{CANCER RESEARCH 50. 4478-4484, August 1. 1990]
Interstitial Pressure Gradients in Tissue-isolated and Subcutaneous Tumors:
Implications for Therapy1
Yves Boucher,2 Laurence T. Baxter,1 and Rakesh K. Jain4
Tumor Microcirculation Laboratory; Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890
ABSTRACT
High interstitial fluid pressure (IFF) in solid tumors is associated with
reduced blood flow as well as inadequate delivery of therapeutic agents
such as monoclonal antibodies. In the present study, IFF was measured
as a function of radial position within two rat tissue-isolated tumors
(mammary adenocarcinoma R3230AC, 0.4-1.9 g, n = 9, and Walker 256
carcinoma, (1.5 5.0 g, n = 6) and a s.c. tumor (mammary adenocarcinoma
R3230AC, 0.6-20.0 g, n = 7). Micropipettes (tip diameters 2 to 4 ^m)
connected to a servo-null pressure-monitoring system were introduced to
depths of 2.5 to 3.5 mm from the tumor surface and IFF was measured
while the micropipettes were retrieved to the surface. The majority (86%)
of the pressure profiles demonstrated a large gradient in the periphery
leading to a plateau of almost uniform pressure in the deeper layers of
the tumors. Within isolated tumors, pressures reached plateau values at
a distance of 0.2 to I.I mm from the surface. In s.c. tumors the sharp
increase began in skin and levelled off at the skin-tumor interface. These
results demonstrate for the first time that the IFF is elevated throughout
the tumor and drops precipitously to normal values in the tumor's
periphery or in the immediately surrounding tissue. These results confirm
the predictions of our recently published mathematical model of intersti
tial fluid transport in tumors (Jain and Baxter, Cancer Res., 48: 70227032, 1988), offer novel insight into the etiology of interstitial hyperten
sion, and suggest possible strategies for improved delivery of therapeutic
agents.
INTRODUCTION
The therapeutic efficacy of systemically administered antineoplastic agents ranging from chemotherapeutic drugs to bi
ological response modifiers such as monoclonal antibodies and
cytokines depends upon the ability of these molecules to reach
their target in adequate quantities. The limited therapeutic
success of current systemic antineoplastic regimens may be
attributed to their inability to reach all regions of a solid tumor
in optimal quantities. For example, monoclonal antibodies have
been demonstrated to accumulate mainly in the perivascular
regions and in the peripheral regions of several solid tumors.
In addition to heterogeneous blood supply, we have recently
proposed another mechanism for the nonuniform delivery of
therapeutic agents: high IFF5 in tumors (1-3). The elevated
Received 12/12/89; revised 4/2/90.
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.
1Supported by the NCI (CA-36902) and NSF (CBT-88-16062). This work
was presented at the American Institute of Chemical Engineers Annual Meeting
in San Francisco, November 6-10, 1989; the Microcirculation Society Meeting
in Washington, DC, March 31 to April 1, 1990; the BiomédicalEngineering
Society Meeting in Washington. DC, April 2-5,1990; and the Radiation Research
Society Annual Meeting in New Orleans. April 8-12, 1990.
! Recipient of Fonds de la Recherche en Santédu Quebec Postdoctoral Fellow
ship (1988-1990).
3 Recipient of NSF Predoctoral Fellowship (1986-1989).
4 To whom requests for reprints should be addressed.
5The abbreviations used are: IFF, interstitial fluid pressure; t.i., tissue isolated;
P,, effective vascular pressure, P, —a A ir; P,, vascular pressure; a A *•,
osmotic
reflection coefficient x osmotic pressure difference between blood vessel and
interstitium;
where S/Kis exchange vessel surface area per unit volume, ¿pis vascular hydraulic
conductivity, K is interstitial hydraulic conductivity, and R is tumor radius.
IFF restricts the access of therapeutic agents to neoplastic cells
by (a) reducing the driving forces for extravasation of fluid and
macromolecules and (b) generating a convective flux of fluid
and solute towards the periphery of tumors. For a tumor of 1cm radius (4.2 g), the value of the radially outward fluid velocity
at the tumor periphery is on the order of 0.1 ^m/s (2).
Since the original work of Young et al. (4) in 1950 it has
been known that IFF is significantly higher in tumors compared
to normal tissues. Generally, in normal tissues IFPs are subatmospheric or just above atmospheric values (5), whereas in
tumors the upper range of pressure is between 10 and 30 mm
Hg (1). Several studies have also demonstrated that intratumor
pressure increases as a tumor grows (4, 6-9). Intratumor pres
sure gradients have been evaluated by Wiig et al. (6). The
micropuncture technique was used to measure IFF in the su
perficial layers and the wick-in-needle technique was used for
measurements in the deeper regions. IFF was found to decrease
gradually from the center to the periphery of the tumor. Similar
results were also obtained in our laboratory by Misiewicz (9).
Recently, we have developed a mathematical model to describe
the interstitial pressure as a function of depth in a tumor (3,
10). The results of this model suggest that the pressure is
elevated throughout the tumor, except for a sharp drop in the
periphery of tissue-isolated tumors (Fig. 1) and at the tumornormal tissue interface in tumors embedded in normal tissue.
While these predictions are plausible, there are no data on
precise spatial distribution of IFF in isolated or embedded
tumors to test this model. Therefore, the goal of this work was
to measure IFF as a function of depth in tumors.
The micropuncture technique was used to measure pressure
within two rat t.i. tumors (mammary adenocarcinoma
R3230AC and Walker 256 carcinoma) and a s.c. tumor (mam
mary adenocarcinoma R3230AC). The t.i. tumor was used as a
model of tumors growing in the body cavities (e.g., métastases
on the peritoneal wall and colon carcinoma exposed to the
lumen), and the s.c. tumor was used as a model of solid tumors
surrounded by normal tissue. The results were compared with
our mathematical model, and the implications for therapy are
discussed.
MATERIALS
AND METHODS
Animals and Tumors. Walker 256 carcinoma and mammary adeno
carcinoma R3230AC were transplanted in female Sprague-Dawley and
Fisher 344 rats, respectively. The two tumors were grown as ovarian
t.i. preparations with a single artery and vein, following the procedure
of Cullino and Grantham (11) as adapted by Sevick and Jain (12). In
brief, the ovary was removed and tumor slurry was injected in the fat
pad which is linked to the rest of the body by the ovarian artery and
vein. The preparation was enclosed in a Parafilm bag and when the
tumor reached the desired size it was exteriorized for pressure meas
urements. Subcutaneous tumors were prepared by injecting tumor
slurry from the mammary adenocarcinoma R3230AC line in the flank
of Fisher 344 rats.
Pressure Measurement. Systemic arterial pressure was measured by
a pressure transducer (model P23Gb; Gould Inc., Pittsburgh, PA) filled
with heparinized saline, connected to a preamplifier (model 11-4113-
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TUMOR INTERSTITIAL
0.0
Normalized Depth (r/R)
Fig. 1. The pressure profiles generated with the mathematical model for a t.i.
tumor are shown here as normalized pressure as a function of normalized depth
(where 0 = tumor surface, 1 = tumor center, and r/R = depth/tumor radius).
Profiles are shown for different values of the parameter a2, which is the dimensionless hydraulic conductivity ratio. For physiological parameters within a
tumor. «2is approximately 1350 (3, 10). Adapted from (Ref. 3). The profiles for
a tumor grown s.c. are given in Ref. 10.
TUMOR
MICROMAMPIXATOR
Fig. 2. A diagram of the major components of the experimental system. A
micropipette (length, ~5 cm; tapered portion. 5-mm long; with a tip diameter of
2-4 HU) is positioned with a micromanipulator inside the tumor. A servo-null
system is used to counterbalance the tissue pressure with an external motor (14).
This counter-pressure is then measured with a pressure transducer, and the
amplified output is sent to a chart recorder.
PRESSURE
artery was cannulated and the rats were placed on a temperatureregulated heating pad. Following a small skin incision, the Parafilm
bag enclosing the isolated tumor was removed. To minimize tumor
movements due to respiration, two 23-gauge needles were passed
through the skin on opposite sides of the tumor pedicle. The needles
were fixed to a cork which was taped to the heating pad. Subcutaneous
tumors were immobilized in a similar way after shaving the fur overlying
the tumor mass. During the measurement of IFP with the micropuncture technique, warm isotonic saline was dripped continuously on
isolated tumors and the intact skin overlying s.c. tumors.
After zero pressure was recorded in the saline film covering the
tumor, the micropipettes were introduced perpendicularly to the surface
to depths of 2.5 to 3.5 mm and then retracted to the surface. Individual
pressure measurements between 3.5 and 1.5 mm and from 1.5 mm to
the surface were made at intervals of 0.1 to 0.3 mm and 0.05 to 0.15
mm, respectively. Each pressure measurement was monitored for at
least 10 s. The IFP measurements were accepted when (a) no visible
distortion of the tumor or skin surface was observed, (h) the fluid
communication between the micropipette and the interstitial space
could be demonstrated electrically, and (c) the zero pressure in the
saline at the surface was not modified during the insertion and with
drawal of the micropipette (15). Micropipettes were advanced to full
depth and retrieved to the surface, since repeated insertion and with
drawal to a given depth led to frequent pipette breakage and clogging.
At least two good tracks were required in an animal to validate the
results. Generally, IFP measurements were restricted to one or two
small (5-mm x 5-mm) regions/tumor.
After the animals were sacrificed, s.c. tumors were removed and the
mean skin thickness was measured. For skin measurements two small
skin incisions were made in the same region where IFP was estimated.
Under a stereomicroscope, the distance between the surface of the skin
and the tumor was measured with a micropipette and the graded
micromanipulator. The mean skin thickness was obtained from at least
five measurements/tumor.
Control Experiments. To validate the present experimental approach,
the following control studies were performed.
(a) The influence of tissue compression and penetration depth of
micropipettes was evaluated in the thigh muscle of anesthetized rats
and in dead tumor tissue. After the removal of the overlying skin,
micropipettes were introduced up to depths of 3.0 to 5.0 mm from the
surface of the muscle. Pressure measurements were obtained at intervals
of 0.2 mm as the micropipettes were withdrawn.
(¿>)
IFP stability in the plateau and in the region of a steep pressure
gradient was tested by continuously monitoring pressure in each region
for 5 to 10 min.
(c) To test if the micropipette could create a "tunnel effect," whereby
01; Gould Inc., Cleveland, OH). Interstitial pressure measurements
were performed using micropipettes and a servo-null device (model 5;
Instrumentation for Physiology and Medicine, Inc., San Diego, CA)
(13, 14). The counter-pressure generated by this system was sent to an
amplifier (model 13-4615-50; Gould Electronics, Cleveland. OH). The
amplified signals from both pressure-measurement devices were sent to
a dual-channel chart recorder (model 595; Omega Engineering, Stam
ford, CT). This type of active system was required due to the extremely
the IFP could equilibrate, IFP measurements obtained as described
slow response time of a passive system for a tip diameter of 2 ^m. The
above were compared to measurements taken up to 200, 400, 800,
system was calibrated by determining the linear relationship between
1200, and 1600 Mm from the surface after the micropipettes were
imposed pressure and measured pressure in a saline test chamber. Fig.
inserted, respectively, to 350. 600, 1000, !400, and 1800 ^m. After one
2 shows a schematic diagram of the experimental setup.
measurement was obtained at each depth, the micropipette was re
A graded micromanipulator was used to maneuver the micropipette
trieved to the surface. These control measurements were compared to
and to measure the depth of insertion. The micropipette, in parallel
deeper measurements in the same region of the tumor.
with the micromanipulator, was positioned to penetrate the tumor
(d) To evaluate if the IFP measured in the superficial layers (1-2.5
perpendicularly. Pipette insertion was aided by the use of a stereomicroscope (Nikon SMZ-1; Charles Seifert Associates, Carnegie, PA). A mm from the surface) of tumors is similar to or different from that of
magnification of X20-45 was used to determine tissue distortion. The
the central regions, micropuncture and micropore chamber pressure
measurements were compared simultaneously. Micropore chambers
tissue was illuminated by a fiberoptic light source (Nikon MKII fiber
surrounded by mammary adenocarcinoma R3230AC were prepared
optic light source and bifurcated light guide: Charles Seifert Associates,
following the procedure of Butler et al. (16). In brief, the micropore
Carnegie, PA).
Micropipette Preparation. A thick-walled capillary tubing was used chambers were coated with tumor slurry and installed under the skin.
Fluid communication between the chamber and the pressure-measuring
(0.86-mm o.d. x 0.38-mm i.d., 0.24-mm wall thickness) to make micropipettes with a horizontal pipette puller (Narishege PN-3; Medical
device was established by PESO tubing ( 16). The pressure was measured
when the chamber was surrounded completely by tumor tissue. With
Systems, Corp., Great Neck, NY). The micropipettes were filled with
1 M NaCl solution prepared from twice filtered, twice distilled, deionthe rats prepared as described previously, the chambers were connected
ized water. The tip diameter ranged from 2 to 4 ^m; the diameter at 1, to a pressure transducer, amplifier, and chart recording system. Micro2, and 3 mm from the tip was 30-35, 75-85, and 150-225 urn,
pipettes were introduced to the depth of 2.5 mm from the surface. As
respectively.
the micropipettes were retrieved, IFP measurements obtained with the
Experimental Procedure. The rats were anesthetized with sodium
two techniques were compared.
pentobarbital (40-50 mg/kg). For subsequent injections of anesthetic,
(e) Finally, IFP decay after death was studied in t.i. tumors. Systemic
an i.p. line was installed. To monitor systemic pressure, the left carotid
and tumor interstitial pressures were recorded simultaneously before
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TUMOR INTERSTITIAL
and after the animals were sacrificed with ether or a Nembutal overdose.
Data Analysis. The experimental results were compared with our
mathematical model (3, 10). An important feature of this model is that
the pressure profile in a uniformly perfused tumor depends on two
parameters: P,, the effective vascular pressure, and a, the dimensionless
parameter which incorporates the ratio of vascular to interstitial hy
draulic conductivities and the blood vessel surface area/unit tissue
volume. Statistical analysis of the data was performed with a one-tailed
Student t test. The mathematical model was tested by fitting the data
to the model to yield these two parameters, using nonlinear least squares
regression. The resulting parameters were compared with literature
values.
PRESSURE
t.i.D Walker
5-ol
i.i.0 Mammary
s.c.lU1 Mammary
3-crU.
g.1
-_nL•
0
RESULTS
In the majority of tumors studied (86% of pressure profiles),
IFF rose rapidly in the tumor periphery in t.i. tumors (Fig. 3A)
and within the skin or in the skin-tumor interface in s.c. tumors
(Fig. 35). In both cases the pressure reached a maximum and
remained relatively uniform throughout the tumor. To charac
terize the IFF profiles as a function of depth, the depth at which
the pressure was 90% or more of the mean plateau pressure
was considered as the end point of the sharp rise. In isolated
tumors the increase in pressure ended at a distance of 0.15 to
1.2 mm from the surface. The relative frequency of the end
point of the pressure rise versus the radial position is illustrated
in Fig. 4. The steepness of the increase in IFF was variable. In
t.i. Walker 256 carcinoma and mammary adenocarcinoma
3230AC the profiles rose, respectively, from atmospheric pres
sure to 90% of maximum pressures within 0.1 to 0.3 mm and
2"'S*iPressuree.Sen
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1.5
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Depth (mm)
Fig. 3. Typical experimental pressure profiles for mammary adenocarcinoma
tumors. A, t.i. tumor (tumor 10, 0.4 g). Maximum pressures between 10 and 15
mm Hg were found between 0.15 and 2.0 mm from the tumor surface. The sharp
rise occurred between 0.2 and 0.8 mm from the surface. B, s.c. tumor (tumor 6.
2.6 g), with a mean skin thickness of 300 firn, represented by the vertical line. In
tracks 2 and 3 the sharp rise in pressure occurs in skin. The steep rise in pressure
seems to occur completely in the tumor for track 4.
1
2
3
4
5
6
7
8
9101112
Depth (x 100 microns)
Fig. 4. Histograms representing the relative frequency (number of pressure
profiles) of the distance from the tumor surface to the beginning of the IFF
plateau in Walker 256 t.i. tumors, mammary adenocarcinoma R3230AC t.i.. and
R3230AC s.c.
Table 1 Types of interstitial pressure profiles
Shapes of the pressure profiles are described in the text.
No. of profiles
Plateau and
steep gradient
U shape
Double
plateau
Mammary adenocarcinoma
(t.i.)
Mammary adenocarcinoma
(S.C.)
Walker 256 carcinoma (t.i.)
29(87.9%)
3(9.1%)
1(3.0%)
18 (94.7%)
1 (5.3%)
15(75.0%)
0
2(10.0%)
3(15.0%)
Total
62(86.0%)
4(6.4%)
3(4.8%)
3(4.8%)
Gradual
increase
0
O
O
0.1 to 0.8 mm. Within s.c. tumors (mammary adenocarcinoma
R3230AC) plateau pressures were reached at 0.1 to 0.8 mm
from the skin surface.
The mean skin thickness was 0.3 mm (n = 7), with a range
of 0.12 to 0.55 mm. The minimum and maximum skin thick
ness values overlying a given tumor could vary by more than
100% in some cases. Because of this large variation in skin
thickness, it was not possible to determine the exact location
of the pressure increase for the different pressure profiles ob
tained. However, to obtain a relative estimate, the pressure
profiles were divided into three categories: those starting their
rise at depths smaller than the minimum skin thickness, those
rising at a depth between the minimum and maximum skin
thickness, and those rising at a depth greater than the maximum
skin thickness. Based on these criteria, 29% of the pressure
profiles started their rise in the skin, 54% at the skin-tumor
interface, and 17% in the tumor.
Three types of atypical pressure profiles were also seen: (a)
a U shape formed by high pressures followed by a sharp drop
and a rapid increase; (b) a gradual decrease in pressure from
the deeper to the peripheral regions of the tumor; and (c) a
profile formed by two rapid increases and two plateaus. The U
shapes had a width of 300 to 700 urn and were observed within
a distance of 200 to 1400 ^m from the surface. Table 1 shows
the relative frequency of occurrence of the pressure profiles.
The mean systemic pressure was 103 mm Hg (SD = 15.4).
The maximum IFPs ranged from 2 mm Hg (Walker 256 t.i.)
to 37 mm Hg (mammary adenocarcinoma R3230AC t.i.). The
magnitudes of the mean plateau pressures in tumors are given
in Table 2. A strong correlation was found between the mean
interstitial pressure and the mass of the tumor for Walker 256
t.i. (Fig. 5); however, the mammary adenocarcinoma tumors
(both s.c. and t.i.) did not show a significant correlation between
the mass and IFF. The mean pressure was almost equal in
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TUMOR INTERSTITIAL
PRESSURE
Table 2 Mean plateau pressures
(mmHg)Small
'MES.2y1hCQ£¿B4035-30-zs-20-15
tumors
Large tumors
g)7.8 (<1 g)
(>1
±3.8" (n = 2)
6.1±2.9(n = 5)
Mammary adenocarcinoma s.c.
9.1±3.9(n = 5)
16.1 ±7.9 (n = 4)
Mammary adenocarcinoma t.i.
Walker 256 carcinoma t.i.Pressure 3.4 ±1.2(n = 3)* 10.8±4.6(n = 3)*
°Mean ±SD of pressures at depths of 1.0, 1.2, and 1.4 mm.
* Statistically significant difference in means (P < 0.05).
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rj
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Controlw^Lw.
A
o
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,—.—,—!—
,—;=•
Depth (mm)
Fig. 6. Pressure profiles obtained in a mammary adenocarcinoma R3230AC
t.i. tumor. Note that after a deep insertion of the micropipettes (tracks I to 4)
the sharp drop occurs at a greater distance from the surface compared to
measurements (Control) obtained following an insertion to a depth of 1 mm or
less. Error bars, SD of four measurements for the control (see "Discussion" for
explanation of the tunnel effect).
2S
•a
Interstitial
0.5
1.0
1.5
Systemic
.«
Depth (mm)
en
E
20
£ "8
15•.»
10-
_ -s'
—
"<3
1234
Time post-mortem (min)
Fig. 7. As a control experiment the interstitial (R3230AC t.i.) and systemic
arterial pressures were monitored following death of the animal by an ether
overdose. Solid gray line, arterial pressure, ranging from 0 to 100 mm Hg; dashed
black line, interstitial pressure, ranging from 0 to 10 mm Hg. Note that the IFP
decreased in parallel with the systemic pressure.
0123456
Tumor Mass (g)
Fig. 5. In A, the mean IFP is plotted versus the depth of the measurement for
all Walker 256 t.i. tumors. •,large tumors, >1.2 g (n = 3); O, small tumors,
<0.6 g (n = 3). Error bars, S.D. The difference in the mean plateau pressures
between large and small tumors is statistically significant (/' •0.05). In B, in the
Walker carcinoma t.i. the mean central IFP (depth, >1.25 mm) was linearly
related to the tumor mass (r2 = 0.94; IFP = 3.05 x mass (g) + 3.02 mm Hg) for
weights up to 6 g. However, no such correlation was found for other tumor
preparations (see Table 2).
the two pressures decayed in parallel after death (Fig. 7).
The simultaneous measurement of IFP with two techniques
demonstrated that the pressure was very similar in central and
superficial regions. Mean values of 8.7 and 8.8 mm Hg were
found, respectively, in the center of the tumor by micropore
chamber measurements and in the superficial regions of the
tumor (1-1.5-mm deep) with micropipettes.
DISCUSSION
small and large s.c. mammary adenocarcinoma
mors.
R3230AC tu
Control Studies
The measurement of IFP as a function of radial position in
the thigh muscle showed that IFP was not influenced by the
depth of penetration. IFP was found to vary between 0 and 0.4
mm Hg. The pressure in dead tumor tissue was 0 mm Hg.
IFP measurements taken after the micropipettes were intro
duced no more than 0.2 mm past the desired depth showed that
micropipettes introduced to greater depths (e.g., 2.0 to 3.5 mm
from the surface) may underestimate the pressures at a distance
of 200 to 1000 urn from the tumor surface (Fig. 6). In some
cases the controls showed that deep pressure measurements
shifted the profiles up to 400 /¿mtoward the center of the
tumor.
The IFP was stable in both the flat and steep gradient regions
for periods of up to 10 min. The simultaneous recording of
interstitial pressure and systemic blood pressure showed that
The objective of this study was to experimentally determine
the spatial distribution of interstitial pressure within solid tu
mors. These experiments were motivated by predictions of steep
pressure gradients in the tumor periphery by our recent math
ematical model (3, 10). The principal result of this study is that
IFP is elevated throughout the tumor and drops precipitously
in the periphery of isolated tumors or at the skin-tumor inter
face in s.c. tumors. This finding is in general agreement with
our model.
Two previous experimental studies showed that interstitial
pressure was highest in the center of a tumor and decreased
toward the periphery (6, 9). Wiig et al. (6) used a combination
of micropipette and wick-in-needle methods, while Misiewicz
(9) used micropipettes for all measurements. In both studies,
unlike the current study, a sharp pressure drop in the periphery
was not seen. In the study of Wiig et al. (6), mean IFP meas
urements were made at the "surface," "outer," "middle," and
"center" of the tumor, as opposed to obtaining a precise radial
distribution. The "surface" measurements were obtained up to
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TUMOR INTERSTITIAL
a depth of 800 urn with micropipettes. No IFF measurements
were reported between 0.8 and 2.0 mm from the tumor surface,
and in the deeper layers measurements were made with the
wick-in-needle method. Because of the lack of spatial resolution
in the superficial layer, it was difficult to discern the flat and
the steep gradient regions of the pressure profiles. In the deeper
layers, no significant differences in pressure were found between
the central and middle layer; however, IFF was significantly
higher in the center as compared to the outer region. While it
is possible that the difference in pressure between outer and
central regions represents a true biological phenomenon, tech
nical artifacts might also explain the difference in IFF between
the two regions. Higher pressures in the center could be due to
compression caused by penetration of the needle. Alternatively,
the lower pressure in the outer layer could be due to the "tunnel"
formed by the insertion of the needle used to measure pressure.
The IFF in the tunnel could partially equilibrate with the
atmospheric pressure or skin pressure, yielding a lower meas
urement. In addition, the 23-gauge needle (diameter, 0.63 mm)
used by Wiig et al. (6) is of larger diameter than a micropipette
and could make a larger tunnel. This argument is supported by
our current studies with the micropuncture technique.
In the present study, in the majority (86%) of pressure tracks
the IFF rose rapidly from 0 mm Hg to its maximum value,
ranging from 2 to 37 mm Hg, in less than 1 mm from the
surface. However, in many of the pressure tracks, zero pressure
was maintained for depths of 200 to 800 urn into the tumor.
This may in part be a function of the insertion depth of the
micropipette. The depth of micropipette insertion was found to
be important for the evaluation of IFF at 1 mm from the
surface. When micropipettes were inserted at distances of 1.0
mm or less, pressures were higher in the periphery as compared
to deeper insertions (Fig. 6). This control shows that the pres
sure close to the surface may not be zero but, in fact, closer to
the maximum pressure seen in the center of the tumor. Since
the measured pressure was non-zero when the pipette was being
inserted but near zero when withdrawn from a depth of 2.0 mm
or more, it is probable that the zero pressure tail seen near the
surface might be due to a tunnel being produced by the thicker
part of the capillary tube away from the tip. This would result
in a region of zero pressure where the tissue fluid is in equilib
rium with the pressure on the outside of the tumor. This result
suggests that in reality the pressure gradient may be steeper in
the periphery of the tumor. Difference in the tissue composition
between the periphery and the more central parts of t.i. tumors
could also explain the variations in pressure observed at 1 mm
from the surface. Histological examination of rapidly growing
t.i. tumors has demonstrated that the surface may be covered
by a layer of fibrin of variable thickness.6 If the fibrin layer is
noncompliant, then the hole left by the micropipette will not
close, and the measured pressure will be zero. Further inside
the tumor there does not seem to be any error associated with
a tunnel produced by the pipette. The pressure was recorded
for 10 min in areas of the tumor where the pressure was
changing with radial position. This pressure remained constant
with time, and high values were maintained.
The change in interstitial pressure due to a decrease in blood
pressure resulting from animal death was quite evident within
minutes after death. There was a rapid initial drop in the first
few minutes to a few mm Hg, followed by a slower decay
towards zero pressure. These results suggest that the driving
force for elevated IFF in tumors is the systemic pressure. The
' P. M. Cullino, personal communication.
PRESSURE
decrease in IFF following animal death was also seen by Wiig
et al. (6); however, they did not measure systemic pressure
simultaneously.
Limitations
Depth of penetration is the main limitation of the micropuncture technique. In normal tissues this is not a major
drawback if one assumes that IFF is uniform throughout the
tissue. Because of the presence of pressure gradients in tumors,
IFPs have to be evaluated from the superficial to the central
regions. By comparing central (micropore chamber) and super
ficial (micropuncture) pressures, we found that IFF was similar
in the two regions. This is additional evidence for relatively
uniform interstitial hypertension in the deeper layers of tumors.
Since it was practically impossible to relate the skin thickness
to the exact location where the micropipette was inserted,
measurements of the mean skin thickness were made in the
region where two or more pressure profiles were obtained.
However, due to the large variation of the skin thickness it was
not possible to determine precisely the exact location of the
pressure rise for the different pressure profiles. While it appears
that, in some cases the pressure increase was in the skin or the
tumor periphery, in other cases it was not possible to give a
definitive answer because of the proximity of the pressure
gradient to the skin-tumor interface. The variations in skin
thickness between the minimum and the maximum value were
often 300 ¿¿m.
The tunnel effect described previously could also
modify the exact location of the pressure gradient. Although of
minor importance, the 50-100-^m intervals between pressure
measurements could also be considered as a limiting factor.
Comparison with Mathematical Model
Fig. 8 shows the comparison of our mathematical model with
experimental data for one set of pressure readings. In this
experiment there is excellent agreement between theory and
experiment. In addition, the best fit values for the parameters
(a2 = 1210 ±420, Pc = 10.2 ±1.2 mm Hg) are in good
agreement with average values based on the literature («2=
1356, Pe = 11.5; see Ref. 3). The fit was obtained by minimizing
the unweighted sum of squares between the model and data by
varying the two parameters. Table 3 gives the estimated param
eters for a number of pressure profiles from different tumor
types. These profiles were selected to be fit by the model based
Depth (mm)
Fig. 8. This figure shows the comparison of theory to a typical pressure profile
(tumor 7. mammary adenocarcinoma s.c.). •.data points;
. theoretical profile
fitted for two parameters: the effective vascular pressure. P., and the hydraulic
conductivity ratio. <t2.Error bars, estimates of the precision of pressure and depth
measurements. The error in depth measurements was obtained from the variation
in the location of the surface between insertion and withdrawal. The error in
pressure measurements was a result of the fluctuation of pressure due to animal
respiration. The estimated parameters are very close to the expected physiological
values (see text).
4482
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TUMOR INTERSTITIAL
Table 3 Model parameters
All parameter values were obtained by least squares fit to the mathematical
model, as: parameter ±SE of estimate.
PRESSURE
o-ATT).These results also provide novel insights into the etiology
of interstitial hypertension in tumors, as discussed below.
Implications
typeWalker
Tumor
mass(g)0.470.522.35.00.842.65.06.7200.350.400.400.460.60P,
/K1
(mmHg)2.7 °1900±
The results
obtained here have significant implications in two
areas: the etiology of interstitial hypertension and the delivery
of therapeutic agents.
Etiology of Interstitial Hypertension. As reviewed recently ( 1),
at least three pathophysiological mechanisms have been asso
ciated with the elevated interstitial pressure in tumors: the lack
adenocarci-noma
320830
±
0.24.7
±
2604500
±
0.311.0±
±
s.c.Mammary
of functioning lymphatics, high vascular permeability, and vas
0.411.3
1300570
±
AB78AB9
cular
collapse resulting from cells proliferating in a confined
1701200
±
±0.710.2
space. It is evident that the lack of functioning lymphatics is
±4002800
0.66.8
±
essential for interstitial hypertension. If the net fluid extrava0.95.9
±
17004000
±
800850
±1
0.66.9
±
sating from the exchange vessels could be continuously removed
1.48.0
±
7203200
±
ABIO11
by lymphatic vessels or some other mechanism, the interstitial
1.212.8
±
67006200
±
pressure would not rise to the effective vascular pressure, Pf
adenocarci-noma
1.05.2
±
36009500
±
(=/", —a ATT).The next question is: what leads to values of P,
7100660
±
±0.36.3
t.i.Tumor123AB45AB6AB12»13»14
0.84.1
±
4006900
±
greater than 30 mm Hg in tumors? If the vessels were not
±
±0.110.7± 13002800
highly permeable, the osmotic term (a A TT)could be comparable
0.46.4
6805000
±
to P„and hence the IFF would be much lower than Pv (or even
0.48.6
±
25005600
±
ABTumor
±0.2a1
±1000
close to zero). However, large vascular permeability would tend
to reduce the osmotic pressure contribution and would make Pc
8.5 ±5.l g 3090 + 2540
Overall mean
3.1
(and hence the IFF) close to the hydrostatic pressure in ex
L. S = ratio of vascular to interstitial hydraulic conductivities X
a a /R = —•—
change vessels A.
K V
The measured value of /\ in tumor exchange vessels (<20surface area of exchange vessels per unit tissue volume.
* The tracks from animals 12 and 13 were obtained by inserting the pipette
/jm diameter) is 10-15 mm Hg (17). Therefore, the maximum
only 200 f<mpast the desired measurement depth, as opposed to over 2 mm deep.
value of IFF in a tissue with highly permeable vessels and
nonfunctional lymphatics would be 10-15 mm Hg, considerably
on two criteria: (a) the region of near-zero pressure, if one
less than 30 mm Hg measured previously and in this investi
existed, was less than 300 ¿itnand (b) there were at least four gation. As shown clearly in Fig. 7, the driving force for IFF is
data points at or near the value of the plateau pressure.
the vascular pressure. Therefore, the vascular pressure in some
As discussed in Ref. 10 there are slight differences in the exchange vessels in tumors has to be equal to or greater than
mathematical model for s.c. versus t.i. tumors. However, the 30 mm Hg. Such large vascular pressures are only found in
s.c. and isolated tumor data were analyzed in the same way for precapillary arterioles in tumors (17). This observation leads to
three reasons: (a) including a layer of skin in the theoretical
two hypotheses: precapillary arterioles in tumors are themselves
model requires four additional parameters: skin thickness, ef
functioning as exchange vessels (i.e., become permeable) or
fective normal vascular pressure, and interstitial and vascular
somehow the pressure in the exchange vessels is being raised
hydraulic conductivities in normal tissue; (b) the length over to the arteriolar level. There is no direct evidence in the litera
which the pressure drop occurred is greater than the uncertainty
ture to date supporting the former hypothesis. There is, how
in the measurement of pipette depth and skin thickness; and ever, a plausible mechanism supporting the latter hypothesis.
If proliferation of cancer cells in the proximity of an exchange
(c) the shape of the experimental profiles is qualitatively simi
lar. The theory predicts that the pressure increase in a s.c. vessel causes it to collapse, the pressure on the proximal side
tumor occurs in the skin rather than in the tumor. In some of the vessel would go up to the level of its feeding arteriole
experiments the pressure did rise to a near maximum value and result in a proportionately higher interstitial pressure. This
hypothesis also explains the relationship between elevated in
within the skin, in agreement with the model. However, in other
terstitial pressure and vascular stasis and the correlation be
tumors the uncertainties in skin thickness and depth measure
ments did not permit us to discern whether the pressure rose tween IFF and growth in some tumors. At the periphery, the
IFF must equilibrate with the surrounding pressure. The pres
in the tumor or in surrounding tissue. Unlike model predictions,
sure
drops over a small distance due to the rapid removal of
the IFF did not rise immediately in the periphery of some
fluid from the periphery of the tumor.
isolated tumors. As stated earlier, this apparent discrepancy is
Delivery of Therapeutic Agents. The results of the interstitial
probably due to the tunnel effect or a layer of fibrin on the
pressure experiments also have some important implications
tumor surface.
for cancer detection and treatment. The results show a relatively
Another qualitative agreement between theory and experi
high and uniform pressure in the center of the tumor and a
ment was the relationship between blood pressure and intersti
sharp gradient of pressure in the periphery of the tumor. This
tial pressure. The decay of the interstitial pressure to 0 mm Hg leads to very little filtration of macromolecules from blood
within minutes after animal death and the correlation between
vessels in the center, as well as a convective flow in the tumor
reduced systemic pressure and reduced interstitial pressure
periphery which tends to push the solute towards the edge of
support our model, in which a main determinant of tumor
the tumor (3). These conclusions are also supported by the
interstitial pressure is the effective vascular pressure. Our math
recent data of Dvorak et al. (18), who have shown that small
ematical model predicts that the IFF is directly related to the molecules can readily penetrate small tumors, whereas macroeffective vascular pressure (microvascular pressure minus on- molecules are limited primarily to the normal tissue-tumor
cotic pressure contribution in the exchange microvessels; /•„
— interface region. Considering these factors, novel approaches
t.i.Mammary
256 carcinoma
±0.32.6
0.39.7
±
1.118.8
±
1.322.8
±
6.04.3
±
1600670
3501700±
±
17003100±
1300190
170830
±
4483
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TUMOR INTERSTITIAL
to drug treatment using a low molecular weight agent in com
bination with antibodies have been suggested, e.g., bifunctional
antibody in conjunction with a low molecular weight chelate
and antibody-directed catalysis in conjunction with prodrugs
(19).
Another effect of the large pressure gradient at the periphery
of the tumor is that it leads to a large convective flow radially
outward near the periphery. Therefore, material that has extravasated in the periphery of the tumor will be washed out of the
tumor as the solute is cleared from the bloodstream. It is also
important to note that there is a large intertumor variation in
pressure. Two tumors of the same type and mass, grown in the
same environment, may have greatly different IFPs. A typical
range of variation is 10-40 mm Hg in large solid tumors. The
heterogeneities in blood flow and other physiological parame
ters may be much greater, ranging from a perfusion rate of zero
to perfusion rates greater than in the brain (20, 21). For these
reasons the response of tumors to therapeutic agents may vary
considerably.
Although technically difficult, other approaches suggested by
the results of this study show that delivery could be enhanced
by (a) decreasing the interstitial pressure in the center of the
tumor by physical or enzymatic means, (b) increasing the
diffusional permeability of the tumor blood vessels, allowing a
pathway for extravasation which does not depend on the differ
ence between vascular and interstitial pressure, or (c) increasing
the dose of unlabeled antibody to increase concentration gra
dients to aid transcapillary and interstitial diffusion (19).
ACKNOWLEDGMENTS
We thank Brenda Bartel for her assistance in tumor implantation
and Dr. Helge Wiig for his helpful comments on the manuscript.
ADDENDUM
Recently in collaboration with Drs. J. M. Kirkwood and W. D.
Bloomer, we have measured interstitial hypertension in human tumors.
In superficial melanomas the interstitial pressure varied between 39
and 45 mm Hg [Y. Boucher, D. Opacic, J. M. Kirkwood, and R. K.
Jain. Elevated interstitial fluid pressure in human melanomas (Ab
stract). 16th Gray Conference—Vasculature as a Target for AntiCancer Therapy, Manchester, England, September 17-21, 1990.) and
in cervical carcinomas between 10 and 30 mm Hg [H. D. Roh, Y.
Boucher, W. D. Bloomer, and R. K. Jain. Interstitial hypertension in
human cervical carcinomas: Effect of radiation (Abstract). 16th Gray
PRESSURE
Conference—Vascular as a Target for Anti-Cancer Therapy, Manches
ter, England, September 17-21, 1990.].
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Interstitial Pressure Gradients in Tissue-isolated and
Subcutaneous Tumors: Implications for Therapy
Yves Boucher, Laurence T. Baxter and Rakesh K. Jain
Cancer Res 1990;50:4478-4484.
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