ArticlePDF Available

Original Article The Laboratory Opossum (Monodelphis domestica) as a Natural Mammalian Model for Human Cancer Research

Authors:

Abstract and Figures

This study established that human cancer cells (A375 melanoma, HT-29 colon cancer, PC-3p prostate cancer) that were xenografted into suckling opossums could proliferate and globally metastasize as early as 11 days after injection. Light and electron microscopic examinations (HT-29 colon cancer) determined that the cellular features exhibited by the xenogeneic human tumors grown in laboratory opossums were consistent with those observed in tumors removed from humans. The tumor induction rate, patterns of tumor growth and regression, and types of host immune responses against the xenografted tumors were influenced by injection dosages, injection sites and injection ages of suckling opossums. The results highlight the value of the opossum model as a natural in vivo system for investigating human cancer growth, metastasis and apoptosis at the cellular and molecular levels; enhancing identification of tumor associated antigens or T cell epitopes through use of humoral and cellular expression cloning techniques; elucidating mechanisms utilized by tumor cells to evade host immunosurveillance; and devising diagnostic and therapeutic methods for cancer treatment.
Human colon cancer cells xenografted into suckling opossums. A. Suckling pups at 2-w.o. (litter 4). All five pups carried s.c. tumors (arrows) induced by s.c. injection of 0.25 × 10 6 HT-29 colon cancer cells into 2-d.o. pups. B: HT-29 colon cancer established two weeks after injection of 1.0 × 10 6 cells into a 0-d.o. pup (litter 7). The s.c. tumor appeared to be well-established with no signs of regression. C: The s.c. tumor established five weeks after injection of 1.0 × 10 6 cells into a 1-d.o. neonate (litter 9). The tumor was intermixed with regressing whitish tumor streaks. D: The intramuscular (chest wall) tumor found in the same pup as in C. On gross examination, the tumor looked newly established in comparison with the regressing s.c. tumor. E. A 2-w.o. tumor established in a 3-d.o. pup (litter 11) by s.c. injection of 1.0 × 10 6 cells. There was essentially no lymphocytic or other cellular response to the 2-w.o. tumor. HE, bar = 0.2 mm. F. A 3-w.o. tumor established in a 0-d.o. pup (litter 7) by s.c. injection of 1.0 × 10 6 cells. A granulomatous response and lymphocytic infiltration were detected in this tumor sample. HE, bar = 0.2 mm. G. A 4-w.o. tumor established in a 9-d.o. pup (litter 18) by s.c. injection of 2.5 × 10 6 cells. There was a moderate inflammatory infiltrate associated with this neoplasm, which, in contrast to tumors induced by injection of neonatal pups, was primarily lymphocytic (L). Additionally, it was supported by moderate interstitial fibrosis and neovascularization. HE, bar = 0.1 mm. H. A 3-w.o. tumor that had metastasized to the meninges of a pup injected at 0 days of age with 1.0 × 10 6 cells (litter 7). Neoplastic cells were evident in the vessels of meninges. No immune response was detected. HE, bar = 0.2 mm. I. Multifocal embolic metastatic cells in the septa of lungs (arrows) of a pup of litter 13 that were necropsied at the eleventh day after injection. The window in the upper left corner shows one of the metastatic foci at a higher magnification. HE, bar = 1.0 mm.
… 
Content may be subject to copyright.
Int J Clin Exp Pathol (2009) 2, 286-299
www.ijcep.com/IJCEP 810004
Original Article
The Laboratory Opossum (Monodelphis domestica) as a
Natural Mammalian Model for Human Cancer Research
Zhiqiang Wang1, 2, Gene B. Hubbard2, Fred J. Clubb Jr4 and John L. VandeBerg2
1Department of Pathology, The Methodist Hospital, Houston, Texas, 77030; 2Department of Genetics, Southwest
Foundation for Biomedical Research, San Antonio, TX 78227; 3Department of Comparative Medicine, Southwest
Foundation for Biomedical Research, San Antonio, TX 78227 and 4Texas Heart Institute, Houston, TX 77030
Received 08 October 2008; Accepted in revision 13 October 2008; Available online 06 November 2008
Abstract: This study established that human cancer cells (A375 melanoma, HT-29 colon cancer, PC-3p prostate
cancer) that were xenografted into suckling opossums could proliferate and globally metastasize as early as 11
days after injection. Light and electron microscopic examinations (HT-29 colon cancer) determined that the
cellular features exhibited by the xenogeneic human tumors grown in laboratory opossums were consistent with
those observed in tumors removed from humans. The tumor induction rate, patterns of tumor growth and
regression, and types of host immune responses against the xenografted tumors were influenced by injection
dosages, injection sites and injection ages of suckling opossums. The results highlight the value of the opossum
model as a natural in vivo system for investigating human cancer growth, metastasis and apoptosis at the cellular
and molecular levels; enhancing identification of tumor associated antigens or T cell epitopes through use of
humoral and cellular expression cloning techniques; elucidating mechanisms utilized by tumor cells to evade host
immunosurveillance; and devising diagnostic and therapeutic methods for cancer treatment.
Keywords: animal model, human cancer, Opossum, Monodelphis domestica
Introduction
Equipped with improved knowledge and
advanced technologies, investigators are now
able to make rapid progress in cancer
immunology. With the successful devising and
application of serological expression (SEREX)
and cytotoxic T lymphocyte (CTL) based
expression techniques, many human tumor
associated antigens (TAAs) and T-cell specific
epitopes have been identified and cloned [1,
2]. The utility of these approaches, however,
has been limited by the fact that human
tissues are required to conduct the
experiments and that tumor cells can evade
host immunosurveillance. As a result, many
TAAs and T cell epitopes remain to be
identified. The potential of these technologies
has been further limited by lack of an animal
model that can complement this deficiency.
The widely used athymic nude mice are
immunoincompetent. Mice that are
immunologically suppressed as a
consequence of treatment by
immunosuppressors (e.g. cortisone,
cyclosporine A) also are inappropriate because
the xenografted cells also are suppressed by
the same drugs [3].
Another intensively investigated topic in
oncology is metastasis, which is the biggest
threat to patient survival. The most widely
used in vivo model to study metastasis is
again the athymic nude mice. However,
malignant tumors xenografted into the nude
mice rarely metastasize spontaneously [4]. In
addition, the clinical predictability of antitumor
drugs screened in the nude mice is low [5].
The best animal model to simulate human
cancer cell behavior would be receptive to
xenografts of human cancer cells in a more
natural immunological environment. Ideally,
the model would initially exhibit
immunodeficiency (to allow tumor
establishment) but would eventually acquire
immunocompetency. Another characteristic of
a good model would be the capacity to support
metastasis of human cells and preferably an
initial micrometastasis stage. Finally, an
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
optimal animal model would eventually
generate tumor specific markers and would
show a predictable translation of model
behavior into corresponding effects within
humans.
The gray short-tailed laboratory opossum,
Monodelphis domestica (family: Didelphidae),
is a small (80-120 g), docile animal which
breeds throughout the year and produces
large litters [6]. Newborn opossums are at a
stage approximating that of 14-day fetal rats
or 40-day-old human embryos. Unlike most
marsupials, Monodelphis females lack a
pouch and neonatal pups are exposed and
therefore can be easily manipulated
experimentally. Monodelphis are
predominately used as the prototype
laboratory marsupial for research on normal
developmental processes early in life (i.e.,
those that occur after birth in Monodelphis
and before birth in rats and mice), and on
experimental perturbations of those processes.
Baker et al [7] reported a developmental delay
in the maturity of the immune system of the
brushtail possum, Trichosurus vulpecula.
Although the immune system is not fully
mature until after the age of weaning, it begins
to mature rapidly after 50 days of age, which is
approximately equivalent to 14 days of age for
Monodelphis. Functionally, Monodelphis
exhibit an atypical secondary response to
particulate antigens such as sheep red blood
cells [8]. Peripheral blood lymphocytes
respond by proliferation to Concanavalin A and
other mitogens, but are stimulated weakly or
not at all by allogeneic or xenogeneic (mouse)
cells in mixed lymphocyte culture (MCL) [9, 10].
Despite the weak MLC response, which was
not due to genetic homogeneity, allogeneic tail
skin grafts were rejected promptly, suggesting
that the cellular immune response of
Monodelphis is similar to that of eutherian
mammals with the exception of a weak MLC
response [11].
We hypothesized that at early developmental
stages, the opossum’s incompetent T cell-
mediated self-recognition may 1) provide an
opportunity for xenogeneic tumors to establish,
and 2) lead to immunotolerance to xenogeneic
tumors. The use of neonatal opossums to grow
allografted and xenografted (murine)
melanoma cells was successful [12, 13]. In
this article, we report and discuss our findings
on the growth and regression patterns of
xenogeneic human tumors; the influence of
dosage, sites of injections, and age of animals
at the time of injection; the cellular features at
the light microscopic and ultrastructural levels;
the host immune responses against
xenografted tumors, as reflected by the
histopathological features of the tumors; and
the differential host immune responses
against rapidly growing colon cancer cells
versus dying colon cancer cells as a result of
mitomycin treatment.
Materials and Methods
Experimental Animals
The animals used in this study were produced
in the colony maintained at the Southwest
Foundation for Biomedical Research (SFBR),
San Antonio, Texas. All animals were
maintained and bred as described [6].
Mothers with litters older than five days were
anesthetized by conventional halothane
inhalation [14]. This method, which involves
putting the mother with litter in a beaker pre-
equilibrated with halothane, frequently results
in loss of newborn pups that release the
nipples during anesthesia, or after the mother
recovers from anesthesia and behaves in an
agitated manner. Therefore, mothers carrying
pups that were younger than five days were
anesthetized by a newly devised halothane
inhalation method [15]. By placing over the
mother’s head a 50 ml conical tube, which
was pre-equilibrated with 1 ml halothane on a
cotton ball, we were able to induce rapid
anesthesia of the mother while sparing the
babies from contact with the anesthetic.
Because opossum mothers lack a pouch,
when a mother is laid on her dorsal surface,
the neonates are fully exposed, each affixed to
a nipple. A 29-gauge needle attached to a 0.3
ml insulin syringe was used for injection of
tumor cells. After completion of the injections,
the mother was returned to the original cage.
The litters were observed weekly for survival
and tumor growth.
Pups bearing tumors were euthanized by CO2
inhalation for necropsy. Tumors and peripheral
lymph nodes, including inguinal, suprascapular
and subaxillary groups, and visceral organs
were dissected and fixed in formaldehyde for
pathology examination. Bodies of small pups
were fixed in 10% neutral buffered formalin
287 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
Table 1 Induction, growth, metastasis and regression of human tumor cells in opossums as observed by clinical inspection and pathology
A. A375 melanoma
Litter
No.
Initial litter
size
Injection
age (days)
Dose
No. of pups with tumor/total remaining no. of pups at the indicated weeks after injection (1-10)
1
2
3
4
5 6
7
8
9
10
1 12 1 0.1×106
0/9
0/9
0/7
0/7
2/7*
1/7
1/7†
0/0
2 11 0 0.25×106
0/10
10/10
10/10
6/6
6/6*
4/6
4/6**
0/0
3 9 0 0.25×106
0/5
5/5
5/5
5/5
5/5*§
2/2§
0/0
4 8 0 0.25×106
0/5
5/5
5/5
5/5
5/5§
0/0
5 10 0 0.25×106
3/7
7/7
7/7
6/6*
4/6†
0/0
6 13 2 0.25×106
0/9
7/9
7/9
7/9†
4/6†*‡
1/3*
1/3*
0/3¶
7 7 2 1.0×106
0/4
4/4
4/4
4/4
4/4*†‡
3/3*
0/3†
0/0
8 6 6 2.0×106
0/6
2/6
5/5
5/5†‡
0/4*†
0/2†
0/0
B. PC-3p prostate cancer
Litter
No.
Initial litter
size
Injection
age (days)
Dose
No. of pups with tumor/total remaining no. of pups at the indicated weeks after injection (1-10)
1
2
3
4
5 6
7
8
9
10
1 11 0 0.25×106
0/11
2/11
4/10
7/8†‡
3/5*†‡
1/3
0/3
0/3†
0/0
2 12 0 0.25×106
0/12
0/11
2/11
3/4
2/3*
0/2†
0/1
0/1
0/1
0/1
3 9 1 0.5×106
2/8
3/8
7/8
6/6
3/6*
2/6
0/6†
0/4¶
4 10 2 1.0×106
0/9
7/7
7/7
4/4
4/4†‡
2/2*
0/2
0/2
0/2†
0/1††
5 7 3 1.0×106
0/4
4/4
4/4
4/4
2/4*
2/4†
0/0
6 9 8 1.0×106
2/9
4/8†
4/7†
3/5
3/5†*
0/0
7 9 3 1.5×106
7/7
7/7
5/5
3/4*†‡
0/0
8 9 3 2.0×106
2/2
2/2†‡
0/0
9 6 3 2.0×106
0/0
10 10 7 2.0×106
3/10
10/10
10/10
10/10
10/10
10/10*
2/10
0/10¶
288 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
C. HT-29 colon cancer
Litter
No.
Initial litter
size
Injection
age (days)
Dose
No. of pups with tumor/total remaining no. of pups at the indicated weeks after injection (1-10)
1
2
3
4
5 6
7
8
9
10
1 11 0 0.25×106
0/7
0/7
0/7
0/7
1/7 1/7*
0/7
0/7
0/7†
0/0
2 9 1 0.25×106
0/9
0/8
1/8
3/8
3/8* 0/8†
0/0
3 10 2 0.25×106
0/10
0/10
2/10
2/10*§
0/7 0/7†
0/0
4 8 2 0.5×106
5/5
5/5
5/5
5/5* 3/5
0/5
0/5†
0/3†
0/0
5 11 8 0.5×106
0/11
0/11
0/10
0/10†
0/5¶
6 12 10 0.5×106
0/12
0/12
0/11
0/11†
0/6¶
7 7 0 1.0×106
4/4
4/4†‡
0/0
8 9 0 1.0×106
9/9
9/9
5/9* 5/9†
0/4
0/4†
0/0
9 7 1 1.0×106
5/5
5/5
5/5*†‡
0/0
10 10 2 1.0×106
2/3
2/3
2/3
2/3* 2/3†
0/0
11 9 3 1.0×106
4/6
4/6†
0/0
12 9 3 1.0×106
5/6
5/6
5/6
5/6*†
0/0
13 8 5 1.0×106
3/7†‡
2/6†
0/3*
0/3† 0/2†
0/0
14 8 1 2.0×106
0/0
15 8 2 2.0×106
8/8
0/0
16 9 9 2.0×106
0/9
0/8
0/8
1/8
1/8† 0/4¶
17 8 6 2.5×106
0/8
0/8
3/8†
2/5
0/5* 0/5†
0/3¶
18 9 9 2.5×106
0/8
0/8
2/8†
0/5* 0/5†
0/0
19 5 20 3×106 1/5†
3/4*
2/4
2/4
2/4* 0/4
0/4†
0/0
* Tumor regression had occurred. † Necropsy was conducted. ‡ Metastasis had occurred. § Reduction in number was due to cannibalization by agitated
mother due to light halothane anesthesia or by forced detachment of suckling pups for photographing and tumor growth monitoring. ¶ The remaining
pups were used for other research purposes. Animal died at the age of 23 months. ** Death from unknown causes of mother and litter. ††Animal is
still alive.
The dose as stated does not necessarily denote the actual dose retained by the pups because a fraction of the injected cells could have been lost by
postinjection leakage at the injection site, spontaneously or as a consequence of licking by the mother.
289 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
after their cranial, thoracic and abdominal
cavities were opened. For electron microscopic
examinations, the tumor was cut into 3 mm
slices immediately after dissection and fixed in
3% glutaradehyde at 4ºC.
All animals at the SFBR received humane care
in compliance with the "Principles of
Laboratory Animal Care," formulated by the
National Society for Medical Research, and the
"Guide for the Care and Use of Laboratory
Animals" (NIH Publication No. 85- 23, revised
1996). Prior to initiating this study, the SFBR
Institutional Animal Care and Use Committee
reviewed and approved the protocol related to
this study.
Cell Lines
The human A375 melanoma cells and PC-3p
prostate cancer cells were obtained from the
University of Texas M.D. Anderson Cancer
Center and were cultured in a humidified 5%
CO2/95% air incubator. The A375 cells were
cultured in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum,
and the PC-3p cells were cultured in Ham's
F12K medium containing 10% fetal bovine
serum, respectively.
The human HT-29 colon cancer cells used in
this study were purchased from American Type
Culture Collection (ATCC) and cultured in
McCoy's 5a medium supplemented with 10%
fetal bovine serum in a humidified 5% CO2/95%
air incubator. HT-29 cells grow as colonies in
culture. Because HT-29 cell counts using a
hemocytometer were not accurate due to cell
clumping, the number of cells injected was
estimated by assuming that HT-29 and A375
cells have the same size and comparing the
pellet sizes of the HT-29 cells and A375 cells,
which do not clump and can be accurately
counted. Because the body temperature of
adult opossums is 32.6°C, all cells were
cultured at 33°C.
For the growth inhibition experiment, the HT-
29 cells that were cultured to confluence were
treated with 1 mg/ml mitomycin (Sigma Inc.)
for 24 hr. The mitomycin-treated HT-29 cells
and untreated HT-29 cells were harvested at
the same time, and injected in the same doses
to the same animals at different sites (lower
dorsal sides). All three cell lines grow well at
33°C. The cells were cultured to confluence in
100 × 20 mm culture dishes before being
harvested for injection. Harvested cells were
washed two times with 1 X phosphate buffered
saline (PBS) and resuspended in a volume of
PBS equal to the volume of the pellets for
injection.
Results
Establishment, Growth, Metastasis and
Pathological Features of Xenografted Tumors
As shown in Table 1, 335 neonatal suckling
opossums of different ages and belonging to
37 litters were injected s.c. into the dorsal side
with different doses of human A375
melanoma cells, PC-3p prostate cancer cells,
or HT-29 colon cancer cells.
A375 melanoma
The dosage of 0.1 × 106 A375 melanoma cells
injected into 12 1-day-old (1-d.o.) pups of litter
1 (Table 1A) was inadequate to consistently
induce tumors for at least five weeks after
injection although two had observable tumors
which quickly regressed. Necropsies at week 7
showed that one pup had a 0.5 x 0.75 cm s.c.
tissue mass, but all others were negative.
When we injected 0.25 × 106 A375 cells into
51 0-2-d.o. pups belonging to five litters (litters
2-6, Table 1A), a 100% tumor-take rate was
observed among the 27 surviving pups (litters
2-5) that were injected at 0 days old. Average
tumor size was approximately 0.5 x 0.75 cm
by week 4, and the tumors started to regress
by week 5. In comparison, when the same
dose was injected into 13 2 d.o. pups (litter 6),
the tumor-take rate was 78% by week 2
(Figure 1A); by week 4, one larger tumor had
grown to 1.25 x 1.5 cm; by week 5, the tumors
started to regress (Figure 1B).
Necropsies of the six pups of litter 5 (Table 1A)
at week 6 showed that one pup had tumor
invasion into the spinal column, which caused
bony distortion of the pup’s body; three pups
exhibited regressing s.c. tumors, measuring
0.25 x 0.25 cm; two other pups were negative.
Necropsies of six tumor-bearing pups (litter 6)
at weeks 4 and 5 showed that while the 4-
week-old (4-w.o.) tumors looked newly
established, the 5-w.o. tumors showed signs of
regression, i.e., whitish dots and streaks
scattered on the surface of the tumors (Figure
1B). Upon further examination, one of the
three pups exhibited a large solid fresh tumor,
290 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
Figure 1 Human melanoma cells xenografted into suckling young opossums. A. Suckling pups at two weeks of
age (litter 6, Table 1A). Amelanocytic tumors, as pointed by arrows, were induced by s.c. injection of 0.25 × 106
A375 cells at the age of 2 days. B. Regressing xenografted human melanoma at week 5 after injection (litter 6).
Whitish dots and streaks were evident in the tumors. C. A large solid tumor located in the muscular tissues of
chest wall in a pup of litter 6. This 5-w.o. tumor appeared to have been newly established by comparison to the
regressing s.c. tumor shown in B. D. Tumor of 4-w.o. Monodelphis (litter 6). Proliferation of tumor cells was
associated with considerable cell death and mineralization. Minimal host inflammatory cells were noted.
Hematoxylin and eosin (HE), bar = 0.2 mm.
measuring 1.25 x 1.25 cm, in the left chest
wall (Figure 1C).
Microscopic examination of the six necropsied
pups of litter 6 revealed that tumor cell
proliferation associated with considerable cell
death and mineralization in the 4-w.o. tumor
(Figure 1D). Inflammatory responses, however,
were not remarkable. Findings on the 5-w.o.
tumors were similar, except for moderate host
inflammatory reactions. Metastasis was
detected in two 5-w.o. pups, one was found in
the meninges and the other was found in the
lungs. Pathologic examination of a 6-w.o.
tumor-bearing pup (litter 5) did not reveal any
metastatic foci.
After 1.0×106 cells were injected into seven 2-
d.o. pups (litter 7), four survived. Tumors,
measuring 0.25 x 0.5 cm, became observable
during week 2, and continued to grow to 0.5 x
1.0 cm during two more weeks before
regression occurred at week 5. Necropsy of
one pup at week 5 exhibited a regressing 0.5 x
0.5 cm s.c. tumor mass and exhibited
multifocal lung metastasis. Necropsies of the
other three pups that carried regressed tumors
at week 7 exhibited no positive findings.
Pups at 6 days old (litter 8) tolerated 2.0 × 106
cells. The average tumor size was 0.25 x 0.25
cm at week 3 and grew to 0.5 x 0.5 cm by
week 5. In contrast to the 4-w.o. tumor of litter
6 (see above), pathology of one pup at week 4
showed good tumor cell growth with moderate
primary neutrophilic inflammation. The lung
contained a single inflammatory neutrophilic
focus.
PC-3p Prostate Cancer
291 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
Figure 2 Human prostate cancer cells xenografted into suckling opossums. A. Suckling opossums at 3 weeks old
(litter 1, Table 1B). The tumors (arrows) were induced by s.c. injection of 0.25 × 106 PC-3p cells on the day of birth
(0-d.o.). During week 4, one pup of litter 1 (white arrow) appeared sick and was euthanized (window). Two fresh-
looking tumors exhibiting aggressive growth were observed at necropsy. B. Lung tissue with multifocal metastatic
prostate cancer from the same pup as shown in the window of A. Prominent central necrosis was associated with
some proliferative foci. HE, bar = 0.2 mm. C. Prostate cancer of a 5-w.o. pup (litter 1). Tumors cells are
proliferative and there is no indication of host rejection. HE, bar = 50 μm. D. Prostate cancer induced by s.c.
injection of 2 × 106 PC-3p cells into a 3-d.o. pup (litter 8). In contrast to the proliferative and viable 5-w.o. tumor
shown in C, this 3-w.o. tumor exhibited resorption and death of the tumor cells. HE, bar = 100 μm.
The 0-d.o. pups that were injected with
0.25×106 PC-3p cells (litters 1-2, Table 1B)
exhibited observable tumors during week 2; by
week 3, the average tumor size was 0.5 x 0.5
cm (Figure 2A). During week 4, some tumors
grew to 0.75 x 0.75 cm and one pup of litter 1
(Table 1B) appeared sick and was euthanized
(Figure 2A, pop-up window). Necropsy
exhibited two fresh-looking tumors. One
measured 0.5 x 0.5 cm. The other, which
measured 1.0 x 1.0 cm, exhibited aggressive
growth. It occupied the entire left chest wall,
which compressed the chest cavity.
Pathological examination of this pup revealed
viable neoplastic cells with local invasion into
soft tissues behind the skull and in skeletal
muscles of the forearm with extension into the
thoracic cavity, pericardial sac, paravertebral
thoracic skeletal muscle and meninges.
Metastatic tumor foci were evident throughout
the lungs. Some of these proliferative foci had
prominent central necrosis (Figure 2B). By
week 5 (litter 1), aggressive tumor growth was
still observed in a pup of litter 1, showing no
signs of host rejection (Figure 2C). Metastases
to the meninges and lungs were also detected
in this pup. Two pups died during each of week
4 and week 5 and necropsies of the three 8-
w.o. pups exhibited no positive findings (litter
1, Table 1B). One pup in litter 2 is still alive.
When 0.5 × 106 PC-3p cells were injected into
nine 1-d.o. pups (litter 3), tumors were induced
in all six surviving pups by week 4, measuring
292 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
0.5 x 0.75 cm. Tumors started to regress
during week 5. Necropsy of two pups at week
7 exhibited regressed s.c. tumor tissue streaks.
The dosage of 1 × 106 PC-3p cells resulted in
a 100% tumor-take rate when injected into 2
3-d.o. pups (litters 4-5, Table 1B), but only 50%
in the 8-d.o. pups (litter 6). The average tumor
size was 0.5 x 0.75 cm by week 4, with the
largest measuring 1.0 x 1.5 cm. Tumor
regression was observed at week 5 and after.
Necropsy of two pups of litter 4 at week 5
revealed a 1.0 x 1.5 cm s.c. tumor, which
appeared newly established; necropsy of a
third pup at week 9 exhibited a completely
regressed s.c. tumor. The fourth pup (litter 4)
died at the age of 23 months. Pathology of a 5-
w.o. pup (litter 4) revealed that the tumor
exhibited marked growth and expansion both
through the skin with ulceration and into the
deep skeletal muscle as well as the sternum,
and with penetration into the chest cavity. The
minimal inflammatory infiltrates in this tumor
were essentially all lymphocytic. Metastatic
sites were seen in both kidney and liver of this
pup. Pathology of a 6-w.o. pup in litter 5
revealed granulomatous reaction and no
viable prostate cancer cells.
Similarly, 1.5 × 106 cells injected into nine 3-
d.o. pups (litter 7) also led to a 100% tumor-
take rate. The tumor growth and regression
patterns were similar to those of litters 4-6
(Table 1B). In contrast to the proliferative 5-
w.o. tumors induced by 1.0 × 106 cells (litter
4), pathology of two of the four necropsied 5-
w.o. pups (litter 7) showed that the tumor cells
were essentially being destroyed, with only a
few viable cells seen. Metastasis to the
meninges, lungs and kidneys were detected in
these animals.
The dosage of 2 × 106 PC-3p cells was poorly
tolerated by 3-d.o. pups (litters 8-9, Table 1B):
only two of the 11 injected pups survived more
than two weeks. Necropsy of the two surviving
pups at week 3 (litter 8) revealed 0.5 x 1.0 cm
s.c. tumors that appeared to have been newly
established. However, the relatively large
tumor burden impacted the viability of tumor
cells: pathology of two 3-w.o. tumor samples
(litter 8, Table 1B) revealed resorption and
death of the tumor cells at the original
injection sites with meningeal metastasis in
one pup (Figure 2D).
The same dosage was well tolerated by 7-d.o.
pups (litter 10, Table 1B), and the tumor-take
rate was 100%. Tumors measured about 0.25
x 0.5 cm by week 2; by week 4, the average
tumor size had more than doubled, with the
largest measuring 1.5 x 1.5 cm. Tumors
started regressing remarkably during week 6;
by week 8, the tumors had become non-
observable.
HT-29 Colon Cancer
For the 30 pups (litter 1-3, Table 1C) injected
by 0.25 × 106 HT-29 cells, six pups developed
observable tumors by week 5. The tumor size
was relatively small, measuring about 0.25 ×
0.25 cm.
An increased dosage of 0.5 × 106 cells
induced tumors in all five surviving pups of
litter 4, which grew to 0.5 × 0.75 cm before
starting to shrink by week 5 (Figure 3A). This
dosage, however, did not induce tumors in 8-
10-d.o. pups (litters 5-6).
The dosage of 1.0 × 106 cells was injected into
the pups of 7 litters (litters 7-13) consisting of
59 pups of 0-5 days of age. Tumors were
analyzed at necropsy at ages 2, 3, 4, 5, 6 and
8 weeks. Necropsy of six 2-w.o. pups (litter 11)
and four 3-w.o. pups (litter 7) exhibited well-
established s.c. tumors (Figure 3B). Necropsy
of six 4-w.o. pups (litter 12) at week 4 and five
5-w.o. pups (litter 9) exhibited tumors that
were intermixed with regressing whitish tumor
streaks (Figure 3C). In comparison, necropsy
of the nine 6-8-w.o. pups (litter 8) showed
regressed tumors. It is noteworthy that, like
the A375 melanoma (Figure 1C), one 5-w.o.
pup (litter 9) carried two contrasting tumor foci:
one regressed s.c. tumor and another fresh-
looking intramuscular (chest wall) tumor,
which measured 0.5 × 1.25 cm (Figure 3D).
Histologically, 2-w.o. tumor cells were
extremely viable in appearance, and there
were no infiltrations of host inflammatory cells
(Figure 3E). At week 3, host inflammatory
responses started to appear (Figure 2F). At
week 4, pyogranulomatous formation and
necrosis of tumor cells were seen, but a few
viable tumor cells were still present. By week 5,
the pyogranulomatous responses and tumor
cell death were more remarkable. Finally, the
6-w.o. tumor was dissected into numerous
granulomas, indicating that the tumor was
undergoing resolution by the host.
293 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
Figure 3 Human colon cancer cells xenografted into suckling opossums. A. Suckling pups at 2-w.o. (litter 4). All
five pups carried s.c. tumors (arrows) induced by s.c. injection of 0.25 × 106 HT-29 colon cancer cells into 2-d.o.
pups. B: HT-29 colon cancer established two weeks after injection of 1.0 × 106 cells into a 0-d.o. pup (litter 7).
The s.c. tumor appeared to be well-established with no signs of regression. C: The s.c. tumor established five
weeks after injection of 1.0 × 106 cells into a 1-d.o. neonate (litter 9). The tumor was intermixed with regressing
whitish tumor streaks. D: The intramuscular (chest wall) tumor found in the same pup as in C. On gross
examination, the tumor looked newly established in comparison with the regressing s.c. tumor. E. A 2-w.o. tumor
established in a 3-d.o. pup (litter 11) by s.c. injection of 1.0 × 106 cells. There was essentially no lymphocytic or
other cellular response to the 2-w.o. tumor. HE, bar = 0.2 mm. F. A 3-w.o. tumor established in a 0-d.o. pup (litter
7) by s.c. injection of 1.0 × 106 cells. A granulomatous response and lymphocytic infiltration were detected in this
tumor sample. HE, bar = 0.2 mm. G. A 4-w.o. tumor established in a 9-d.o. pup (litter 18) by s.c. injection of 2.5 ×
106 cells. There was a moderate inflammatory infiltrate associated with this neoplasm, which, in contrast to
tumors induced by injection of neonatal pups, was primarily lymphocytic (L). Additionally, it was supported by
moderate interstitial fibrosis and neovascularization. HE, bar = 0.1 mm. H. A 3-w.o. tumor that had metastasized
to the meninges of a pup injected at 0 days of age with 1.0 × 106 cells (litter 7). Neoplastic cells were evident in
the vessels of meninges. No immune response was detected. HE, bar = 0.2 mm. I. Multifocal embolic metastatic
cells in the septa of lungs (arrows) of a pup of litter 13 that were necropsied at the eleventh day after injection.
The window in the upper left corner shows one of the metastatic foci at a higher magnification. HE, bar = 1.0 mm.
It is noteworthy that pathological examination
of one 4-w.o. tumor sample (litter 18, Table 1C)
induced by injection with 2 × 106 cells into a 9-
d.o. pup showed that the tumor was expansile,
invasive and had a moderate mitotic rate.
There was a moderate inflammatory infiltrate
associated with this neoplasm, which was
primarily lymphocytic (Figure 3G). Additionally,
294 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
it was supported by moderate interstitial
fibrosis and neovascularization.
To assess metastasis, systemic pathology was
carried out on four pups with 3-w.o. tumors
(litter 7), one pup carrying a 5-w.o.
intramuscular tumor in the chest wall (litter 9),
and five pups with 6-w.o. tumors (litter 8). The
results showed that 1) in the four pups of litter
7, neoplastic cells were detected within the
meninges of the brain and spinal column in
one of the four pups (Figure 3H) and no
metastasis was detected in the other three
pups; 2) meningeal adenocarcinoma was
observed in the pup carrying the intramuscular
tumor in the chest wall (litter 9); 3) no
metastasis was detected in the five pups of
litter 8. It is noteworthy that one pup of litter
13 appeared very sick by the 11th day after
injection and was euthanized. Pathological
examination showed multifocal metastatic
cells in the lungs and abdominal cavity (Figure
3I).
The dosage of 2.0 × 106 cells was excessive
for 1-2-d.o. opossums (litters 14-15), but was
well tolerated by 9-d.o. pups (litter 16). The
tumor-take rate, however, was only 13% for
this litter (1 of 8 surviving pups).
An experimental dosage of greater than 2 ×
106 cells injected into 35 pups belonging to
litters 17-19. Although only two pups of litter
17 carried observable tumors by week 3,
necropsy of three non-tumor bearing pups of
this litter showed that one was positive. In
comparison, when the same dosage was
injected into eight 9-d.o. pups (litter 18), two
pups exhibited tumors by week 4. Necropsies
of these two pups and one littermate at that
time exhibited s.c. tumors, measuring 0.25 ×
0.25 cm in one pup and 0.75 × 1.0 cm in
another. There was no positive finding in the
third pup. Necropsies of the other five pups
during week 6 were unremarkable. Tumors
started to regress by week 5 after injection.
When 3 × 106 cells were injected into five 20-
d.o. pups, no tumors were observed one week
after injection. Necropsy of one pup at this
time, however, revealed tumor induction. By
week 2, tumors were observable in three of
four remaining pups; one had regressed during
the following week; the other two persisted
until week 5 after injection.
Ultrastructural Cellular Features of
Xenografted Human Colon Cancer
To determine if the xenogeneic tumors grown
in opossums can exhibit cellular features that
are consistent with the human condition, we
studied a 3-w.o. tumor, measuring 0.75 x 0.75
cm, from a pup that was injected with 1 x 106
HT-29 cells at the age of 3 days. Using
electron microscopy, we examined a number
of ultrastructural cellular features, which
included morphology of nuclei and nucleoli,
mitochondria, junctions, lumens, microvilli,
and secretory granules.
Ultrastructural evaluation revealed neoplastic
cells that showed clusters and glandular-like
formations. Morphologically, the cells varied
from oval to columnar with large nuclei and
distinct nucleoli. Nuclei varied from round to
oval with irregular to cleaved margins. Nucleoli
were prominent and, in scattered nuclei, were
in doublets; mitoses were present.
Mitochondria were present and varied from
scattered clusters (most cells) to completely
filling the cytoplasm (rare cells) (Figure 4A).
Junctional complexes were prominent (Figure
4A). The cytoplasm of the cells contained
ribosomes, glycogen, and to lesser extent
intermediate filaments (Figure 4A). Scattered
mitotic spindles were present (Figure 4B).
Microvilli were present and tended to show
apical orientation in areas with formed lumina
(Figure 4C). These findings were consistent
with an adenocarcinoma.
Host Immune Response against Vital versus
Dying Colon Cancer Cells
Because the laboratory opossum is capable of
rejecting allografted skin tissue [11], it is
reasonable to assume that regression of the
xenografted tumors is a result of natural
rejection of foreign tissues. However, it
remains to be determined whether active
tumor growth, which leads to differential
expression of TAAs at different tumor
progression stages [16], also contributes to
rejection.
We prepared two sets of HT-29 cells: one was
treated with mitomycin and one was untreated.
Treatment with mitomycin drastically changed
the growth pattern of the HT-29 cells. Growth
of the mitomycin-treated cells was slowed, but
not stalled, because the cells were still able to
grow by forming large colonies for about one
week. Then growth of the cells stopped, and
295 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
Figure 4 Ultrastructural cellular features of xenografted 3-w.o. colon cancer cells injected with 1 x 106 HT-29 cells
at the age of 3 days. A. Mitochondria are present in scattered clusters (arrow), and junctional complexes are
prominent (square). Black arrow is pointing to intermediate filaments. B. Arrow is pointing to a mitotic spindle. C.
Microvilli are present and tend to show apical orientation in areas with formed lumen.
the cells started to detach from the culture
dishes (Figure 5A).
We injected two 30-d.o. pups and four 41-d.o.
pups with treated and untreated cells at a
dose of 5 × 106 cells. At these ages, the
inflammatory responses of the host against
the treated and non-treated xenografted cells
did not exhibit any obvious differences.
Then we injected three pups at the age of 51
days. One week after injection, we euthanized
one pup, which by inspection, showed a
reddish inflammatory sign at the site of
injection of the untreated HT-29 cells. Upon
necropsy, we found that the untreated HT-29
cells elicited a stronger inflammatory reaction
from the host than the treated HT-29 cells.
When we euthanized the other two pups four
days later, we found that one pup showed a
similar response, i.e., the untreated cells were
more inflammatory than the treated cells.
However, the other pup showed no difference
between the sites injected with treated and
untreated cells, and thus the inflammation in
this pup was self-resolving.
We proceeded to inject seven 57-d.o. pups.
One week after injection, we euthanized five
pups and found that, similar to the 51-day-old
pups, the untreated HT-29 cells elicited a
much stronger inflammatory reaction from the
host than the treated HT-29 cells (Figure 5B).
However, by the time of necropsy of the
remaining two pups at two weeks after
injection, the inflammatory responses were
resolved and no differences between the two
injection sites were detectable.
Discussion
Host tolerance to grafted foreign tissue
296 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
Figure 5 Host immune response against viable vs. dying colon cancers. A. HT-29 colon cancer cells (left) and HT-
29 cells at one week after mitomycin treatment (see text for details). Mitomycin-treated HT-29 cells were still able
to attach and form colonies. However, the growth of the mitomycin-treated cells was stalled one week after
treatment and the cells then started to detach. B. A 57-d.o. opossum injected with HT-29 colon cancer cells and
mitomycin-treated HT-29 colon cancer cells and necropsied one week later. The untreated HT-29 cells elicited
greater inflammatory reaction (right lower dorsal region; reddish inflammation can been seen) than the treated
HT-29 cells (left lower dorsal region).
remains a complicated topic in immunology. It
is well-known that down-regulated MHC-1
expression in cancer cells plays a role in the
induction of host tolerance [17], and this
characteristic of cancer cells probably
contributed to the results of this study.
However, the delayed developmental window
period, as well as other characteristics
displayed by the marsupial immune system [7,
8, 9, 10, 11], probably made a more important
contribution to the induction of tolerance in
this study. The extrapolation from results
obtained with brushtail possums [7] that
immunological incompetence is most
pronounced in the first two weeks of
Monodelphis life is consistent with results
from this study. It also is consistent with
results from other related studies, e.g., UV-
induced opossum melanoma cells allografted
to opossums less than two weeks old display a
capacity of aggressive tumor growth and
metastasis despite MHC-1 mismatch [12], and
murine B16 melanoma cells xenografted
within the first two weeks of life remain viable
through to the adulthood [13]. Studies using
non-tumor tissues also support this finding:
murine neural progenitor cells, whose MHC-1
expression is also down-regulated [18], have
been xenografted into the eyes of suckling
young opossums (5-10 days of age) and,
unlike in opossums of more advanced age
(>35 days of age), became established without
eliciting any host immunological responses [19,
20].
297 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
Despite tolerance to early grafted tumor
tissues, regression of xenografted tumor
tissues eventually took place as the
opossum’s immune system matured,
indicating the roles of aberrant expression of
TAAs during growth of cancer cells at different
stages [16, 21]. The differential host immune
responses against different tumor types (e.g.,
prominent inflammation against colon cancer,
moderate inflammation against melanoma,
and minimal inflammation against prostate
cancer), and against non-mitomycin treated
colon cancer cells vs. mitomycin treated cells,
provide evidence supporting this hypothesis.
A host immune response against cancer cells
involves the interaction of many different cell
types and cell products, including granulocytes
[22], monocytes/macrophages [23], natural
killer (NK) cells [24], dendritic cells (DCs) [25],
and cytotoxic lymphocytes (CTLs) [26]. In
addition to the obvious presence of
granulocytes, lymphocytes and macrophages
within the tumor tissues detected by light
microscopic examinations, the presence of
DCs and NKs infiltrating the xenogeneic colon
cancer was also indicated by electron
microscopic studies (unpublished data). It is
noteworthy that the HT-29 cells xenografted at
later ages could escape the host’s innate
immune attack; the tumors became
observable at a much later time after injection.
The histopathology results of these tumors
showed predominant lymphocytic infiltration,
indicating that detection and destruction of
these tumors is accomplished by a different
mechanism. Because CTLs constitute one of
the most important effector mechanisms of
antitumor immunity, the opossum model could
be the only natural mammalian model for
investigating the use of CTLs to combat human
cancer.
We do not know if the pup from litter 9 that
exhibited a regressed s.c. tumor, but had a
fresh-looking intramuscular tumor, had cells
inadvertently injected into both sites, or if the
intramuscular tumor was a metastasis. In
either case, the results suggest that s.c.
tumors exhibit different growth patterns by
comparison with intramuscular tumors
because of different distributions of immune
cells, e.g., the distribution of DCs in the s.c.
and muscular tissue [27].
It is noteworthy that the s.c. xenografted HT-29
colon cancer exhibited a benign pattern of
growth in most opossums, metastasis was
detected in the central nervous system of only
two opossum pups despite the relative large
size of the established tumors, and in the
lungs and abdominal cavity of one pup.
Nevertheless, the relatively short time period
for the HT-29 cells to spontaneously
metastasize (within two weeks after injection)
makes the opossum an ideal in vivo model for
investigating metastasis. By comparison, the
PC-3p prostate cancer cells and A375
melanoma cells demonstrated much more
aggressive growth and a much higher rate of
spontaneous metastasis: six out of seven
systemically examined prostate cancer-bearing
pups had metastases; four out of nine
melanoma-bearing pups had metastases.
These results highlight the value of the
opossum model as an in vivo model system to
investigate metastasis.
In summation, the establishment of human
cancers in Monodelphis and the
demonstration of metastatic phenotypes in a
relatively short period of time indicate that
Monodelphis will complement the murine
model as a valuable resource for cancer
related research. The distinct regression
patterns consequent to the innate immune
response and the cellular immune response of
the host establish the opossum model as a
novel natural system for tumor
immunobiological research. Further
development of this model system could lead
to identification of immunological components
in recognizing and combating cancer cells and
devising diagnostic and therapeutic methods
for cancer treatment.
Acknowledgements
We thank Donald E. Taylor, Susan Collins,
Gerardo Colon, and Ernesto Morin for
maintenance of the animals, Marie V. Silva
and Antonio Perez for anatomic pathology
support, Ralph Nichols for TEM support, and
Dr. Sen Pathak for providing the human A375
melanoma cells and PC-3p prostate cancer
cells. We also thank April W. Hopstetter for
assistance in editing and preparing the
manuscript. This work was supported by a
grant from The Robert J. Kleberg, Jr. and Helen
C. Kleberg Foundation.
Please address all correspondences to Zhiqiang
Wang, M.D., Ph.D., Department of Pathology, The
Methodist Hospital, Houston, Texas, 77030. Tel:
713-441-3490; Fax: 713-793-1630;
298 Int J Clin Exp Pathol (2009) 2, 286-299
Wang Z et al/Monodelphis domestica: a Natural Model for Human Cancer Research
299 Int J Clin Exp Pathol (2009) 2, 286-299
Email: zwang@tmhs.org
References
[1] Pfreundschuh M, Shiku H, Takahashi T, Ueda R,
Ransohoff J, Oettgen HF and Old LJ. Serological
analysis of cell surface antigens of malignant
human brain tumors. Proc Natl Acad Sci USA
1978;75:5122-5126.
[2] Knuth A, Danowski B, Oettgen HF and Old LJ. T-
cell–mediated cytotoxicity against autologous
malignant melanoma: analysis with interleukin
2-dependent T cell cultures. Proc Natl Acad Sci
USA 1984;81:3511-3515.
[3] Aramant R and Turner JE. Cross-species
grafting of embryonic mouse and grafting of
older postnatal rat retinas into the lesioned
adult rat eye: the importance of cyclosporin A
for survival. Brain Res 1988:469:303-307.
[4] Fidler IJ. Rationale and methods for the use of
nude mice to study the biology and therapy of
human cancer metastasis. Cancer Metastasis
Rev 1986;5:29-49.
[5] Kerbel RS. What is the optimal rodent model
for anti-tumor drug testing? Cancer Metastasis
Rev 1998-1999;17:301-304.
[6] VandeBerg JL. The laboratory opossum
(Monodelphis domestica). In: Poole T and
English P (eds). UFAW Handbook on the
Management of Laboratory Animal. 7th edition,
volume 1: Terrestrial Vertebrates. Blackwell
Science Ltd., Oxford, U.K. 1999; pp. 193-209.
[7] Baker ML, Gemmell E and Gemmell RT.
Ontogeny of the immune system of the
brushtail possum, Trichosurus vulpecula. Anat
Rec 1999;256:354-365.
[8] Stone WH, Bruun DA, Manis GS, Holste SB,
Hoffman ES, Spong KD and Walunas, T. The
immunobiology of the marsupial, Monodelphis
domestica. In: Stolon TC, Fletcher CJ, Bayne CJ,
Secombes JT, Zelikoff LE, Twerdok DP and
Anderson JS (eds). Modulators of Immune
Responses, the Evolutionary Trail,
Breckenridge Series 2. SOS Publications, Fair
Haven, NJ. 1996;11:149-165.
[9] Infante AJ, Samples NK, Croix DA, Redding TS,
VandeBerg JL and Stone WH. Cellular immune
response of a marsupial, Monodelphis
domestica. Dev Comp Immunol 1991;15: 189-
199.
[10] Stone WH, Brunn DA, Foster EB, Manis GS,
Hoffman ES, Saphire DG, VandeBerg JL and
Infante AJ. Absence of a significant mixed
lymphocyte reaction in a marsupial
(Monodelphis domestica). Lab Anim Sci 1998;
48:184-189.
[11] Stone WH, Manis GS, Hoffman ES, Saphire DG,
Hubbard GB and VandeBerg JL. Fate of
allogeneic skin transplantations in a marsupial
(Monodelphis domestica). Lab Anim Sci 1997;
47:283-287.
[12] Robinson ES and Dooley TP. A new allogeneic
model for metastatic melanoma. Eur J Cancer
1995;31A:2302-2308.
[13] Wang Z, Hubbard GB, Pathak S and VandeBerg
JL. In vivo opossum xenograft model for cancer
research. Cancer Res 2003;63:6121-6124.
[14] Robinson ES and VandeBerg JL. Blood
collection and surgical procedures for the
laboratory opossum (Monodelphis domestica).
Lab Anim Sci 1994;44:63-68.
[15] Wang Z and VandeBerg JL. Survival anesthetic
and injection procedures for neonatal
opossums. Contemp Top Lab Anim Sci 2003;
42:41-43.
[16] Old LJ and Chen YT. New paths in human
cancer serology. J Exp Med 1998;187: 1163-
1167.
[17] Bubenik J. MHC class I down-regulation:
tumour escape from immune surveillance? Int
J Oncol 2004;25:487-491.
[18] Dyer CA, Hickey WF and Geisert EE Jr.
Myelin/oligodendrocyte-specific protein: a
novel surface membrane protein that
associates with microtubules. J Neurosci Res
1991;28:607-613.
[19] Sakaguchi DS, Van Hoffelen SJ and Young MJ.
Differentiation and morphological integration of
neural progenitor cells transplanted into the
developing mammalian eye. Ann NY Acad Sci
2003;995:127-139.
[20] Van Hoffelen SJ, Young MJ, Shatos MA and
Sakaguchi DS. Incorporation of murine brain
progenitor cells into the developing
mammalian retina. Invest Ophthalmol Vis Sci
2003;44:426-434.
[21] Brenner PC, Rettig WJ, Sanz-Moncasi MP,
Reuter V, Aprikian A, Old LJ, Fair WR and Garin-
Chesa PT. AG-72 expression in primary,
metastatic and hormonally treated prostate
cancer as defined by monoclonal antibody
CC49. J Urol 1995;153:1575-1579.
[22] Koga Y, Matsuzaki A, Suminoe A, Hattori H and
Hara T. Neutrophil-derived TNF-related
apoptosis-inducing ligand (TRAIL): a novel
mechanism of antitumor effect by neutrophils.
Cancer Res 2004;64:1037-1043.
[23] Lesimple T, Moisan A and Toujas, L. Autologous
human macrophages and anti-tumour cell
therapy. Res Immunol 1998;149:663-671.
[24] Soloski MJ. Recognition of tumor cells by the
innate immune system. Curr Opin Immunol
2001;13:154-162.
[25] Cerundolo V, Hermans IF and Salio M.
Dendritic cells: a journey from laboratory to
clinic. Nat Immunol 2004;5:7-10.
[26] Greenberg PD. Adoptive T cell therapy of
tumors: mechanisms operative in the
recognition and elimination of tumor cells. Adv
Immunol 1991;49:281-355.
[27] Bonnotte B, Gough M, Phan V, Ahmed A, Chong
H, Martin F and Vile RG. Intradermal injection,
as opposed to subcutaneous injection,
enhances immunogenicity and suppresses
tumorigenicity of tumor cells. Cancer Res 2003;
63:2145-2149.
ResearchGate has not been able to resolve any citations for this publication.
Article
Embryonic day-15 mouse retinas were grafted into an adult rat retinal lesion site and allowed to survive for 9 and 30 days. Grafted animals received either no Cyclosporin A treatment, treatment for the first 10 days of transplantation or continuous treatment. In a second experimental set up, postnatal day-21 rat retinas were grafted under the same conditions and received either no Cyclosporin A administration, treatment during the first 24 h or continuous treatment over a 6-day survival period. Our results show that continuous Cyclosporin A treatment is necessary for successful cross-species grafting as well as to maintain for some time partly viable day-21 rat retinal grafts.
Article
The numbers and distribution of T and B cells in the thoracic thymus, spleen and intestinal tissue and the proliferation of T lymphocytes were examined during pouch life and in the adult to determine when the developing brushtail possum reaches immunological maturity. CD3-positive cells were observed in the thoracic thymus at day 2 post-partum indicating that the thymus produces T lymphocytes at or soon after birth. By day 25 the thymus was fully populated with CD3-positive T lymphocytes and they were observed in distinct regions of the cortex and medulla. By day 48 postpartum, B and T lymphocytes were identified in the follicles and parafollicular areas of the spleen. Although the numbers of T and B cells in the spleen increased significantly from day 25 to day 100 post-partum (P < 0.005), fewer cells were present at day 150 post-partum than in the adult (P < 0.05). Peyer's patches were not observed in the intestines up to day 73 postpartum. However, both T and B cells were observed in the intestinal lymph nodes. Although the T lymphocytes at weaning showed a proliferative response, the response was not as great as that observed in the adult possum. Thus, the immune system of the possum is not fully developed at weaning but continues its development after pouch life. Anat Rec 256:354-365, 1999. (C) 1999 Wiley-Liss, Inc.
Article
Sera from 30 patients with astrocytoma were tested for antibody reacting with cell surface antigens of cultured autologous astrocytoma cells. Ten percent of the patients had antibody detectable by mixed hemadsorption assays, approximately 50% by immune adherence and protein A assays, and 100% by anti-C3-mixed hemadsorption assays. Absorption analysis of reactive sera with autologous, allogeneic, and xenogeneic cells permitted the definition of three classes of astrocytoma cell surface antigens. Class I antigens showed an absolute restriction to autologous astrocytoma cells. Class II antigens were shared by all astrocytomas tested and could be detected also on neuroblastoma, sarcoma, and some (but not all) melanoma cell lines; these antigens were not found on cell lines derived from carcinomas or normal tissues. Class III antigens were widely distributed on cultured normal and malignant cells of human and animal origin. In this series, sera from 2 patients recognized class I antigens, 4 patients' serum recognized class II antigens, and 13 patients' sera recognized class III antigens. Absorption tests have shown that the AJ (class II) antigen of astrocytoma is serologically related to the previously described AH (class II) antigen of melanoma; in tests of nine melanoma cell lines, there was a correspondence between the AJ and AH phenotypes. This method of autologous typing provides a way to classify the cell surface antigens of astrocytomas and to assess the clinical significance of humoral immunity to these antigens.
Article
Marsupials are interesting subjects for studies of comparative and developmental immunology because they separated from eutherian mammals over 100 million years ago and because the newborns are still in a fetal state. We studied cellular immunity in a fully pedigreed colony of the marsupial, M. domestica (commonly called the gray short-tailed opossum). Peripheral blood lymphocytes were separated on nylon wool columns into adherent cells bearing surface immunoglobulin (B cells) and nonadherent cells (T cells) recovered in the ratio of 1:3. Peripheral blood lymphocytes responded by proliferation to Con A and other mitogens. Nonadherent cells were responsive to Con A, but adherent cells were not. Peripheral blood lymphocytes were stimulated weakly or not at all by allogeneic or xenogeneic (mouse) cells in mixed lymphocyte culture. Despite the weak MLC response, which was not due to genetic homogeneity, allogeneic and xenogeneic tail skin grafts were rejected promptly. These data suggest that the cellular immune response of M. domestica is similar to that of eutherian mammals with the notable exception of weak MLC responses.
Article
This chapter attempts to identify the principles and issues elucidated in animal models that may provide insights for the development of effective approaches to modulate T cell functions to promote the eradication of human tumors. The importance of T cells in surveillance against the outgrowth of tumor cells is probably best demonstrated with virally transformed tumors, such as EBV-induced B cell lymphomas, which occur with markedly increased frequency in transplant patients specifically depleted of T cells, and with UV-induced squamous cell cancers that occur with high frequency in renal transplant patients. Second, the reasons for the growth of presumably immunogenic tumors in immunocompetent hosts have become more apparent with an improved understanding of the function of T cell subsets and the requirements for the induction and expression of T cell responses. The earlier perceptions that cytotoxic T cells recognize foreign proteins integrally inserted in the cell membrane had focused efforts for more than a decade on the use of the powerful new monoclonal antibody technology combined with biochemical purification techniques to isolate tumor antigens expressed on the cell membrane. However, analyses of the requirements for recognition of virally infected cells by CD8+ cytotoxic T cells demonstrated that Class I-restricted Tc do not recognize integral membrane proteins but rather recognize intracellular proteins that are synthesized in the cytoplasm. Tumors containing unique or mutated and potentially immunogenic cellular proteins may selectively or predominantly elicit only CD4+ or CD8+ T cell responses because the antigen is adequately presented in the context of only one MHC molecule, and in this setting only T cells from the subset restricted to that MHC molecule appear effective in tumor therapy.
Article
Only a few proteins are known to be exclusively expressed in central nervous system (CNS) myelin. A novel surface membrane protein expressed only in CNS myelin and oligodendrocytes of higher vertebrates has been identified by a monoclonal antibody. This CNS myelin/oligodendrocyte-specific protein, MOSP, has a molecular weight of 48 kDa and a pI of approximately 6.7. In the presence of the monoclonal antibody, MOSP remains on the surface of cultured oligodendrocytes but becomes associated with cytoplasmic microtubules. Our results suggest that MOSP plays an important role in membrane/cytoskeleton interactions during the formation and maintenance of CNS myelin. MOSP also may play a critical role in the pathogenesis of diseases of CNS myelin.
Article
Human neoplasms are biologically heterogeneous. The extensive cellular diversity found in malignant neoplasms is generated by the rapid emergence of clonal subpopulations of tumor cells with different properties that include invasion, metastasis and responsiveness to treatment. Studies in rodent systems have indicated that cancer metastases can be clonal in their origin and that different metastases can originate from different progenitor cells from the primary tumor. This metastatic heterogeneity of tumor cells has many ramifications for studies of tumor biology, in general, and studies of therapy, in particular. The heterogeneous nature of metastatic human neoplasms can now be studied under defined conditions in healthy athymic nude mice. The neoplasms must be free of mouse pathogens and the mice must be kept in specific-pathogen-free conditions. Careful consideration must be given to the intimate tumor-host relationship for each tumor system studied, because the metastatic potential of human neoplasms can vary with the site of implantation into nude mice. Several methods for studying the biology of human neoplasms in the nude mouse are described as well as techniques to assure the success of these studies. The data show that the healthy young nude mouse can be a useful in vivo model for ascertaining the metastatic potential of human neoplasms, for selecting and maintaining cell variants of high metastatic potential from heterogeneous human tumors, and for studying therapeutic agents directed against metastatic cells proliferating in visceral organs.
Article
The cytotoxic reactivity of lymphocytes for autologous melanoma cells was studied in a group of 13 melanoma patients. No cytotoxicity was observed with lymphocytes freshly isolated from peripheral blood or with lymphocytes cocultured for 7 days with autologous melanoma cells. Growth of lymphocytes previously sensitized with autologous melanoma in vitro in interleukin 2 (IL-2)-containing medium, however, resulted in cytotoxic reactivity for autologous melanoma in 7/13 patients. The reactivity of IL-2-dependent lymphocytes for autologous melanoma was particularly striking in one patient (A.V.) who has had an unexpectedly favorable clinical course and, because of their consistently high reactivity, AV lymphocytes were selected for detailed specificity analysis. After 2-3 weeks in culture in IL-2-containing medium, AV lymphocytes were cytolytic for autologous melanoma cells but not autologous Epstein-Barr virus-transformed B cells, autologous fibroblasts, or allogeneic tumor targets. Specificity of autologous melanoma reactivity was confirmed by competitive inhibition assays. The IL-2-dependent AV lymphocytes formed rosettes with sheep erythrocytes and expressed OKT 3 and Ia antigens. After longer periods of culture, AV lymphocytes were found to react with a wider range of target cells, and repeated attempts to isolate cultures with restricted reactivity to autologous melanoma by resensitization with autologous melanoma and limiting-dilution techniques were unsuccessful. The restricted reactivity of early cultures could be preserved, however, in frozen storage, but shifted again toward broader reactivity after several weeks in culture. The recognition of cytotoxic T cells with initial restricted reactivity for autologous melanoma suggests reinvestigation of the question of specific cellular immunity to human cancer.