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[CANCER RESEARCH 62, 661– 664, February 1, 2002]
Advances in Brief
Pulmonary and Lymph Node Metastasis Is Associated with Primary Tumor
Interstitial Fluid Pressure in Human Melanoma Xenografts1
Einar K. Rofstad,2 Siv H. Tunheim, Berit Mathiesen, Bjørn A. Graff, Ellen F. Halsør, Kristin Nilsen, and
Kanthi Galappathi
Group of Radiation Biology and Tumor Physiology, Department of Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
Abstract
Interstitial fluid pressure (IFP) is elevated in many experimental and
human tumors, and high IFP is associated with poor prognosis in human
cancer. The significance of elevated IFP in the development of metastatic
disease was investigated in the present work by using A-07 human melanoma xenografts as models of cancer in humans. IFP was measured with
the micropipette technique (tumor periphery) and the wick-in-needle
technique (tumor center). Tumor hypoxia was studied by immunohistochemistry using pimonidazole as a hypoxia marker and by using a radiobiological assay. High central tumor IFP was found to be associated with
the development of pulmonary (P ⴝ 0.000085) and lymph node
(P ⴝ 0.000036) metastases in small (150 –200 mm3) A-07 tumors. Hypoxic
cells could not be detected in these tumors. Our study suggests that
interstitial hypertension may facilitate tumor cell intravasation and,
hence, promote metastasis by mechanisms independent of tumor hypoxia.
ation between IFP and development of metastatic disease is reported
in the present study. IFP and hypoxic fraction may be correlated in
some tumors (10, 12), and there is experimental and clinical evidence
suggesting that hypoxia may promote cancer metastasis (13). Confounding effects of hypoxia were avoided in the present study by
using small A-07 human melanoma xenografts as tumor models (14).
A-07 tumors are well vascularized and do not develop hypoxic regions at volumes of ⬍200 mm3 as demonstrated here by using
immunohistochemical and radiobiological assays in attempts to detect
hypoxic cells.
Materials and Methods
Mice and Tumors. Adult (8 –10 weeks of age) female BALB/c-nu/nu mice
were used as host animals for xenografted tumors. Tumors were initiated from
exponentially growing A-07 cell cultures. The A-07 cell line, established as
Introduction
described previously (14), was maintained in monolayer culture in RPMI 1640
Most experimental and human tumors develop elevated IFP3 during (25 mM HEPES and L-glutamine) supplemented with 13% bovine calf serum,
50 mg/liter streptomycin. Cells (⬃2.0 ⫻ 105)
growth (1). Measurements of IFP in tumors have yielded values up to 250 mg/liter penicillin, and
2⫹
suspended in 10 ␮l of Ca and Mg2⫹-free HBSS were inoculated intrader110 mm Hg, whereas most normal tissues show IFP values close to 0
mally in the left mouse flank as described earlier (14). Animal experiments
mm Hg (2, 3). The microvascular hydrostatic pressure is the principal were approved by the Institutional Committee on Research Animal Care and
driving force for interstitial hypertension in tumors (4, 5). Tumors were performed according to the Interdisciplinary Principles and Guidelines
generally show high resistance to capillary blood flow, low resistance for the Use of Animals in Research, Marketing, and Education (New York
to transcapillary fluid flow, and impaired lymphatic drainage (6). Academy of Sciences, New York, NY).
Therefore, the microvascular hydrostatic pressure forces fluid from
IFP Measurements. The IFP in the periphery of tumors was measured
the microvasculature into the tumor interstitium where the fluid ac- with micropipettes and a servo-nulling device (Vista Electronics, Ramon, CA)
cumulates, distends the elastic extracellular matrix, and causes an by using a procedure similar to that described by Boucher et al. (4). Micropiincrease in the IFP (5, 7). The resistance to fluid flow in the tumor pettes with tip diameters of 2– 4 ␮m, filled with 1 M NaCl solution, were
interstitium greatly exceeds the resistance to transcapillary fluid flow moved progressively into tumors by using a graded micromanipulator under
stereomicroscopic guidance. Zero pressure was recorded in a drop of saline at
(7), leading to a pseudostable state where the central tumor IFP is
the tumor surface before each micropipette insertion. IFP was measured at
nearly equal to the microvascular hydrostatic pressure (1, 5).
intervals of 0.25 mm up to a tumor depth of 2.5 mm. Each IFP value was
Tumors may be resistant to various categories of cancer treatment recorded for at least 10 s. Measurements were accepted as valid when the fluid
because of interstitial hypertension. Elevated IFP may lead to poor communication between the micropipette and the tissue could be confirmed
and heterogeneous uptake of macromolecular and nanoparticle ther- electrically and the zero pressure in the saline at the tumor surface was not
apeutic agents, and, hence, resistance to some forms of immunother- modified during micropipette insertion and withdrawal.
The IFP in the center of tumors was measured with the wick-in-needle
apy and gene therapy (8). Interstitial hypertension may also cause
impaired blood flow and oxygen supply (9), and, hence, resistance to technique by using a procedure similar to that described by Tufto and Rofstad
radiotherapy (3, 10). Interestingly, a recent prospective study has (15). Each IFP value was recorded for ⱖ5 min. The fluid communication
shown that preradiotherapy IFP can predict disease-free survival in between the pressure transducer and a tumor was tested by compressing and
decompressing the tubing between the needle and the transducer after a stable
patients with cervical carcinoma independent of clinical prognostic
value had been reached. Measurements were accepted as valid when the
factors and tumor oxygenation (11).
readings after these tests did not differ by more than 1 mm Hg. Tumor IFP was
An experimental study demonstrating for the first time an associ- determined by calculating the mean of these two readings. IFP values in
normal tissues, recorded i.m. and subdermally, served as internal controls.
Received 10/16/01; accepted 12/5/01.
The mice were kept under general anesthesia during IFP measurements.
The costs of publication of this article were defrayed in part by the payment of page
Propanidid (Gedeon Richter, Budapest, Hungary), fentanyl/fluanisone (Janscharges. This article must therefore be hereby marked advertisement in accordance with
sen Pharmaceutika, Beerse, Belgium), and diazepam (Dumex, Copenhagen,
18 U.S.C. Section 1734 solely to indicate this fact.
1
Denmark) were administered i.p. in doses of 400, 0.24/12, and 4 mg/kg body
Supported by grants from The Norwegian Cancer Society.
2
To whom requests for reprints should be addressed, at Department of Biophysics,
weight, respectively. The body core temperature of the mice, measured with a
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310
rectal probe, was kept at 36 –38°C by using a heating pad.
Oslo, Norway. Phone: 47-22-93-42-79; Fax: 47-22-93-42-70; E-mail: e.k.rofstad@
Immunohistochemical Detection of Hypoxia. Pimonidazole was used as
labmed.uio.no.
3
a marker of tumor hypoxia. A peroxidase-based immunohistochemical method
The abbreviations used are: IFP, interstitial fluid pressure; pimonidazole, {1-[(2was used to detect hypoxic tumor regions (16). Pimonidazole hydrochloride,
hydroxy-3-piperidinyl)propyl]-2-nitroimidazole}.
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INTERSTITIAL FLUID PRESSURE AND METASTASIS
kindly supplied by Professor James A. Raleigh (Department of Radiation
Oncology, University of North Carolina School of Medicine, Chapel Hill, NC)
was dissolved in 0.9% NaCl and administered i.p. in doses of 30 mg/kg body
weight. The tumors were dissected free from the mice 4 h after the pimonidazole administration and fixed in phosphate-buffered 4% paraformaldehyde.
Slides with tumor tissue preparations were incubated with polyclonal rabbit
antiserum to pimonidazole-protein adducts, a gift from Professor J. A. Raleigh.
Visualization of the antibody complex was achieved with the 3,3-diaminobenzidine chromogen. Hematoxylin was used for counterstaining. Large A-07
tumors, ⬃1000 mm3 in volume, were used as positive controls. The histology
of tumors staining positive for pimonidazole has been displayed elsewhere
(16).
Radiobiological Detection of Hypoxia. A Siemens Stabilipan X-ray unit,
operated at 220 kV, 19 –20 mA, and with 0.5 mm Cu filtration, was used for
irradiation. Cell cultures were irradiated at a dose rate of 3.4 Gy/min and
tumors at a dose rate of 5.1 Gy/min. Hypoxic tumors were obtained by
occluding the blood supply with a clamp 5 min before irradiation. The
detection of hypoxia in tumors with unperturbed blood supply was based on
the paired survival curve method (17). Cell survival was measured in vitro by
using a plastic surface colony assay. Lethally irradiated (30 Gy) feeder cells
were used to ensure a linear relationship between the number of colonies and
the number of plated cells. Single-cell suspensions were prepared from tumors
by using a combined mechanical and enzymatic procedure. The tumors were
minced with scalpels in Ca2⫹ and Mg2⫹-free HBSS before being subjected to
enzymatic treatment at 37°C for 2 h. The enzyme solution consisted of 0.2%
collagenase, 0.05% Pronase, and 0.02% DNase in HBSS. Cells giving rise to
colonies with ⬎50 cells were scored as clonogenic. The experimental procedure has been described in detail elsewhere (16).
Metastasis Assay. Primary tumors were initiated in the left mouse flank as
described above. They were removed surgically when the volume was 150 –
200 mm3, i.e., 7–9 days after initiation. The mice were examined for clinical
signs of metastases twice a week. They were killed and autopsied when they
were moribund or 3 months after the removal of the primary tumor. The lungs
were examined for pulmonary metastases, and mediastinum, abdomen, and the
interscapular, submandibular, axillary, and inguinal regions were examined for
lymph node metastases. Moribund mice were always positive for metastases.
The presence of metastases was confirmed by histological examinations.
Statistical Analysis. Correlations between two parameters were searched
for by linear regression analysis. Statistical comparisons of data were performed by using the Student’s t test. The data sets were verified to comply with
the conditions of normality and equal variance. Probability values of P ⬍ 0.05,
determined from two-sided tests, were considered significant. The statistical
analysis was performed by using SigmaStat statistical software (Jandel Scientific GmbH, Erkrath, Germany).
Results
Studies investigating associations between IFP and metastasis
should make use of tumors without hypoxic regions, because hypoxia
Fig. 1. F, cell survival levels for A-07 cultures irradiated under aerobic conditions in
vitro. Points, means of five experiments; bars, ⫾ SE. Œ, cell survival levels for A-07
tumors irradiated under hypoxic conditions in vivo. Points, means of six tumors;
bars, ⫾ SE. ‚, cell survival levels for A-07 tumors irradiated with 7.5 Gy under
unperturbed conditions in vivo. Points, single tumors. Dashed curve, calculated cell
survival curve for A-07 tumors with a hypoxic fraction of 1% irradiated under unperturbed
conditions in vivo.
Fig. 2. A, intratumor heterogeneity in IFP in A-07 tumors was studied by using the
micropipette technique. Normalized IFP versus depth, measured from the skin surface, is
shown. IFP was normalized by assigning a value of 100% to the highest IFP value
recorded in each tumor. Points, means of 6 tumors; bars, ⫾ SE. B, the reproducibility of
the wick-in-needle technique was investigated by measuring the IFP in the center of A-07
tumors twice. The second measurement was performed 3– 4 h after the first measurement.
IFP (Measurement # 1) versus IFP (Measurement # 2) is shown. Points, individual tumors.
Curve, regression line (P ⬍ 0.00001; R2 ⫽ 0.96).
may promote metastasis (13), and IFP and hypoxic fraction may be
correlated in some tumors (12). Therefore, radiobiological experiments were performed to search for hypoxic cells in 150 –200-mm3
A-07 tumors (Fig. 1). Cell survival curves were determined for
aerobic monolayer cultures and for tumors with occluded blood supply, i.e., tumors with a hypoxic fraction of 100%, to establish a
framework for the experiments. These reference experiments gave cell
survival curves consistent with an oxygen enhancement ratio of ⬃3,
in agreement with classical radiobiological theory (17). The expected
cell survival curve for tumors with a hypoxic fraction of 1%, calculated from these data by using the paired survival curve method (17),
is shown as a dashed curve in Fig. 1. Ten tumors with unperturbed
blood supply were then irradiated with a dose of 7.5 Gy and assayed
for cell survival. The surviving fractions determined for these tumors
were similar to those measured for aerobic monolayer cultures exposed to 7.5 Gy and lower than those expected for tumors having a
hypoxic fraction of 1% (P ⫽ 0.0040). These data demonstrate that the
fraction of radiobiologically hypoxic cells in 150 –200-mm3 A-07
tumors is ⬍1% and is consistent with the tumors having no radiobiologically hypoxic cells. In comparison, similar experiments have
shown that the mean fraction of radiobiologically hypoxic cells in
200 – 400-mm3 A-07 tumors is 6% (16).
Intratumor heterogeneity in IFP was studied by moving micropipettes progressively into tumors from the outward surface and recording the IFP at intervals of 0.25 mm. The IFP increased with increasing
depth until a plateau was reached at a depth of 0.75 mm, measured
from the skin surface (Fig. 2A). Six tumors were included in the study,
and all of the tumors showed a steep IFP gradient in the periphery and
relatively uniform IFP values at depths beyond 0.75 mm. The IFP
profiles did not differ noticeably among the tumors, apart from the
fact that the absolute values were highly different, ranging from 5 to
15 mm Hg in the central plateau. Therefore, intertumor heterogeneity
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INTERSTITIAL FLUID PRESSURE AND METASTASIS
Fig. 3. IFP in metastatic and nonmetastatic A-07 primary tumors. The lungs
were examined for pulmonary metastases, and mediastinum, abdomen, and the
interscapular, submandibular, axillary, and inguinal regions were examined for
metastases in lymph nodes. Points, single tumors. A, lungs and lymph nodes. B,
lungs only. C, lymph nodes only.
in IFP was studied by recording the IFP in the center of tumors with
the wick-in-needle technique. The reproducibility of such measurements was investigated by measuring the IFP in 18 tumors twice. The
second measurement was performed 3– 4 h after the first measurement. The correlation between the first and the second measurement
was excellent (P ⬍ 0.00001; R2 ⫽ 0.96); the difference between the
two IFP values never exceeded 2 mm Hg (Fig. 2B).
An association between IFP and metastasis was searched for by
performing an experiment involving 50 mice. The primary tumors
were removed when the volume was within the range of 150 –200
mm3. IFP was measured in the tumor center with the wick-in-needle
technique immediately before tumor removal, and pimonidazole was
administered to the host mice 4 h earlier. Twenty-one mice developed
metastases, whereas 29 mice did not. Nine of the metastasis-positive
mice showed both pulmonary and lymph node metastases, 10 showed
pulmonary metastases only, and 2 showed lymph node metastases
only. The metastatic primary tumors had ⬃1.7-fold higher IFP than
the nonmetastatic primary tumors (Fig. 3A; P ⫽ 0.000026). Moreover,
IFP was ⬃1.7-fold higher in the primary tumors that metastasized to
the lungs than in those that did not give rise to pulmonary metastases
(Fig. 3B; P ⫽ 0.000085) and ⬃1.8-fold higher in the primary tumors
that metastasized to lymph nodes than in those that did not form
lymph node metastases (Fig. 3C; P ⫽ 0.000036). Hypoxic regions,
i.e., positive pimonidazole staining, could not be detected in any of the
primary tumors. In contrast, five large A-07 control tumors, ⬃1000
mm3 in volume, showed hypoxic fractions of 6 –10%, consistent with
previous studies (16).
Discussion
Metastatic spread of malignant cells from the primary tumor to
distant sites is the major cause of death of patients with cancer. The
metastatic process is composed of a cascade of linked, sequential, and
highly selective steps involving multiple host-tumor interactions.
These steps include invasion of tumor cells into blood or lymphatic
vessels, survival in the circulation, arrest in a secondary organ, extravasation into the secondary organ interstitium and parenchyma,
proliferation in the secondary organ, and induction of angiogenesis.
Metastatic cell phenotypes are, because of the complexity of the
dissemination process, believed to have accumulated several stable
and/or unstable genomic changes affecting growth regulation and
tissue homeostasis. Associations between metastatic spread and gene
expression have been demonstrated for many gene products (18).
The study reported here demonstrates that metastasis also is associated with the IFP of the primary tumor. The development of metastases from small A-07 tumors was studied, and the primary tumors
that metastasized to the lungs showed ⬃1.7-fold higher IFP than those
that did not form pulmonary metastases. Moreover, IFP was ⬃1.8fold higher in the primary tumors that metastasized to lymph nodes
than in those that did not give rise to lymph node metastases. However, it should be noticed that a substantial fraction of the nonmeta-
static tumors showed IFP values within the same range as those of the
metastatic tumors, suggesting that high IFP is not a sufficient condition for metastasis in small A-07 tumors. High IFP is probably not a
necessary condition either, because primary tumors with IFP values as
low as 5 mm Hg were capable of forming both pulmonary and lymph
node metastases.
IFP was measured in a single location in the tumor center by using
the wick-in-needle technique. Micropipette studies of the intratumor
heterogeneity in IFP justified this procedure. The IFP was relatively
uniform throughout the tumors and dropped precipitously to normal
tissue values at the tumor surface, in accordance with results from
theoretical considerations and experimental studies of murine tumors
(4). Moreover, IFP measurements with the wick-in-needle technique
gave highly reproducible results, demonstrated by performing repeated measurements in the same tumors, and control measurements
in normal tissues gave IFP values ranging from ⫺1 to ⫹1 mm Hg,
consistent with the normal tissue values reported by others (19).
Tumors with low resistance to transcapillary fluid flow, such as the
A-07 tumors studied here, develop a central IFP that is nearly equal to
the microvascular hydrostatic pressure (5, 15). The microvascular
hydrostatic pressure is determined by the resistance of the capillary
network to blood flow (9, 12), which is elevated in tumors for several
reasons, including high blood viscosity, abnormal capillary structure
and organization, and capillary compression because of tumor cell
proliferation in a confined space (6). Therefore, intertumor heterogeneity in central IFP in tumors with low resistance to transcapillary
fluid flow is mainly a consequence of intertumor heterogeneity in the
microvascular parameters determining the resistance to capillary
blood flow. The A-07 tumors used in the present work were initiated
in the same site in identical hosts from the same cell culture. The
intertumor heterogeneity in resistance to capillary blood flow and,
hence, the intertumor heterogeneity in central IFP was, therefore, most
likely a result of stochastic processes in the development of the
capillary network rather than genetic differences among the tumors.
Consequently, the association between metastasis and IFP in A-07
tumors is unlikely to reflect that the most metastatic cell phenotypes
developed the tumors with the highest IFP; the association rather
reflects that high IFP, caused by stochastic processes, increased the
probability of metastasis in genetically similar tumors.
There is significant evidence from clinical and experimental studies
that tumor hypoxia may promote metastasis by activating hypoxiainducible factor-1 and other transcription factors, and, hence, increase
the expression of genes playing an important role in the metastatic
process (13). Studies of carcinoma of the uterine cervix have suggested that IFP and hypoxic fraction may be correlated in tumors (3,
10). Confounding effects of hypoxia were avoided in the present work
by using small A-07 tumors as models of human cancer. None of the
tumors included in the study had developed hypoxic regions at tumor
removal, as was revealed by using pimonidazole as a hypoxia marker.
Radiobiological experiments showed that the fraction of hypoxic cells
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INTERSTITIAL FLUID PRESSURE AND METASTASIS
in small A-07 tumors is ⬍1%. The radiobiological data were consistent with the assumption that the tumors have no hypoxic cells and
were, thus, in agreement with the immunohistochemical observations.
Consequently, the association between metastasis and IFP reported
here is unlikely to reflect an association between metastasis and
hypoxia. If A-07 tumors are representative models of tumors in
humans, the present study suggests that interstitial hypertension may
promote metastatic disease in human cancer by mechanisms independent of tumor oxygenation.
The mechanisms by which interstitial hypertension promotes metastasis in A-07 tumors cannot be established from the experiments
reported here. However, it is tempting to speculate that mechanical
forces may facilitate tumor cell intravasation and, hence, metastatic
spread in tumors with high IFP. Pulmonary metastases in A-07 tumors
most likely originate from tumor cells entering blood vessels within
the primary tumor. Studies of experimental tumors have shown that
abrupt decreases in the microvascular hydrostatic pressure leads to
fluid flow from the interstitium into the microvasculature (7). A-07
tumors show substantial intermittent blood flow at the microvascular
level (16), implying that abrupt decreases in the microvascular hydrostatic pressure may be a frequent phenomenon, and it is, thus,
possible that the central tumor IFP for short periods of time is
substantially higher than the hydrostatic pressure of a microvessel,
particularly in tumors with high central IFP, and that the subsequent
fluid flow facilitates intravasation of tumor cells adjacent to the
microvessel. Lymph node metastases in A-07 tumors most likely
originate from tumor cells spread by lymphatics. Functional lymphatics cannot be detected in the interior of A-07 tumors but can be
observed close to the tumor periphery in the normal skin tissue
surrounding the tumors. Studies of experimental tumors have shown
that interstitial fluid is forced from the periphery of tumors into
adjacent normal tissue where it is collected and removed by lymphatics (4, 5). This fluid flow is driven by the difference in IFP between
the tumor and the adjacent normal tissue. The IFP gradient in the
periphery of A-07 tumors is steep, and it is, thus, possible that in
tumors with high central IFP, the outward fluid flow is sufficiently
strong that the migration of peripheral tumor cells toward lymphatics
is promoted by convection.
Only two studies of the IFP of melanomas in humans have been
reported thus far, one by Boucher et al. (19) and the other by Curti et
al. (2). These studies revealed that the IFP in general is higher in
melanoma than in human tumors of other histological types, including
cervical carcinoma, breast carcinoma, lymphoma, and colorectal carcinoma (2, 3, 10, 11, 20). Boucher et al. (19) measured IFP values as
high as 48 mm Hg in some large melanomas, and even higher values,
ⱕ110 mm Hg, were recorded by Curti et al. (2). In comparison, the
highest IFP measured in the A-07 melanomas included in our metastasis study was only 20 mm Hg. Therefore, it is possible that metastasis promotion by interstitial hypertension is an even more extensive
problem in human melanomas than the present experimental study
suggests. Interestingly, Curti et al. (2) showed that elevated tumor IFP
was associated with poor prognosis in melanoma patients treated with
interleukin 1␣ or interleukin 2 immunotherapy.
In summary, pulmonary and lymph node metastasis is associated
with the IFP of the primary tumor in A-07 human melanoma xe-
nografts. The association does not reflect that the most aggressive cell
phenotypes develop the primary tumors with the highest IFP but
suggests rather that interstitial hypertension increases the probability
of metastatic dissemination in genetically similar tumors. The mechanism by which elevated IFP promotes metastasis in A-07 tumors is
independent of tumor oxygenation.
Acknowledgments
We thank Olav Groven for excellent technical assistance. We also thank
Professor James A. Raleigh, Department of Radiation Oncology, University of
North Carolina School of Medicine, Chapel Hill, NC, for supplying pimonidazole hydrochloride and antipimonidazole rabbit antiserum.
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Pulmonary and Lymph Node Metastasis Is Associated with
Primary Tumor Interstitial Fluid Pressure in Human Melanoma
Xenografts
Einar K. Rofstad, Siv H. Tunheim, Berit Mathiesen, et al.
Cancer Res 2002;62:661-664.
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