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§
§
From the Departments of Medical Biophysics,*
Microbiology and Immunology,
and
Oncology,§
University of Western Ontario,
London Regional Cancer Centre,
London,
Ontario, Canada
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our goal in the present study was to investigate the multistep nature of metastatic inefficiency in a mouse liver model, by quantifying what proportions of B16F1 melanoma cells injected intraportally 1) survive and extravasate, 2) divide and form micrometastases, 3) develop into macroscopic tumors, and 4) remain as solitary dormant cells. We used in vivo videomicroscopy12,13 to observe individual cells directly and quantify cell extravasation, a novel cell accounting assay to quantify the survival of injected cancer cells,10 and immunohistochemistry to assess proliferation (Ki-67) and apoptosis (TUNEL). We found that >80% of the injected cells survived and had extravasated by day 3. However, few extravasated cells began to grow, with only 1 in 40 forming micrometastases (4 to 16 cells) by day 3. Furthermore, few micrometastases continued to grow, with only 1 in 100 progressing to form macroscopic tumors by day 13; in fact, by then most micrometastases had disappeared. Surprisingly, 36% of injected cells remained by day 13 as solitary cancer cells, 95% of which were shown to be dormant; in contrast, within macroscopic tumors, only 3% of cells were dormant. Thus, in this model, metastatic inefficiency is principally determined by two distinct aspects of cell growth after extravasation: the failure of solitary cells to initiate growth and the failure of early micrometastases to continue growth into macroscopic tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B16F1 murine melanoma cells14
were maintained in
tissue culture (37°C, 5% CO2 humidified atmosphere) in
Alpha minimal essential medium plus ribonucleosides (
-plus MEM; Life
Technologies, Burlington, Ontario, Canada) supplemented with 10% fetal
calf serum (FCS; Hyclone, Logan, UT). Cells were routinely subcultured
as subconfluent monolayers every 3 days and were not kept in culture
for more than five passages.
Cells for injection were fluorescently labeled (yellow-green) using
Fluoresbrite carboxylated polystyrene nanospheres of 48 nm diameter
(Polysciences, Warrington, PA). Nanospheres were prepared in a sterile,
monodispersed suspension diluted 1:50 in Opti-MEM serum-reduced medium
(Life Technologies). Cells were labeled by replacing the -plus MEM
with the nanosphere suspension for 1 hour as described
previously.15
All cells spontaneously incorporated these
virus-sized fluorescent nanospheres into their cytoplasm and retained
them.10
Cells were harvested by trypsinization and
resuspended in -plus MEM/10% FCS to a final concentration of
1.5 x 106
cells/ml. Tests of the labeled cells showed
that the labeling procedure does not affect the plating efficiency or
growth of B16F1 cells in vitro or the metastatic behavior
in vivo. Before injection, it was determined by fluorescence
microscopy that 
95% of the cells excluded ethidium bromide,
indicating that membrane integrity was maintained.10,16
Additionally, 10.2-µm-diameter microspheres were added to the cell
suspension (~5:1 cells:microspheres) to allow for monitoring of cell
survival as described in the cell accounting procedure given below.
Experimental Metastasis Assay
Female C57Bl/6 mice (Harlan Sprague-Dawley, Indianapolis, IN) aged
6 to 8 weeks, syngeneic to B16F1 cells, were cared for in accordance
with standards of the Canadian Council on Animal Care, under an
approved protocol of the University of Western Ontario Council on
Animal Care. Mice were anesthetized using a ketamine/xylazine mixture
(1.6 mg of ketamine and 0.08 mg of xylazine per 15 g of body mass)
administered by intraperitoneal injection. A suspension of 3 x
105
fluorescently labeled B16F1 cells and 6 x
104
microspheres in 0.2 ml of -plus MEM supplemented
with 10% FCS was injected into the superior mesenteric vein of each
mouse to target the liver as described.13
Buprenorphrine
analgesic (0.02 to 0.04 mg/kg) was administered subcutaneously as mice
awoke and also 18 hours after surgery.
At 13 days after injection, mice were sacrificed by CO2 asphyxiation. Livers were examined for visible surface tumors (mass, tumor number, and tumor size) and then fixed in 10% neutral buffered formalin (pH 7.6). Three of the livers were randomly chosen and grossly sectioned (~1 mm thick) using a scalpel to determine whether there were any tumors present in the interior that were not visible when examining the liver surface. The remaining livers were examined using the cell accounting procedure described below.
Intravital Videomicroscopy
The procedure for intravital videomicroscopy of mouse liver has been described previously.12,17 Briefly, mice were anesthetized with sodium pentobarbitol (60 mg/kg intraperitoneally), after which the liver was exposed and the mouse placed on a viewing platform on the stage of an inverted epifluorescence microscope (Nikon Diaphot TMD). A fiber optic light source provided oblique transillumination, resulting in high-contrast views of the liver microvasculature along with associated cells and tissues. Images were obtained using a video camera, viewed on a video monitor, and recorded on SVHS videotape. Body temperature was monitored and maintained at 37°C, and anesthesia was maintained with supplemental administration of sodium pentobarbitol as required. At the end of each experiment, the animal was killed by anesthetic overdose, and the liver was removed and fixed in 10% neutral buffered formalin.
Mice that had been injected with cancer cells, as described above for the experimental metastasis assay, were observed at either of two time points: immediately (15 to 90 minutes) after injection or 3 days later. The injected B16F1 cells were assessed as being wholly intravascular, in the process of extravasating, or extravascular. Individual cells were positively identified by their fluorescence and/or melanin content. Intravital videomicroscopy was also used for cell accounting10 (see below) in the superficial regions of the liver, as it is possible to optically slice to a depth of ~50 µm below the surface. The numbers of multicellular foci (4 to 16 cells) present at day 3 were also quantified. Although other workers have previously visualized such early micrometastases from lacZ-transfected gastric carcinoma cells using ß-galactosidase-stained liver sections,18 in the present study we were able to quantify early micrometastases in vivo, visible due to their melanin content, using intravital videomicroscopy.
Cell Accounting in Tissues
To determine the proportions of the injected cancer cells that extravasate and survive in the tissue, form micrometastases, or develop into tumors, it is necessary to express the number observed in a tissue sample relative to the number of cells originally entering that volume. We recently developed a cell accounting technique for this purpose, based on the standard method for measuring distribution of blood flow,19 and used it with in vivo videomicroscopy to determine the 24-hour survival of melanoma cells in chick embryo CAM.10 Inert microspheres (nonfluorescent) that remain trapped by size restriction within the microcirculation are injected together with the cancer cells, providing a reference standard for monitoring cell survival at various times later. In mouse liver, cancer cells injected intraportally become arrested by size restriction in periportal sinusoids,13 which have a diameter of 5.9 ± 0.87 µm (mean ± SD).20 Therefore, to ensure trapping of all of the reference microspheres in sinusoids, the microspheres should be not less than ~8.5 µm in diameter. (This size represents 3 SDs above sinusoidal mean diameter, and therefore only 1 in 1000 vessels would be expected to allow a microsphere to pass through.) We were able to obtain 10.2-µm polystyrene microspheres with a very narrow range of diameters (±0.1 µm SD; Bangs Laboratories, Fishers, IN), and using in vivo videomicroscopy we verified, by direct observation, that all microspheres entering the liver microcirculation after intraportal injection immediately became trapped in sinusoids. No microspheres were ever observed passing through the sinusoids to the hepatic venous outflow.
To confirm that the microspheres remained trapped in the liver on a long-term basis, livers from four mice at each of three different time points (90 minutes and 3 and 13 days after injection) were examined to determine whether the number of microspheres per unit volume of tissue stayed constant over time. Formalin-fixed livers were cut to 30-µm-thick sections using a Vibratome Series 1000 sectioning system (Technical Products International, St. Louis, MO). Sections were mounted on a number 1 coverglass and viewed using the microscope described above. One section from each lobe of the liver was analyzed to count the number of microspheres. The 10.2-µm nonfluorescent microspheres were readily seen due to their high refractive index as well as their distinctive spherical shape. The area, and consequently the volume, of each section was determined, allowing a microsphere density to be calculated.
For cell accounting experiments, microspheres were included in the
melanoma cell suspension to be injected into the mouse, at a
concentration of 3 x 105
microspheres/ml (~5:1
cells:microspheres). We estimate that after the injection (0.2 ml per
mouse) less than 1% of all periportal sinusoids are blocked by a
microsphere; no general disruption of blood flow occurs, as downstream
sinusoids are supplied by collateral flow. To determine the exact ratio
of cells:microspheres in the syringe before injection, a drop of the
suspension was placed on a coverglass and the numbers of cells and
microspheres observed in eight fields of view (20x objective) were
recorded. Cells remained uniformly dispersed and cell clumping was not
observed. Only those cells that maintained membrane integrity, tested
by exclusion of ethidium bromide10
(95%) were used in
calculating the cell:microsphere ratio. To quantify the percentage of
injected melanoma cells surviving in the liver, the cell:microsphere
ratio in the organ at 90 minutes and 3 and 13 days after injection was
compared with the ratio in the syringe before injection.10
The percentage cell survival was calculated as the (cell:microsphere
ratio in liver after injection)/(cell:microsphere ratio in syringe
before injection) x 100.
To determine cell survival throughout the liver, at the three time points after injection, formalin-fixed livers were cut to 30-µm-thick sections and examined as described above; a minimum of four sections per liver, transecting the whole organ at different positions, were examined. The numbers of B16F1 cells and 10.2-µm microspheres, along with micrometastases present at days 3 and 13, were recorded. Individual melanoma cells were positively identified by their fluorescence and/or melanin content; micrometastases observed at days 3 and 13 always displayed melanin. It should be noted that it is the ratio of cells/microspheres that matters, not the actual numbers of cells or microspheres. The actual numbers counted will depend on the total areas of the sections of liver obtained, and there is typically a certain amount of variability in the distribution within the liver of any cell suspension injected intraportally. However, in our cell accounting procedure, this variability is controlled for by the co-injection of the microspheres with the cells. Confirmation of the technique using thick sections was accomplished by performing cell accounting in vivo using videomicroscopy immediately after injection and at day 3. This allowed for a comparison of cell survival values determined from intact livers in vivo versus those from liver sections. Whenever multicellular foci were observed, either as micrometastases or macroscopic tumors, it was assumed that they originated from a single cell, as metastases have been shown to be clonal in origin.21-23 Thus, the percent cell survival data obtained for days 3 and 13 represent minimal values.
Immunohistochemistry
Livers from three mice that had been injected with B16F1 cells 2 weeks previously (as described above) were fixed in 10% NB formalin for 24 hours and embedded in paraffin according to standard histological procedures. Serial sections (4 µm) were cut from two blocks from each liver and stained as follows: section 1 with Harris' hematoxylin and eosin (H&E), section 2 using the TUNEL assay (TdT-mediated dUTP nick-end-labeling) to assess apoptosis, section 3 with S100 (polyclonal antibody; Dako Z311) to identify melanoma cells, and section 4 with Ki-67 (monoclonal antibody; Novocastra NCL-Ki67-MM1) to assess proliferation. First, the sections stained with S100 were examined to identify melanoma cells. These cells were then assessed in the adjacent serial sections for markers of apoptosis and proliferation. Eight such sets of serial sections were examined from each tissue block, the six blocks yielding a total of 48 sections for each stain. The percentages of melanoma cells staining positive for TUNEL or Ki-67 were determined for 1) solitary cells within the tissue and 2) cells within tumors.
TUNEL Assay (after Gavrieli et al)24
Deparaffinized sections were pretreated with proteinase K (20 µg/ml) for 15 minutes, endogenous peroxidase was inactivated using 3% hydrogen peroxide in methanol for 3 minutes, TdT (0.3 U/µl) and biotinylated dUTP were added, and the sections were incubated at 37°C for 60 minutes. Extra avidin peroxidase was applied for 30 minutes at 37°C. The sections were stained using an AEC (3-amino-9-ethylcarbazole) kit (Sigma), including a positive control that had been treated with DNAse I.
Ki-67 Staining25
Deparaffinized sections were pretreated two times in a microwave oven for antigen retrieval (3 minutes on high power and 7 minutes on low power in 10 mmol/L citrate buffer), 3% hydrogen peroxide was used to block endogenous peroxidase activity, nonspecific binding was blocked by incubating slides with normal goat serum, Ki-67 antibody was applied (1/150 dilution) overnight at room temperature, and positive staining was detected by incubating with a biotinylated secondary antibody followed by streptavidin-biotin peroxidase complex, according to the manufacturer's protocol (LSAB2 kit, Dako). Slides were counterstained with Mayer's hematoxylin. Positive and negative controls were also included.
S100 Staining26
The same protocol was followed as for Ki-67, except that the Envision kit (Dako), employing peroxidase-labeled polymer conjugated to anti-mouse and anti-rabbit immunoglobulins, replaced biotinylated secondary antibody and streptavidin peroxidase reagents. A 1/400 dilution of the primary antibody was applied for 2 hours.
Statistical Analysis
Statistical analysis was performed using SigmaStat for Windows v1.0 (Jandel Scientific, San Rafael, CA). All analyses were based on the number of mice. Differences between means were determined using the t-test when groups passed both a normality test and an equal variance test. When this was not the case, the Mann-Whitney rank sum test was used. A one-way analysis of variance was used to test whether the number of microspheres per unit volume of liver tissue changed with time. A level of P < 0.05 was regarded as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Arrest and Extravasation
The fate of melanoma cells immediately after intraportal injection
and 3 days later was studied by direct observation in vivo
using videomicroscopy. All cells became arrested by size restriction in
liver sinusoids of acinar zone 1, near the ends of terminal portal
venules. A total of 280 cells were observed in vivo during
15 to 90 minutes after injection (n = 5 mice),
at which time only 1 cell had begun the process of extravasation; all
other observed cells were wholly intravascular (Figure 1a)
. A total of 185 cells were observed
in vivo at 3 days after injection (n
= 5 mice), of which only 1 cell remained completely intravascular, 3
were in the process of extravasation, and all other cells were entirely
extravascular. The above numbers were expressed as the percentage of
observed cells and multiplied by percent cell survival (see below) to
convert the results into percentage of injected cells. Thus, at 90
minutes, 87.3% of the injected B16F1 cells remained completely within
the microvasculature, whereas by 3 days, the vast majority of injected
cells (82.1%) had extravasated into the surrounding tissue.
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We were able to express counts of cells, micrometastases, and tumors relative to the absolute number of cancer cells injected, rather than the number observed at any given time in the tissue, by including reference microspheres with the tumor cell suspension injected into the circulation. This cell accounting assay depends on all of the injected microspheres remaining trapped in the liver over the entire course of the study, and to verify this, additional analyses were performed. We determined the numbers of microspheres per cubic millimeter of tissue at 90 minutes (32.7 ± 19.5 (SD)), 3 days (58.6 ± 12.5), and 13 days (37.6 ± 14.5) after injection, from examination of 30-µm-thick sections of livers from four mice at each time point. These results show that there was no overall loss of microspheres during the experimental period (P = 0.10; analysis of variance).
The liver was sampled through its whole thickness by counting cells and
microspheres in thick sections to assess cell survival. The total
percentages of injected B16F1 cells surviving at 90 minutes, 3 days,
and 13 days were 87.4%, 83.4%, and 36.2%, respectively. (The
percentage cell survival values obtained by intravital videomicroscopy
at 90 minutes and 3 days did not differ significantly from the above
values obtained from tissue sections; P 0.31.) The
12.6% loss during the first 90 minutes was significant
(P < 0.01), but the additional 4% loss over
the next 3 days was not (P = 0.21), suggesting
that any early cancer cell loss occurred soon after injection. By day
13, cell survival was significantly lower than at the other two time
points (P < 0.01), showing that additional loss
had occurred after extravasation. Despite this loss, more than
one-third of injected cells still remained in the liver at day 13.
At 90 minutes after injection, all surviving cells were found within
periportal sinusoids as solitary cells. At both days 3 and 13, most of
the injected cells remaining were found in the extravascular tissue as
solitary cells, and very few had formed micrometastases or macroscopic
tumors (see below). The percentages of injected cells present as
solitary cells at the three time points are shown in Figure 2
; 87.4% remained at 90 minutes
(n = 5 mice; the sections used for analysis
contained a total of 6271 solitary cells and 1403 reference
microspheres), 81.4% remained at day 3 (n = 5
mice; 5570 solitary cells, 1299 microspheres), and 36.1% remained at
day 13 (n = 5 mice; 1445 solitary cells, 836
microspheres). Thus, almost 2 weeks after injection, more than
one-third of the cells still survived within the liver as solitary
extravasated cells.
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By day 3, some cells had developed into micrometastases, all
consisting of 4 to 16 cells (see example, Figure 1b
). Although the vast
majority of injected cells successfully extravasated (82.1%), only
2.04% began to replicate (Figure 3
;
n = 5 mice, 126 micrometastases). At day 13, when
36.1% of injected cells were still present in the tissue as solitary
cells, only 0.07% of injected cells were present as micrometastases
(Figure 3
; n = 5 mice, 3 micrometastases). Thus,
between days 3 and 13 the number of early micrometastases decreased by
a factor of 29. In contrast, during this same interval, the number of
solitary cells fell by only a factor of 2.3 (Figure 2)
. These ratios
indicate that the rate of loss of micrometastases was more than an
order of magnitude greater than that for solitary cells.
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; n = 8 mice; numbers of tumors per
liver were 4, 4, 5, 49, 65, 69, 72, and 151). To address whether in our
model tumors also develop in the interior of the liver, three of the
livers (randomly chosen) were grossly sectioned to ~1 mm thick using
a scalpel. Forty of these sections (two sides per section) examined
under a dissecting microscope showed only 1 tumor in the interior of
the liver (not visible by examining the liver surface)
versus 76 at the surface. This confirms that counting the
number of B16F1 tumors on the liver surface gives an accurate picture
of the number present in the whole organ. Dormancy of Solitary Cancer Cells after Extravasation
To determine whether the cancer cells remaining in liver tissue as
solitary cells 2 weeks after injection were dormant, we used
immunohistochemistry to stain serial tissue sections with (in order):
H&E, TUNEL to assess apoptosis, S100 to identify melanoma cells, and
Ki-67 to assess proliferation. When a melanoma cell was identified by
S100, the adjacent serial sections were examined to determine whether
this cell was undergoing apoptosis or proliferation. The micrographs in
Figure 4
show examples of solitary cells (and
tumors) stained with S100, TUNEL, and Ki-67. A total of 174 solitary
cancer cells were found, and of these, only 5 (3%) stained with TUNEL
and 3 (2%) with Ki-67 (Figure 5)
. Thus, 166
of these solitary cells (95%) showed no evidence of either apoptosis
or proliferation, indicating that they were dormant. The sections also
showed six small tumors, ranging in size from 0.45 x 0.15 mm to
2.8 x 1.4 mm (consisting of ~200 to 2000 cells in a section).
The proportion of cells within these tumors that stained with TUNEL was
6 ± 2.7% (mean ± SD) and for Ki-67 was 90.7 ± 6.8%
(Figure 5)
. Thus, only 3.3% of cancer cells within tumors were
dormant, ie, displayed no evidence of either apoptosis or
proliferation. This low level of dormancy for cells within tumors
stands in marked contrast to the very high level of dormancy (95%)
that was found for solitary cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our overall findings are summarized as a flow chart in Figure 6
, showing the multistep nature of metastatic
inefficiency. The majority (>80%) of injected cells survived the
initial phase within the circulation and had successfully extravasated
by day 3. Very few of these extravasated cells divided and formed
colonies; only 1 in 40 had formed micrometastases (4 to 16 cells) by
day 3. Furthermore, few of the micrometastases continued to grow; only
1 in 100 micrometastases progressed to form macroscopic tumors by day
13, whereas most micrometastases had disappeared. More than one-third
of the extravasated cells were still present in the tissue at day 13,
as solitary cancer cells, most of which were dormant. As shown in
Figure 5
, only 5% of solitary cells were undergoing either
proliferation or apoptosis, in contrast to a value of 97% for cells
within macroscopic tumors. Loss of injected cells occurred in two
phases: a rapid loss of just over 10% within the microvasculature by
90 minutes followed by a slow loss of 50% within the extravascular
tissue by 2 weeks (the 4% loss between 90 minutes and 3 days was not
statistically significant).
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Contrary to what has been generally thought, only a small percentage of the cancer cells that succeeded in extravasating went on to form tumors. Only 1 in 40 extravasated cells had formed micrometastases by day 3, and the rest remained in liver tissue as solitary cells. This indicates that a major contributor to metastatic inefficiency was failure of extravasated cells in the target organ to initiate growth. However, initiation of growth cannot fully account for metastatic inefficiency, as only 1 in 100 of the micrometastases that formed by day 3 actually went on to form macroscopic tumors. Thus, another major contributor to metastatic inefficiency was the failure of micrometastases to continue growth into macroscopic tumors.
What, then, is the fate of solitary cells and micrometastases that do not go on to form tumors? Our results show that, although a slow loss of cells occurred with time, by day 13 over one-third of injected cells remained in the liver as solitary extravasated cells, of which only 5% were undergoing either proliferation or apoptosis. Thus, we conclude that 95% of these solitary cancer cells were dormant. It is conceivable that this large pool of dormant cells had the potential to be activated at some later time, which would be consistent with clinical evidence that human malignancies can recur years after apparently successful treatment of a primary tumor.31,32 In contrast to the relatively high survival of solitary cancer cells in tissue at day 13, our results show a very low survival for early micrometastases. Only 3.5% of the micrometastases present at day 3 still remained by day 13, and 1% had developed into macroscopic tumors, whereas the remainder had disappeared. This means that the loss of micrometastases occurred at a 10-fold greater rate than the loss of solitary cells, suggesting that cancer cells that begin to divide in vivo are much more vulnerable to destruction than solitary cells in an inactive state.
Tumor dormancy is a well established concept, referring to the failure
of some small tumors (
1 to 2 mm diameter) to increase further in size
because of an absence of angiogenesis.33
Recent evidence
shows that such dormancy can arise from balanced proliferation and
apoptosis within the tumor.34
Our finding that 95% of the
large pool of solitary cancer cells in the liver at 2 weeks were
neither proliferating nor undergoing apoptosis represents an additional
concept of dormancy, applied to single tumor cells. If these cells have
the potential to be activated at a later time and commence growth, they
would be analogous to time bombs hidden within the tissue. If this
situation also applies clinically, then it will be important to learn
how to control these potentially activatable cells. Because these cells
have a low proliferative index, they would be unaffected by therapies
directed against dividing cells. Growth of tumor cells immediately
after extravasation or after a period of cellular dormancy is regulated
by a combination of factors inherent to individual cells and the
microenvironment in which they are located (eg, growth factors,
hormones, extracellular matrix).35-37
Whether the
continued growth of early micrometastases is regulated similarly is not
yet clear, but it is known that growth of larger metastases (>1 to 2
mm) depends on angiogenesis33
as well as immune
regulation.38
Our results point to the initiation and
maintenance of growth of micrometastases, as well as activation of
dormant solitary cells, as key targets against which therapeutic
strategies should be directed.
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Acknowledgements |
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Footnotes |
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Supported by National Cancer Institute of Canada Grant 8133. A. F. Chambers is a Senior Scientist of Cancer Care Ontario.
Accepted for publication June 25, 1998.
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 A. C. Ferrari, N. N. Stone, R. Kurek, E. Mulligan, R. McGregor, R. Stock, P. Unger, U. Tunn, A. Kaisary, M. Droller, et al. Molecular Load of Pathologically Occult Metastases in Pelvic Lymph Nodes Is an Independent Prognostic Marker of Biochemical Failure After Localized Prostate Cancer Treatment J. Clin. Oncol., July 1, 2006; 24(19): 3081 - 3088. [Abstract] [Full Text] [PDF]
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K. Yamauchi, M. Yang, P. Jiang, M. Xu, N. Yamamoto, H. Tsuchiya, K. Tomita, A. R. Moossa, M. Bouvet, and R. M. Hoffman Development of Real-time Subcellular Dynamic Multicolor Imaging of Cancer-Cell Trafficking in Live Mice with a Variable-Magnification Whole-Mouse Imaging System. Cancer Res., April 15, 2006; 66(8): 4208 - 4214. [Abstract] [Full Text] [PDF]
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C. Spangenberg, E. U. Lausch, T. M. Trost, D. Prawitt, A. May, R. Keppler, S. A. Fees, D. Reutzel, C. Bell, S. Schmitt, et al. ERBB2-Mediated Transcriptional Up-regulation of the {alpha}5{beta}1 Integrin Fibronectin Receptor Promotes Tumor Cell Survival Under Adverse Conditions. Cancer Res., April 1, 2006; 66(7): 3715 - 3725. [Abstract] [Full Text] [PDF]
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J. M. Lloyd, C. M. McIver, S.-A. Stephenson, P. J. Hewett, N. Rieger, and J. E. Hardingham Identification of Early-Stage Colorectal Cancer Patients at Risk of Relapse Post-Resection by Immunobead Reverse Transcription-PCR Analysis of Peritoneal Lavage Fluid for Malignant Cells Clin. Cancer Res., January 15, 2006; 12(2): 417 - 423. [Abstract] [Full Text] [PDF]
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R. J. Epstein Maintenance Therapy to Suppress Micrometastasis: The New Challenge for Adjuvant Cancer Treatment Clin. Cancer Res., August 1, 2005; 11(15): 5337 - 5341. [Abstract] [Full Text] [PDF]
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S. Goodison, J. Yuan, D. Sloan, R. Kim, C. Li, N. C. Popescu, and V. Urquidi The RhoGAP Protein DLC-1 Functions as a Metastasis Suppressor in Breast Cancer Cells Cancer Res., July 15, 2005; 65(14): 6042 - 6053. [Abstract] [Full Text] [PDF]
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M. C. P. Smith, K. E. Luker, J. R. Garbow, J. L. Prior, E. Jackson, D. Piwnica-Worms, and G. D. Luker CXCR4 Regulates Growth of Both Primary and Metastatic Breast Cancer Cancer Res., December 1, 2004; 64(23): 8604 - 8612. [Abstract] [Full Text] [PDF]
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K. Hyoudou, M. Nishikawa, Y. Umeyama, Y. Kobayashi, F. Yamashita, and M. Hashida Inhibition of Metastatic Tumor Growth in Mouse Lung by Repeated Administration of Polyethylene Glycol-Conjugated Catalase: Quantitative Analysis with Firefly Luciferase-Expressing Melanoma Cells Clin. Cancer Res., November 15, 2004; 10(22): 7685 - 7691. [Abstract] [Full Text] [PDF]
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H.-K. Yu, J.-S. Kim, H.-J. Lee, J.-H. Ahn, S.-K. Lee, S.-W. Hong, and Y. Yoon Suppression of Colorectal Cancer Liver Metastasis and Extension of Survival by Expression of Apolipoprotein(a) Kringles Cancer Res., October 1, 2004; 64(19): 7092 - 7098. [Abstract] [Full Text] [PDF]
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R. J. Ludwig, B. Boehme, M. Podda, R. Henschler, E. Jager, C. Tandi, W.-H. Boehncke, T. M. Zollner, R. Kaufmann, and J. Gille Endothelial P-Selectin as a Target of Heparin Action in Experimental Melanoma Lung Metastasis Cancer Res., April 15, 2004; 64(8): 2743 - 2750. [Abstract] [Full Text] [PDF]
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H. Seeliger, M. Guba, G. E. Koehl, A. Doenecke, M. Steinbauer, C. J. Bruns, C. Wagner, E. Frank, K.-W. Jauch, and E. K. Geissler Blockage of 2-Deoxy-D-Ribose-Induced Angiogenesis with Rapamycin Counteracts a Thymidine Phosphorylase-Based Escape Mechanism Available for Colon Cancer under 5-Fluorouracil Therapy Clin. Cancer Res., March 1, 2004; 10(5): 1843 - 1852. [Abstract] [Full Text] [PDF]
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Y. Takahashi, T. Teshima, N. Kawaguchi, Y. Hamada, S. Mori, A. Madachi, S. Ikeda, H. Mizuno, T. Ogata, K. Nojima, et al. Heavy Ion Irradiation Inhibits in Vitro Angiogenesis Even at Sublethal Dose Cancer Res., July 15, 2003; 63(14): 4253 - 4257. [Abstract] [Full Text] [PDF]
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N. Seki, Y. Hayakawa, A. D. Brooks, J. Wine, R. H. Wiltrout, H. Yagita, J. E. Tanner, M. J. Smyth, and T. J. Sayers Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Apoptosis Is an Important Endogenous Mechanism for Resistance to Liver Metastases in Murine Renal Cancer Cancer Res., January 1, 2003; 63(1): 207 - 213. [Abstract] [Full Text] [PDF]
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A. Zijlstra, R. Mellor, G. Panzarella, R. T. Aimes, J. D. Hooper, N. D. Marchenko, and J. P. Quigley A Quantitative Analysis of Rate-limiting Steps in the Metastatic Cascade Using Human-specific Real-Time Polymerase Chain Reaction Cancer Res., December 1, 2002; 62(23): 7083 - 7092. [Abstract] [Full Text] [PDF]
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G. N. Naumov, I. C. MacDonald, P. M. Weinmeister, N. Kerkvliet, K. V. Nadkarni, S. M. Wilson, V. L. Morris, A. C. Groom, and A. F. Chambers Persistence of Solitary Mammary Carcinoma Cells in a Secondary Site: A Possible Contributor to Dormancy Cancer Res., April 1, 2002; 62(7): 2162 - 2168. [Abstract] [Full Text] [PDF]
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H. J. Varghese, M. T. M. Davidson, I. C. MacDonald, S. M. Wilson, K. V. Nadkarni, A. C. Groom, and A. F. Chambers Activated Ras Regulates the Proliferation/Apoptosis Balance and Early Survival of Developing Micrometastases Cancer Res., February 1, 2002; 62(3): 887 - 891. [Abstract] [Full Text] [PDF]
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Y. J. Lee and Y. K. Song Cooperative Interaction between Interleukin 10 and Galectin-3 against Liver Ischemia-Reperfusion Injury Clin. Cancer Res., January 1, 2002; 8(1): 217 - 220. [Abstract] [Full Text] [PDF]
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T. Tokuhara, H. Hasegawa, N. Hattori, H. Ishida, T. Taki, S. Tachibana, S. Sasaki, and M. Miyake Clinical Significance of CD151 Gene Expression in Non-Small Cell Lung Cancer Clin. Cancer Res., December 1, 2001; 7(12): 4109 - 4114. [Abstract] [Full Text] [PDF]
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 Y. Ward, W. Wang, E. Woodhouse, I. Linnoila, L. Liotta, and K. Kelly Signal Pathways Which Promote Invasion and Metastasis: Critical and Distinct Contributions of Extracellular Signal-Regulated Kinase and Ral-Specific Guanine Exchange Factor Pathways Mol. Cell. Biol., September 1, 2001; 21(17): 5958 - 5969. [Abstract] [Full Text] [PDF]
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B.-K. Moon, Y. J. Lee, P. Battle, J. M. Jessup, A. Raz, and H.-R. C. Kim Galectin-3 Protects Human Breast Carcinoma Cells against Nitric Oxide-Induced Apoptosis : Implication of Galectin-3 Function during Metastasis Am. J. Pathol., September 1, 2001; 159(3): 1055 - 1060. [Abstract] [Full Text] [PDF]
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M. Guba, G. Cernaianu, G. Koehl, E. K. Geissler, K.-W. Jauch, M. Anthuber, W. Falk, and M. Steinbauer A Primary Tumor Promotes Dormancy of Solitary Tumor Cells before Inhibiting Angiogenesis Cancer Res., July 1, 2001; 61(14): 5575 - 5579. [Abstract] [Full Text] [PDF]
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C. W. Wong, A. Lee, L. Shientag, J. Yu, Y. Dong, G. Kao, A. B. Al-Mehdi, E. J. Bernhard, and R. J. Muschel Apoptosis: An Early Event in Metastatic Inefficiency Cancer Res., January 1, 2001; 61(1): 333 - 338. [Abstract] [Full Text]
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A. Bratland, K. Risberg, G. M. Mælandsmo, K. B. Gützkow, O. E. Olsen, A. Moghaddam, M.-y. Wang, C. M. Hansen, H. K. Blomhoff, J. P. Berg, et al. Expression of a Novel Factor, com1, Is Regulated by 1,25-Dihydroxyvitamin D3 in Breast Cancer Cells Cancer Res., October 1, 2000; 60(19): 5578 - 5583. [Abstract] [Full Text]
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H. H. Wang, A. R. McIntosh, B. B. Hasinoff, E. S. Rector, N. Ahmed, D. M. Nance, and F. W. Orr B16 Melanoma Cell Arrest in the Mouse Liver Induces Nitric Oxide Release and Sinusoidal Cytotoxicity: A Natural Hepatic Defense against Metastasis Cancer Res., October 1, 2000; 60(20): 5862 - 5869. [Abstract] [Full Text]
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M. D. Cameron, E. E. Schmidt, N. Kerkvliet, K. V. Nadkarni, V. L. Morris, A. C. Groom, A. F. Chambers, and I. C. MacDonald Temporal Progression of Metastasis in Lung: Cell Survival, Dormancy, and Location Dependence of Metastatic Inefficiency Cancer Res., May 1, 2000; 60(9): 2541 - 2546. [Abstract] [Full Text]
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A. H. Ree, M. M. Pacheco, M. Tvermyr, O. Fodstad, and M. M. Brentani Expression of a Novel Factor, com1, in Early Tumor Progression of Breast Cancer Clin. Cancer Res., May 1, 2000; 6(5): 1778 - 1783. [Abstract] [Full Text]
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G. Vona, A. Sabile, M. Louha, V. Sitruk, S. Romana, K. Schutze, F. Capron, D. Franco, M. Pazzagli, M. Vekemans, et al. Isolation by Size of Epithelial Tumor Cells : A New Method for the Immunomorphological and MolecularCharacterization of Circulating Tumor Cells Am. J. Pathol., January 1, 2000; 156(1): 57 - 63. [Abstract] [Full Text] [PDF]
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A. H. Ree, M. Tvermyr, O. Engebraaten, M. Rooman, O. Rosok, E. Hovig, L. A. Meza-Zepeda, O. S. Bruland, and O. Fodstad Expression of a Novel Factor in Human Breast Cancer Cells with Metastatic Potential Cancer Res., September 1, 1999; 59(18): 4675 - 4680. [Abstract] [Full Text] [PDF]
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J. E. Testa, P. C. Brooks, J.-M. Lin, and J. P. Quigley Eukaryotic Expression Cloning with an Antimetastatic Monoclonal Antibody Identifies a Tetraspanin (PETA-3/CD151) as an Effector of Human TumorCell Migration and Metastasis Cancer Res., August 1, 1999; 59(15): 3812 - 3820. [Abstract] [Full Text] [PDF]
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J. M. Jessup, P. Battle, H. Waller, K. H. Edmiston, D. B. Stolz, S. C. Watkins, J. Locker, and K. Skena Reactive Nitrogen and Oxygen Radicals Formed during Hepatic Ischemia-Reperfusion Kill Weakly Metastatic Colorectal Cancer Cells Cancer Res., April 1, 1999; 59(8): 1825 - 1829. [Abstract] [Full Text] [PDF]
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A.-M. Khatib, M. Kontogiannea, L. Fallavollita, B. Jamison, S. Meterissian, and P. Brodt Rapid Induction of Cytokine and E-Selectin Expression in the Liver in Response to Metastatic Tumor Cells Cancer Res., March 1, 1999; 59(6): 1356 - 1361. [Abstract] [Full Text] [PDF]
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G. Naumov, S. Wilson, I. MacDonald, E. Schmidt, V. Morris, A. Groom, R. Hoffman, and A. Chambers Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis J. Cell Sci., January 6, 1999; 112(12): 1835 - 1842. [Abstract] [PDF]
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) in the region
immediately downstream from the cell; sinusoids further downstream are
being supplied by collateral flow. b: Micrometastasis
(M) at the liver surface
3 days after cell injection, displaying melanin. A microsphere
(
