Tail vein assay of cancer metastasis.

Abstract

The most damaging change during cancer progression is the switch from a locally growing tumor to a metastatic killer. This switch involves numerous alterations that allow tumor cells to complete the complex series of events needed for metastasis. In considering steps required for successful metastasis, extravasation from blood vessels in target organs is regarded as a critical process. Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining of blood vessels and degrade its underlying basement membrane in order to escape into the extravascular tissue where they establish metastasis. This unit describes the most common assay applied to evaluate the metastatic potential of blood-borne tumor cells. The protocol is often called "experimental metastasis", distinct from "spontaneous metastasis", where the tumor cells are first allowed to form a primary tumor in the site of injection and then escape into lymphatic or blood circulation. Cultured tumor cells are injected into the tail vein and allowed to circulate. After 12 to 20 days the recipients are euthanized and the lungs are evaluated for the presence of metastatic tumors.

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UNIT 19.2
Tail Vein Assay of Cancer Metastasis
BASIC
PROTOCOL
The most damaging change during cancer progression is the switch from a locally growing
tumor to a metastatic killer. This switch involves numerous alterations that allow tumor
cells to complete the complex series of events needed for metastasis (UNIT 19.1). In
considering steps required for successful metastasis, extravasation from blood vessels in
target organs is regarded as a critical process. Circulating tumor cells arrested in the
capillary beds of different organs must invade the endothelial cell lining of blood vessels
and degrade its underlying basement membrane in order to escape into the extravascular
tissue where they establish metastasis. This unit describes the most common assay applied
to evaluate the metastatic potential of blood-borne tumor cells. The protocol is often called
“experimental metastasis”, distinct from “spontaneous metastasis”, where the tumor cells
are first allowed to form a primary tumor in the site of injection and then escape into
lymphatic or blood circulation. The Basic Protocol is to apply intravenous injection as a
method for introducing tumor cells into circulation. For this purpose, cells maintained in
tissue culture are dissociated into a single cell suspension, counted, and injected into the
tail vein of mice or rats. After 12 to 20 days, the animals are euthanized and their lungs
are removed, rinsed, and fixed. The number of pulmonary tumor colonies is then counted
with the aid of a dissecting microscope. When an organ containing extensive secondary
foci is seen, it is also removed and fixed for subsequent enumeration of tumor foci.
NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic techniques should be used accordingly.
NOTE: All culture incubations should be performed in a humidified 37°C, 7% CO2
incubator unless otherwise specified. Some media (e.g., DMEM) may require altered
levels of CO2 to maintain a pH 7.4.
NOTE: All protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee (IACUC) or must conform to governmental
regulations regarding the care and use of laboratory animals.
Materials
Cells of interest growing in tissue culture (e.g., B16 mouse melanoma,
ATCC#CRL-1619; 3LL Lewis lung carcinoma; A375 human melanoma,
ATCC#CRL-6322; 13762 MAT rat mammary adenocarcinoma)
Supplemented high-glucose DMEM-10 (UNIT 10.4)
Trypsin/EDTA solution (UNIT 10.4)
CMF-DPBS: divalent cation-free Dulbecco’s PBS (CMF-DPBS; Life
Technologies; APPENDIX 2A)
Mice for injection (or rats)
70% ethanol
3.8% (v/v) formaldehyde
Bouin’s solution (see recipe; optional)
6-cm tissue culture dishes
15-ml polypropylene tubes
Sorvall RT6000D centrifuge and H-1000B rotor or equivalent
Beaker containing warm water (∼50° to 60°C)
Lamp with 150-W light bulb (optional)
Restraining device (restrainer)
1-ml syringe, tuberculin type
27-G, 3⁄4-in. needles
Dissecting microscope
Supplement 12
Contributed by Michael Elkin and Israel Vlodavsky
Current Protocols in Cell Biology (2001) 19.2.1-19.2.7
Copyright © 2001 by John Wiley & Sons, Inc.
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Additional reagents and equipment for counting cells with hemacytometer (UNIT 1.1)
Prepare cells
1. Culture a sufficient number of cells in supplemented high-glucose DMEM-10 in 6-cm
tissue culture dishes in a humidified 37°C, 7% CO2 incubator.
Other size dishes (i.e., 35-mm, 10-cm) can be used.
2. Aspirate medium from subconfluent cultures (0.5–1 ×105 cells/cm2), add 2 ml
trypsin/EDTA solution per 6-cm tissue culture dish, and incubate 2 to 3 min at 37°C,
or until the cells have rounded up, but are not detached.
3. Aspirate trypsin/EDTA solution, resuspend cells in 5 ml warm supplemented high-
glucose DMEM-10, transfer into a 15-ml polypropylene tube, centrifuge 7 min at
∼350 ×g (1000 rpm Sorvall RT6000D centrifuge, H-1000B rotor, or equivalent), room
temperature, and resuspend in 5 ml warm CMF-DPBS.
Trypsin/EDTA solution is commonly used to detach adherent cells. Its action is terminated
by resuspending the cells in supplemented high-glucose DMEM-10. Prolonged (and
unnecessary) trypsinization of tumor cells can alter their survival and metastatic behavior
in vivo. Trypsinization >3 min may affect lung colony formation and should be avoided.
Serum-free DMEM can be used instead of CMF-DPBS.
4. Count cells on a hemacytometer (UNIT 1.1), and prepare working cell suspension at 2.5
× 105 to 1 × 106 cells/ml, depending on the metastatic potential of the cells (e.g., 2.5
× 105 B16F10 or A375SM cells/ml; 1 × 106 A375 cells/ml).
It is important to guard against clumping or aggregation of cells. The number of lung
colonies is affected not only by the number of tumor cells injected, but also by the
aggregation of cells and the tumor embolus size. To avoid clumping of cells, the cell
preparations must be free of serum, and cells should be injected in a divalent cation- free
balanced salt solution, which also serves to decrease clumping.
Obviously, the trypsinization step is not applied when nonadherent cells (e.g., mouse Eb
T-lymphoma) are tested. Cells growing in suspension are washed in CMF-DPBS, or
serum-free medium, counted, and suspended at the desired cell density.
Intravenously inject tumor cells
5. Dilate the lateral tail veins by immersing the tail for 1 to 2 min in warm (50° to 60°C)
water.
The tail vein is dilated to facilitate the insertion of a 26- or 27-G needle. Alternatively,
animals housed in a well-ventilated cage are placed for ∼5 min under a lamp with a 150-W
light bulb. The distance of the lamp from the cage can vary, but 4 to 8 in. is sufficient.
6. Place the mouse in a restrainer by grasping the mouse by the tail out of the cage and
placing with tail protruding through the opening in the wall of a restraining device.
7. Fill 1-ml syringe (tuberculin type) with the cell suspension and remove air bubbles.
8. Hold the distal third part of the tail between thumb and middle finger, wipe clean
with 70% ethanol, and rotate to position the lateral vein. Stabilize the vein with the
index finger. Apply slight pressure to straighten the tail and further dilate the lateral
vein.
9. Hold the 1-ml syringe with a 27-G, 3⁄4-in. needle in the other hand. Direct the bevel
of the needle upward. Force the needle gently through the skin (at a slight angle) and
then immediately thread it almost parallel into the vein. When the vein is canulated,
advance the needle into the lumen an additional 5 mm and slowly inject 0.2 to 0.3 ml
of the cell suspension.
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No significant pressure should be applied at this stage. If the needle is not in the vein, bleb
formation and tissue resistance to the inoculum can easily be felt, indicating failure to
canulate the vein. Perform the first injection at the distal third of the tail. In case of failure,
additional attempts may be made proximally in the same or opposite lateral vein. Intrave-
nous injection of anaesthetized mice, or of mice that struggle violently should be avoided.
Tail vein injection is delicate and requires pretraining, usually, 20 to 30 mice may suffice.
Locating the lateral vein is relatively difficult with C57BL mice compared to Balb/c mice.
Enumerate pulmonary tumor colonies
10a. After 2 to 3 weeks post-injection, euthanize mice (or rats) by CO2 asphyxiation or
cervical dislocation (or other approved method), remove the lungs, rinse in tap water,
fix in 3.8% formaldehyde, and count colonies with the aid of a dissecting micro-
scope.
An illuminated colony–counting magnifying lens can be used instead of a dissecting
microscope.
When an organ containing extensive secondary foci is observed, it is also removed, rinsed,
and fixed for subsequent enumeration of tumor foci. Most pulmonary tumor colonies in
mice are found near or on the surface of the lungs. Therefore, pigmented colonies that differ
in color from the lung parenchyma (such as melanoma) can be counted when adequately
magnified.
Tumor colonies that are not pigmented and do not differ in color from the pulmonary
parenchyma are more difficult to enumerate. To induce contrast between tumor colonies
and the lung parenchyma perform step 10b.
10b. Optional: Remove lungs (or other organs) and rinse in tap water. A few minutes
later, place the lungs in a beaker containing Bouin’s solution. After 24 hr, rinse the
lungs in water to remove excess Bouin’s solution and count the tumor colonies with
the aid of a dissecting microscope.
The lung parenchyma will immediately turn yellow. After 24 hr, the white tumor colonies
can readily be distinguished from the yellow lung parenchyma.
11. Specify the actual number of pulmonary colonies counted for each mouse, or show
the range of counts by indicating the lowest and highest numbers of colonies observed
within a given experimental group. Present the average number of nodules per mouse
± standard error or standard deviation, using the two-tailed student’s t-test. Murine
lungs with >300 colonies are reported as such.
Instead of actual counting of pulmonary colonies, one can remove and weigh the lungs of
each mouse and express the results as average lung weight ± standard deviation. Normal
lung weight is ∼200 mg.
REAGENTS AND SOLUTIONS
Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Bouin’s solution
0.9% (v/v) picric acid
9% (v/v) formaldehyde
5% (v/v) acetic acid
Store up to 1 year at room temperature
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COMMENTARY
Background Information
Metastasis is the foremost cause of death
from cancer. Fortunately, metastasis is an ex-
tremely inefficient process, with few of the
many cells shed from a primary tumor success-
fully forming secondary tumors (Hart and
Fidler, 1980; Liotta, 1986; Fidler and Ellis,
1994; Ruoslahti, 1996). Sequential steps in the
metastatic process are as follows: escape of
cells from the primary tumor, entry of cells into
lymphatic or blood circulation (intravasation),
survival and transport in circulation, escape of
cells from circulation (extravasation), and
growth of cells to form secondary tumors in a
new organ environment (Hart and Fidler, 1980;
Liotta, 1986; Fidler and Ellis, 1994; Ruoslahti,
1996). The majority of cells that successfully
escape from a primary tumor will not complete
all of the steps necessary to give rise to metas-
tatic tumors. Steps that have been considered
to be major contributors to this inefficiency, and
thus, rate limiting for metastasis, include cell
survival in, and escape from, circulation (Hart
and Fidler, 1980; Liotta, 1986; Fidler and Ellis,
1994; Ruoslahti, 1996). Once in circulation, the
tumor cells often attach at or near junctions
between adjacent endothelial cells, followed by
retraction of the endothelial cells borders, and
migration toward the exposed underlying base-
ment membrane (BM). The invading tumor
cells then degrade the subendothelial BM in
order to migrate out of the vascular compart-
ment. These steps are collectively addressed by
the tail vein assay, commonly applied in vivo
as a route for introducing cells into blood cir-
culation (Fidler, 1973a, b).
Experimental metastasis, measured by the
tail vein assay, is inhibited by molecules (i.e.,
RGD-containing peptides, laminin-derived
peptides, sulfated polysaccharides) and anti-
bodies which inhibit cell-cell and cell-matrix
interactions (Humphries et al., 1986; Iwamoto
et al., 1987). Because basement membranes
underlying epithelial and endothelial cells ap-
pear to be clear barriers to metastasis, enzymes
degrading BM proteins and glycosaminogly-
cans are regarded as promising targets for can-
cer therapy (Stetler-Stevenson et al., 1993;
Finkel, 1999; Vlodavsky et al., 1999). Invasion
through a reconstituted basement membrane
(Matrigel; UNIT 10.2) is most often used to model
extravasation (UNIT 12.2). Cell invasiveness in
vitro correlates with the metastatic potential
(i.e., lung colonization) of intravenously in-
jected cells. Moreover, inhibitors of ECM de-
grading enzymes inhibit both BM invasion in
vitro and experimental metastasis in vivo, fur-
ther emphasizing the significance of the BM as
a prominent physical barrier to metastasis (Par-
ish et al., 1987; Stetler-Stevenson et al., 1993;
Vlodavsky et al., 1994). Some of these mole-
cules are being examined in cancer patients
(Kohn and Liotta, 1995). Taking into account
the complexity of the metastatic process, it is
now understood that the mechanistic conclu-
sions on the significance of a certain step and/or
action of a given molecule cannot be drawn
based on the endpoint outcome of the tail vein
assay (i.e., number of metastases counted at the
end of the experiment; Chambers and Ma-
trisian, 1997). A procedure, intravital videomi-
croscopy (IVVM), for direct in vivo observa-
tion of early steps in metastasis, has recently
provided evidence that some of the assump-
tions about mechanisms of metastasis need to
be revised (Chambers and Matrisian, 1997;
Al-Mehdi et al., 2000; Cameron et al., 2000).
Unlike early studies, direct IVVM observation
of intravenously injected cancer cells revealed
that a large proportion of the injected cells not
only survive injection and arrest in a target
organ, but also succeed in extravasation (Cham-
bers and Matrisian, 1997; Al-Mehdi et al.,
2000; Cameron et al., 2000). It was suggested
that, unlike previous assumptions, extravasa-
tion may be a relatively easy process and that
metastatic inefficiency depends, to a large ex-
tent, on the inhibition of growth in a subset of
extravasated cells (Cameron et al., 2000).
Availability of a favorable microenvironment
in the target organ is critical for seeding and
proliferation of the disseminated cells (“seed
and soil” theory; Killion et al., 1998).
Previous research has concentrated on the
contribution of individual genes to metastasis.
Initially, there was hope that a single metasta-
sis-specific gene could be identified to be re-
sponsible for conversion to a metastatic pheno-
type. Although some cells could be converted
to a metastatic phenotype by DNA transfection
of certain genes, including those encoding
ECM-degrading enzymes (Stetler-Stevenson et
al., 1993; Vlodavsky et al., 1999), there is no
single master “metastasis” gene that regulates
cellular abilities necessary for cancer metasta-
sis (Chambers and Matrisian, 1997). Gene-ex-
pression profiling, using high-density DNA mi-
croassays combined with the tail vein assay, led
to the identification of several genes involved
in ECM assembly and in regulation of the
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actin-based cytoskeleton that are selectively
upregulated in metastatic mouse and human
melanoma cells (Clark et al., 2000). A particu-
lar significance was ascribed to RhoC, which
may regulate metastasis by controlling cy-
toskeletal events essential for cell motility in
response to extracellular factors (Clark et al.,
2000).
Critical Parameters and
Troubleshooting
General considerations for metastasis. The
pathogenesis of metastasis begins with the in-
vasion of tissues, blood vessels, and/or lym-
phatics by cells originating from a primary
cancer. Following their release into circulation,
most tumor emboli are initially arrested in the
lung tissue, simply because the lung capillary
bed is the first to be encountered (Fidler, 1973a;
Hart and Fidler, 1980), although some cells
recirculate and are trapped in other organs. It is
because of this that the majority of intrave-
nously injected cells extravasate and form me-
tastatic colonies in the lungs, as in fact evalu-
ated by the tail vein assay described in this unit.
It should be noted that this assay, also known
as “experimental metastasis”, is restricted to
evaluate the metastatic potential of circulating
blood-borne cells. In contrast, the more com-
plete cascade can be studied using the “sponta-
neous metastasis” assay in which the tumor
cells are injected subcutaneously to first form
a primary tumor. The tumor is either allowed
to grow (Vlodavsky et al., 1999), or is resected
when it reaches a certain size (Fitzer-Attas et
al., 1997). The latter protocol better mimics the
situation in patients who undergo resection of
their primary tumor, a procedure which is often
associated with stimulated vascularization and
growth of already spread metastatic nodules
(O’Reilly et al., 1994). Briefly, for measure-
ments of spontaneous metastasis, mice are most
often inoculated in the footpad with 2 × 105
cells per mouse. Local tumors are measured
with calipers. When individual tumors reach a
diameter of 8 mm, tumor-bearing legs are am-
putated (Fitzer-Attas et al., 1997). Two to 3
weeks post-amputation, the mice are euthan-
ized and the number of pulmonary colonies or
lung weights are determined as described for
intravenously injected tumor cells (Fitzer-Attas
et al., 1997).
Single cell suspension. A major problem
likely to be encountered with the tail vein assay
is the broad variation in the number of pulmo-
nary colonies obtained among mice of the same
group. In some experiments, these may vary
between 10 and 100 nodules per mouse. This
variation may primarily be due to inappropriate
standardization of the cell suspension. Every
effort should be made to obtain a single-cell
suspension since cell clumping and aggrega-
tion often result in an increased number of lung
colonies (Fidler, 1973b). Clearly, cell aggrega-
tion and clump (embolus) size influence the
outcome of experimental metastasis assays. For
example, mice injected with 10,000 clumps of
4 to 5 cells will develop more pulmonary foci
than mice injected with 50,000 single tumor
cells (Fidler, 1973b). The significance of cell
aggregation is also demonstrated by the greatly
increased number of tumor foci obtained when
the tumor cells are injected simultaneously with
lethally irradiated tumor cells, or with live
syngeneic embryonic cells (Fidler, 1973b; Hart
and Fidler, 1980). To avoid clumping, trypsini-
zation and centrifugation of the cultured cells
should be gentle and the dissociated cells
should be suspended in serum-free, Ca2+/Mg2+-
free balanced salt solution. Routine viability
tests (e.g., trypan blue exclusion) and even-
plating efficiency in vitro do not necessarily
predict or correlate with the in vivo metastatic
potential of over-trypsinized cells (Fidler,
1973b). Also, the 15-ml tube, as well as the
syringe containing the cell suspension should
be gently rotated from time to time and not be
left standing for long periods of time (i.e., >30
min) to prevent the cells from settling and
aggregating at the bottom of the tube or in the
syringe. The cells should be pipetted gently
prior to being taken up into the syringe for
injection. Failure to obtain a single-cell suspen-
sion will result in a diverse and broad range of
colony number among mice of the same experi-
mental group, and in differences in colony size
in the same mouse.
Cultures. An important parameter is the
health of the cells. Cultures should be actively
growing, but should be passaged at least 48 hr
prior to the day of the tail vein assay. Also, the
use of early passaged cultures (<20) is recom-
mended. Relatively poor metastatic potential is
often observed with late-passage cells. If the
cells tend to loose their metastatic ability, it is
advisable to go back and select for highly me-
tastatic cells by means of a repeated isolation
of pulmonary metastases and subsequent
growth in culture, according to the scheme first
described by Fidler (1973a) and applied by
many other groups (Clark et al., 2000). Briefly,
to select for highly metastatic tumor cells, pul-
monary metastatic nodules are removed asep-
tically, minced, grown in vitro, and re-injected
Current Protocols in Cell BiologySupplement 12
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