Cancer Cell Biology
Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion
Article first published online: 16 AUG 2002
Copyright © 2002 Wiley-Liss, Inc.
International Journal of Cancer
Volume 101, Issue 5, pages 415–422, 10 October 2002
How to Cite
Harmey, J. H., Bucana, C. D., Lu, W., Byrne, A. M., McDonnell, S., Lynch, C., Bouchier-Hayes, D. and Dong, Z. (2002), Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion. Int. J. Cancer, 101: 415–422. doi: 10.1002/ijc.10632
- Issue published online: 3 SEP 2002
- Article first published online: 16 AUG 2002
- Manuscript Accepted: 8 JUL 2002
- Manuscript Revised: 12 JUN 2002
- Manuscript Received: 4 FEB 2002
- UICC ICRETT. Grant Number: 1019/99
- Royal Irish Academy Senior Visiting Fellowship
- Enterprise Ireland International Collaboration Award. Grant Number: IC/1999/050
Endotoxin/lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, is a potent inflammatory stimulus. We previously reported that LPS increased the growth of experimental metastases in a murine tumor model. Here, we examined the effect of LPS exposure on key determinants of metastasis—angiogenesis, tumor cell invasion, vascular permeability, nitric oxide synthase (NOS) and matrix metalloproteinase 2 (MMP2) expression. BALB/c mice bearing 4T1 lung metastases were given an intraperitoneal (i.p.) injection of 10 μg LPS or saline. LPS exposure resulted in increased lung weight and incidence of pleural lesions. LPS increased angiogenesis both in vivo and in vitro. Vascular permeability in lung tissue was increased 18 hr after LPS injection. LPS increased inducible nitric oxide synthase (iNOS) and MMP2 expression in lung tumor nodules. 4T1 cells transfected with green fluorescent protein (4T1-GFP) were injected via lateral tail vein. LPS exposure resulted in increased numbers of 4T1-GFP cells in mouse lung tissue compared to saline controls, an effect blocked by the competitive NOS inhibitor, NG methyl-L-arginine (NMA). LPS-induced growth and metastasis of 4T1 experimental lung metastases is associated with increased angiogenesis, vascular permeability and tumor cell invasion/migration with iNOS expression implicated in LPS-induced metastasis. © 2002 Wiley-Liss, Inc.
Most cancer patients ultimately succumb to metastatic disease, and up to 50% of cancer patients already have metastatic deposits at the time of diagnosis.1 In many cases, the primary tumor can be successfully treated by surgery, radiotherapy, chemotherapy or a combination but the subsequent growth of previously dormant or clinically undetectable metastatic deposits presents a serious obstacle to the complete eradication of the disease. Elucidating factors that influence the development and progression of metastatic disease is critical to the development of effective therapies for patients with metastatic deposits.
Metastatic tumor growth involves a complex series of sequential events involving a number of cell types, cytokines and pathways. After the initial transformation event, growth of a primary tumor is accompanied by extensive angiogenesis. Cells with a metastatic phenotype invade the tissue stroma and penetrate the blood vessels to enter the circulation. The majority of tumor cells entering the circulation are rapidly destroyed but those that do survive can then become trapped in organ capillary beds and extravasate into the organ parenchyma. Cell proliferation and vascularization of the secondary deposit completes the metastatic process.2
Endotoxin/lipopolysccharide (LPS), a cell wall constituent of Gram-negative bacteria, is released during growth or lysis of bacteria and acts as a potent inflammatory stimulus, eliciting a range of cytokines, growth factors and inflammatory mediators. LPS and some bacteria have been shown to have angiogenic activity.3, 4, 5 Inflammation has been linked with angiogenesis, resulting in changes in permeability, activation of endothelium and vessel remodeling.6 In support of a link between inflammation and tumor progression, there is a growing body of evidence that anti-inflammatory agents such as the nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclo-oxygenase activity, inhibit both tumorigenesis and growth of colon and mammary tumors.7 Endotoxin is ubiquitously present in air, and we previously implicated endotoxin in surgically induced tumor growth.8, 9 Endogenous gut bacteria are a major source of endotoxin, which can translocate across the gut into the circulation following surgical trauma or thermal injury.10, 11, 12
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a potent angiogenic cytokine stimulating growth and differentiation of endothelial cells.13 By increasing vascular permeability, resulting in the leaky vasculature characteristic of tumor angiogenesis, VEGF facilitates extravasation of tumor cells, an early event in the metastatic cascade.14 The matrix metalloproteinases (MMPs), a family of related proteinases, are responsible for degradation and remodelling of the extracellular matrix. These enzymes are overexpressed in many tumors and there is solid evidence that they play a key role in the malignant progression and invasiveness of many tumors.15
The role of nitric oxide (NO) in tumor growth and metastasis remains paradoxical. Although it can be cytotoxic to tumor cells,16 NO could facilitate metastasis by increasing vasodilation and permeability.17 NO is synthesised by both constitutive (eNOS) and inducible (iNOS) nitric oxide synthase enzymes. iNOS is induced in response to a range of stimuli, including LPS.18 Comparison of highly and weakly metastatic variants of a spontaneous murine mammary carcinoma demonstrated that eNOS-derived NO increased tumor cell migration, invasion and angiogenesis.19 In contrast, a highly metastatic melanoma clone lost its metastatic ability when transfected with functional iNOS.20
We previously reported increased plasma LPS in mice following surgery and that surgery or LPS exposure increased metastatic tumor growth in an experimental murine metastasis model.9 To clarify the mechanisms underlying LPS-induced metastatic growth, we have extended those studies to examine the effect of LPS on key determinants of the metastatic process, namely angiogenesis, tumor cell invasion, vascular permeability, NOS and MMP expression. In addition, we assessed the effect of NO blockade on tumor cell invasion in vivo.
MATERIAL AND METHODS
Tumor cells and culture conditions
4T1 tumor cells, a spontaneously metastasizing mammary adenocarcinoma cell line, were a generous gift from Dr. Fred Miller, Duke University. Cells were maintained as monolayer cultures in DMEM supplemented with 10% FBS, sodium pyruvate, nonessential amino acids, L-glutamine and vitamins (Life Technologies, GIBCO BRL, Gaithersburg, MD.). GFP (green fluorescent protein)-transfected 4T1 cells were maintained in the same medium supplemented with 800 μg/ml G418 (Life Technologies). Cell cultures were incubated in an atmosphere of 5% CO2 in air at 37°C and were free of Mycoplasma and the following pathogenic viruses: reovirus type 3, pneumonia virus, K virus, Theiler's encephalitis virus, Sendai virus, minute virus, mouse adenovirus, mouse hepatitis virus, lymphocytic choriomeningitis virus, ectromelia virus and lactate dehydrogenase virus (assayed by M.A. Bioproducts, Walkersville, MD). Tumor cells were harvested from subconfluent cultures with 0.25% trypsin-0.02% EDTA. Trypsin was neutralized with medium containing 10% FBS, washed 3 times in PBS and resuspended in PBS at 2.5 × 105/ml for injection. Only single-cell suspensions of greater than 90% viability as determined by Trypan Blue exclusion were used for injections.
Transfection of 4T1 cells with green fluorescent protein
The pEGFP-C1 vector (Clontech, Palo Alto, CA) contains enhanced green fluorescent protein (GFP) and neomycin resistance genes. An amount of 3 × 105 4T1 cells at 50% confluence were plated in 2 ml MEM containing 5% fetal bovine serum in a 35 mm2 plate. Twenty-four hours later, cells were transfected with 1 μg pEGFPC1 using FuGene™ 6 transfection reagent according to the manufacturer's instructions (Roche Diagnostics, Indianopolis, IN). Cells were harvested with trypsin-EDTA 24 hr after transfection and subcultured at a ratio of 1:15 in medium containing 200 μg/ml G418. The level of G418 was increased step-wise to 800 μg/ml. GFP-expressing clones were isolated with cloning rings (Bel-Art Products, Pequannock, NJ) using trypsin-EDTA and amplified by conventional culture methods.
Female BALB/c mice were purchased from the Animal Production Area, National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD). Mice were maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services and National Institutes of Health. Mice were used at 8–10 weeks according to institutional guidelines.
Additional groups of animals for permeability assays, Western blot analysis and migration of GFP-transfected cells were purchased from Charles River Institute (Margate, Kent, UK) and maintained in the Biomedical facility of the Royal College of Surgeons in Ireland licensed and approved by the Department of Health. All procedures were carried out under animal license guidelines of the Department of Health in Ireland.
Mice received 5 × 104 4T1 cells via lateral tail vein injection. Seven days later, mice received an intraperitoneal (i.p.) injection of 10 μg lipopolysaccharide (serotype 055:B5; Sigma Chemical, St. Louis, MO). Control mice received an i.p. injection of PBS. At different time intervals after LPS injection, mice were anaesthetized, exsanguinated and euthanased by cervical dislocation. Lungs were harvested and lung weight and incidence of pleural and other lesions recorded. Lungs from 5 mice/group were fixed in Bouins solution to identify and measure tumor lesions. Number and size of experimental metastases was determined with the aid of a dissecting microscope.
In vivo permeability
Eighteen hours after i.p. administration of LPS or saline, mice (n = 3/group) were injected i.v. with 0.2 ml of 2.5 mg/ml FITC-labelled bovine albumin (Sigma Chemical, St. Louis, MO). Thirty minutes later, mice were sacrificed and lungs harvested. Hand-cut sections of formalin-fixed tissue (approx. 1 mm thick) were mounted under coverslips with immersion oil on glass slides and viewed under a fluorescence stereomicroscope.21
A Miles assay was performed as described with minor modifications.22 Eighteen hours after LPS or saline, mice (n = 5/group) received an i.v. injection of 0.2 ml 5 mg/ml Evans Blue dye in PBS. Fifteen minutes later, ears were removed, weighed and dye extracted in 0.5 ml formamide at 37°C for 48 hr. Data is expressed as OD650/mg tissue.
In vivo tumor cell migration
Seven days after injection of 5 × 104 4T1 cells via lateral tail vein, mice (n = 3/group) received another injection of 5 × 104 GFP-transfected 4T1 cells and i.p. injection of either 10 μg LPS or PBS. NO-blockade was achieved by i.p. administration of 60 μM NMA (NG methyl-L-arginine) (Sigma Chemical 16 hr before and at the time of LPS injection. Eight hours later, mice were sacrificed and lungs removed and placed in PBS or embedded in OCT compound for frozen sections. GFP-expressing cells were visualized in hand-cut sections (approx. 1 mm thick) of fresh tissue by fluorescence microscopy. The presence of GFP-expressing cells in lung tissue was confirmed by immunohistochemical staining of frozen sections for GFP as described below. The number of GFP-expressing cells in each of 6 random high-power fields [×20 objective and ×10 ocular]) per sample was quantified.
In vitro angiogenesis assay
Angiogenesis in response to LPS was determined using a commercial angiogenesis assay (TCS Biologicals, Buckingham, UK) essentially according to the manufacturer's instructions. In this system, human endothelial cells are co-cultured with stromal cells in a culture matrix, mimicking the in vivo cell interactions and basement matrix. Cells were cultured in the presence or absence of 10 μg/ml LPS for 11 days. At the end of the culture period, tubules were fixed and stained with an antibody to CD31. Images were captured from low magnification fields of view (×4 objective and ×10 ocular) using a microscope (Nikon Eclipse E600) with CCD camera and framegrabber Lucia ScMeas Version 4.5 software (©Laboratory Imaging). After background correction, digitised images were converted to binary form. A threshold value, set below blood vessel optical density to differentiate vessels from stromal cells and matrix, was applied to all images, and relative tubule area calculated as the number of dark pixels. Image processing algorithms were written using the Image Processing Toolbox within the numeric computing environment of MATLAB® (The MATH WORKS, Natick, MA). Assays were carried out 3 times and 3 random fields per sample analysed. Sample wells containing 2 ng/ml VEGF were included as a positive control.
Eighteen hours after i.p. administration of LPS or saline, mice (n = 3/group) were sacrificed and tumor nodules dissected from lungs using an 18 G needle and flash frozen in liquid nitrogen. Nodules were homogenized in 500 μl lysis buffer (10 mM Tris-HCL, pH 7.5, 10 mM EDTA, 1 mM EGTA, 0.2% Triton X-100, 1 mM PMSF and protease inhibitor cocktail [Sigma Chemical]) using a Dounce homogenizer. Lysates were centrifuged at 12,000g for 15 min at 4°C. Total protein concentration was determined by the BCA assay according to the manufacturer's instructions (Pierce Chemical, Rockford, IL).
Fifty micrograms of protein was separated on a 10% denaturing polyacrylamide gel. Following electrophoresis, proteins were transferred to nitrocellulose membrane. The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 (TBST) and 5% nonfat dried milk for 1 hr at room temperature and incubated overnight with anti-iNOS or eNOS (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:10,000 in TBST containing 1% nonfat dried milk, or anti-MMP2 (a gift from Andrew Docherty, Celltech, Slough, UK) diluted 1:1,000 in TBST. The membrane was washed 3 times in TBST, incubated for 90 min with horseradish peroxidase-conjugated anti-mouse IgG (1:2,000 in TBST) for iNOS, eNOS and MMP2 (DAKO, Glostrup, Denmark) and washed 6 times in TBST. Bound antibody complexes were visualized using the ECL enhanced chemiluminescence system (Pierce Chemical). For MMP2 bound antibody complexes were detected using diaminobenzidine, β-actin expression was detected using a β-actin antibody kit (Oncogene Research Products, San Diego, CA) according to the manufacturer's recommendations.
Quantification of microvessel density
Harvested lungs were embedded in OCT compound (Miles, Elkhart, IN), snap-frozen in liquid nitrogen and stored at −70°C. Eight micrometer sections were fixed in cold acetone for 5 min, 1:1 acetone:chloroform for 5 min, acetone for 5 min and washed 3 times in PBS for 5 min each. Endogenous peroxidase activity was blocked by incubating slides for 12 min with 3% H2O2 in methanol. Sections were washed 3 times in PBS and incubated in blocking solution (5% normal horse serum, 1% normal goat serum) for 20 min. Sections were incubated overnight at 4°C with 1 μg/ml MECA32 (rat anti-mouse panendothelial antigen [Pharmingen, San Diego, CA]) diluted in blocking solution. Sections were washed 3 times in PBS followed by blocking solution for 10 min before incubation with the second antibody (horseradish peroxidase-conjugated goat anti-rat IgG; Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:200 in blocking solution for 90 min at room temperature. Sections were washed 3 times in PBS and bound antibody complexes visualized by staining with AEC (3-amino-ethylcarbazole; BioGenex, San Ramon, CA). Sections were washed with PBS, counterstained with Gills 3 haematoxylin (Sigma Chemical) and mounted with Universal Mount (Research Genetics, Huntsville, AL).
For each section, vessels were counted in 5 high-power fields (200× magnification [×20 objective and ×10 ocular]) as described.23 Lung sections from each of 5 mice/group were analysed. Data are expressed as mean ± SEM.
Frozen lung sections (8 μm) were fixed for 3 min in 4% paraformaldehyde, washed 3 times in PBS, permeabilised in 0.1% Triton X-100 for 3 min, then washed 3 times in PBS. Staining was as above except blocking solution was 5% normal goat serum and anti-GFP (abcam, Cambridge, UK) was used at 1:100 in protein block. Second antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; Jackson ImmunoResearch Laboratories) was diluted 1:1,000 in blocking solution. Bound antibody was detected with DAB (diaminobenzidine; Sigma Chemical). For each section, GFP-positive cells were counted in 5 high-power fields (200× magnification). Lung sections from each of 3 mice/group were analysed. Data are expressed as mean ± SEM.
Unpaired Student's t-test was used to analyse data from in vitro angiogenesis, quantification of microvessel density and Miles assay. In vivo data were analysed by Fischer's exact test or unpaired Student's t-test (2-tailed) for incidence of pleural lesions and lung weight, respectively. In vivo tumor cell migration data were analysed by Mann-Whitney U test. Data were taken to be significant where p < 0.05.
Endotoxin-induced metastatic growth
We previously demonstrated that endotoxin increased metastatic tumor growth in a murine model of experimental metastasis.9 Table I shows that LPS exposure 1 week after tumor cell injection resulted in increased lung tumor burden, evidenced by increased lung weight (0.262 ± 0.05 vs. 0.229 ± 0.03 g in controls) and incidence of lesions in the pleural cavity or on chest wall and ribs (10/15 vs. 1/13 in the saline control group). The normal lung weight of 8-week-old female BALB/c mice is 0.139–0.177 g; thus these lung weights represent a significant tumor burden. There was no obvious difference in the topography of lung lesions between the 2 groups with the majority of nodules on the lung surface and no significant difference in the number of visible tumor nodules in the 2 groups. However, the lung nodules in the LPS group were significantly larger than those in the control mice (mean diameter 1.02 ± 0.05 mm vs. 0.8 ± 0.03 mm). These data indicate that LPS increased the growth of existing nodules and facilitated tumor cell migration/invasion into the pleural cavity.
|Group||Lung weight (g) Mean ± S.D.||Incidence of pleural lesions||Number of lung nodules Mean ± S.D.||Lung nodule diameter (mm) Mean ± S.D.|
|Saline||0.229 ± 0.03||1/13||70.6 ± 28.13||0.80 ± 0.21|
|LPS||0.262 ± 0.051||10/152||74.33 ± 25.9||1.02 ± 0.283|
Angiogenesis following endotoxin exposure
Since angiogenesis is central to both tumour growth and metastasis, we examined angiogenesis within lung lesions of mice exposed to LPS and saline controls. Although CD31 is the most commonly used marker of endothelial cells, antibodies to CD31 resulted in diffuse staining at the border between normal lung tissue and lung lesions and high background staining in the uninvolved lung tissue, making it difficult to clearly identify tumor vessels (data not shown). We therefore stained lung sections with mAb MECA32, which recognises an epitope constitutively expressed on vascular endothelium of most organs with the exception of vessels in cardiac and skeletal muscle and vessels in the central nervous system.24 This antibody clearly identified vessels within the tumor nodules, although it too resulted in some background staining in the uninvolved lung tissue. Representative lung sections from LPS and control mice (n = 5/group) are shown in Figure 1. Increased angiogenesis within lung tumors of mice exposed to LPS 5 days earlier is clear relative to the saline controls. In lung tumors from endotoxin-exposed and saline control mice, the average number of MECA32-positive vessels/mm2 was 96.01 ± 9.31 vs. 65.67 ± 5.91, respectively (p = 0.019).
Angiogenesis in vitro
We then investigated the direct effects of LPS on angiogenesis in vitro. Cells were cultured in the presence or absence of 10 μg/ml LPS for 11 days. As a positive control, cells were incubated in the presence of 2 ng/ml VEGF. The resulting tubules were stained with an antibody to CD31. Representative fields are shown in Figure 2a. Relative tubule area was calculated as the number of dark pixels/field. Figure 2b shows that 10 μg/ml LPS significantly increased tubule formation in this assay, 31.38 × 103 ± 3.28 × 103vs. 9.54 × 103 ± 1.10 × 103 pixels/mm2 in the control samples, representing 45% of the culture area vs. 10% in control wells (p ≤ 0.0001). Tubule formation in positive controls containing the known angiogenic factor, VEGF (2 ng/ml), was 35.27 × 103 ± 7.47 × 103 pixels/mm2, occupying 54% of the culture well area.
Endotoxin-induced vascular permeability
Tumor cells must invade the blood vessels as part of the metastatic process. It is likely that many of the events facilitating LPS-induced metastasis and angiogenesis occur soon after LPS exposure. We therefore examined lung vessel permeability using whole-tissue mounts of lung from mice injected with FITC-BSA, 18 hr after LPS or saline injection. Representative sections are shown in Figure 3a. Increased leakage of FITC-albumin can be seen as focal puffs of extravasated fluorescence in the lungs of mice exposed to LPS relative to the saline control mice. Increased vascular permeability in response to LPS was confirmed by Miles assay (Fig. 3b). Dye leakage in LPS-treated mice was 9.81 ± 1.02 vs. 3.22 ± 0.414 OD650 units/mg tissue in control mice.
NOS and MMP expression
Figure 3 clearly demonstrated increased vascular permeability in mice exposed to LPS. Since NO is a powerful vasodilator, we examined the expression levels of both the constitutive and inducible nitric oxide synthase(s)—eNOS and iNOS, respectively—in lung tumor nodules 18 hr after exposure to LPS (Fig. 4). LPS strongly induced iNOS expression in lung nodules (lanes L1, 2 and 3) relative to control mice (S1, 2 and 3). Although eNOS was expressed in lung tumor nodules of both control and LPS-treated mice, LPS did not alter its expression. As MMPs are critical in the degradation of ECM and therefore tumor cell invasion, we examined the effect of LPS on MMP2 expression. 4T1 cells constitutively produce high levels of MMP9, making it very difficult to load sufficient extract to visualize MMP2 on zymograms (Harmey, data not shown); MMP2 expression was examined by Western blot. LPS induced high levels of MMP2 in lung tumor nodules (L1, 2 and 3), and MMP2 appeared to be absent in lung tumors of control mice (S1, 2 and 3), although the antibody does not discriminate between pro- and active MMP2. Infiltrating host cells are the most likely source of LPS-induced MMP2 and iNOS, as we did not detect MMP2 or iNOS expression in control or LPS-treated 4T1 cells in vitro (data not shown). A Western blot of the same samples probed for β-actin is shown as control.
Tumor cell migration and invasion
To determine whether the increased incidence of extra-pulmonary metastases in mice exposed to LPS was associated with increased tumor cell invasion, we evaluated the effect of LPS on the migration of GFP-transfected 4T1 tumor cells into the lung tissues of tumor-bearing mice. To reproduce any tumor-derived influences present when LPS was injected in earlier experiments (Table I), injection of GFP-expressing cells and LPS was preceded by tumor implantation 7 days earlier. Since tumor cell invasion is an early event in the metastatic process, lungs were harvested 8 hr after injection of 50,000 GFP-4T1 cells and examined for the presence of GFP-expressing cells. Hand-cut sections were examined for GFP-expressing cells using an epifluorescent microscope. The presence of GFP-expressing cells in lung tissue was confirmed by immunohistochemistry of frozen sections examined under a light microscope (Fig. 5a). We scored GFP-expressing cells in each of 6 high-power fields (0.096 mm2)/mouse in 3 independent experiments. To normalise for differences in absolute cell numbers between experiments, the pooled results are expressed as percent of control, where the number of GFP-expressing cells in lungs of control animals is taken as 100% (Fig. 5b). LPS injection (10 μg) at the time of injection of GFP-4T1 cells significantly increased the number of tumor cells migrating into the lung tissue (185.75 ± 20.36% of control, p = 0.01 vs. control). As we had identified increased lung vascular permeability (Fig. 3) and increased iNOS expression (Fig. 4) in lung tumor nodules after LPS exposure, we examined the effects of NOS blockade on the migration of GFP-4T1 cells into lung tissue. NMA, a competitive inhibitor of all NOS isoforms, was given to mice 16 hr before and at the time of LPS and GFP-4T1 injection. NMA prevented LPS-induced migration of 4T1 cells into lung tissues (108.75 ± 30.82% of control, p = 0.03 vs. LPS) but did not alter tumor cell migration in control mice (117 ± 18.34% of control).
We have demonstrated that LPS increases growth of experimental lung metastases and incidence of pleural metastases in an experimental murine model of metastasis. The development of pleural metastases is analogous to the development of malignant pleural effusions in cancer patients. We previously reported that LPS exposure resulted in increased circulating VEGF in the same murine model used here.9 Pleural effusions are associated with increased VEGF and vessel permeability and inhibition of VEGF receptor tyrosine phosphorylation reduced the formation of pleural effusions suppressing vascular hyperpermeability in a murine model of lung adenocarcinoma.25
LPS has previously been reported to have angiogenic activity in a number of angiogenesis models.3, 4 LPS increased angiogenesis in tumor nodules and directly increased tubule formation in an in vitro model of angiogenesis. We have previously shown that LPS increased VEGF production by both macrophages and tumor cells.9, 26 Although LPS has previously been reported to have angiogenic activity in a number of angiogenesis models,3, 4 this is the first demonstration that LPS increases tumour angiogenesis in vivo.
LPS is known to induce a wide range of cytokines, growth factors and mediators relatively rapidly. In order to elucidate the molecular events underlying LPS-induced tumor growth and invasion, we examined a number of parameters at early time points. LPS has previously been shown to increase microvascular permeability.27 We demonstrated that LPS increased the permeability of lung vasculature in keeping with the normal physiologic response to an inflammatory stimulus.28 However, increased vascular permeability facilitates metastasis.14
LPS increased the migration of GFP-transfected 4T1 cells into the lung tissues of mice bearing 4T1 lung metastases. Not all cells seeded in secondary tissues go on to form metastatic deposits;29 however, increased seeding is likely to result in increased metastatic growth. In LPS-exposed mice, treatment with NMA returned GFP-4T1 cell invasion into the lung to the levels of controls. As NMA inhibits all NOS isoforms, it is difficult to conclusively implicate either the constitutive or inducible NOS isoforms. However, the fact that LPS increased iNOS expression without altering eNOS suggests that iNOS mediates LPS-induced tumor invasion. However, invasion is only part of the metastatic process and it is likely that other mediators play a role in LPS-induced growth of metastases. For example, VEGF and MMP2, both of which are induced by LPS,9 are likely to play a role in vascularization. Future experiments will investigate the role of these factors using specific blocking agents.
The precise role of NO in tumor growth remains unclear with evidence for both pro- and antitumor effects. For example, NO production from the constitutive eNOS of C3-L5 murine mammary adenocarcinoma promoted tumor cell invasion in vitro by downregulating TIMPs (tissue inhibitors of the matrix metalloproteinases) and iNOS induced by LPS/IFNγ (interferon γ) upregulated MMP2.30 In agreement with these findings, LPS induced iNOS expression in 4T1 lung tumor nodules, increased MMP2 expression and increased tumor cell invasion. eNOS activity has been implicated in the promotion of tumor cell migration, invasion and angiogenesis19 as well as VEGF-induced angiogenesis and vascular permeability31 and iNOS has been implicated in melanoma metastasis and pleural effusions.32
In contrast, a synthetic bacterial lipopeptide, JBT 3002, activated the tumoricidal properties of macrophages and potentiated the therapeutic efficacy of irinotecan against human pancreatic carcinoma in nude mice, an effect directly correlated with macrophage infiltration into the tumors and iNOS expression by these macrophages.16 Furthermore, in vitro cytoxicity of macrophages incubated with JBT 3002 and IFNγ was partly mediated by NO. In another study, transfection of K-1735 murine melanoma cells with iNOS suppressed tumorigenicity and reduced metastasis.20
It is possible that the effects of NO on tumor growth and metastasis are context-specific, namely dependent on tumor type, tumor model, interactions with other cytokines and growth factors, NOS isoform(s) expressed or cell type expressing NOS-tumor or host cells. However, a recent review has suggested a number of explanations for the positive and negative associations of NO with tumor progression.33 The genetic constitution of tumor cells, in particular p53 status, appears to be important in determining NO sensitivity or resistance. Furthermore, NO resistance may be conferred by cyclo-oxygenase 2 (COX-2) activation.34 COX-2 has been implicated in the growth of a number of tumour types.35 COX-2 is likely to play a role in the tumor-promoting activities induced by LPS, as LPS has previously been shown to induce COX-2.36 We have recently demonstrated that COX-2 inhibition decreased 4T1 tumour growth in vivo.37 Furthermore, COX-2 inhibition blocked LPS-induced 4T1 tumour growth and metastasis in vivo and decreased prostaglandin E2 production by 4T1 cells in vitro (data not shown). Thus COX-2 inhibitors may have value in blocking downstream tumor-promoting activities of NO and LPS. Indeed, co-administration of a COX-2 and an iNOS inhibitor demonstrated more potent chemopreventive activities than either agent alone in a model of chemically induced colon cancer.38
Major mechanisms promoting bacterial translocation are disruption of ecologic equilibrium of gastrointestinal tract to allow overgrowth of intestinal bacteria, suppression of host immune defenses, surgical trauma and increased permeability of the intestinal mucosal barrier.39 We have shown that LPS creates a permissive environment for metastatic disease progression by altering critical factors in the metastatic process, namely increasing tumor cell invasion, vascular permeability and angiogenesis. Thus any event that results in increased circulating endotoxin may be a risk factor for the progression of metastatic disease in patients with a previous history of cancer. Anti-inflammatory agents, in particular those that block COX-2, may be of use in patients at risk of metastatic disease particularly if they are also at risk of endotoxaemia.
Supported by UICC ICRETT fellowship 1019/99, Royal Irish Academy Senior Visiting Fellowship and Enterprise Ireland International Collaboration Award IC/1999/050 to J.H. We thank Dr. I.J. Fidler for his support and critical reading of the manuscript, Dr. J. Walsh for developing software to quantify in vitro angiogenesis results and Ms. D. Reynolds, Ms. M. Lloyd, Ms. B. Knighton and Mr. D. Fan for excellent technical assistance.
- 26Lipopolysaccharide increases vascular endothelial growth factor production by macrophages and tumor cells. FASEB J 1998;166(suppl 8): 432., , , .
- 28Comparative effects of betamethasone, cyclosporin and nedcromil sodium in acute pulmonary inflammation and metalloproteinase activities in bronchoalveolar lavage fluid from mice exposed to lipopolysaccharide. Pulmon Pharmacol Therapeut 1999; 12: 165–71., , , , , .