Establishment, Growth, Metastasis and Pathological Features of Xenografted Tumors
As shown in , 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.
Induction, growth, metastasis and regression of human tumor cells in opossums as observed by clinical inspection and pathology
The dosage of 0.1 × 106 A375 melanoma cells injected into 12 1-day-old (1-d.o.) pups of litter 1 () 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 × 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, ), 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 × 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 (); by week 4, one larger tumor had grown to 1.25 × 1.5 cm; by week 5, the tumors started to regress ().
Figure 1 Human melanoma cells xenografted into suckling young opossums. A. Suckling pups at two weeks of age (litter 6, ). Amelanocytic tumors, as pointed by arrows, were induced by s.c. injection of 0.25 × 106 A375 cells at the age of 2 days. (more ...)
Necropsies of the six pups of litter 5 () 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 × 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 (). Upon further examination, one of the three pups exhibited a large solid fresh tumor, measuring 1.25 × 1.25 cm, in the left chest wall ().
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 (). 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 × 0.5 cm, became observable during week 2, and continued to grow to 0.5 × 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 × 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 × 0.25 cm at week 3 and grew to 0.5 × 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
The 0-d.o. pups that were injected with 0.25×106 PC-3p cells (litters 1-2, ) exhibited observable tumors during week 2; by week 3, the average tumor size was 0.5 × 0.5 cm (). During week 4, some tumors grew to 0.75 × 0.75 cm and one pup of litter 1 () appeared sick and was euthanized (, pop-up window). Necropsy exhibited two fresh-looking tumors. One measured 0.5 × 0.5 cm. The other, which measured 1.0 × 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 (). By week 5 (litter 1), aggressive tumor growth was still observed in a pup of litter 1, showing no signs of host rejection (). 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, ). One pup in litter 2 is still alive.
Figure 2 Human prostate cancer cells xenografted into suckling opossums. A. Suckling opossums at 3 weeks old (litter 1, ). 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, (more ...)
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 0.5 × 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, ), but only 50% in the 8-d.o. pups (litter 6). The average tumor size was 0.5 × 0.75 cm by week 4, with the largest measuring 1.0 × 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 × 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 (). 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, ): 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 × 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, ) revealed resorption and death of the tumor cells at the original injection sites with meningeal metastasis in one pup ().
The same dosage was well tolerated by 7-d.o. pups (litter 10, ), and the tumor-take rate was 100%. Tumors measured about 0.25 × 0.5 cm by week 2; by week 4, the average tumor size had more than doubled, with the largest measuring 1.5 × 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, ) 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 (). This dosage, however, did not induce tumors in 8-10-d.o. pups (litters 5-6).
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 (more ...)
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 (). 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 (). In comparison, necropsy of the nine 6-8-w.o. pups (litter 8) showed regressed tumors. It is noteworthy that, like the A375 melanoma (), 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 ().
Histologically, 2-w.o. tumor cells were extremely viable in appearance, and there were no infiltrations of host inflammatory cells (). At week 3, host inflammatory responses started to appear (). 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.
It is noteworthy that pathological examination of one 4-w.o. tumor sample (litter 18, ) 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 (). Additionally, 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 () 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 ().
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 × 0.75 cm, from a pup that was injected with 1 × 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) (). Junctional complexes were prominent (). The cytoplasm of the cells contained ribosomes, glycogen, and to lesser extent intermediate filaments (). Scattered mitotic spindles were present (). Microvilli were present and tended to show apical orientation in areas with formed lumina (). These findings were consistent with an adenocarcinoma.
Figure 4 Ultrastructural cellular features of xenografted 3-w.o. colon cancer cells injected with 1 × 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 (more ...)
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 the cells started to detach from the culture dishes ().
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, (more ...)
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 (). 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.