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Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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and an additional, as yet
unidentified, soluble factor. These data underscore the importance of
tumor/stromal interaction in the production of angiogenic peptides such
as IL-8 in NSCLC. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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We have previously used a NSCLC/fibroblast coculture assay to demonstrate that tumor/stromal interactions promote the stromal-cell release of bFGF (5 , 6) . Whereas NSCLC and pulmonary fibroblasts that are cultured separately release no detectable bFGF, fibroblasts cocultured with NSCLC release readily detectable quantities of the peptide (5 , 6) . These results suggest that normal pulmonary fibroblasts may be an important microenvironmental source of angiogenic factors in NSCLC.
IL-8 is one of the most important angiogenic peptides in NSCLC (7, 8, 9) . In recent studies, NSCLC cell lines such as A549 and Calu-1 were shown to produce IL-8 (7 , 8) . NSCLC-derived IL-8 stimulated the chemotaxis of endothelial cells and promoted angiogenesis in corneal pocket assays (7) . IL-8 also enhanced the growth and angiogenesis of the two NSCLC cell lines in murine tumor models (8) . In these assays, the administration of neutralizing IL-8 antibodies to tumor-bearing mice reduced NSCLC size by >40% and decreased tumor vascularity (8) . However, the addition of recombinant IL-8 or neutralizing IL-8 antibodies did not alter the proliferation of NSCLC cell lines in vitro, suggesting that IL-8 had an indirect effect on tumor cell growth via its role in tumor angiogenesis (8) . It was not possible to assess stromal IL-8 production in these murine tumor models because there is no identified murine IL-8 homologue (10) .
In associated analyses of primary NSCLCs, the tumors were found to have
4-fold more abundant IL-8 transcripts and protein than normal
lung specimens (7)
. When primary NSCLC specimens were
immunostained for IL-8, there was heterogeneous IL-8 expression in
tumor cells. However, in certain NSCLCs, particularly squamous cell
lung cancers, stromal cells also expressed increased IL-8
(7)
.
Tumor-derived cytokines stimulate the release of IL-8 by fibroblasts in vitro (11) , raising the possibility that tumor/stromal interactions have similar effects on IL-8 release in vivo. To determine whether stromal cells produce IL-8 as a consequence of interaction with NSCLC cells, we have examined IL-8 expression in the tumor/fibroblast coculture model (5) .
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Fibroblast/Tumor Cell Coculture.
Fibroblasts were grown to confluence in six-well plates. NSCLC cells
(2.5 x 105) were added either
directly onto the fibroblasts or into the upper chamber of a transwell
apparatus (Costar, Cambridge, MA), which physically separated the tumor
cells from the fibroblasts but allowed for interaction between the
cells via soluble factors. After an overnight culture in DMEM/10% FCS,
cells were washed twice in serum-free medium and incubated for
24 h in 2 or 3 ml of serum-free DMEM. Selected cocultures were
treated with neutralizing antibodies to IL-1, IL-1ß, TNF-, or
normal goat IgG at 1 µg/ml (R&D Systems, Inc., Minneapolis, MN),
catalase (100 units/ml, Sigma), or NMA (0.75 mg/ml, Sigma). The
conditioned media were harvested and centrifuged to remove cellular
debris. Supernatants were frozen at -20°C until ELISA.
IL-8 ELISA.
IL-8 protein levels in conditioned media were measured by ELISA (R&D
Systems) per the manufacturer’s protocol. Briefly, 50 µl of sample
or standard in 100 µl of diluent were added to each well coated with
a murine monoclonal antibody against IL-8. To be within range,
supernatants from A549 and H441 cultures were diluted 1:4 in serum-free
DMEM. Other supernatants were analyzed undiluted. An IL-8 polyclonal
antibody conjugated to horseradish peroxidase was added, and samples
were incubated for 2.5 h. After six washes, substrate solution was
added for 30 min. Absorbance was measured on a microplate reader
(Bio-Rad) at 450 nm, and IL-8 concentration was determined in each
sample by comparison to a standard curve that was generated by log-log
plot (Bio-Rad software). Duplicate samples were analyzed for each
coculture condition.
Northern Blot.
Fibroblasts and tumor cells (2.5 x 106) were cocultured in 100-mm transwell dishes
(Costar) under similar culture conditions described above. After
24 h of culture in 20 ml of serum-free DMEM, the conditioned media
were collected, and RNAs from both the fibroblasts and tumor cells were
isolated separately by acid guanidinium thiocyanate/phenol chloroform
extraction (RNA-STAT-60, Tel-Test, Inc., Friendswood, TX),
size-fractionated on a 1% agarose gel under denaturing conditions, and
transferred to a nylon membrane (Hybond N+, Amersham Pharmacia
Biotech). The blots were then hybridized with a
32P-labeled IL-8 cDNA probe (gift of Barrett
Rollins, Dana-Farber Cancer Institute, Boston, MA) or a
32P-labeled glyceraldehyde-3-phosphate
dehydrogenase cDNA probe.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, T) and pulmonary fibroblasts (Fig. 1
, F)
cultured separately or together (Fig. 1
, F/T). As described
previously, the A549 NSCLC line constitutively expressed readily
detectable levels of IL-8 protein, whereas the CCL-210 fibroblasts
released negligible amounts of the chemokine (Fig. 1A
, T,
F). However, when A549 cells were cocultured with pulmonary
fibroblasts, 4 times more IL-8 was detected in the resulting
conditioned medium (Fig. 1A
, F/T).
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). The NSCLC cell line H441 also
constitutively expressed moderate levels of the IL-8 protein (Fig. 1B
), whereas the other NSCLC cell lines produced minimal
quantities of the cytokine (Fig. 1, C–F
). However, when all
five additional NSCLC cell lines were cocultured with stromal cells,
increased levels of IL-8 were detected in the resulting conditioned
media (Fig. 1, B–F
). Similar results were also obtained
when the NSCLC cell lines were cocultured with another pulmonary
fibroblast line (CCL-153, data not shown). Taken together, these data
indicate that enhanced IL-8 release is a common feature of
NSCLC/stromal cocultures.
IL-8 Release Does Not Require Direct Tumor/Stromal Cell Contact.
To determine whether the increased IL-8 release in NSCLC/stromal
cocultures requires physical contact between the tumor cells and
fibroblasts, the A549 and Calu-1 cell lines were also cocultured
with fibroblasts in a transwell apparatus that permits only the
diffusion of soluble factors. As indicated in Fig. 2, A and B,
IL-8 release was also augmented when
either of two representative NSCLC cell lines (A549 or Calu-1) was
cocultured with pulmonary fibroblasts in transwell.
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). In transwell cocultures of A549 and pulmonary
fibroblasts [CCL-210 (Fig. 2C
) or CCL-153 (Fig. 2E
)], IL-8 transcripts were significantly more abundant in
fibroblasts cocultured with A549 than fibroblasts cultured alone;
tumor-derived A549 IL-8 transcripts were abundant in both conditions
(Fig. 2, C and E
). In transwell cocultures of
Calu-1 NSCLC cells and pulmonary fibroblasts [CCL-210 (Fig. 2D
) or CCL-153 (Fig. 2F
)], IL-8 transcripts were
more abundant in both the NSCLC cells and the fibroblasts. Taken
together, these data indicate that: (a) IL-8 transcripts are
induced in pulmonary fibroblasts and certain NSCLC cell lines as a
consequence of tumor/stromal interaction; and (b) the
observed IL-8 induction did not require direct cell-cell contact,
implicating a soluble mediator.
Characterization of the Soluble Factor(s) Mediating IL-8 Induction
in NSCLC/Stromal Coculture.
In previous studies, a variety of soluble factors, such as IL-1,
IL-1ß, TNF-, H2O2, and
nitric oxide, have been shown to induce IL-8 in epithelial cells and
fibroblasts (11, 12, 13)
. To assess the possibility that one
of these factors was responsible for the IL-8 induction in
tumor/stromal cocultures, the cocultures were plated in the presence or
absence of neutralizing antibodies directed against IL-1, IL-1ß,
or TNF or inhibitors of oxidant stress (NMA or catalase; Fig. 3
). Neutralizing IL-1, IL-1ß, or TNF antibodies alone or in
combination were unable to block the induction of IL-8 in
A549/pulmonary fibroblast cocultures; inhibitors of oxidant stress (NMA
and catalase) were similarly ineffective in blunting IL-8 release (Fig. 3A
). In similar cocultures of SL-6, H520, and H125 NSCLC
cell lines with pulmonary fibroblasts, neutralizing IL-1, IL-1ß,
and TNF- antibodies had little effect on IL-8 induction (Fig. 3,
B, C, and E
). In contrast, the induction of IL-8
in Calu-1/stromal cocultures was specifically blocked by an IL-1
neutralizing antibody but was not inhibited by neutralizing antibodies
to IL-1ß or TNF (Fig. 3D
). Similar results were obtained
when Calu-1 was cocultured with either CCL-153 or CCL-210 pulmonary
fibroblasts (Fig. 3D
and data not shown).
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on IL-8 induction in Calu-1
NSCLC/stromal cocultures, additional cocultures were plated in
transwell in the presence or absence of the neutralizing IL-1
antibody. Thereafter, RNAs from Calu-1 NSCLC cells alone, pulmonary
fibroblasts alone, or transwell cocultures plated with or without the
IL-1 antibody were prepared and analyzed for IL-8 transcripts. As
indicated in Fig. 4
, Calu-1/stromal coculture resulted in a 4-fold increase in IL-8
transcripts in the pulmonary fibroblasts and a 2-fold increase in IL-8
transcripts in Calu-1 cells. The addition of neutralizing IL-1
antibodies to the Calu-1/stromal cocultures reduced both IL-8
transcripts (Fig. 4A
) and secreted protein (Fig. 4B
) to baseline levels.
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plays an important
role in the induction of IL-8 in cocultures of fibroblasts with Calu-1
NSCLC cells. However, additional, as yet unidentified soluble factors
induce or enhance IL-8 release in cocultures of pulmonary fibroblasts
and the other examined NSCLC cell lines. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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IL-8 directly promotes endothelial cell proliferation, chemotaxis, and tubular morphogenesis (14, 15, 16) . The chemokine may also have indirect effects on angiogenesis resulting from the induction of MMP-2 and enhanced migration of endothelial cells through extracellular matrix (17) . These observations are of particular interest because IL-8 is a major angiogenic factor in primary NSCLC tumors and murine models of NSCLC (7 , 8) .
In previous studies, NSCLC cell lines such as A549 were shown to produce IL-8 and to enhance angiogenesis in an IL-8 specific manner (7 , 8) . Although tumor-derived IL-8 clearly promoted angiogenesis in these in vitro assays and in vivo murine models, the contribution of stromal cells to IL-8 induction and production could not be examined in the model systems. That stromal cells might contribute to IL-8 production in NSCLC is of particular interest because pulmonary fibroblasts are the dominant source of IL-8 in other angiogenesis-dependent disease states, such as idiopathic pulmonary fibrosis (4) .
In our own studies, two of six NSCLC cell lines (A549 and H441)
expressed high basal levels of IL-8, whereas the remainder had low to
negative IL-8 expression. These results are consistent with previous
analyses in which 8 of 13 NSCLC cell lines produced high levels of IL-8
(18)
. Although only two of the NSCLC cell lines used in
our studies had high basal levels of IL-8, all cocultures of NSCLC and
pulmonary fibroblasts secreted increased amounts of the cytokine (Fig. 1
). In representative tumor/stromal cocultures, IL-8 was always induced
in pulmonary fibroblasts and was also induced in certain NSCLC cell
lines (Fig. 2
). These data raise the interesting possibility that
pulmonary stroma may potentiate IL-8 production and related tumor
angiogenesis in NSCLC.
These observations are consistent with recent studies suggesting a comparable stromal influence on tumor cell production of IL-8 in a melanoma nude mouse model (19) . In these studies, IL-8 transcripts were significantly more abundant in melanoma cells metastatic to lung than in melanoma cells cultured alone in vitro. Furthermore, IL-8 transcripts were abundant in s.c. melanoma deposits but less abundant in liver metastases, suggesting that stromal effects on IL-8 may be organ dependent.
Earlier studies have led to the assumption that the cancer cells are the source of IL-8 in primary tumor specimens. However, our data indicate that stromal cells produce increased IL-8 as well as enhance tumor cell IL-8 production in tumor/stromal cocultures. Tumor/stromal interactions have also been shown to influence the production of other angiogenic factors, such as VEGF and hepatocyte growth factor (HGF). For example, the VEGF promoter was strongly activated in tumor-associated fibroblasts in a murine model (2) . Furthermore, HGF expression was increased in fibroblasts cocultured with tumor cells in additional analyses (3) .
The mechanism by which IL-8 is induced in tumor/stromal cocultures
remains to be defined. In several in vitro models, the
induction of IL-8 required cell/cell contact (20
, 21)
.
Although IL-8 induction was most efficient in direct cocultures (Fig. 2
), the cytokine was also induced when tumor cells were separated from
stromal cells by a transwell membrane. These data implicate a soluble
factor in IL-8 induction in the tumor/stromal cocultures. Antibodies
directed against known soluble IL-8 mediators such as IL-1ß and
TNF-, and inhibitors of oxidant stress did not blunt IL-8 release in
the NSCLC/pulmonary fibroblast cocultures. Neutralizing IL-1
antibodies inhibited IL-8 induction in Calu-1/stromal cocultures but
had no effect in cocultures containing other NSCLC cell lines. Taken
together, these data suggest that additional as yet unidentified
soluble factors modulate IL-8 induction in the majority of
NSCLC/stromal cocultures.
Our results are consistent with mounting evidence that normal surrounding stromal cells may promote the growth and dissemination of tumors by modulating the release of critical angiogenic peptides. Further understanding of the interactions between tumor cells and adjacent normal stromal elements may lead to new therapeutic strategies.
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Acknowledgments |
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FOOTNOTES |
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1 This study was funded by National Institutes of
Health Grants 1KO8CA69133-01 and 1RO1CA76286-01. 
2 Present Address: EPI INSERM 9911, Faculte de
Medecine, Pasteur Avenue de Vallombrose 06107, Nice Cedex 2,
France.
3 Present Address: CJF-INSERM 96005, Faculte de
Medecine, Pasteur Avenue de Vallombrose, 06107, Nice Cedex 2,
France.
4 To whom requests for reprints should be
addressed, at Dana-Farber Cancer Institute, 44 Binney Street, Boston,
MA 02115. Phone: (617) 632-3874; Fax: (617) 632-4734; E-mail: margaret_shipp{at}dfci.harvard.edu
5 The abbreviations used are: bFGF, basic
fibroblast growth factor; VEGF, vascular endothelial growth factor; IL,
interleukin; NSCLC, non-small cell lung cancer; NMA,
NG-methyl-L-arginine.
Received 9/22/99. Accepted 11/19/99.
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