High-Energy Shock Waves Induce Blood Flow Reduction in Tbmors1

[CANCER RESEARCH 53. 1590-1595. April I. 19931
High-Energy Shock Waves Induce Blood Flow Reduction in Tbmors1
Fernando Gamarra, Fritz Spelsberg, Gerhard E. H. Kuhnle, and Alwin E. Goetz2
Institute of Surgical Research IF. G.. F. S.. G. E. H. K.j and Institute of Anesthesiology ¡A.E. G.j. Luilwif>~Ma.*iniilians-llni\:ersit\ Munich, Klinikum Griisshtidem. Munich.
Germani
ABSTRACT
We have studied the effect of extracorporeally applied high-energy
shock waves (HESW) on blood flow in amelanotic melanomas (A-Mel-3).
Two tumors were implanted in the dorsal skin of 21 Syrian golden ham
sters. One of the tumors was treated with 200 HESW, and the other served
as an intraindividual control. Mean blood flow in the whole tumor, or the
tumor excluding necrotic areas, was quantitatively measured using autoradiography
with iodo['4C]antipyrine at 30 min (n = 5), l h (n = 5), 3 h
(n = 5), and 12 h (n = 6) after HESW treatment. As measured for the
whole tumor, blood flow in the controls was 23.4 ±7.9 ml/100 g/min
(median ±SE) and thus in the range reported in the literature. Thirty min
or l h after the application of HESW, tumor perfusion was reduced to 6
±4% or 5 ±4% (median ±SE) of the corresponding controls, respec
tively. Three h after treatment, perfusion increased slightly to 7 5% and
after 12 h increased significantly to 55 ±25% of the corresponding
controls. Values measured excluding the necrotic areas were higher in all
groups. Temporary reduction of tumor perfusion after treatment with
HESW was interpreted as a consequence of HESW-induced damage to
tumor microcirculation. These effects should be taken into account for
maximizing the therapeutic efficiency of HESW on tumors and for com
bining HESW treatment with other therapeutical modalities.
INTRODUCTION
Extracorporeally generated HESW1 have become the standard
treatment for fragmentation of kidney stones ( 1) and have been ap
plied for lithotripsy of gallstones (2). This was made possible because
shock waves can be focused on targets within the body with a mini
mized effect on the tissue surrounding those targets.
The use of HESW as a means for nonsurgical, local tumor treatment
has also been suggested (3). First investigations have been carried out
demonstrating the cytotoxic effects of HESW on tumor cells in vitro
(3, 4). Furthermore, treatment of experimental tumors in vivo with
HESW has been shown to induce the delay of tumor growth (3, 5, 6).
HESW are known to have damaging effects on the vasculature of
normal tissues; side effects of shock wave lithotripsy of renal stones
or gallstones include edema formation, hemorrhage, and reduction of
tissue perfusion (7, 8). Damage to microcirculation has been con
firmed by histology, electron microscopy, and intravital microscopy
(9, 10).
Similar effects of HESW have been observed on tumor vasculature
(11, 12). This is of particular interest if HESW are tobe used for tumor
therapy. HESW-induced damage of tumor vasculature with subse
quent impairment of perfusion would contribute to tumor cell death,
in addition to the direct cytotoxic effects of the shock wave itself. Cell
death secondary to perfusion defects would depend on their extent and
duration. Such mechanisms of action have been elucidated for mo
dalities of tumor therapy like hyperthermia (13) or photodynamic
therapy (14, 15). Moreover, perfusion changes after HESW have to be
Received 10/16/92: accepted 1/25/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
' Supported by grants of the Bundesministerium fürForschung und Technologie
(0706903 A5) and the Kurt KörberStiftung.
2 To whom requests for reprints should be addressed, at Institute of Anesthesiology,
Klinikum Grosshadern. Ludwig-Maximilians-University. Marchioninistrasse 15, 8000
Munich 70. Germany.
' The abbreviations used are: HESW, high-energy shock waves; IAP. 4-iodo|iV-mi-r/iv/-
l4C)antipyrine; MAP. mean arterial blood pressure: ROI. region of interest.
taken into account for therapeutic approaches combining HESW and
chemotherapy or other treatment modalities.
The aim of this study was therefore to quantify the extent and
duration of perfusion changes in tumors after the application of
HESW.
MATERIALS AND METHODS
Animals and Tumors. The experiments were performed in 21 male Syrian
golden hamsters (70-80 g) bearing two amelanotic hamster melanomas (AMel-
3) ( 16) in the dorsal skin. During tumor implantation. HESW application,
and blood flow measurements the animals were anesthetized with pentobarbital
(Nembutal; Sanoft-Ceva. Hannover. Germany; 60 mg/kg i.p.). For tumor im
plantation the dorsal skin was shaved and chemically depilated (Pilcamed;
Schwarzkopf GmbH. Lübeck.Germany). About 5 x IO6 A-Mel-3 cells were
inoculated s.c. at two paravertebral sites (thoracic and lumbar regions) in the
dorsal skin. Seven days after implantation tumors had grown to diameters of
7-10 mm, corresponding to volumes of 160-250 mm1 [volumes were calcu
lated as described by Weiss et al. (5)1.
HESW Application. HESW were electrohydraulically generated with the
Dornier lithotripter model XL1 (Dornier Medizintechnik. Germering. Germa
ny), as described previously (1). Briefly, an underwater spark discharge pro
duces a radially expanding shock wave which is reflected on a metal semiellipsoid
(Fig. 1). Thus the shock wave is concentrated at a focal site where
maximum pressures of 800 bar are reached within I ns. The 50% isobar of the
pressure field is currently defined as the shock wave focus; it has a diameter
of 5 mm and measures 22 mm in the longitudinal axis (17).
At day 7 of tumor growth the animals were placed into plexiglas tubes
which were covered with 2 mm of styrotbam on the inner side to protect the
body from the high-pressure field of the shock waves. The tumor-hearing
dorsal skin was elevated through a slit in the tube, fixed with three sutures
along a plastic arc (Fig. 1). and sealed watertight with a surgical incision drape
(Opraflex; Lohmann GmbH, Neuwied. Germany) shielding the space between
the skin and the tube. This arrangement made it possible to submerge the
tumors under water (water bath temperature. <36°C) while the rest of the
animals remained dry inside the tube.
One of the tumors was randomly chosen for HESW treatment. It was
positioned at the shock wave focus, which was localized by two low-energy
laser beams intersecting there. Two hundred HESW were applied on the tumor
at a fixed discharge voltage of 15 kV, a condenser capacity of 80 nF, and a
HESW application frequency of 2.3 Hz. Thus the overall treatment time was
87 s. The second tumor, located at a distance of about 30 mm from the first one,
was not exposed to HESW (Fig. 1).
Measurement of Tumor Blood Flow. Tumor blood flow was measured
with the autoradiographic tissue equilibration technique developed by Kety
(19) and Sakurada (18). Polyethylene catheters (Portex. Ltd., Hythe, Kent,
England) were implanted into the right carotid artery (outer diameter, 0.96 mm;
inner diameter. 0.58 mm), femoral artery, and superior vena cava (outer di
ameter. 0.61 mm: inner diameter. 0.28 mm). Forty jiCi of the inert, readily
diffusible compound IAP (NEN Research Products. Du Pont de Nemours.
Dreieich, Germany) were evaporated to dryness and redissolved in 0.5 ml 0.9%
NaCI solution. The carotid catheter was cut at a length of 35 mm. and two
arterial blood samples of approximately 20 (jl were drawn into heparinized
glass capillaries. The IAP solution was then injected through the superior vena
cava catheter by means of an infusion pump (Harvard Appliance. Ltd., Kent,
England) with a constant flow over 30 s. During the infusion period, further
arterial blood samples (approximately 20 ul each) were withdrawn from the
freely flowing carotid catheter every 2-3 s. MAP in the femoral artery was
registered continuously during the experiment. Exactly 30 s after the start of
the IAP infusion both tumors were rapidly resected, immediately frozen in
liquid nitrogen, and stored at -70°C.
1590
TUMOR PERFUSION AFTER SHOCK WAVES
lasor
semiallipsoidal
reflector
spark gap
Fig. 1. Experimental setup for application of HESW under water. The animals were
placed in a plexiglas tube. The tumor-bearing back skin was extended through a slit in the
tube. After randomization, one of the tumors was positioned in the shock wave focus with
the help of two low-energy laser beams crossing there. The second tumor was beyond the
focus area and served as an untreated control. HESW were electrohydraulically generated
and fix-used after reflection on a metal semiellipsoid. Water flowing into the tube was
continuously evacuated by a suction device.
Arterial blood samples were weighed and mixed with scintillation fluid to
determine MC activity with a beta counter (Rack Beta 1219; LKB Wallac.
Turku, Finland) and calculate the 14C concentration in the blood.
The MC concentration in tissue was visualized autoradiographically. The
tumor was cut alternately into 20-um and 7-um cryosections (cryostat at
-20°C): a 20-um section for autoradiography was followed by a 7-um section
for histology and a 100-um slice, which was discarded. Twenty-um sections
were placed on X-ray film (NMC: Eastman Kodak. Rochester, NY) for 2
weeks together with calibrated UC tissue standards (I4C microscales; Amersham
Buchler GmbH. Braunschweig. Germany). Seven-urn sections were
stained with hematoxylin and eosin.
The autoradiograms showing tissue I4C distribution in tumor sections were
evaluated densitometrically with an image analysis system (IPS Autoradiog
raphy Software Package; Kontron GmbH. Eching. Germany). Images of the
transilluminated autoradiograms of tumor sections and the corresponding UC
tissue standards were acquired with a CCD videocamera (XC-77; Sony. Co
logne, Germany) coupled to a macroviewer, digitized, and displayed on a
monitor. The images were compared to the corresponding hematoxylin and
eosin-stained histology. By means of a digitizer table. ROIs were selected as
follows: ROI I included the whole cross-section of the tumor without the
surrounding normal tissue, and ROI 2 included the cross-section of the tumor
without the surrounding normal tissue and without necrotic areas in the tumor.
The demarcation of the ROIs was performed by comparing the autoradiograms
with histology. The limits between normal and tumor tissue were easy to
establish. Tumor necrosis was identified as an area with no cellular structures
in hematoxylin and eosin histology.
Average blood flow within each ROI was calculated by an iterative poly
nomial regression with a computer program integrated into the image analysis
system (IPS Autoradiography Software Package) according to Kety's equation
(18):
c/m =AX C,(t) X , 'dt
where C,( T) is the tissue concentration of IAP at the end of the infusion period
(T = 30 s). Ca(f ) is the arterial concentration at time t after beginning with the
infusion of IAP. A is the blood tissue partition coefficient of IAP in A-Mel-3
tumors, and A"is a parameter which is related to blood flow F as follows:
F = K X A/m
where m is a value between 0 and I defining the extent to which IAP diffusional
equilibrium is established between tissue and blood. We assumed no
diffusion barriers for IAP between tumor vessels and interstitial space and
therefore chose m = 1.
Twenty autoradiograms corresponding to 20 levels of each single tumor
were evaluated, and the mean blood flow value of each tumor was calculated.
The blood tissue partition coefficient A was determined in separate exper
iments. Two hamsters, each bearing three tumors in the dorsal skin, were
tracheotomized and artificially ventilated with a 70"7r N2O/30% O; mixture.
Anesthesia was maintained by 1.5% enflurane. Catheters were placed in the
right carotic and femora] arteries and superior vena cava. To avoid metabolic
degradation of IAP during the time needed for equilibration between blood and
tissue, laparotomy was performed and both renal arteries and veins, the hepatic
portal vein, and hepatic artery were ligated (18. 20). Spleen, stomach, and the
small and large intestine were carefully removed, and the abdominal wall was
closed. After 30 min. 40 uCi IAP were injected i.v. Twenty-ul arterial blood
samples were drawn before and every 15 min following IAP. Ninety min after
injection, the last blood samples were drawn, and the tumors were resected and
deep frozen. iaC concentrations in blood and tumor tissue were determined as
described above. The tissue-blood partition coefficient of IAP was calculated
as:
A= Ci(T)/Ca(T)
Experimental Protocol. In hamsters bearing two A-Mel-3 tumors, one of
the tumors was randomly choosen for treatment with HESW; the other served
as an imraindividual. untreated control. After exposure to HESW the animals
were randomly assigned to four groups. Tumor blood tlow was measured 30
min after treatment with HESW in the first group (n = 5). l h after HESW in
the second (n = 5). after 3 h in the third (n = 5). and after 12 h in the fourth
group (n = 6).
Statistics. For each investigated group the median blood flow ±SE was
calculated using the mean values of each single tumor.
Blood flow values in the control tumors of the different groups or in the
different groups of HESW-treated tumors were analyzed for statistical signif
icance using the Kruskal-Wallis test for nonparametric one-way analysis of
variance and multiple comparisons on ranks for independent samples (21).
Tumors treated with HESW and their corresponding controls were statistically
compared with the Wilcoxon matched pairs signed rank test. This test was also
used to compare values measured in ROI l and 2 of the same tumors (22). The
relationship between blood flow in the control tumors and MAP was examined
by linear regression and correlation analysis (22). P < 0.05 was regarded to be
significant.
RESULTS
Tissue-Blood Partition Coefficient of Iodo['4C]antipyrine. Be
tween 60 and 90 min after injection of IAP. its concentration in blood
did not change further. It was assumed therefore that an equilibrium
had been reached in the IAP distribution between blood and tissue. As
assessed by autoradiography. the distribution of IAP within the tumors
was homogeneous.
The blood-tissue partition coefficient (A) of IAP in the tumors was
0.86 ±0.06 (mean ±SD). A = 0.86 was later used for the determi
nation of tumor blood flow in control and HESW-treated tumors.
MAP during Blood Flow Measurements. MAP values in the
different groups during the injection of IAP are shown in Table I in
detail. At the beginning of IAP injection the MAP was 92.5 ±4.9 mm
Hg (median ±SE of all animals). MAP and blood flow of the control
tumors correlated significantly. Measurements reflected no signifi
cant differences in MAP between the experimental groups. MAP
remained unchanged during the injection of IAP and withdrawal of
blood samples.
1591
TI MOR PERFUSION AFTER SHOCK WAVES
Table I Middle arterial Mood pressure during injection of IAP Imm Hgl
MAP as measured through a catheter in the femoral artery at the beginning (/ = 0 si,
during U = 15 s), and the end (/ = 30 s) of IAP injection and release of arterial blood
samples. The values are given as medians ±SE in mm Hg. No significant differences
between the experimental groups or between the values for different times during IAP
injection were measured.
Experimental
groups30
min after HESW in = 5)
1 h after HESW («= 5)
3 h after HESW (n = 5)
51)2Ahll after HESW In =
together (n = 20)Time
injection/ during IAP
Os=80
±7.8
97 ±17.3
94±11.5
129.7592Â.±5
±4.9=
s84 15
±6.6
92 ±16.2
93 ±8.7
149.148Â8.±5
±2.9/
30=s80
±7.2
90 ±16.2
94 ±6.9
169.158Â7.±5
±4.0
Table 2 Blood flow in control tumors
Blood flow (ml/100 g/min) in control tumors as measured in ROÕI (whole tumor) and
ROI 2 (tumor without necrolic areas). Values are medians ±SE.
Time after
application of
HESW30
5)1min (n =
h (n = 5)
3 h (n = 5)
61)2Ahll (n =
control tumors (n = 21)Blood
flow
g/min)R(OmÃl/•100
I19.0
±15.3
23.4 ±8.9
60.0 ±18.7
1245.252Â3.±4
±7.9ROI
226.7
±22.4
32.4 ±10.6
79.4 ±19.3
1327.683Â2.±5
±5.6
Blood Flow in the Control Tumors. Blood flow of the untreated
control tumors is summarized in Table 2. No statistically significant
differences were found between the groups of control tumors. Taking
all control tumors together (Fig. 2), 23.4 ±7.9 ml/100 g/min (median
±SE) were measured in ROÕ1, corresponding to the whole tumor,
and 32.5 ±5.6 ml/100 g/min in ROI 2, corresponding to the tumor
without necrotic areas. Blood flow in the control tumors and MAP
correlated significantly (P < 0.05; Spearman's correlation coefficient
= 0.57). Values measured in ROI l were significantly less than those
measured in ROI 2 (P < 0.001). Blood flow as measured in ROI l
ranged between 2 and 80 ml/100 g/min in the different tumors. Within
one tumor, maximum and minimum values ranged between 0 and 110
ml/100 g/min if measured in small ROIs including about 1 mm2 of a
tumor cross-section.
Blood Flow in the HESW-treated Tumors. We observed that the
tumors became hemorrhagic and edematous even during the applica
tion of HESW. Tumor and tumor overlaying skin maintained their
macroscopic structure and were not ulcerated after the application of
HESW. The following results are given as median ±SE (Table 3).
Treatment with HESW induced a breakdown of tumor perfusion. As
measured in ROI 1 (Fig. 3«),tumor blood flow was reduced to 1.7 ±
0.7 ml/100 g/min 30 min after application of HESW and to 1.1 ±0.9
ml/100 g/min 1 h after HESW. In ROI 1 tumor blood flow 3 h after
HESW was slightly increased to 4.1 ±1.4 ml/100 g/min. Twelve h
after exposure to HESW tumor perfusion was 11.5 ±6.9 ml/100
g/min and thus significantly higher as compared to 30 min, l h, and
3 h after treatment (P < 0.01).
The following perfusion values were measured in ROI 2 (Fig. 3b).
Thirty min and l h after exposure to HESW tumor blood flow was 2.7
±1.2 and 2.0 ±1.5 ml/100 g/min, respectively. A significant increase
of tumor perfusion (P <0.05) was assessed 3 h after HESW: 4.0 ±1.6
ml/100 g/min. Twelve h after treatment tumor perfusion further in
creased to 24.9 ±12.8 ml/100 g/min (P < 0.01 versus 30 min and 1
h after HESW). Some tumors had blood flow values exceeding those
measured in the corresponding control tumors.
Measurements of tumor perfusion in ROI l after treatment with
HESW were always significantly lower than perfusion in the corre
sponding controls (P < 0.05). In ROI 2 blood flow in the tumors
exposed to HESW was significantly reduced at 30 min, l h, and 3 h
after treatment as well (P < 0.05), whereas in the group 12 h after
HESW no significant differences of perfusion were measured between
untreated and treated tumors.
DISCUSSION
The objective of this study was to quantify changes of tumor
perfusion during the first hours after a single treatment with HESW.
We chose the amelanotic hamster melanoma A-Mel-3 (16) for our
experiments because previous studies performed on this tumor model
in our laboratory had addressed the effects of HESW on tumor mi
crocirculation (12) and tumor growth (5). A-Mel-3 is a rapidly grow
ing and well-vascularized tumor (16, 23). Vascularization occurs be
tween 4 and 10 days after implantation, with necrotic areas appearing
on the fourth day.
The experimental Dornier lithotripter XL1 used here is similar to
other commercially available Dornier models (like the MPL 9000 or
HM3) for disintegration of kidney stones or gallstones in patients.
Maximal shock wave pressures of the XL1 are higher than those of the
MPL 9000 or HM3 (factor 1.25 or 2.6) (17), but for each stone
disintegration up to 10 times more HESW than was used in our
experiments are currently applied (24).
The major advantage of the autoradiographic tissue equilibration
technique to measure blood flow is its high spatial resolution, which
100
00
oo
o
•¿u
oom
80
60
40
20
O
I
t
JROI
1 ROI 2
Fig. 2. Blood tlow values (ml/HX) g/min) of all control tumors together as measured in
ROI l (•;corresponding to the whole tumor) or 2 (V; corresponding to the whole tumor
without necrotic areas). Each symbol <•.V) represents mean blood flow in one tumor.
Horizontal lines, median values.
Table 3 Blood flow in tumors after application of HESW
Blood tlow (ml/100 g/min) in tumors at different times after application of HESW as
measured in ROI I (whole tumor) and 2 (tumor without necrotic areas). Values are
medians ±SE.
Time after HESW
treatment30
min (n = 5)
1 h (n = 5)
3 hi« = 5)
12 h (n = 6)Blood
tlow
g/min)ROI(ml/100
l1.7
±0.7
Â1.±10.94.1
±1.4
11.5 ±6.9'ROI
22.7
±1.2"
2.0 ±1.5"
4.0 ±1.6"'*
24.9 ±12.8d
" P < 0.05 versus corresponding control tumors.
* P < 0.05 versus tumors 30 min or l h after treatment.
' P < 0.01 versus tumors 30 min. l h, or 3 h after treatment.
'' P < 0.01 versus tumors 30 min or I h after treatment.
1592
TUMOR PERFUSION AI-TER SHOCK WAVES
gg
00
oo
100
80
2 so
—¿ 40
ì
C
"O
oom
20
0
100
#
80
G
l
00
o
° 60
40
O
O
3
20
0
30 min Ih 3h 12 h
B
30 min 1h 3h 12 h
Fig. 3. Blood flow values (ml/100 g/min) of tumors treated with HESW 30 min. l h.
3 h, and 12 h after treatment, a. measurements in ROI l (corresponding to the whole
tumor). Each symbol (•) represents mean blood flow in one tumor. Horizontal lines.
median values of each group. #, P < 0.01 versus tumors 30 min. l h. and 3 h after
treatment, b, measurements in ROI 2 (corresponding to the tumor without necrotie areas).
Each symbol (T) represents mean blood flow in one tumor. Horizontal lines, median
values of each group. #.Pversus tumors 30 min or l h after treatment.
allows blood now determination in demarcated tissue volumes (25).
Horton et al. (26) compared this method with the microspheres tech
nique and found that both provide comparable perfusion values in the
brain. This autoradiographic technique has been applied to measuring
blood flow in some brain tumors (27-29) and RT-9 tumors implanted
s.c. (30). Recently, Tozer and Morris (31) measured blood flow in
LBDS fibrosarcomas implanted s.c. and different tissues of rats with
1AP autoradiography and stated that the technique provides "reason
able values" for tumor and normal tissues.
For assessing blood flow in A-mel-3 tumors we took into account
the recommendations of Patlak (32) and Williams et al. (33) for
accurate measurements: use of a short, freely flowing catheter for the
withdrawal of arterial blood, an experimental time of T = 30 s, and
fast removal and freezing of the tissue samples. Theoretically, errors
in the estimation of blood flow with IAP have to be expected in tissues
under ischemie conditions. Potential error sources are changes in the
factors A (i.e., the blood-tissue partition coefficient of IAP) and m
(i.e., the extent to which IAP diffusional equilibrium is established
between tissue and blood) because of modifications in tissue compo
sition or changes in vascular permeability, respectively (34). Marked
differences in Awould also lead to a heterogeneous distribution of IAP
between perfused and ischemie regions in untreated tumors after 90
min of equilibration time. Since we did not detect any regional het
erogeneities in IAP concentration between necrotie and vital regions
of untreated tumors in the experiments for determination of A, we
exclude major changes of A in ischemie tissues. Inaccuracy in the
determination of A(10-15%) would result in small, tolerable errors in
the calculation of blood flow (27, 32, 35). Changes in m during
ischemia are more difficult to assess. An increase in the permeability
of vessels would not affect the results since this would just shift m
toward unity, whereas m = 1 had been already assumed. If there is an
incomplete mixing of the tracer along vessels with low flow condi
tions, reduction of the true m would lead to underestimation of blood
flow (34). To date we are not aware of any changes of m during
ischemia.
The blood flow values we measured in control tumors were com
parable to those reported for other experimental tumors implanted s.c.
(30, 36. 37). Mean blood flow values of the control tumors were found
to vary within a wide range. Perfusion was also regionally heteroge
neous within each tumor. Both findings are characteristic for tumor
blood flow (36, 38). By intravital microscopy. Endlich et al. (23)
determined the following total perfusion values for A-Mel-3 tumors:
40.4 and 21.1 ml/100 g/min on the 4th and 12th days after tumor
implantation, respectively. These data correspond to our measure
ments. In control tumors as well as in tumors exposed to HESW the
blood flow values assessed in ROI l (whole tumor) were significantly
different from those of ROI 2 (tumor without necrotie regions) since
necrotie areas were in general characterized by low perfusion values.
Such relations between histology and blood flow have been described
by Tozer and Morris (31 ), Kuhnle et al. (25), and Walenta et al. (39).
Measurements in ROI 2 reflect perfusion of the vital tumor regions.
In this study, HESW had been focused on one tumor at a distance
of 3 cm from the intraindividual control tumor in the same animal. The
possibility cannot be completely excluded, however, that the control
tumor and/or the tissue surrounding the control tumor were affected
by HESW. The assumption that HESW had no relevant effect on the
perfusion of the control tumors is supported by the facts that their
blood flow values were in the range as expected for tumors implanted
s.c. and that no significant differences in perfusion rates were mea
sured that depended on the time after application of HESW.
Thirty min and l h after application of HESW tumor blood flow
was significantly reduced to values which were not clearly discernible
from the background level. Ischemia was induced in the whole tumor,
and maximum blood flow values within one tumor did not exceed 7
ml/100 g/min as measured in small ROIs (1 mm2; data not shown in
detail). Reduction of tissue perfusion is a consequence of HESWinduced
damage of tumor microcirculation. Some of the effects of
HESW on renal and other tissues are hemorrhage, edema, venous
thrombosis, and focal necrosis (7, 40). Histological and electron mi
croscopic studies have revealed defects and loss of endothelial cells,
rips in capillaries and venular walls with extravasation of red blood
cells and leukocytes, and formation of platelet plugs (10). These
morphological changes of renal vasculature cause the reduction of
renal plasma flow (8). which may become permanent (24, 41). By
intravital microscopy of the microcirculation of the dorsal skin of
hamsters, arteriolar vasoconstriction, venular hemorrhages, and
thrombus formation have been documented following exposure to
HESW (9). Similar damaging effects of HESW on tumor microcircu
lation have to be expected. Indeed, interstitial hemorrhage and vessel
damage in tumors after the application of HESW have been described
(5, 11, 12,42).
1593
TUMOR PERFUSION AFTER SHOCK WAVES
Three h after treatment a significant increase of blood flow was
measured in ROI 2 as compared to the values 30 min or l h after
HESW. This finding might be interpreted as a beginning of tumor
reperfusion. It should be noted, however, that measurements in ROI 2
might overestimate blood flow after HESW. This is due to the fact that
we were not able to determine whether the necrotic areas, which are
excluded in ROI 2, had increased in size as a consequence of HESW
or not. Thus ROI l might be more reliable for the analysis of tumor
perfusion in the treated tumors. As measured in both ROI l and 2,
tumor blood flow 12 h after the application of HESW was signifi
cantly higher as compared to earlier measurements. Values obtained in
ROI 2 indicate no differences in perfusion rates of treated and control
tumors beyond 12 h after exposure to HESW. leading to the conclu
sion that tumor reperfusion had started between 3 and 12 h after
treatment. Possible explanations for the reperfusion of the A-Mel-3
tumors are the relaxation of long-lasting vasoconstriction in the sup
plying arterioles or recanalization of thrombosed vessels. Changes in
tumor blood flow after the application of HESW were not dependent
upon macrohemodynamic parameters as suggested by MAP, which
was the same in all groups. To exclude the influence of systemic
effects on blood flow measurements, tumors after the application of
HESW were compared to intraindividual control tumors; the break
down of tumor perfusion and early reperfusion between 3 and 12 h
after HESW were confirmed.
Blood flow reduction after HESW is probably one of the main
mechanisms leading to the delay of tumor growth. The findings of
Oosterhof et al. (6) that HESW are more effective on well vascularized
tumors support this statement. Tumor cell death secondary to
ischemia plays an important role in therapeutic modalities like hyperthermia
(13, 15, 36) or photodynamic therapy (14. 15) and could be
essential for tumor therapy with HESW. In addition to focusing the
shock waves on the tumor, an increased sensitivity of tumor vasculature
to the treatment could constitute a factor enhancing its selective
action.
We postulate that repeated applications of HESW in short intervals,
i.e., before tumor reperfusion after each exposure occurs, would pro
long tumor ischemia and have a more pronounced therapeutic effect.
Indeed, the same number of HESW is more effective if applied in
many fractionated doses, as shown by Oosterhof et al. (6) and Hoshi
et al. (42) or Weiss et al. (5) for A-Mel-3 tumors. However, complete
tumor remission after repeated applications of HESW has not been
achieved yet. Since the extent of perfusion defects and the time
needed for reperfusion had not been considered in those studies we
suppose that the intervals chosen between the exposures to HESW
(12, 24, or 48 h) had been too long.
The effects of HESW on tumor blood flow must also be taken into
account for combined treatment with other agents like chemotherapeutics.
Several studies have demonstrated additive and/or synergistic
effects of HESW and chemotherapeutic agents or biological response
modifiers (43-45). According to our results, the chemotherapeutic
agent must be given prior to the application of HESW to make
possible its intravascular transport into the tumor. On the other hand,
if HESW are applied after the chemotherapeutic agent has accumu
lated in the tumor the blood flow reduction induced would contribute
to a slower washout of the agent. Based on our knowledge, only
agents that are active under ischemie conditions should be considered.
We conclude that HESW have significant effects on tumor perfu
sion which most probably determine their therapeutic efficiency. Per
fusion changes should be taken into account to optimize tumor therapy
with HESW and/or the combined treatment with HESW and other
therapeutic strategies.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the valuable comments of Prof. Dr. K.
Messmer, Prof. Dr. W. Mueller-Klieser, and S. Walenta on the manuscript.
REFERENCES
1. Chaussy, C.. Brendel, W., and Schmiedt, E. Extracorporeally induced destruction of
kidney stones by shock waves. Lancet. 2: 1265-1268. 1980.
2. Sackmann. M.. Delius, M., Sauerbruch. T., Holl. J., Weber, W., Ippisch. E.. Hagelauer,
U., Wess. O.. Hepp. W., and Brendel. W. Shock-wave lithotripsy of gallbladder stones.
The first 175 patients. N. Engl. J. Med., 318: 393-397, 1988.
3. Russo. P.. Stephenson. R. A.. Mies. C.. Huryk. R.. Heston. W. D.. Melamed. M. R..
and Fair, W. R. High energy shock waves suppress tumor growth in vitro and in vivo.
!. Urol.. 135: 626-628. 1986.
4. Oosterhof, G. O., Smits, G. A., de Ruyter, J. E., van-Moorselaar, R. J.. Schalken, J.
A., and Debruyne. F. M. The in vitro effect of electromagnetically generated shock
waves (Lithostar) on the Dunning R3327 PAT-2 rat prostatic cancer cell-line. A
potentiating effect on the in vitro cytoloxicity of vinblastin. Urol. Res., 17: 13-19,
1989.
5. Weiss, N.. Delius. M., Gambihler, S.. Dirschedl. P., Goetz, A., and Brendel. W.
Influence of the shock wave application mode on the growth of A-Mel 3 and SSK2
tumors in vivo. Ultrasound Med. Biol., 16: 595-605. 1990.
6. Oosterhof. G. O.. Smits. G. A., de Ruyter, A. E., Schalken. J. A., and Debruyne, F. M.
In vivo effects of high energy shock waves on urological tumors: an evaluation of
treatment modalities. J. Urol.. 144: 785-789, 1990.
7. Ackaert. K. S., and Schröder.F. H. Effects of extracorporeal shock wave lithotripsy
(ESWL) on renal tissue. A review. Urol. Res.. 17: 3-7. 1989.
8. Kaude, J. V.. Williams, C. M.. Millner. M. R., Scott. K. N.. and Finlayson. B. Renal
morphology and function immediately after extracorporeal shock-wave lithotripsy.
Am. J. Roentgenol., 145: 305-313, 1985.
9. Brendel. W.. Delius. M.. and Goetz. A. E. Effect of shock waves on the microvasculature.
Prog. Appi. Microcirc.. 12: 41-50. 1987.
10. Karlsen. S. J.. Smevik. B.. and Hovig, T. Acute morphological changes in canine
kidneys after exposure to extracorporeal shock waves. A light and electron micro
scopic study. Urol. Res., 19: 105-115, 1991.
11. Russo. P., Mies. C.. Huryk, R.. Heston, W. D. W., and Fair, W. R. Histopathologic and
ultrastructural correlates of tumor growth suppression by high energy shock waves. J.
Urol.. 137: 338-341. 1987.
12. Goetz, A. E.. Königsberger, R., Feyh. J., Conzen, P. F.. and Lumper, W. Breakdown
of tumor microcirculation induced by shock-waves or photodynamic therapy. In: K.
Messmer and A. Baethmann (eds.K Surgical Research: Recent Concepts and Results,
pp. 81-93. Berlin and Heidelberg: Springer Verlag, 1987.
13. Song, C. W. Effect of local hyperthermia on blood flow and microenvironment: a
review. Cancer Res., 44: 472ls-i730s. 1984.
14. Star. W. M.. Marijnissen, H. P. A.. Berg-Blok. A. E.. Versteeg. J. A. C., Franken. K.
A. P.. and Reinhold, H. S. Destruction of rat mammary tumor and normal tissue
microcirculation by hematoporphyrin derivative photoradiation observed in vivo in
sandwich observation chambers. Cancer Res.. 46: 2532-2540. 1986.
15. Chaplin, D. J. The effect of therapy on tumour vascular function (invited review). Int.
J. Radial. Biol., 60: 311-325. 1991.
16. Former. J. G.. Mahy, A. G., and Schrodt, G. R. Transplantable tumors of the Syrian
(Golden) hamster. Pan I: Tumors of the alimentary tract, endocrine glands and
melanomas. Cancer Res., 21: 161-198. 1961.
17. Müller. M. Domier-Lithotripter im Vergleich. Vermessung der Sto:dswellenfelder und
Fragmentationswirkungen. Biomed. Tech., 35: 250-262, 1990.
18. Sakurada, O.. Kennedy. C., Jehle, J., Brown, J. D.. Carbin, G. L., and Sokoloff. L.
Measurement of local cerebral blood flow with iodo[14C|antipyrine. Am. J. Physiol.,
234: H59-H66. 1978.
19. Kety, S. S. Measurement of local blood flow by the exchange of an inert, diffusible
substance. Methods Med. Res.. 8: 228-236, 1960.
20. Möller,v. K. Pharmakologie, p. 574. Basel and Stuttgart: Benno Schwabe and Co.,
1961.
21. Theodorsson-Norheim. E. Kruskal-Wallis lest: BASIC computer program to perform
nonparametric one-way analysis of variance and multiple comparisons on ranks of
several independent samples. Comput. Methods Programs Biomed.. 23: 57-62, 1986.
22. Sachs, L. Angewandte Statistik. Ed. 6. pp. 244-246. 308-311. Berlin. Heidelberg, and
New York: Springer Verlag. 1984.
23. Endrich. B.. Hammersen. F.. Goetz. A., and Messmer. K. Microcirculatory blood flow,
capillary morphology, and local oxygen pressure of the hamster amelanotic melanoma
A-Mel-3. J. Nati. Cancer Inst., 68: 475^(85. 1982.
24. Williams. C. M.. and Thomas, W. C. J. Permanently decreased renal blood flow and
hypertension after lithotripsy. N. Engl. J. Med., 321: 1269-1270. 1989.
25. Kuhnle. G. E. H., Dellian. M., Walenta, S.. Mueller-Klieser, W.. and Goetz. A. E.
Simultaneous high-resolution measurement of adenosine triphosphate levels and
blood flow in the hamster amelanotic melanoma A-Mel-3. J. Nati. Cancer Inst.. 84:
1642-1647. 1992.
26. Horton. R. W., Pedley. T. A., and Meldrum. B. S. Regional cerebral blood How in the
rat as determined by particle distribution and by diffusible tracer. Stroke, //: 39-44,
1980.
27. Groothuis, D. R.. Blasberg, R. G.. Molnar. P.. Bigner. D., and Fenstermacher, J. D.
Regional blood flow in avian sarcoma virus (ASV)-induced brain tumors. Neurology,
33: 686-696. 1983.
28. Groothuis. D. R., Pasternak. J. F.. Fischer. J. M.. Blasberg, R. G.. Bigner, D. D.. and
Vick, N. A. Regional measurements of blood flow in experimental RG-2 rat gliomas.
1594
TUMOR PERFUSION AFTER SHOCK WAVES
Cancer Res., -13: 3362-3367. 1983.
29. Blasberg, R. G., Molnar. P., Horowitz. M.. Kornblith. P.. Pleasants. R., and Fenstermacher.
J. Regional blood flow in RT-9 brain tumors. J. Neurosurg.. 5#: 863-873.
1983.
30. Blasberg, R. G., Horowitz. M.. Strong. J.. Molnar, P., Patlak. C. Owens. E.. and
Fenstermacher. J. Regional measurements of [l4C]misonidazole distribution and
blood flow in subcutaneous RT-9 experimental tumors. Cancer Res.. 45: 1692-1701.
1985.
31. Tozer, G. M., and Morris. C. C. Blood flow and blood volume in a transplanted rat
fibrosarcoma: comparison with various normal tissues. Radiother. Oncol., 17: 153-
166. 1990.
32. Patlak. C. S., Blasberg. R. G., and Fenstermacher, J. D. An evaluation of errors in the
determination of blood flow by the indicator fractionation and tissue equilibration
(Kety) methods. J. Cereb. Blood Row Metab.. 4: 47-60. 1984.
33. Williams, J. L., Shea. M., Furlan. A. J.. Little. J. R., and Jones. S. C. Importance of
freezing time when iodoantipyrine is used for measurement of cerebral blood flow.
Am. J. Physiol.. 261: H252-H256. 1991.
34. Tornita. M., and Gotoh. F. Local cerebral blood flow values as estimated with
diffusible tracers: validity of assumptions in normal and ischemie tissue. J. Cereb.
Blood Flow Metab., /: 403^tll, 1981.
35. Ekloef, B., Lassen, N. A.. Nilsson. L., Norberg. K.. Siesjoe, B. K.. and Torloef. P.
Regional cerebral blood flow in the rat measured by the tissue sampling technique: a
critical evaluation using four indicators [14]C-antipyrine, [14]C-ethanol. [3]H-water
and [133|xenon. Acta Physiol. Scand.. 91: 1-10. 1974.
36. Jain. R. K., and Ward-Hartley. K. Tumor blood flow—characterization, modifications,
and role in hyperthermia. IEEE Trans.. SU-3I: 504-526. 1984.
37. Tozer, G. M.. Lewis, S., Michalowski. A., and Aber. V. The relationship between
regional variations in blood flow and histology in a transplanted rat fibrosarcoma. Br.
J. Cancer. 61: 250-257. 1990.
38. Vaupel. P.. Kallinowski. F.. and Okunieff. P. Blood flow, oxygen and nutrient supply,
and metabolic microenvironment of human tumors: a review. Cancer Res.. 49: 6449-
6465. 1989.
39. Walenta. S.. Dellian. M.. Goetz. A. E.. Kuhnle. G. E. H.. and Mueller-Klieser. W.
Pixel-to-pixel correlation between images of absolute ATP concentrations and blood
flow in tumours. Br. J. Cancer. 66: 1099-1102. 1992.
40. Delius. M.. Enders. G., Xuan. Z. R.. Liebich, H. G., and Brendel. W. Biological effects
of shock waves: kidney damage by shock waves in dogs—dose dependence. Ultra
sound Med. Biol., 14: 117-122, 1988.
41. Williams. C. M.. Kaude. J. V.. Newman. R. C.. Peterson. J. C.. and Thomas. W. C.
Extracorporeal shock-wave lithotripsy: long-term complications. Am. J. Roentgenol.,
ISO: 311-315. 1988.
42. Hoshi. S.. Orikasa, S.. Kuwahara, M.. Suzuki. K.. Yoshikawa, K.. Saitoh. S.. Ohyama,
C.. Satoh, M.. Kawamura. S.. and Nose. M. High energy underwater shock wave
treatment on implanted urinary bladder cancer in rabbits. J. Urol.. 146: 439^43,
1991.
43. Oosterhof, G. O., Smiths. G. A., de Ruyter, J. E.. Schalken. J. A., and Debruyne. F.
M. Effects of high-energy shock waves combined with biological response modifiers
or Adriamycin on a human kidney cancer xenograft. Urol. Res., 18: 419-424, 1990.
44. Holmes. R. P.. Yeaman. L. !.. Li. W. J.. Hart. L. J.. Wallen. C. A.. Woodruff. R. D.. and
McCullough. D. L. The combined effects of shock waves and cisplatin therapy on rat
prostate tumors. J. Urol., 144: 159-163. 1990.
45. Hoshi, S., Orikasa, S., Kuwahara, M., Suzuki, K.. Shirai, S., Yoshikawa, K.. and Nose,
M. Shock wave and THP-Adriamycin for treatment of rabbit's bladder cancer. Jpn. J.
Cancer Res.. 83: 248-250. 1992.
1595