any changes in vessel
numbers, hematocrit, or rheology in the current study. That is, blood
flow rate is proportional to the fourth power of vessel radius
(Poisseuille’s law; Ref. 35
). This means that a reduction
in arteriolar radius to 80% would reduce flow rate through the system
to 41%, which is very close to the actual data for red cell velocity
(Fig. 3b)
and blood flow rate (data not shown). For the
group treated with CA-4-P alone, there was no reduction in arteriolar
or venular diameter at early times, which could account for the
profound blood flow reduction observed in these tumors (comparing Fig. 4, a
and b, with Fig. 3b
). The small
arteriolar constriction, to 90% of the starting value, at 1 h
posttreatment (Fig. 4a)
, would contribute to the maintenance
of blood flow reduction at this time, but it is only sufficient to
reduce blood flow to 66% of the starting value (Poisseuille’s law).
Clearly, this does not account for the full effect of the drug. The
combination treatment produced a profound effect on arteriolar
diameter, reducing it to 60% of the starting value by 45 min after
the start of CA-4-P treatment. However, even this major
vasoconstriction is only sufficient to reduce blood flow rate to 13%
of the starting value, which clearly is not sufficient to account for
the changes shown in Fig. 3
.
The increase in vascular permeability to albumin, observed in the inguinal fat pad tumors at 1 and 10 min after CA-4-P treatment, is likely to be a key factor leading to blood flow shut-down. This could occur not only via physical collapse of a subpopulation of small venules (see above) but also via an increase in intravascular hematocrit, leading to an increase in blood viscosity consequent to fluid loss from the vasculature. Indeed, there was some evidence for an increased hematocrit in large venules of the lower tumor surface at early times after treatment. In the rat, vascular shut-down by CA-4-P is specific for tumors, with most normal tissues minimally affected by the drug (15) . It will be important to determine whether this is attributable to the absence of permeability changes in normal tissue blood vessels after CA-4-P, or whether blood flow in normal tissues can be sustained in the face of increases in permeability. This study is currently under way. An increase in blood viscosity could also be caused by any direct effect of CA-4-P on red cell shape, but this has not been investigated. It has been suggested that small changes in red cell deformability may occur more readily and have a greater consequence in tumors than in normal tissues because of the unique tumor metabolic environment and vascular morphology (36) .
The behavior of red cells at low flow rates in vivo has been
described recently (37)
. It was found that, below a
critical red cell velocity (250 to 300 µm/s), red cell movement
became erratic, with significant radial movements away from the main
direction of flow. This was associated with orientation changes
involving the long axis of the cell becoming rather perpendicular to
the direction of flow. These conditions lead to an energy dissipation
of flow and an enhanced collisions of red cells with each other. This,
in turn, leads to the stacking of red cells to form rouleaux and a
massive increase in viscosity and vascular resistance to flow. Rouleaux
may completely block microvessels. Mean pretreatment velocities in
venules of the upper surface of tumors were 500 µm/s, and rouleaux
formation was consistently observed as flow rates decreased after
CA-4-P treatment. Thus, these rheological changes are likely to play a
major part in the vascular shut-down observed in the current study, and
there will be a positive feed-back, with blood flow reduction leading
to the stacking behavior, which leads to additional blood flow
reduction. In addition, as blood flow reduces, the tumor blood vessels
will become more hypoxic, and this can increase the suspension
viscosity of red cells (38)
and increase rouleaux
formation, the latter via exposure of adhesion molecules on the surface
of the cells. This may be more apparent in the central vessels, which
tend to be more hypoxic than the peripheral ones (39
, 40)
.
A summary of the putative mechanisms leading from CA-4-P-induced damage
to the vascular endothelium to blood flow collapse is shown in Fig. 7
and is reminiscent of an acute inflammatory reaction.
|
Blood flow at the periphery of tumors was consistently less affected than at the center of tumors, and this is consistent with the sparing of the tumor periphery in terms of tumor cell toxicity (10) . Vessels at the periphery may be more resistant to the primary damage induced by CA-4-P than those at the center as a result of putative differences in vascular/endothelial structure between the two compartments. Alternatively, the peripheral vasculature may be more resistant to some of the secondary effects of CA-4-P treatment. For instance, the peripheral vessels tend to be larger and faster flowing than the central vessels, which would make them less susceptible to blood flow collapse via diameter changes and would make a small blood flow reduction less catastrophic. Similarly, the interstitial fluid pressure of tumors tends to drop precipitously at the tumor periphery (41) , and this would favor vascular collapse in the central region. The fact that hemorrhage was apparent at the periphery of tumors after CA-4-P treatment is an indication that the peripheral vessels may be just as sensitive to the primary effects of CA-4-P as the central vessels. Whatever the cause, it is apparent that the reduced response of the peripheral vessels limits the effectiveness of CA-4-P and causes rapid regrowth of tumors after treatment. Combination of CA-4-P with treatments designed specifically to target the tumor cells in the periphery have shown particular promise (42) .
The effect of acute systemic NOS inhibition, using L-NNA, was to decrease red cell velocity and blood flow rate by 50% throughout the observation period, which is consistent with previous studies for this tumor growing s.c. (31) . Similar results have been reported for other tumors (32) . There was significant arteriolar vasoconstriction, which would account for the blood flow changes induced by L-NNA alone. An increase in leukocyte-endothelial interactions is also a likely consequence of NOS inhibition (32) , although this was not investigated in the current study. NO is known to influence vascular permeability, although its effects in tumors are variable. Fukumura et al., (32) found that systemic NOS inhibition reduced vascular permeability to albumin in one experimental tumor model but not in a second. A reduction in tumor vascular permeability after the administration of L-NNA would tend to protect against vascular damage by CA-4-P, if our hypothesis regarding the role of vascular permeability changes in CA-4-P-induced vascular damage is correct. Clearly this was not the case, indicating that additional experiments are required to clarify potential interactions between NOS inhibition and CA-4-P.
Qualitatively, the vascular effects of L-NNA alone were very different
from those of CA-4-P alone, arteriolar vasoconstriction playing only a
minor part in the latter case, as described above. The vascular effects
of the combination of L-NNA with CA-4-P were qualitatively very similar
to those for CA-4-P alone, but the effects were greater for the
combined-treatment group. At the end of the observation period,
vascular resistance seemed to be higher, and there were more stagnant,
distended vessels in the combined-treatment group than in the
CA-4-P-alone group. However, there was no evidence for a potentiating
effect of L-NNA on CA-4-P, which was found previously for the prodrug
of L-NNA, N-nitro-L-arginine
methyl ester, on tumor blood flow and vascular resistance in the P22
tumor grown s.c. (15)
. There could be several reasons for
this disparity relating to the differences in experimental design, and
this requires additional investigation. In any case, the current study
reveals that CA-4-P induces a reduction in tumor blood flow, which is
qualitatively different from that obtained with NOS inhibition, and it
suggests that the combination of the two approaches may be
advantageous. Tumor growth and toxicity studies are required to test
this possibility.
In conclusion, this paper describes the tumor vascular response to systemic treatment with CA-4-P and suggests a mechanism of action leading from initial tubulin binding to vascular shut-down. This mechanism is reminiscent of a classical acute inflammatory reaction, involving a very rapid increase in vascular permeability to plasma proteins. It suggests that acute NOS inhibition, in combination with CA-4-P treatment, has an additive effect that may be possible to exploit therapeutically. CA-4-P is now in clinical trial as one of the few primarily antivascular, as opposed to antiangiogenic, drugs currently available. It will be important to fully elucidate its mechanism of action to be able to use it effectively and to develop this approach further.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by the Cancer Research
Campaign, the National Lottery Charities Board of the United Kingdom,
and a travel grant from NATO (to M. W. D.). 
2 To whom requests for reprints should be
addressed, at the Gray Cancer Institute, P. O. Box 100, Mount Vernon
Hospital, Northwood, Middlesex, HA6 2JR, United Kingdom. Phone:
44-01923-828611; Fax: 44-01923-835210; E-mail: tozer{at}graylab.ac.uk
3 The abbreviations used are: CA-4-P, disodium
combretastatin A-4 3-0-phosphate; NO, nitric oxide;
L-NNA, N-nitro-L-arginine; NOS, nitric oxide synthase;
ROI, region of interest; MABP, mean arterial blood pressure.
4 V. E. Prise, J. Wilson, D.J. Honess, and G. M. Tozer. Blood flow response of tumors and normal
tissues to treatment with combretastatin A-4-P, manuscript in
preparation.
5 G. M. Tozer and V. E. Prise, unpublished
data.
Received 9/13/00. Accepted 7/ 2/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and large (>10 µm diameter,
) venules. Errors are omitted from the data on large and small
venules for clarity. * represents significant difference
(P < 0.05) between large and small
venules.
); and vehicles
only (
). *, significant difference (P < 0.05) from untreated tumors; 
, significant difference
(P < 0.05) between CA-4-P alone and
CA-4-P plus NOS inhibition.
