Assay of Tumorigenicity in Nude Mice
Tumorigenicity is defined as the ability of viable cultured
cells to give rise to progressively growing tumor
nodules, showing viable and mitotically active cells,
in immunologically nonresponsive animals over a
limited observation period (WHO, 1987). It is,
however, no absolute property in a cell line, but will
always be defined by the assay in which it is tested. In
order to ensure reproducibility, it is therefore very
important that the cells tested are from the same batch
and that the animals used are syngenic. The only way
of testing whether a cell line has achieved all the characteristics
needed for solid tumor growth is by testing
its ability to form such tumors in vivo
. In order to rule
out the interference of the immune defense of the
recipient, testing must take place in a host organism
that will not reject the transplant. If the cells being
tested are originally from mice or rats, syngenic
animals can be used as recipients of cell inocula. Otherwise,
immune-deficient animals that are incapable of
recognizing and rejecting nonself tissue are used. The
nude mouse is ideal in this respect in that its immune
defect affects thymic development, resulting in a lack
of functional T lymphocytes in the animals (Rygaard,
1973). Therefore, a large proportion of mammalian
tumors can be transplanted to nude mice without
being rejected. If a cell line will not produce tumors in
nude mice, it will sometimes help to mix irradiated
mouse or human fibroblasts 1:1 with the tumor cell
inoculum (Wilson et al.
, 1984). The severe combined
immune deficiency (SCID) mouse, in which both T and
B lymphocyte maturation are impaired due to a deficiency
in a gene coding for recombinase, which participates
in the recombination of genes coding for T and B lymphocyte antigen receptors, can also be used
in this kind of assay (Bosma et al.
, 1983). Some reports
say that SCID mice will let some cell lines form tumors
even though they are not tumorigenic in nude mice
(Xie et al.
Testing the tumorigenicity of cells propagated and
perhaps transformed in vitro
is relevant in a number of
different contexts, such as transfection with antisense
DNA in order to decrease tumor formation (Cheng et al.
, 1996) or investigation of the role of specific genes
in increased or decreased tumorigenicity after in vitro
manipulation (Sun et al.
, 1996). In the production of
vaccines, it is important to know that the cells used for
propagating a virus are not tumorigenic in vivo
1987). The potential tumorigenic effect of different
genes can be tested by transfecting immortalized cell
lines with oncogenes and subsequently inoculating
them on nude mice (Chisholm and Symonds, 1992) or
by assaying the baseline expression of a gene in a cell
line and relating this to the tumorigenicity of the cells.
Immortalized cell lines are also used for determining
the carcinogenic effect of chemical compounds by in
vitro treatment and subsequent in vivo
testing in nude mice (Iizasa et al.
, 1993). A number of
phenotypical features are characteristic of malignantly
transformed cells. As opposed to normal cells, they are
immortalized, i.e., they can grow in culture for more
than 100 population doublings. They lose contact
inhibition, resulting in piling up of cultured cells, and
they exhibit high plating efficiency (i.e., a high number
of colonies obtained per 100 cells plated at 2-50
cells/cm2). The growth rate of such cells is normally
higher than for normal cells, and they can exhibit
chromosomal abnormalities such as aneuploidy or
heteroploidy (Shimizu et al.
, 1995). These are all
characteristics that can be observed in in vitro
experimental setups. The ability to form colonies in
soft agar is another characteristic closely connected to
the malignant phenotype. During the multistep process
of malignant transformation, cells become anchorage
independent, probably as a result of cell surface modifications
(Nicolson, 1976). This enables them to grow in
suspension or in semisolid media such as soft agar.
Testing for this characteristic is widely used to evaluate
the possible tumorigenic effect of different manipulations
performed on cells in vitro
Angiogenesis, the ability to induce blood vessel formation,
is characteristic of cancer cells. The angiogenic
potential of a cell line can be assayed by implanting
tumor cells at the edge of rabbit cornea or by incubating
it with tumor cell extracts and observing the
formation of blood vessels. Implantation into the
chorioallantroic membrane of chicken embryos followed
by observation of the formation of blood vessels
is also used for assaying angiogenesis (Folkman, 1985).
Invasiveness is another characteristic of tumor cells,
which can be assayed in in vitro
The in vivo
protocol suggested here is adapted
from the guidelines of WHO given for tumorigenicity
testing of tumor cells used for propagation of the
poliomyelitis virus used in the manufacturing of polio
vaccine (WHO, 1987).
II. MATERIALS AND
Mice used are 6- to 8-week-old Balb/cA nu/nu
females. They are from Taconic M&B, Ry, Denmark,
and are kept at our institute for 1 week before experiments
are initiated. They are fed autoclaved water and
sterilized food pellets and kept in Macrolon II cages
under SPF conditions.
All plasticware used for tissue culture and harvest
of cells is from Greiner GmbH. Tissue culture medium
is RPMI 1640 with UltraGlutamin (BioWhittaker, Cat.
No. 12-702F/U1). The trypsin-EDTA solution (Cat. No.
17-161E) and phosphate-buffered saline (PBS) without
(Cat. No. 17-516F) are also from BioWhittaker.
Syringes for injection of tumor cells are 1-ml Luer
tuberculin from Once (Cat. No. 1202) fitted with a 0.5
× 16 Terumo Neolus needle (Cat. No. NN-2516R).
For fixation of excised tissue, buffered formalin
(Lillies fixative) is prepared: 163.1 g 24.5%
formaldehyde solution, 4.5 g natriumdihydrogenphosphatdihydrate
(Merck 6345), and 8.2 g dinatriumphosphatdihydrate
(Merck 6580). Add sterile water until the
volume reaches 1000 ml.
A positive and a negative control cell line should
always be included in an assay of tumorigenicity in
order to ensure that the procedure has been carried out
correctly. HeLa cells (American Type Culture Collection
CLL-2) can be used as positive controls, and MRC-
5 cells (American Type Culture Collection CLL-171)
can be used as negative controls.
A. Preparation of Cells
- Tissue culture medium: Prepare the same cell
culture medium for each of the cell lines tested that
you would normally use for propagation in vitro. For
this experiment, we use RPMI 1640 with 50ml/500ml
of heat-inactivated calf serum (heat inactivate in a
water bath at 56°C for 30min) and penicillin/streptomycin
to a final concentration of 100 IU/ml penicillin
and 10mg/ml streptomycin.
- PBS without Ca2+ and Mg 2+
- Trypsin-EDTA solution
|FIGURE 1 (A) Subconfluent cells (colon adenocarcinoma
ready to be harvested. (B) Cells kept on ice,
ready to be injected.
(C) Subcutaneous injection of 0.2 ml
cell suspension. (D) Mouse with
(E) Measurement of tumor. (F) Autopsy after 21 days.
Mouse with and without tumor growth. (G) Axillary lymph
(arrow). (H) Organs removed for fixation in
formalin. (I) Fixed tissue
and histological preparations.
(J) Lung metastasis (objective ×).
B. Inoculation and Inspection
- Expand the cell line(s) to be tested for tumorigenicity
and the two control cell lines in culture until
an appropriate number of cells has been obtained. At
least 10 mice should be used for each cell line. Each
mouse must receive 107 cells. Harvest the cells when
they are subconfluent and thus still in the logarithmic
growth phase (Fig. 1A).
- The harvest of cells from culture flasks depends
on whether they grow in suspension or adhere to the
flask. If cells grow adherently, they can be removed
either by scraping with a cell scraper or by trypsination
for 5-10min at 37°C (in an incubator). Prior to
adding trypsin, remove culture medium and wash
cells with 10ml PBS without calcium and magnesium
in order to remove any traces of serum that will
otherwise inhibit the action of trypsin.
- After removal from the flask, centrifuge the cells
for 5 min at 200g, discard the supernatant, and resuspend
the cells in 10 ml PBS or culture medium without
antibiotics and serum. Centrifuge again at 200g for
5min. Discard supernatant and resuspend cells in
10ml PBS or medium as described earlier.
- Dilute a small sample (e.g., 0.5 ml) of the cell suspension
1:10 in PBS or medium. Dilute this solution
1:1 with 0.4% trypan blue. Let the suspension stand
for 2-5 min and then count cells in a hemocytometer.
Keep the rest of the cells on ice while preparing and
counting the sample. Calculate the number of viable cells per milliliter of the original suspension as follows:
viable cells in larges squares counted/number of large
squares counted × dilution of cells × 104.
- Adjust the concentration of the cell suspension to
be inoculated to 5 × 107/ml viable cells in serum-free
culture medium or PBS. This may include one more
step of centrifugation and resuspension in a suitable
volume. Always make sure to have an excess of cell suspension as some of it will be trapped in the syringe.
An extra 0.5 ml for 10 mice to be injected is sufficient.
- Keep cells on ice until use and be sure to
inoculate as soon as possible after the adjustment of
concentration (Fig. 1B).
C. Examination of Recipients and Tumors
- Inject 0.2 ml of the cell suspension subcutaneously in
the right lateral aspect of the thoracic wall (Fig. 1C).
- Mice should be observed daily and inspected for
tumor growth at least three times a week (Fig. 1D).
Prepare Lillies fixative (see Section II) and store at
D. Reporting of Findings
- Tumors are measured in two perpendicular
dimensions (Fig. 1E). After 21 days of observation, sacrifice
mice and autopsy immediately. Inspect the inoculation
site from the deep aspect and excise (Fig. 1F).
In order to describe the metastatic potential of the cell
line tested, excise the ipsilateral axillary lymph node
(Fig. 1G), part of the spleen, one-third of the liver,
and the inferior lobe of the left lung for histological
examination (Fig. 1H).
- Fix tissues in buffered formalin (Lillies fixative),
embed in paraffin, and section in 5-µm sections. Stain
with hematoxylin and eosin and study under a light
microscope (Fig. 1I). Histological examination enables
the investigator to see if the tumor has retained the
morphological characteristics of the primary tumor
from which the cell line was established and whether
it gives rise to metastases (Fig. 1J).
Reports of macroscopic and microscopic findings
should be given for each cell line. Macroscopic findings
include the conditions of mice during the test
period and macroscopic tumor growth, if any. The
macroscopic appearance of the tumor-inoculation site
at autopsy should also be reported. Microscopic findings
should include a description of the tumor tissue
or the inoculation site if no tumor appears. Metastases,
location, and frequency should be reported.
An example of an observation table is given in Table
I for HeLa cells used as a positive control in a tumorigenicity
assay carried out in nude Balb/cA mice.
According to the World Health Organization, the
positive control cell (HeLa) should give rise to progressively
growing tumors in 9 out of 10 mice if the assay is
carried out correctly. Furthermore, when a histopathological
examination of tumor nodules is performed,
mitotic activity must be detected in the tumor tissue.
For the cell line tested for tumorigenicity, at least 9
out of 10 mice should develop a tumor if the cell line
is to be designated tumorigenic. If only some mice in
a group develop tumors, the assay should be repeated
with the same cell dose and with an increased cell
dose, e.g., 5 × 107
. As mentioned in Section I, it is a prerequisite
that the cells used are from the same batch
and that the recipient mice are syngenic. The cell line
used as a negative control should not, of course, give
rise to tumor growth in any of the 10 animals. Some authors use a more differentiated assay, administering
cells in differentiated doses and categorizing cell
lines as high, moderate, low, or nontumorigenic
(Gurtsevitch and Lenoir, 1985).
It is of the utmost importance for the interpretation
of results that the cell lines tested be free of
mycoplasma infection, as mycoplasma can alter their
ability to form tumors (Fogh, 1973). If there is not a
routine of regular mycoplasma testing in the cell
culture laboratory, a mycoplasma test should be
performed and a negative outcome ensured before
cell lines are used in a tumorigenicity assay.
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