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Tumor Biology |
Departments of Pathology and Laboratory Medicine [C. W. W., A. L., L. S., J. Y., Y. D., R. J. M.], Radiation Oncology [G. K., E. J. B.], and Institute for Environmental Medicine [A. B. A-M.], University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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INTRODUCTION |
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Introducing tumor cells i.v. into immune-deficient experimental mice can be used as a model to duplicate the later steps of hematogenous metastasis for a wide variety of tumor cells. When lung colonies develop after i.v. injection, the cell line is regarded as having metastatic potential. In general, lung colonies form only if the cells injected are positive in spontaneous metastasis assays (i.e., if they form metastases after growth as s.c. tumors; Refs. 7 and 8 ). The injection of metastatic tumor cells into the mouse tail vein usually leads to the formation of lung, pleural, or mediastinal colonies, although colonies at other sites sometimes occur. By harvesting colonies from specific sites and selecting through several cycles of injection, it has been possible to select for cells that preferentially metastasize to these sites, such as the ovary or liver (9) . Homing to the lung can be partially attributed to the anatomy of the circulation that obliges all blood to flow through the lungs. Some have proposed that cells lodge mechanically in the pulmonary capillaries due to size constraints, but capillary occlusion cannot be sufficient in itself for growth as a metastasis because not all tumorigenic cells give rise to lung colonies (for review see Refs. 10 and 11 ). Adhesion factors have been identified that are required for some tumor cells to attach to pulmonary endothelium. For example, adhesion of B16F10 melanoma cells in the lung has been shown to depend on Lu-ECAM-1, whereas dipeptidyl peptidase IV (also known as CD26) is required for the attachment of R3230AC rat mammary and RPC-2 rat prostate carcinoma cells (12, 13, 14, 15, 16) . Esb cells derived from a murine T-cell lymphoma that metastasizes to the liver after i.v. injection instead form colonies in the muscle when ß1 integrin is disrupted, but only rarely form colonies in the lung (17) . Furthermore, we have recently directly observed attachment of tumor cells to precapillary arterioles in the lung (18) .
Whereas introduction of metastatic tumor cells into the circulation results in colony formation, the majority of the injected tumor cells do not produce colonies. For example, i.v. injection of 5 x 104 cells may result in, at most, 200 colonies (19) . The failure of the majority of the cells to form colonies has been termed "metastatic inefficiency" (20) . Fidler and Nicolson (21) injected highly metastatic tumor cells labeled with 125IUDR, a radioactive thymidine analogue, into mice. By measuring radioactivity levels in various organs, they were able to determine where the injected cells migrated to after injection. These experiments indicated that the majority of cells was rapidly cleared from the blood and initially arrested in the lungs (21 , 22) . They demonstrated that by 24 h, >85% of the cells initially arrested in the lungs were lost. Two cell lines were used, one with a 8–10-fold greater ability to form pulmonary colonies than the other, yet both led to equivalent counts retained in the lungs at 2 min to 1 day (9 , 21) . These observations have been confirmed with other cell lines and with different methods of radioactive labeling by other groups (23, 24, 25, 26, 27) . The fate of these cells that fail to metastasize has not previously been demonstrated to be due to death by apoptosis.
To directly examine the fate of tumor cells within the lungs, we labeled metastatic tumor cells with GFP3 and observed their fate in the first 1–2 days after tail-vein injection. Our observations indicated that many of the injected cells undergo apoptosis within the lungs. These results directly establish that tumor cells die in the lungs after introduction into the circulation through apoptotic processes.
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MATERIALS AND METHODS |
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2.10.10 and HT1080 cells expressing GFP were isolated after transfection of pEGFP-C2 (Clontech, Palo Alto, CA), 2 weeks of selection with Geneticin, followed by sorting in a flow cytometer. The 5% brightest cells were sorted and subcloned. This resulted in constitutively fluorescent clones. GFP-expressing adenovirus was a kind gift from Meenhard Herlyn (Wistar Institute, Philadelphia, PA). B16F10 cells were infected with the virus at a ratio of 20 plaque-forming units/cell, resulting in at least 90% fluorescent cells.
Radiation Treatment.
Cells (1 x 106) were plated on
10-cm dishes. The next day, they were subjected to 10 Gy of Cesium 137
irradiation (Shephard Mark I Model 68A irradiator). Twenty-four and
48 h following irradiation, cells were trypsinized, pelleted, and
resuspended in 200 µl of DMEM. Cells in the medium and attached cells
were pooled because apoptotic cells frequently detached. Ten
microliters of propidium iodide solution [containing 3.4
mM trisodium citrate, 0.1% NP40, 4.83 mM
spermine tetrahydrochlorate, 0.5 mM Tris, and 0.62
mM propidium iodide (pH 7.6)] was added to 20 µl of cell
suspension, and the cells were examined under a fluorescent microscope.
They were scored for apoptosis as determined by its morphology (ruffled
edges, condensed chromatin and nuclear fragmentation, cell shrinkage,
and formation of apoptotic bodies).
Tumorigenicity and Experimental Metastasis Assays.
Female NCR-nu/nu mice, 4–6 weeks of age, were
obtained from Taconic Farms (Germantown, NY) and housed aseptically
(laboratory animal facilities, University of Pennsylvania). Cells used
for injection were grown to subconfluence, subjected to brief trypsin
treatment, washed, and resuspended in serum-free DMEM. For
tumorigenesis experiments, mice were injected bilaterally s.c. in both
flanks with 5 x 105 cells in a
single cell suspension in 100 µl. Tumors were measured using vernier
calipers for calculation of tumor size. Six tumors were measured for
each time point. For experimental metastasis assays, mice were injected
with a single cell suspension of 1 x 104 cells in 100 µl into the tail vein. Animals
were killed when exhibiting labored breathing or after 19 days. Lungs
were fixed in 10% buffered formalin, and a dissecting microscope was
used to examine lungs for evidence of metastasis.
Intravital Video Microscopy.
This assay was performed as described by Al-Mehdi et al.
(18)
. Briefly, mice were sacrificed by an i.p. overdose of
sodium pentobarbitol at time points ranging from 30 min to 24 h
after injection with cells expressing GFP. The chest was opened, and
pulmonary circulation was cleared of blood by gravity flow of perfusate
through a cannula inserted in the main pulmonary artery, exiting from
the transected left ventricle. The perfusate was Krebs-Ringer
bicarbonate solution [118.45 mM NaCl, 4.74
mM KCl, 1.17 mM
MgSO4.7H2O, 1.27
mM
CaCl2.2H2O, 1.18
mM
KH2PO4, 24.87
mM NaHCO3 (pH 7.4), 10
mM glucose, and 5% dextran]. To visualize lung
vasculature, the lungs were infused with DiI-acetylated low-density
lipoprotein (Molecular Probes, Eugene, OR). The lungs were removed and
examined under an inverted fluorescence microscope.
Fluorescent Microscopy.
Ten-micrometer sections were cut from mouse lung frozen in Histo Prep
(Fisher Scientific, Fair Lawn, NJ). The sections were fixed in 2%
paraformaldehyde and stained with 2.5 µg/ml DAPI (Sigma Chemical Co.,
St. Louis, MO). Green fluorescent cells were confirmed by overlay with
a DAPI-stained nucleus.
Immunohistochemistry.
Ten-micrometer sections were cut from mouse lung frozen in Histo Prep
(Fisher Scientific). The sections were fixed in 2% paraformaldehyde
and postfixed in 2:1 ethanol:acetic acid solution. A Tris NaCl buffer
[1 M Tris, 140 M NaCl, and 0.1% Tween 20 (pH7.6)] was used as
rinsing buffer, and the sections were blocked with 5% goat serum. The
primary antibody, polyclonal rabbit anti-GFP antibody, was obtained
from the University of Alberta, Calgary, Canada (Ref. 31
;
1:1500; overnight at 4°C). The alkaline phosphatase-antirabbit IgG
complex was from PharMingen (San Diego, CA; 1:100; 1 h at room
temperature). Levamisole (10 mg/ml; Sigma Chemical Co.) blocked any
endogenous alkaline phosphatase. The chromagen used to visualize the
reaction was Stable Fast Red/Naphthol Phosphate (Research Genetics,
Huntsville, AL), and the sections were counterstained with aqueous
hematoxylin.
TUNEL Staining.
Female nu/nu mice, 4–8 weeks of age, were given injections
with a single cell suspension of 2 x 106 cells in 100 µl into the tail vein. After
24 or 48 h, the mice were sacrificed and the lungs were frozen in
Histo Prep. Ten-micrometer frozen sections were made, and apoptosis was
identified using the ApopTag kit (Intergen, Gaithersburg, MD) according
to the manufacturer’s protocol. Briefly, sections were quenched in 2%
hydrogen peroxide. The optimal dilution and incubation with the TdT
enzyme was 1:54 for 1.5 h at 37°C. We used an antidigoxigenin
antibody from Boehringer Manheim (Indianapolis, IN; 1:1000; 1 h at
room temperature) in place of the antibody in the kit. The reaction was
visualized by diaminobenzidine tetrahydrochloride (Vector Laboratories,
Burlingame, CA) as the chromogen, followed by a methyl green
counterstain. Spleen sections were used as a positive control with each
preparation. The number of positive cells was determined by light
microscopy at x400 magnification. The slides were scanned using a
digital micrometer (Microcode II; Boeckeler Instruments, Tucson, AZ) to
ensure that all areas were counted only once. To determine the area of
the histological sections, the slides were digitally scanned and the
area of each section was calculated using Openlab software
(Improvision, Coventry, United Kingdom).
Double-labeled apoptosis and anti-GFP sections were first stained with anti-GFP and visualized with Fast Red as described above, rinsed in water, and then stained with the ApopTag kit and visualized by diaminobenzidine tetrahydrochloride, as described above. The double-labeled sections were counterstained with aqueous hemotoxylin.
Immunoblotting.
Western blotting was as described by Maniatis et al.
(32)
. Briefly, 106 cells were plated
in 10-cm tissue culture dishes. The next day, they were lysed with 200
µl of sample buffer [10% glycerol, 2% SDS, 100
mM DTT, and 50 mM Tris (pH
6.8)]. Protein samples were denatured by boiling for 5 min and run on
a 12% SDS-polyacrylamide gel. After transfer onto nitrocellulose
membrane (Life Technologies, Inc.), the membrane was blocked overnight
in 5% milk-PBS. Bcl-2 was detected using a mouse antihuman Bcl-2
monoclonal antibody (Calbiochem, San Diego, CA) at a dilution of 1:200
and a 1:5000 dilution of secondary antibody, goat antimouse IgG
horseradish peroxidase (Boehringer Mannheim). Bands were visualized
using the enhanced chemiluminescence kit (Amersham Pharmacia,
Piscataway, NJ).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Counterstaining with DAPI revealed the intact nuclei of
the tumor cells surrounded by the nuclei of the cells of the lung (Fig. 1B)
. We noted that many of the green fluorescent cells
appeared to have ragged edges, often with blebbing of the surface, and
that the intensity of the fluorescence appeared diminished (Fig. 1C)
. The nuclei of these cells resembled apoptotic bodies
(Fig. 1D)
. HT1080-GFP cells showed a similar morphology
after i.v. injection (data not shown; Ref. 18
).
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. To show that the apoptotic cells were tumor cells, we
used immunohistochemistry with an antibody to GFP to identify the tumor
cells. The anti-GFP antibody was visualized using alkaline
phosphatase-coupled anti IgG as a secondary antibody. The detection
reaction for the alkaline phosphatase gives rise to a red
color. GFP-positive cells could readily be detected in the frozen
sections from the lungs that had been injected with tumor cells, but
not in the uninjected lungs (Fig. 2B)
. Double-staining
revealed that many of the TUNEL-positive cells also contained GFP,
confirming that apoptotic cells within the lung were, indeed, tumor
cells (Fig. 2C)
. In similar experiments, other metastatic
cell lines, including HT1080 and the murine melanoma B16F10, also gave
rise to apoptotic cells within 24 h of i.v. injection into nude
mice (data not shown).
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. These cells proved to be resistant to apoptosis induced by radiation
(Table 1)
. We then asked whether the number of TUNEL-positive cells in the lungs
differed after the injection of the parental cells compared with
injection with cells overexpressing Bcl-2. We counted the number of
apoptotic cells in sections from the lungs harvested 24 or 48 h
after i.v. injection of each cell type and computed the area of the
sections counted (Table 2)
. Nine- to 11-fold fewer apoptotic cells were evident in the lungs of
mice that received the cells overexpressing Bcl-2 at 24 or 48 h
(P = 0.005). Thus, Bcl-2 protected cells from
apoptosis in the lungs following i.v. injection. The number of
macroscopic lung colonies formed, on average, was 5-fold greater
(P 
0.001) after injection of either of two
different clones of the 2.10.10 cells expressing Bcl-2 relative to the
parent line (Table 3)
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. Thus, alteration in tumorigenicity would not seem to account for
these results.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Survival in vivo in the circulation may be affected by mechanical factors. Although it has been proposed that cells are destroyed directly through sheer forces in the vasculature, it is also possible that mechanical stresses lead to apoptosis in susceptible cells (42 , 43) . The effect of the immune system is also unclear. In these experiments, young nude mice were used to reduce the influence of immune reactivity to tumor cells. Furthermore, there is no reason to expect that the immune system would respond within 24 h or that differential effects would be seen between the various isogenic cells used in these experiments.
The expression of genes known to affect apoptosis in culture, such as
p53, Bcl-2, fas, integrin 
6, or nitric oxide production affects
metastasis in experimental assays. Takaoka et al.
(44)
observed that Bcl-2 overexpression in B16 melanoma
cells strongly enhanced pulmonary metastasis, but they did not examine
the effect of Bcl-2 on apoptosis in vivo. Del Bufalo
et al. (45)
hypothesized that Bcl-2
overexpression enhances tumorgenicity and metastatic potential of
MCF7ADR cells by inducing metastasis-associated proteins. However, we
found no significant differences in tumorgenicity by cells
overexpressing Bcl-2 (Fig. 4)
. There are inverse correlations between
the extent of induction of apoptosis in culture and metastases. The
question raised by these studies is whether the signals used in culture
bear any relationship to the actual signals received in animals.
Nikiforov et al. (46)
injected a mixture of two
mouse fibroblasts, one expressing Bcl-2 and the other not expressing
Bcl-2, into the tail vein of nude mice. They observed a significant
enrichment of Bcl-2-expressing cells recovered 2 h after
incubation in the lungs. At the same time, PCR analysis of total lung
DNA revealed no change in the ratio of the injected cells. These data
suggest that the depletion of non-Bcl-2-expressing cells was due to
loss of viability rather than escape from lungs. However, Bcl-2
overexpression failed to alter the frequency of experimental metastasis
in this case (46)
. McConkey et al.
(47)
also reported that a nonmetastatic prostate carcinoma
cell line was more susceptible to apoptosis in tissue culture, when
compared with a metastatic prostate carcinoma cell line that expressed
twice as much Bcl-2. Owen-Schaub et al. (48)
similarly found that melanoma cells resistant to fas-mediated apoptosis
were more prone to metastasize, and that fas knockout mice had higher
numbers of metastasis. Xie et al. (49)
have
demonstrated that nitric oxide production results in apoptosis in
culture and that there is an inverse correlation between apoptosis in
culture and metastasis. In this study, we extend these results
examining apoptosis 24–48 h in vivo after injection and
show that Bcl-2 overexpression inhibits this early apoptosis. We also
showed that the difference in metastatic potential between two paired
cell lines correlated with the difference in apoptosis in
vivo at 24–48 h after injection. Thus, the effect is in addition
to the possible effects on cell proliferation, dormancy, and apoptosis
that will determine the rate of nodule growth.
In other studies of metastatic inefficiency, Luzzi et al.
(50)
observed only 20% cell death of B16F1 after
injection into the superior mesenteric vein. They attributed metastatic
inefficiency to the failure of dormant solitary cells to initiate
growth and failure of early micrometastases to continue growth into
macroscopic tumors (50)
. These results are significantly
different with the results obtained after injection of radiolabeled
B16F1 in that 
70–80% of radioactivity located in the liver is lost
after 24 h, suggesting extensive cell loss (21
, 22)
.
Cameron et al. (51)
observed only 25% cell
loss in the lung of mice 3 days after injection of B16F10 cells into
the inferior vena cava, but 75% cell loss after 3 days. Although this
correlates with the study by Luzzi et al. (50)
,
their observation regarding cell loss conflicts with the observations
of Fidler and Nicolson (21)
, also using B16F10 cells and
the same mouse strain, that >90% of cells are lost within 1 day of
injection (9
, 22
, 36 , 50)
.
Additional data linking apoptosis to metastasis include studies examining the effect of CD44. Overexpression of a soluble CD44 fragment in a metastatic murine mammary carcinoma cell line blocked metastasis. The cells secreting this CD44 fragment underwent considerably more apoptosis in the lungs than the parental cells at 24–48 h after injection (52) .
The ability of apoptosis to modulate long-term growth of a metastatic tumor colony may be separate from the apoptotic regulation that determines early survival (24–48 h) after attachment to the pulmonary endothelia. These studies indicate that the extent of apoptosis early after pulmonary attachment correlates with the outcome in terms of formation of lung colonies.
In several cases, genes that affect apoptosis and metastasis have been
found to affect the number of TUNEL staining cells when the cells were
grown as tumors. Kimchi’s (53, 54, 55)
laboratory isolated a
gene, DAP, that bears a death domain that can interact with
signals through fas or tumor necrosis factor and whose expression
affects the growth of lung carcinoma cells as tumors or as metastases.
Similarly, metastatic breast carcinoma cells in which 6ß1
heterodimerization is prevented grow poorly as tumors with increased
apoptosis and have decreased lung colonization (56)
. In
these cases, whether the effect is on the ability of the cells to grow
as a tumor or whether survival in the circulation is also affected has
not been established. Our methods allow a distinction between survival
in the circulation and later growth as a tumor. They could be applied
to directly evaluate the effect of specific genes on survival in the
circulation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Funded by National Cancer Institute Grant
CA-46830. 
2 To whom requests for reprints should be
addressed, at University of Pennsylvania, 269a John Morgan Building,
3620 Hamilton Walk, Philadelphia, PA 19104. Phone: (215) 898-8401; Fax:
(215) 573-4243; E-mail: muschel{at}mail.med.upenn.edu
3 The abbreviations used are: GFP, green
fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole; TUNEL,
terminal deoxynucleotidyl transferase-mediated nick end labeling.
Received 11/ 5/99. Accepted 11/ 1/00.
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