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Tumor Biology |
Pathophysiology Unit [K .K., Y. T., Ta. T.], Laboratory of Immunology [Te. T., To. T.], and Laboratory of Ultrastructure Research [Ta. T.], Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464-8681, Japan; Departments of Internal Medicine, Aichi Cancer Center Hospital [T. H.], Nagoya, Aichi 464-8681, Japan; and Laboratory of Pathology, Department of Basic Gerontology, National Institute for Longevity Sciences [O. M.], Ohbu, Aichi 474-8522, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression of certain molecules, such as adhesion receptors and ligands (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) as well as metalloproteinases (18, 19, 20, 21, 22) , has been suggested to play a role in the development of metastatic lesions. Metastasis occurs via two distinct pathways, and tumor cells spread through blood and LVs4 . Although a large number of studies have been conducted, yielding considerable information about the metastatic processes, very little is known about how cancer cells propagate lymphogenous metastasis. Identification of molecules with a crucial role in the lymphogenous spread of cancer cells has been hampered by lack of an appropriate experimental model system. To date, a few cell lines derived from several types of human malignancies have been reported to have a high potential to metastasize regional lymph nodes when the tumor cells were injected at orthotopic sites (23, 24, 25, 26, 27, 28, 29) , although they were found to metastasize to a much lesser extent when propagated s.c. However, no cell lines have been reported thus far as being useful for studies of the lymphogenous metastasis of human lung cancers.
In the present study, we describe an in vivo selection resulting in the establishment of a human lung cancer cell line, NCI-H460-LNM35 (LNM35), which consistently and spontaneously metastasizes to lymph nodes when injected either s.c. or orthotopically. Comparisons between LNM35 and its parental H460 cell lines are also made with regard to expression levels and/or activities of various molecules that are thought to play a part in the metastatic processes. We show that expression of COX-2 is increased in LNM35 and that a specific COX-2 inhibitor, nimesulide, can inhibit the invasion of LNM35 in vitro through Matrigel containing basement membrane components.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Vivo Selection.
In vivo selection was carried out to establish a
high-lung-metastatic subline of H460, using the procedures described by
Fidler (35)
and also in a previous study of ours
(22)
. In brief, 1.0 x 107 of the parental H460 cells in 100 µl of
serum-free RPMI 1640 medium were injected in the s.c. tissue of the
left abdominal wall of 7-week-old female SCID mice. Lung tissues
containing the metastatic tumor cells were excised, minced, and
reimplanted in the abdominal wall of new recipient mice for the
selection of high-metastatic tumor cells. After two rounds of in
vivo selection by means of sequential implantations, metastatic
nodules in the lung tissues were harvested to initiate in
vitro culture of the metastatic tumor cells. Further selection was
then carried out by injecting the resulting cell line into s.c. tissue,
followed by in vitro propagation of tumor cells obtained
from spontaneous ALN metastasis. A clonal cell line was established
from the cultured cell mixture with the limiting dilution method and
use of 96-well culture plates. These cell lines were then maintained in
RPMI 1640 medium with 10% fetal bovine serum.
Spontaneous Metastasis Assay.
Cells (1.0 x 107) in 100
µl of serum-free RPMI 1640 medium were implanted in the s.c. tissues
of the left abdominal wall of 7-week-old female SCID mice or nude mice.
At 45 days (SCID mice) or 55 days (nude mice) after s.c. implantation,
mice were sacrificed by cervical dislocation under deep anesthesia, and
internal organs, including lung, liver, kidney, and spleen as well as
lymph nodes and s.c.T.s, were resected. The resected specimens were
weighed, fixed with 4% paraformaldehyde, and processed for light
microscopic examination of the paraffin-embedded sections stained with
H&E. The lung-metastatic nodules were examined and counted under a
dissecting microscope.
Orthotopic Implantation.
Mice were anesthetized by i.p. injection with 0.28 mg/g body weight of
2.2.2.tribromoethanol (Aldrich Chemical Company, Milwaukee, WI). A 1-cm
long ventral midline incision was made in the neck to expose the
trachea for direct inspection of the orotracheal intubation of a
20-gauge catheter/needle unit (Terumo, Tokyo, Japan), which was
advanced through the oral cavity to a depth of 2.1 cm from the incisor
teeth under visual inspection through the exposed trachea. The needle
was then pulled out, leaving only the outer 20-gauge catheter, through
which a blunt-ended 25-gauge needle (Top, Tokyo, Japan) was inserted a
depth of 4.4 cm. Next, 1.0 x 107 of the cultured LNM35 cells in 50 µl of
serum-free RPMI 1640 medium were directly inoculated through the
inserted needle into the bronchioloalveolar cavity, and the skin
incision was closed with two stitches. The mice were sacrificed as
described above 28 days after orthotopic implantation, and lung and
mediastinum were removed en block and fixed with 4%
paraformaldehyde. Metastasis to the MLNs was examined under a
dissecting microscope and confirmed by histological examination of
paraffin-embedded sections stained with H&E.
Growth Curves.
Cells (1.0 x 105) were
inoculated onto 3.5-cm dishes. At daily intervals, triplicate samples
were harvested and counted with a Coulter counter (Coulter Electronics,
Luton, United Kingdom), and cell numbers were averaged for each time
interval.
Antibodies.
SNH-3 (specific to sialyl Lewis X), 2F3–6 (specific to sialyl Lewis
X-variant), and 2D-3 (specific to sialyl Lewis A) MoAbs (36
, 37)
were generous gifts of Dr. R. Kannagi (Aichi Cancer Center
Research Institute). Anti-E-cadherin and anti-CD44 MoAbs as well as
anti-integrin MoAbs used in this study were obtained from Medical and
Biological Laboratories, Inc. (Nagoya, Japan) except for TS2/7 and
J143, which were the generous gifts of, respectively, Dr. J. L.
Strominger (Harvard University) and Dr. L. J. Old (Memorial
Sloan-Kettering Cancer Institute). The following anti-integrin MoAbs
were used (17)
: ß1, K20; ß2, BL5; ß3, SZ21; 
1,
TS2/7; 2, Gi9; 3, J143; 4, HP2/1; 5, SAM1; 6, GoH3; and
v, AMF/7. Rabbit anti-TIMP-2 polyclonal antibody was obtained from
Chemicon International Inc. (Temecula, CA), affinity-purified
FITC-conjugated goat antimouse and antirat IgG were obtained from
Protos Immunoresearch (San Francisco, CA), and FITC-conjugated rabbit
antimouse IgM was obtained from Cappel Inc. (Malvern, CA).
Biotin-conjugated affinity-purified goat antirabbit IgG and horseradish
peroxidase-conjugated streptavidin were obtained from Vector
Laboratories (Burlingame, CA).
Fluorescence-activated Flow Cytometry.
Aliquots (100 µl) containing 1.0 x 106 cells were subjected to indirect
immunofluorescence staining for the detection by FACScan (Becton
Dickinson Immunocytometry Systems, Mountain View, CA) of the expression
of various adhesion molecules and carbohydrate determinants. MoAbs were
used at a concentration of 10 µg/ml and incubated for 30 min at room
temperature.
Detection of TIMP-2 and Gelatinases A and B.
Cells (1 x 105) were
cultured for 2 days in 1 ml of serum-free RPMI 1640 culture medium in
culture dishes 3.5 cm in diameter followed by harvesting of the culture
supernatant. Each medium sample was concentrated 10-fold with the aid
of Centricon-10 (Amicon, Beverly, MA). TIMP-2, which had been secreted
into the serum-free conditioned medium, was detected by means of
Western blot analysis using anti-TIMP-2 polyclonal antibody and a POD
immunostaining kit (Wako Pharmaceutical Industries, Ltd., Osaka,
Japan), as previously described (38)
. Gelatinases A
(MMP-2, Mr 72,000 type IV collagenase)
and B (MMP-9, Mr 92,000 type IV
collagenase), which had been secreted into the serum-free-conditioned
medium, were detected by means of zymography with gelatin (2 mg/ml) as
the substrate, as described previously (38)
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Northern Blot Analysis of COX-2.
Extraction of RNA from cell lines and Northern blot analysis were
conducted according to the standard procedures. A human COX-2 cDNA
probe was generated by PCR with the aid of a sense primer,
5'-TTCAAATGAGATTGTGGGAAAATTGCT, and an antisense primer,
5'-AGATCATCTCTGCCTGAGTATCTT.
In Vitro Motility and Invasion Assay.
To quantify the in vitro motility and invasion assay,
transwell-chamber culture systems were used. The upper surface of
filters 6.4 mm in diameter with 8-µm pores (Becton Dickinson Labware,
Franklin Lakes, NJ) were coated with 100 µl of 0.1 mg/ml Matrigel
(Collaborative Research Inc., Bedford, MA) in the case of the invasion
assay. Filters were filled with 0.5 ml of serum-free RPMI 1640 medium
and placed on culture plates with 24 wells filled with 1 ml of the
medium. LNM35 cells (1 x 104
cells in motility assay; 1 x 105
cells in invasion assay) were then added to the upper chambers and
cultured. After 24 h of incubation, the filters were fixed with
70% ethanol and stained with Giemsa, and the cells on the lower
surface of the filters were counted in triplicate. Nimesulide was
provided by Hisamitsu Pharmaceutical Co. (Tosu, Japan).
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RESULTS |
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Morphological and Growth Characteristics of LNM35 in
Vitro.
Under a phase-contrast microscope, both parental H460 and in
vivo-selected LNM35 cells demonstrated the polygonal shape typical
of epithelial cells, although adhesion of H460 to the culture dish and
intercellular junction tended to be tighter than that of LNM35. No
significant differences were observed in in vitro cell
growth rates between parental H460 and LNM35 cell lines. Furthermore,
microsatellite analysis using D17S250 and D17S513 to confirm derivation
of LNM35 from H460 showed identical patterns for H460 and LNM35 (data
not shown).
Spontaneous Metastatic Properties of LNM35.
Spontaneous metastatic properties were examined by means of s.c.
injection of 1 x 107 cells of
LNM35. Three independent experiments were carried out, and the injected
mice were sacrificed after 45 or 55 days. At autopsy, s.c. injection of
parental H460 cells as well as of in vivo-selected LNM35
cells consistently yielded a similar sized tumor mass with similar
microscopic features of large cell undifferentiated carcinoma (Fig. 1
, s.c.T.; Table 1
). Markedly enlarged LVs (Fig. 2
, arrowheads) draining to the ALNs (Fig. 2
, ALN) were observed in all
mice injected with LNM35 cells, implying the occurrence of
carcinomatous lymphangitis. Upon histological examination, the enlarged
LVs were shown to be filled with the tumor cells (data not shown),
while lymph node metastasis was also confirmed (Fig. 1
, ALN).
Furthermore, LNM35 cells characteristically caused carcinomatous
lymphangitis on the visceral pleura and in intrapulmonary LVs
surrounding lung-metastatic nodules (Fig. 1
, lung, arrow). Metastasis
to the inguinal lymph nodes was detected in a few cases, and tumor
infiltration of LNM35 cells into blood vessels was detected in some
cases (data not shown). In contrast, no lymphatic involvement was
detected in any mice injected with parental H460 cells. It was also
noted that the number of lung-metastatic nodules in mice inoculated
with LNM35 cells was markedly larger than that seen in mice injected
with parental H460 cells (Table 1)
. No spontaneous metastasis in any
other organ was observed in either parental H460 or LNM35 cells. These
experiments were carried out, and both macroscopic and histological
examinations confirmed 100% occurrence of lymph node metastases of
LNM35 in marked contrast to the complete absence of such occurrence in
the case of parental H460 (Table 1)
. LNM35 cells have been maintained
in culture for >1 year without noticeable changes in their
lymphogenous metastatic potential.
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summarizes the observed frequencies of successful tumor propagation in
the lung and those of histologically confirmed metastases to the MLNs
in cases with i.b.-implanted tumors (Table 2
and Fig. 3
). Histological examination also showed intravasation of LNM35 cells
into the LVs (Fig. 3
, i.b.T., arrows) and blood vessels (data
not shown) of the lung as well as into the lymphatic afferent vessels
of MLNs (LV in Fig. 3
, MLN). These macroscopic and microscopic features
of this orthotopic propagation model are similar to those seen in lung
cancer cases, which suggests that the lymphogenous metastatic processes
of LNM35 mimic those occurring in patients.
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2, 3, 5, 6, v, and ß1 subunits of
integrins were expressed in both LNM35 and H460 at similar levels,
whereas neither expressed the 1, 4, ß2, and ß3 subunits (data
not shown). Expression levels of CD44 were similar for LNM35 and H460,
whereas neither cell line expressed E-cadherin or the ligands for
E-selectin, sialyl Lewis X, sialyl Lewis X-variant, or sialyl Lewis A
(data not shown). We conclude that the observed significant differences
between the metastatic potentials of LNM35 and H460 could not be
accounted for by the differences in expression patterns of either the
gelatinases and their specific inhibitor or of the adhesion molecules
and carbohydrate chains examined thus far.
COX-2 Expression and Effect of Nimesulide on in
Vitro Motility and Invasion.
Because our previous immunohistological studies of COX-2 expression in
human lung cancer patients suggested a possible association of the
increase in COX-2 expression with invasion and metastasis as well as
with poor prognosis, expression of COX-2 was also examined in LNM35 and
H460. Northern blot analysis showed that COX-2 expression was
significantly increased in LNM35 when compared with that in H460 (Fig. 4A)
. As an initial step toward elucidation of the potential
relationship of the increased expression of COX-2 with the highly
metastatic phenotype of LNM35, we examined the effects of a specific
COX-2 inhibitor, nimesulide, in LNM35 in vitro. Nimesulide
was shown to be potent in the inhibition of invasion through Matrigel
as well as of cell motility in a dose-dependent manner at
concentrations significantly lower than that required for the
inhibition of cell growth (Fig. 4B)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The LNM35 cell line is, to the best of our knowledge, the first human
lung cancer cell line to be reported as having such a biological
property. Moreover, LNM35 has significant advantages over previously
reported cell lines. LNM35 spontaneously metastasizes at a 100%
incidence not only as a result of orthotopic i.b. propagation but also
as a result of conventional s.c. injection. This proven highly
reproducible nature of lymphogenous metastases makes LNM35 very
different from others because s.c. inoculation of other lines
reportedly yielded lymphogenous metastases at frequencies ranging from
0 to 57%. In addition, LNM35 has a relatively short latency period,
making it possible to obtain experimental results within 28 days after
i.b. inoculation and 45 days after s.c. inoculation. It is also
noteworthy that LNM35, orthotopically propagated by means of our
modified i.b. technique, closely mimics the clinical manifestations of
human lung cancer patients, infiltrates into lymphatic vessels, and
metastasizes to the MLNs.
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The present extensive search for differentially expressed molecules, however, allowed us to identify that COX-2 expression was significantly increased in LNM35. We could also show that motility and invasion of LNM35 could be inhibited significantly at least in vitro by treatment with nimesulide. These findings are of considerable interest because we previously found that COX-2 is expressed intensely in lung cancer cells infiltrating into the surrounding stromal tissues and in the corresponding lymph node metastases (45) and that an increase in COX-2 expression may be associated with a poor prognosis in patients undergoing surgical resection of early stage lung adenocarcinomas (46) . Although an increase in expression of COX-2 has been suggested to play a significant role in the carcinogenesis of colorectal and lung adenocarcinomas (45 , 47 , 48) , it has also been reported that introduction of the COX-2 gene may intensify invasiveness of colon cancer cells but that this invasiveness could be reduced by treatment with sulindac sulfide, a known COX inhibitor (49) . Although further in vivo studies are necessary, the present study together with previous observations suggest the potential involvement of COX-2 in the metastatic processes of lung cancers. Studies such as expression profiling using LNM35 and its parental H460 are also warranted to search for additional differentially expressed molecules to elucidate the complex mechanisms of tumor metastasis.
In conclusion, we successfully established for the first time a human lung cancer cell line, LNM35, with high capability for developing spontaneous lymphogenous metastasis when inoculated in mice. The highly reproducible metastasis of LNM35, even as a result of conventional s.c. inoculation, as well as its short latency period should prove to be suitable for the screening of agents active in the suppression of metastasis in human lung cancer, which may ultimately lead to the development of novel means of diagnosis and nonsurgical therapy of this fatal disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a Grant-in-Aid for
Scientific Research on Priority Areas from the Ministry of Education,
Science and Culture of Japan. 
2 Present address: Laboratory of Pathology, Aichi
Cancer Center Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya
464-8681, Japan.
3 To whom requests for reprints should be
addressed, at the Laboratory of Ultrastructure Research, Aichi Cancer
Center Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya 464-8681,
Japan.
4 The abbreviations used are: LV, lymphatic
vessel; MLN, mediastinal lymph node; COX-2, cyclooxygenase 2; i.b.,
intrabronchial; i.b.T., i.b. tumor; MMP, matrix metalloproteinase; ALN,
axillary lymph node; s.c.T., s.c. tumor; TIMP, tissue inhibitor of
metalloproteinase; MoAb, monoclonal antibody.
Received 9/ 7/99. Accepted 3/ 2/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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