Download Quantitative Analysis of Microvascular Structure

(CANCER RESEARCH 41, 1898-1904,
0008-5472/81/0041-OOOOS02.00
May 1981]
Quantitative Analysis of Microvascular Structure and Function in the
Amelanotic Melanoma A-Mel-31
Kazuaki Asaishi,2 Bernhard Endrich,3 Alwin Götz,and Konrad Messmer
Institute for Surgical Research. Klinikum Grosshadern,
University of Munich. Munich, Federal Republic of Germany
ABSTRACT
Blood cell velocity, capillary diameter, and capillary length
were determined in the microcirculation
of the amelanotic
hamster melanoma A-Mel-3 as well as in s.c. tissue of tumorfree animals. Studies were carried out using a dorsal skin flap
chamber, intravital microscopy, and television techniques after
transplantation of a 0.5-cu mm piece of tumor tissue.
The tumor revealed a special microvascular configuration of
short, thin-walled, sometimes dilated capillaries running around
the edge of the tumor. Large avascular areas appeared in the
center part approximately 5 days after tumor transplantation.
Although mean capillary blood cell velocity was not different
in tumor-containing and tumor-free preparations, localized ir
regularities of blood flow were observed close to points of
endothelial sacculations. Huge platelet conglomerates were
consistently noted in capillaries of the tumor, blocking the
blood stream temporarily.
Due to discrepancies in microvascular morphology and lack
of visible vascularization, large parts of this tumor seem to be
inaccessible to tumor treatment. This implies that better vas
cularization of these regions might enhance the efficiency of
cancer treatment. The chamber technique, intravital micros
copy, and television methods combined with the subsequent,
quantitative microvascular analysis may provide a unique
means for direct evaluation of local therapy, particularly during
early melanoma growth.
INTRODUCTION
Malignant cells can generate new tumor nodules when they
are trapped in capillary networks apart from the location of the
primary tumor. Transplants of tumor material are generally
accepted as experimental models for embedment and growth
of such deposits (1, 2, 8, 15, 18, 21); thus, observations and
measurements within these growing deposits could provide
detailed knowledge on vascular function, particularly during
very early tumor growth.
Based on transparent chamber techniques and observation
of the microcirculation,
angiogenesis into a melanoma was
shown to be markedly unlike that in microcirculatory
blood
vessels undergoing endothelial proliferation in the event of an
inflammatory response (16-18, 20). Microvascular structures
in the melanoma are extremely tortuous during early stages of
tumor development (18, 20). Capillaries and collecting venules
1 Supported by Deutsche Forschungsgemeinschaft
Grant EN 114/2.
2 Present address: Sapporo Medical College and Hospital. First Department
of Surgery. South 1. West 16, Chuoku, Sapporo. Japan.
3 To whom requests for reprints should be addressed, at the Institute for
Surgical Research. Klinikum Grosshadern, University of Munich, Marchioninistrasse 15. 8000 Munich 70. Federal Republic of Germany.
Received June 10, 1980; accepted January 20. 1981.
1898
appear to be redundant in number and show many sacculations
and microaneurysms (20).
However, despite extensive studies, most findings reported
are still descriptive. No quantitative microcirculatory measure
ments and, in particular, no direct analysis of hemodynamic
function have been performed in the melanoma, although this
information is of importance for planning and evaluating local
physical or systemic chemical tumor treatment after a wide
spread dissemination of malignant cells.
Therefore, an attempt was made to quantify blood vessel
diameter, capillary length, and microvascular blood cell velocity
in an amelanotic hamster melanoma, A-Mel-3. Intravital micros
copy and video techniques were used in situ in conjunction
with a newly designed skin flap chamber.
MATERIALS
AND METHODS
Preparation for Microvascular Observation. Experiments
were performed on 11 male hamsters (65 to 70 g) fitted with
aluminum chambers (11). This design, implanted in a dorsal
skin flap, permitted a transplanted piece of tumor tissue to
grow in a sheet-like fashion. For the experiments on hamsters,
the chamber was modified to stabilize the thin, translucent skin.
Spacers made of stainless steel were used yielding a frame-toframe distance of 400 to 450 jum (3).
All animals were housed in single cages under carefully
controlled temperature (Intensive Care Incubator 7510; Dräger,
Lübeck,Federal Republic of Germany; 35°; relative humidity,
50%; room air atmosphere). For direct observation, as well as
video and photographic recordings, the hamster was taught to
crawl into a transparent plastic tube with approximately the
same diameter as the animal in its crouched position. The tube
had a small slot which ran lengthwise and which allowed the
skin flap and the chamber to remain outside. The tube provided
support which was sufficiently rigid for placement on the mi
croscope stage with the possibility of subsequent photography
and/or video recordings in awake animals (Fig. 1). Special
care was taken to avoid any artificial constraint of the prepa
ration.
Preparation of Tumor Material. Tumor cells for transplan
tation were received from a solid virus-induced amelanotic
hamster melanoma, A-Mel-3, established by Fortner ef al. (7).
After injection of 1 x 106 cells s.c. in the back of a hamster, a
solid tumor 2 cm in diameter grew within 14 days. By that time,
the tumor began to become necrotic and killed the animal
within 3 weeks.
Prior to necrosis, tumor fragments were minced, and a 0.5cu mm tissue piece was transplanted 48 hr after implantation
of the chamber (5 animals). Observation and measurements
were started prior to and again 1 day after transplantation of
A-Mel-3.
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Microcirculation in Melanoma
yielding a magnification of approximately x500 on the televi
sion monitor. Vessel diameters were measured separately at
several sites under high optical magnification (10).
RESULTS
Fig. 1. Transparent
skin flap chamber as adapted to the hamster's back. For
intravital microscopy, the animal was taught to crawl into a transparent
tube (diameter of the area under observation, 12 mm).
plastic
Measurements in the Microcirculation. Direct measure
ments in the microcirculation
of tumor-bearing and control
animals were carried out as described previously (6, 8). Briefly,
a photographic overview of the entire chamber was made daily.
To obtain blood flow measurements, different areas of the
preparation were transluminated by a flexible halogen fiber
optic system and were observed microscopically under a mod
ified intravital focusing unit (Orthoplan microscope; Leitz,
Wetzlar, Federal Republic of Germany) using a X10 longworking-distance
objective with a variotubus (Factor 1-3.2;
Leitz) and a x4 eyepiece. The image of the microcirculatory
bed was televised (COHU 4400 video camera; Prospective
Measurements, Inc., San Diego, Calif.) and stored on a Sony
AV 3620 CE video tape (Interberg Electronic, Munich, Federal
Republic of Germany). The final magnification on the television
monitor (Barco RM2400; Interberg Electronic, Munich, Federal
Republic of Germany) was about X1200 when the variotubus
was positioned at Factor 3.0.
Blood flow measurements in the microcirculation of tumor,
as well as in s.c. tissue of tumor-free animals, were preformed
by the dual-window video method (9). Video recordings of
blood cell flow were obtained from selected areas of the
microcirculation,
and the transit time of blood cells between
upstream and downstream locations in a given vessel was
measured by on-line cross-correlation
(9). Video recordings
were carried out without the use of anesthetics; the newly
designed microscope stage provided the degree of immobili
zation needed for proper functioning of our flow-measuring
instrumentation. Microscopic vessels were categorized follow
ing the nomenclature of Zweifach (22); accordingly, the microcirculation was divided into 5 categories using midcapillaries
as reference and dividing the arterial and venous vessels on
both sides into 2 categories. Proximal to the capillary network,
vessels of near capillary dimension showing on occasion vas
omotor activity were considered as precapillaries. The term
"arteriole" was used for vessels with a diameter of 20 to 30
mm that began to distribute precapillary side branches. This
category included in our study microscopic blood vessels with
a diameter as small as 22 /¿m.
On the venous side, a distinction
was made between wide, confluent capillaries (10 to 20 (iim),
postcapillaries, and the larger collecting venules (20 to 40 /urn).
All measurements of capillary length were carried out directly
from the television monitor with the variotubus adjusted to 1
MAY
Morphology. Photography and videotape recordings were
carried out when the microcirculatory
blood vessels became
visible, proving that the melanoma was growing (Figs. 2 and
3). Twelve to 14 hr after tumor cell implantation, an exúdate
appeared which sometimes contained erythrocytes. When this
exúdate became smaller, rosette-shaped deposits of RBC and
petechial bleeding scattered through the area of implantation
were consistently observed. After approximately 4 days, a
typical vascular configuration of short, thin-walled, sometimes
dilated capillaries was seen primarily at the edge of the tumor
(Fig. 4). The appearance of these new blood vessels was
associated with rapid growth of the malignant tissue. Seven
days after transplantation, the tumor portion reached a diam
eter of 4880 ±2205 (S.E.) /¿m.Due to continuous growth, the
tumor obscured the preparation after 13 days, and observa
tions in the microvasculature
of the tumor were no longer
possible.
During earliest tumor growth, arteriolar and precapillary
blood vessels in the tumor increased in diameter, and the
microcirculation of the melanoma seemed to carry a greater
volume of blood.
The diameter of tumor capillaries was of the order of 7.90
± 0.89 firn (n = 39) 4 days after chamber implantation and
10.62 ±1.38 firn (n = 49) (Chart 1) 4 days later. The capillary
diameter in the s.c. tissue of tumor-free animals was 6.14 ±
0.44 jum (n = 22) on Day 4 and 7.70 ±0.85 (n = 16) 8 days
after chamber implantation. Capillary lengths revealed a statis
tically significant difference ( p < 0.02 to < 0.005; paired f test)
between tumor and control animals; i.e., the average capillary
length was 198.8 ±66.7 urn (n = 70) on Day 4 and 269.0 ±
103.9 /xm (n = 49) 4 days later (Chart 2).
Capillary length ¡nthe s.c. tissue of control animals was
578.4 ±127.8 firn (n = 49) 4 days after and 576.0 ±132.0
firn (n = 32) 8 days after implantation of the aluminum chamber.
Large, necrotic, avascular areas appeared in the center part
approximately 5 days after tumor transplantation. At this time,
platelet conglomerates were consistently noted in capillaries of
the amelanotic melanoma, blocking the blood flow temporarily.
100
ao
2
g3
er
ï
40
•
—•
¿daysafter implantation
•
—•
6days after implantation
i—i Sdays after implantation
20
10
12
16
18 [Hm]
Chart 1. Cumulative histograms of capillary diameter in the amelanotic mela
noma A-Mel-3. Tumor growth was associated with a right shift in frequency
distribution and hence an overall increase of capillary diameter (number of
determinations: Day 4, 39; Day 6, 55; Day 8, 49).
1981
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K. Asaishi et al.
CAPILLARY
200 ¿00'
LENGTH:
TUMOR FREE ANIMALS
AMELANOTIC
MELANOMA
A-MEL-3
800 1000
Chart 2. Frequency distribution of capillary length in the tumor as well as in s.c. tissue of tumor-free animals at different days after implantation
chamber (n = number of determinations).
As the melanoma grew further, the vasculature continued push
ing already existing microvascular channels toward the growing
edge while obliterating more centrally placed blood vessels.
Hemodynamic Characteristics in the Microcirculation of
the Amelanotic Melanoma A-Mel-3. In the great majority of
microvessels, the direction of blood flow was around the center
of the tumor. Only very few of the capillary blood channels
showed either intermittent or régurgitant blood flow. This phe
nomenon was strongly related to the existence of platelet
conglomerates in tumor blood vessels. Tables 1 and 2 sum
marize measurements of blood cell velocity on Day 6 after
tumor transplantation and in tumor-free control preparations.
Even though mean blood cell velocities in capillaries and
postcapillaries were not significantly different, blood flow in
single capillaries of A-Mel-3 revealed localized Irregularities in
those segments where microaneurysms or platelet conglom
eration were observed. However, due to the progressive dila
tion of arterioles and precapillaries, blood cell velocity in precapillaries of the tumor was significantly different when com
pared to tumor-free preparations. As the tumor continued to
grow, large areas of the amelanotic melanoma did not show
any blood flow. Petechial hemorrhage developed in those seg
ments where blood flow resumed after a period of very slow
(less than 0.10 mm/sec) or stagnant flow.
DISCUSSION
The first attempt to describe microcirculatory events in the
amelanotic melanoma A-Mel-3 was made by Witte and Goldenberg (20). Subsequent studies have established the morphol
ogy of amelanotic melanomas (17, 18) and other malignant
tumors such as rat hepatoma (21) and rhabdomyosarcoma (4,
5). With intravital microscopy, the microvascular configuration
can be evaluated, in particular, neovascularization, blood flow
through single vessels, and their relationship to the degree of
1900
of the skin flap
Table 1
Diameter range and blood cell velocity across successive segments of the
microcirculation in the amelanotic melanoma A-Mel-3 (Day 6)
(IMP)21.1-26.3
range
(4)"
Terminal arterioles
8.2-19.4 (6)
Precapillaries
4.4-15.0(55)
Capillaries
5.6-20.0 (50)
Postcapillaries
Collecting venulesDiameter 15.0-27.0(20)Blood
a Numbers in parentheses, number of determinations.
b Mean ±S.E. of values for 5 animals.
cell velocity
(mm/sec)1.41
±0.28b
0.90
0.33
0.31
0.36
±0.27
±0.05
±0.05
±0.05
Table 2
Diameter range and blood cell velocity across successive segments of the s.c.
microcirculation in tumor-free preparations (Day 6)
Terminal arterioles
Precapillaries
Capillaries
Postcapillaries
Collecting venulesDiameter
(¿im)17.5-23.1
range
(5)"
9.1-19.3(15)
5.0-10.0(48)
7.5-19.4(55)
16.3-36.4(21)Blood
cell velocity
(mm/sec)0.95
±0.05o
0.49 ±0.07°
0.35 ±0.07
0.37 ±0.06
0.55 ±0.10
Numbers in parentheses, number of determinations.
6 Mean ±S.E. of values for 6 animals.
c Significance versus blood vessels of the amelanotic melanoma A-Mel-3; p
< 0.025 (Student's paired f test).
necrosis in the tumor.
In general, these tumors are vascularized within 4 to 10 days
after implantation. At this time, blood cell velocities in terminal
arterioles and precapillaries of the amelanotic melanoma AMel-3 were elevated when compared to tumor-free prepara
tions. Blood cell velocity measurements at the capillary level
revealed lower values, but striking inhomogeneities of mean
blood cell velocity were not delineated. However, blood flow in
capillaries was inhibited when blood cell conglomerations, in
particular platelet plugs, were present in the microcirculation
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Microcirculation in Melanoma
of A-Mel-3. These findings were not observed in tissue adjacent
to the tumor or in s.c. tissue of tumor-free animals. In addition,
significant differences in capillary length and a broader range
of capillary diameters were found in the amelanotic melanoma.
Since nutritional supply at the capillary level depends on
blood cell velocity, capillary length, and diameter as well as
intercapillary distances, discrepancies of capillary length, dif
ferences in capillary alignment, and blood cell conglomerations
within the capillary network subsequently cause inhomogeneities of tissue perfusion in the melanoma. Measurements of
capillary length and diameter suggest that inhomogeneities of
tissue perfusion originate from discrepancies of morphological
parameters. Hemodynamic alterations are the result of a very
specialized microvascular configuration of this malignant tu
mor.
On the other hand, development of blood-borne métastases
inevitably requires movement of tumor cells into the vascular
compartment leading to a tumor embolus that is carried on and
finally métastasesin distant organs (16, 17). The intravasation
of tumor cells into the microvascular lumina was demonstrated
by Warren ef al. (17) using electron microscopy. They found
that tumor cells did not invade arteries or arterioles. The
capillary endothelium, however, appeared to be extremely frag
ile, allowing intravasation of adjacent tumor cells. Platelet con
glomerates were also seen at the luminal surface of microcirculatory blood channels in the tumor, adhering primarily to
deficiencies in the endothelial lining of very large, thin-walled
"giant capillaries."
The findings of Warren (16) are consistent with our obser
vations; they indicate that cell attachment and cell movement
at the endothelial wall have a significant effect on microvascular
hemodynamics in the amelanotic melanoma A-Mel-3. The angiogenic activity in A-Mel-3 may reflect a dynamic interaction
of metabolically active tissue cells with the existing vasculature
(13, 14, 19), resulting in defects of the endothelial lining, thus
favoring platelet adherence.
By comparing the geometry of the terminal vascular bed in
A-Mel-3 to microcirculatory structures of the malignant neurilemmoma in hamsters (2), as well as to an adenocarcinoma
(12) and a sarcoma in rats (4, 5), typical features of a tumor
microcirculation can be established even though some of the
information relies on descriptive or semiquantitative methods
(2,12).
In general, the terminal vascular bed of a malignant tumor is
characterized by: (a) an irregular, chaotic capillary, postcapillary, and venular network; (b) collecting venules outnumbering
arterioles and precapillaries; (c) a general arteriolar, capillary,
and venular dilation; (d) no vasomotion, i.e., no measurable
closing or opening of arterioles in the tumor; (e) inhomogeneous capillary flow due to regional discrepancies of capillary
length and capillary alignment; and ( f) an increase in mean
capillary length with tumor growth.
From our study, some characteristics typical for A-Mel-3 can
also be derived since they were observed in this tumor only:
(a) platelet conglomerates in the capillary network; (b) numer
ous microaneurysms along the capillary wall. Densely packed
erythrocytes were trapped in these sacculations, underwent a
vortex-like motion for a few seconds, but disappeared into the
main bloodstream almost one by one after a certain time
interval; (c) a large percentage of short capillaries aligned
around the edge of the tumor.
MAY
Alignment of capillaries around the edge of this tumor and
the appearance of necrotic areas 4 days after tumor transplan
tation indicate that only small parts of the melanoma would be
susceptible to chemical treatment. This response is further
limited during the obstruction of blood flow by platelets trapped
in the tumor microcirculation.
Due to discrepancies in microvascular morphology and lack of visible vascularization during
very early stages of growth, large regions of this tumor seem
to be inaccessible to tumor treatment, a fact which might
explain the poor results of chemotherapy in the melanoma.
ACKNOWLEDGMENTS
The authors appreciate the expert and valuable technical assistance
Pfeiffer and A. Kreisle and wish to thank I. Moll for typing the manuscript.
of R.
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1981
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Fig. 2. Typical preparation
1902
prior to the implantation of the amelanotic melanoma A-Mel-3. Approximately
x 10.
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Microcirculation in Melanoma
Fig. 3. The same preparation
MAY
6 days after tumor transplantation.
The amelanotic melanoma A-Mel-3 is located in the right lower part of the skin flap chamber.
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K. Asaishi et al.
Fig. 4. Microcirculatory
pattern in tumor-free prepa
rations (Ai and at the edge of the amelanotic melanoma
A-Mel-3 (ß).Capillaries were visualized after an i.v. injec
tion of 0.2 ml fluorescein isothiocyanate covalently bound
to Dextran 150,000 (Pharmacia AB, Uppsala, Sweden).
Pictures taken from the TV-Monitor. Approximately x 120.
1904
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VOL.
41
Quantitative Analysis of Microvascular Structure and Function
in the Amelanotic Melanoma A-Mel-3
Kazuaki Asaishi, Bernhard Endrich, Alwin Götz, et al.
Cancer Res 1981;41:1898-1904.
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