Biologic instability of pancreatic cancer xenografts in the nude mouse

Bruno M. Schmied1,2, Alexis B. Ulrich1,3, Hosei Matsuzaki1,4, Tarek H. El-Metwally5, Xianzhong Ding5, Mirabella E. Fernandes6, Thomas E. Adrian5, William G. Chaney6, Surinder K. Batra6 and Parviz M. Pour1,7,8

1 UNMC Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA,
2 Department of Visceral and Transplantation Surgery, Insel Hospital, Bern, Switzerland,
3 Department of Surgery, Rheinische Friedrich-Wilhelms-University, Bonn, Germany,
4 Department of Surgery II, Kumamoto University, Kumamoto, Japan,
5 Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE,
6 Department of Biochemistry and Molecular Biology and
7 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor transplants into nude mice (NM) may reveal abnormal biological behavior compared with the original tumor. Despite this, human tumor xenografts in NM have been widely used to study the biology of tumors and to establish diagnostic and therapeutic modalities. Clearly, precise differences in the biology of a given tumor in human and in NM cannot be assessed. We compared the growth kinetics, differentiation pattern and karyotype of an anaplastic Syrian hamster pancreatic cancer cell line in NM and in allogenic hamsters. As with the original tumor, transplants in hamsters grew fast, were anaplastic and expressed markers related to tumor malignancy like galectin 3, TGF-{alpha} and its receptor EGFR at high levels. However, tumors in the NM were well-differentiated adenocarcinomas, grew slower, had increased apoptotic rate and had a high expression of differentiation markers such as blood group A antigen, DU-PAN-2, carbonic anhydrase II, TGF-ß2 and mucin. Karyotypically, the tumors in the NM acquired additional chromosomal damage. Our results demonstrate significant differences in the morphology and biology of tumors grown in NM and the allogenic host, and call for caution in extrapolating data obtained from xenografts to primary cancer.

Abbreviations: BOP, N-nitrosobis(2-oxopropyl)amine; NM, nude mouse; SGH, Syrian golden hamster.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since their introduction to the medical world in 1966 (1) nude mice (NM), with their ability to host cancer cells without rejection, have gained popularity as a model to study human tumor biology (24). They are also used to examine the effects of therapeutic drugs or radiation (58) particularly in cancers difficult to manage, such as pancreatic cancer (9). Tumor transplants into NM normally maintain the phenotype of the original tumor (1012) but lose the metastatic potential of even aggressive cancer (13). Moreover, in some instances the differentiation-state of the xenografts deviates from the original tumor (14,15), possibly due to a genetic instability of heterogeneous tumors and the microenvironment of the host. However, to what extent these changes occur is unknown.

Clearly, precise differences in the biology of a given tumor in humans and in NM cannot be assessed. However, experimentally induced cancer in species other than NM offer a possibility for comparison. We compared the growth pattern of anaplastic Syrian golden hamster (SGH) pancreatic cancer cells (16) in the allogenic hamster and NM host, and found significant differences in the morphology, biology and cytogenetics between the tumors grown in the two species.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor cell line
The KL5B tumor cell line was established from hamster pancreatic cells treated with N-nitrosobis(2-oxopropyl)amine (BOP) in vitro (16). Cells at passage 34 were harvested with 0.05% trypsin–EDTA solution (Sigma, St Louis, MO) and cells were washed twice with phosphate buffered saline (PBS), pH 7.4, and resuspended in fresh PBS.

Animals and experimental design
Eight-week-old female SGH from the Eppley Institute colony and 8-week-old female BEIGE DX 1NM from Harlan (Indianapolis, IN) were housed in a controlled, germ-free environment as recommended by the animal research committee.

Viable KL5B cells (1.0x106) (determined by trypan blue dye exclusion) from the same culture dish, were injected subcutaneously into the upper left, upper right, lower left and lower right quadrant of the abdomen of six SGH and six NM. The body weight of the animals and the size of the growing tumors were monitored weekly. At days 9, 17 and 26, the largest tumor from each animal was excised under phenobarbital anesthesia (0.5ml/100 g/body wt i.p.), carefully freed from skin and peritumoral tissue, weighed, and the ratio of weight to the animal body weight (mg/g/body wt) was calculated. One part of each tumor was fixed in 10% buffered formalin for 12 h and prepared for histological/immunohistochemical examination, while the other part was frozen immediately in liquid nitrogen for determination of K-ras mutation. At day 33, when just one tumor was left in each animal, tritiated thymidine was given (1 µCi/g/body wt) i.p. four times each, at 2-h intervals. One hour after the last injection, all animals were killed by phenobarbital overdose, the tumor removed, weighed and prepared as described above. Autoradiography was performed as described earlier (17) and in each slide the labeling of 500 cells in five different randomly-selected tumor areas were counted by two independent observers. The labeling index was determined by the percentage of cells containing more than five grains over the nuclei.

To determine the invasiveness of the induced tumors, all animals were subjected to a thorough necropsy and examined for tissue abnormalities. Liver, peritoneum, lung, axillary and mesenteric lymph nodes were fixed in 10% buffered formalin, embedded in paraffin, cut into 4 µm step sections, and stained with haematoxylin and eosin for microscopic examination.

Tissues from one tumor grown in NM and SGH (day 26) were minced finely with scissors in ice-cold RPMI-1640 supplemented with FBS, penicillin G (100 g/ml) and streptomycin (100 mg/ml). The chopped tissue was digested in 4 ml serum-free Hank's balanced salt solution (Gibco BRL, Gaithersburg, MD) containing 4 mg/ml collagenase P (Boehringer-Mannheim, Indianapolis, IN) for 15 min at 37°C. Digestion was stopped by washing the material twice in ice-cold HBSS containing 0.05% EDTA, and centrifuged for 1min at 1500 r.p.m. The supernatant was discharged, the pellet resuspended in 10% RPMI-1640 and cultured in 25 cm2 plastic culture flasks (Corning, Cambridge, MA). To eliminate fibroblasts in culture, 50 µM geneticin (Gibco BRL) was added daily for 7 days into the fresh culture medium as described elsewhere (18). The cell line of the hamster tumor was designated KL5BSGH, and the NM cell line was designated KL5BNM.

Contrary to the KL5BSGH cells, which presented a monomorphic cell population, the KL5BNM cells were pleomorphic. Some cells resembled the morphology of KL5BSGH cells and others were of a polygonal type. Single-cell clones were generated from the KL5BNM cells by suspending the cells at high dilution. The single cells were handpicked under a light microscope (magnification x40) and seeded into a 24-well plate at a concentration of 1cell/well. Monoclonal cell growth was monitored in a 6-h interval. In six wells the attached single cell formed a monomorphic polygonal cell colony. One of the colonies was picked for further study and was designated as KL5BNMc. After 8 weeks in culture, KL5BSGH, KL5BNM and KL5BNMc cell lines were prepared for analysis for k-ras mutations, cytogenetics, cell growth and cell cycle analysis. They were also processed for immunohistochemistry, growth factor analysis, receptor binding studies and determination of differentiation markers.

In an in vivo crossover experiment, KL5BSGH cells were subcutaneously injected into three NM according to the method described above. KL5BNM and KL5BNMc cells were injected into three SGH. After 33 days inoculation, all tumors were removed and prepared for histological examination.

Mutation of K-ras and cytogenetic analysis
The mutations of the K-ras sequences at codon 12 or 13 were determined by direct sequencing of the PCR product of exon 1 as described previously (19).

Cytogenetic analysis of >40 cells each of KL5BSGH, KL5BNM and KL5BNMc was performed as described earlier (19,20).

In vitro cell growth, cell cycle analysis and apoptosis
A cell growth curve and the cell doubling time of KL5BSGH, KL5BNM and KL5BNMc cells were established as reported (16).

For cell cycle analysis and apoptosis, cells of all the three cell lines were processed in triplicates using the Telford method (21) with propidium iodide. Cell cycle analysis and apoptosis were performed with a FACStar flow cytometer (Becton Dickinson, CA).

Histochemical and immunohistochemical examinations
The Avidin–peroxidase complex (ABC) method (22) was used for all immunohistochemical examinations. Antibodies to PCNA, Cyclin A and Cyclin D1 were purchased from Novocastra Laboratories (Newcastle upon Tyne, UK); vimentin, {alpha}-1-anti-trypsin, anti-p53 and anti-Ki-67 from Biogenex (San Ramon, CA). DU-PAN-2 and carbonic anhydrase II (CA II) antibodies were donated from Dr M.Hollingsworth (Eppley Cancer Center, NE). Antibody against blood group A was purchased from DAKO (Carpinteria, CA), anti-TGF-{alpha} and anti-EGFR antibodies from Oncogene Research (Cambridge, MA); anti-pancytokeratin, anti-laminin and anti-TGF-ß1 antibodies from Sigma. Phaseolus vulgaris leucoagglutinin and tomato lectin were obtained from Vector (Burlingame, CA). PAS staining was performed using the McManus method and AgNOR staining.

Radioimmunoassay and EGF receptor binding assay
The TGF-{alpha} assay used a commercial polyclonal anti-serum (Penninsula, Belmont, CA) without cross reactivity with other EGF-related peptides. TGF-{alpha} levels were measured in conditioned media as described in detail elsewhere (23).

For the EGF receptor binding assay, KL5BSGH, KL5BNM and KL5BNMc cells were growing in triplicates and proceeded following the protocol published elsewhere (22). Radioactivity was determined in a gamma counter (LKB Wallac, Gaithersburg, MD). Non-specific binding was determined by incubations containing [125I]hEGF and 100 nM unlabeled hEGF.

Total CA activity
Cell differentiation was assessed biochemically measuring the total CA activity following the procedure published by El-Metwally and Adrian (24). To stimulate the CA activity, all-trans retinoic acid (Sigma) was added once at a concentration of 5 µM and the cells were incubated in absolute darkness for 4 days.

Secretion of TGF-ß2 and galectin 3 expression
The immunoassay-kit (Promega, Madison, WI) detects biologically active TGF-ß2 (cross reactivity with TGF-ß1 <5% and TGF-ß3 at 10 ng/ml) in an antibody-sandwich format (25). Following the manufacturer's protocol, the amount of specifically bound TGF-ß2, was quantitated against a standard curve generated with known amounts of TGF-ß2.

Galectin 3 was determined by western blot analysis after transfer of SDS–PAGE-separated extracts to Immobilon as described previously (26). Blots were quantified by densitometry and statistically analyzed.

Statistics
A non-paired Student's t-test was used for statistical evaluation.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor growth and histology
Tumors developed at all four inoculation sites of all animals without significant differences in the tumor size between the four inoculation sites and among the animals of both species. Tumors in SGH grew considerably faster than in NM and were significantly heavier 9 days after tumor cell inoculation (Figure 1Go). The ratio of SGH:NM tumor weight increased with time and was 5.2 at day 33, when the tumors in all six SGH had invaded the visceral peritoneum. In two cases the intestine, and in one case the pancreas, stomach and liver also were invaded by the tumors. In the NM, however, all tumors were encapsulated, well circumscribed, and only one tumor had locally invaded the abdominal muscles, but not the visceral tissues.



View larger version (8K):
[in this window]
[in a new window]
 
Fig. 1. Tumor growth of KL5B cells after s.c. injection into SGH and NM. The tumor weight is calculated in relationship to the body weight (mg tumor wt/g body wt). Hamster tumor growth (black bars) shows significant difference (*P < 0.05, **P < 0.005) to the tumor growth in NM (white bars) already 9 days after inoculation.

 
Histologically, as observed for the original KL5B tumors (16), all tumors in the SGH were anaplastic showing many mitotic figures (>10 mitotic figures per x40 magnification field), multifocal necrosis and haemorrhage (Figure 2AGo). However, all tumors in the NM were well-differentiated adenocarcinomas with desmoplastic reaction (Figure 2BGo) and a low mitotic rate (0–1 mitotic figure per x40 magnification field).




View larger version (299K):
[in this window]
[in a new window]
 
Fig. 2. (A) KL5B tumor s.c. in SGH. Anaplastic tumor showing multifocal necrosis and haemorrhage. H&E, x120. (B) Subcutaneous tumor growth of KL5B cells in NM. A well-differentiated adenocarcinoma with marked glandular formation and intense desmoplastic reaction. H&E, x200.

 
The labeling index of tumor cells in the SGH was significantly higher (>5 grains/nucleus in 13% of the cells) than in NM tumor cells (>5 grains/nucleus in 7% of the cells) with an interobserver variability of 1%.

K-ras mutational analysis and karyotype
Mutation of the K-ras gene at codon 12 (GGT->GAT) was found in both the SGH and NM tumors, and in all their cell lines, as expected since the parental KL5B cells showed this mutation (16).

KL5BSGH cells (Figure 3Go) showed an identical chromosomal pattern as the KL5B cells (16). The KL5BNM cells presented two different clones: one with identical chromosomal changes to the KL5B cells and the other with numerous additional monosomies and rearrangements (Figure 3Go). The karyotype of all KL5BNMc cells was identical to the second clone of the KL5BNM cells.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3. Chromosomal pattern of KL5BSGH and KL5BNM cells. All 40 KL5BSGH cells tested represented an abnormal clone characterized by a missing Y, monosomy 7 and 11, one copy of two markers and two copies of another marker. Thirty-one tested KL5BNM cells showed the same karyotype as the KL5BSGH cells. A second clone of 36 cells was characterized by a missing copy of chromosomes X and Y, monosomy 3, 5, 7, 9, 10, 11, 12, 19 and 20, additional unknown material on chromosomes 1 and 6, a deletion of the short arm of chromosomes 2 and the presence of 10 marker chromosomes. The karyotype of the KL5BNMc cells was identical to the second clone of the KL5BNM cells.

 
In vitro cell growth, cell cycle analysis and apoptosis
The KL5BSGH cells grew faster (doubling time 15 h) than the KL5BNM cells (doubling time 19 h) and much faster than the KL5BNMc cells (doubling time 24.5 h). The KL5BSGH cells preserved the monomorphic, spindle-shaped cytology with distinct nuclei (Figure 4AGo). On the other hand, KL5BNM cells were pleomorphic with some cells resembling the spindle-shaped KL5BSGH cells and other cells being cuboidal to polygonal (Figure 4BGo). The KL5BNMc cells were monomorphic polygonal in their appearance. The KL5BNMc cells and the polygonal KL5BNM cells, but not the spindle-shaped KL5BNM or the KL5BSGH cells, formed ductular-like structures in the confluent state.




View larger version (257K):
[in this window]
[in a new window]
 
Fig. 4. (A) Paraformaldehyde-fixed KL5BSGH cells growing on Permanx chamber slides (Nalgene, Naperville, IL). Pleomorphic cells with elongated, PAS negative cytoplasm. There are a few giant cells with polynucleated nuclei (arrowheads). PAS, x400. (B) Similarly processed KL5BNM cells. Cuboidal to polygonal mucin-producing PAS positive cells (black in colour). PAS, x400.

 
About 34% KL5BSGH, 62% KL5BNM and 69% KL5BNMc cells were in the G0–G1 phase (Figure 5Go), 38, 15 and 7%, respectively, in the G2–M phase. While 25, 12 and 8%, respectively, were in the S-phase. The apoptotic rate was 16 (KL5BNMc), 10 (KL5BNM) and 1% (KL5BSGH).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Cell cycle analysis of the KL5BSGH, KL5BNM and KL5BNMc cells established from the tumors growing in SGH and NM. The black bars represent the KL5BSGH cells, white bars the KL5BNM cells, and striped bars the KL5BNMc cells. In the G0-G1 phase significant differences were found between KL5BSGH and KL5BNM cells (P < 0.005), and KL5BSGH and KL5BNMc cells (P < 0.005). In the G2-M phase a significant higher number of KL5BSGH cells was found than in the KL5BNM (P < 0.05) and KL5BNMc cells (P < 0.005). On the other hand the percentage of KL5BNMc cells in the S-phase was significantly lower than in the KL5BSGH cells (P < 0.05). The apoptotic rate was almost undetectable in the KL5BSGH cells, high in the KL5BNM (P < 0.05), and highest in the KL5BNMc cells (P < 0.05).

 
Immunohistochemical findings
SGH tumors showed a higher proliferation rate (indirectly by Cyclin A, Cyclin D1, Ki-67, PCNA, AgNOR) and higher expression of TGF-{alpha} and its receptor than the NM tumors (Table IGo). Each tumor within the same species presented identical patterns. The glandular structures within the NM tumors and the polygonal KL5BNM and KL5BNMc cells, but not the SGH tumors or the KL5BSGH cells, had PAS-positive mucin and expressed DU-PAN-2 (Figure 6Go) and blood group A antigen (Table IIGo). CA II was moderately positive in the glandular areas of the NM tumors and strongly positive in the polygonal KL5BNM and KL5BNMc cells. However, CA II was almost undetectable in SGH tumors and KL5BSGH cells. P53 protein was found in tumors of both species and in their cell lines with a stronger nuclear staining in the SGH tumors and KL5BSGH cells. TGF-ß1, pancytokeratin, laminin, vimentin, {alpha}-1-antitrypsin, phaseolus vulgaris leucoagglutinin and tomato-lectin were positive in all samples without a significant difference (data not shown).


View this table:
[in this window]
[in a new window]
 
Table I. Expression of tumor proliferation markers and TGF-{alpha} and its receptor in KL5B tumor cells grown in hamster and NM
 


View larger version (153K):
[in this window]
[in a new window]
 
Fig. 6. Expression of DU-PAN-2 antigen in glandular structures of NM tumors. ABC method x200.

 

View this table:
[in this window]
[in a new window]
 
Table II. Differences in the reactivity of antibodies with KL5B cells grown in SGH and NM and in the cell lines derived from the respective tumors
 
Radioimmunoassay and EGF receptor binding assay
TGF-{alpha} was detected in the conditioned medium from the KL5BSGH, KL5BNM and KL5BNMc cells (Figure 7Go). KL5BSGH cells secreted the highest concentrations of TGF-{alpha} (14 fmol/ml/106 cells), followed by KL5BNM cells (4.5 fmol/ml/106 cells) and KL5BNMc cells (3.4 fmol/ml/106 cells). The KL5BSGH, KL5BNM and KL5BMc cells showed different densities of EGF receptors (Figure 8Go). The competitive binding experiment demonstrated that [125I]hEGF bound with a high affinity to the EGF receptors of the KL5BSGH cells, with a moderate affinity to KL5BNM cells, and with the lowest affinity to the KL5BNMc cells. In the KL5BNM and KL5BNMc cells, the binding was completely inhibited by 100 nM unlabeled hEGF and in the KL5BSGH cells by 300 nM unlabeled hEGF.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7. Secretion of TGF-{alpha} by KL5BSGH, KL5BNM and KL5BNMc cells. A significantly (P < 0.05) higher amount of TGF-{alpha} was secreted by KL5BSGH cells compared to KL5BNM or KL5BNMc cells.

 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 8. Competitive binding of EGF receptors in KL5BSGH, KL5BNM and KL5BNMc cells. The competitive binding curve on top presents the KL5BSGH cells, the middle curve the KL5BNM cells, and the bottom curve the KL5BNMc cells. Data present the mean ± SEM from four individual experiments.

 
Total CA activity
The total basal CA activity was highest in the KL5BNMc cells, followed by the KL5BNM cells, and was almost undetectable in KL5BSGH cells (Figure 9Go). As in a previous study (27), incubation with all-trans retinoic acid increased the basal total CA activity in the control Capan-2 cells; in our study 4.5-fold (from 0.77 U/µg to 3.46 U/µg protein). The increase of the basal CA activity in the KL5BNMc cells was ~4.3-fold (from 0.54 U/µg to 2.32 U/µg protein) and in the KL5BNM cells 3.8-fold (from 0.05 U/µg to 0.19 U/µg protein). No significant increase was observed in KL5BSGH cells.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 9. Basal total CA activity (U/µg protein) and after stimulation with retinoic acid in Capan-2, KL5BSGH, KL5BNM and KL5BNMc cells in vitro. Significant increase (P < 0.005) of CA activity occurred after stimulation with 5 µM retinoic acid in Capan-2, KL5BNM and KL5BNMc, but not in KL5BSGH cells.

 
Secretion of TGF-ß2 and galectin 3 expression
The concentration of TGF-ß2 in conditioned medium was 1662 pg/ml/106 cells in KL5BNMc cells, 1003 pg/ml/106 cells in KL5BNM and 385 pg/ml/106 cells in the KL5BSGH cells (Figure 10Go).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 10. Concentrations of secreted TGF-ß2 in conditioned medium of KL5BSGH, KL5BNM and KL5BNMc cells. A significantly higher amount of TGF-ß2 was secreted by the KL5BNMc and KL5BNM cells compared to the KL5BSGH cells (P < 0.005).

 
Both the KL5BNMc and KL5BSGH cells expressed galectin 3 as seen by western blot analysis (Figure 11Go). Densitometric analysis of blots on four independent cell extracts for each cell line showed a 75% increase in galectin 3 expression in the KL5BSGH cells.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 11. Western blot analysis of galectin 3 expression in KL5BSGH and KL5BNMc cells. The bands at 29 kDa are indicative for galectin 3. Quantitative densitometry shows about two times higher levels of galectin 3 in KL5BSGH cells than the KL5BNMc cells.

 
Crossover experiment
When injected into the NM, the KL5BSGH cells grew in the same manner as the KL5B cells. The KL5BNM cells injected into the hamster formed predominantly anaplastic tumors with areas of glandular structures. The KL5BNMc cells in the hamster formed an entirely well-differentiated adenocarcinoma with extensive desmoplastic reaction.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The NM model has become an established tool to investigate the biology and pathophysiology of human cancers, and to develop diagnostic and therapeutic strategies (5). Although in general xenografts in NM retain their original tumor morphology and biology (11,12,28) and show a high degree of genetic integrity (29), in some cases they differentiate into a lower grade (30), and in rare instances also into a higher grade (14,15,31) compared with the primary tumors. Moreover, subcutaneous NM xenografts lack the invasive and metastatic potential of the original tumors (28,32,33). Immunological and local factors, including cell-to-cell and cell-to-matrix interactions, growth factors, cytokines, hormones, locally active enzymes and other yet unknown mechanisms could influence cell growth and morphology of xenografts (3437). Consequently, interpolation of findings in the NM regarding tumor biology to the human conditions requires careful evaluation.

Biological comparison between the original human cancer and its xenografts in NM cannot be made. However, experimental cancer induced in species other than NM could clarify the extent of cytogenetic and biological changes in xenografts. We compared the biology, growth, and differentiation pattern of a well-characterized anaplastic SGH pancreatic cancer cell line (16) after subcutaneous injection into the hamster and NM under identical conditions. We found significant differences in the morphology, biology and cytogenesis between tumors growing in these hosts. KL5B cells in hamsters formed rapidly growing and invasive anaplastic tumors that overexpressed markers associated with malignancy and progression including TGF-{alpha} and its receptor EGFR (38) and galectin 3, which has been shown to be overexpressed in advanced tumor stages and metastasis (39,40). The tumors in NM, however, were well differentiated, circumscribed, grew significantly slower than hamster tumors, and showed a lower proliferation and mitotic rate. The tumors in NM also expressed differentiation markers such as DU-PAN-2 (41), blood group A antigen (4244) and CA II (45). Morphological differentiation features and the higher expression of differentiation markers in the NM tumor were maintained in vitro. The cloned KL5BNMc cells and the parental polyclonal KL5BNM cells showed a higher degree of differentiation than the KL5BSGH cells as was indicated by the increased expression of basal total CA activity, which is a marker for the terminal differentiation of pancreatic ductal cells (45). As in Capan-2 cells (46), which were used by us as a control, all-trans retinoic acid increased the CA activity in the KL5BNMc and polygonal KL5BNM tumor cells, but not in the KL5BSGH tumor cells, indicating that these cells have the potential for a yet higher degree of differentiation.

The desmoplastic reaction in the NM tumors, imitating human pancreatic cancer, seems to be related to the secretion of TGF-ß2, which has been shown to play a role in the interaction between tumor cells and stromal cells and in the stimulation of matrix production (47). KL5BNM tumor cells secreted a much higher level of this cytokine than the KL5BSGH tumor cells.

It appears that differences in the biology of tumors grown in immunocompetent and immunoincompetent hosts are dictated by the chromosomal damage. Confirming previous observation (48), we found that passage of cancer cells through the NM causes genetic instability as reflected by additional chromosomal aberrations in tumor cells growing in NM. Unexpectedly, the cells with more extensive chromosomal alterations showed a more benign behavior than the hamster tumor cells with less chromosomal changes and grew as a well-differentiated adenocarcinoma when reinjected into the hamster. Nevertheless, this observation is in line with human studies showing that extensive chromosomal damage is not necessarily associated with an increased tumor malignancy (49,50). It is possible that extensive damage to chromosomes with break points in certain loci alters the function of genes involved in cell cycle and production of growth factors or their receptors.

K-ras mutation, found in SGH and NM tumors and in all cell lines derived from them, does not seem to play a role in the biology of experimental pancreatic cancer as it seems to be the case in human pancreatic cancer (51).

In conclusion, our results demonstrate significant differences in the morphology and biology of tumors grown subcutaneously in allogenic hamster and in NM. The causative factor(s) are presently unknown. The present model offers a useful system to investigate tumor differentiation and malignancy. Whether or not the changes in tumors grown in NM alter the tumor cell response to therapeutic agents remains to be seen. Nevertheless, the present finding is of a great concern since the NM model has become a standard model for preclinical studies.


    Notes
 
8 To whom correspondence should be addressed Email: ppour{at}unmc.edu Back


    Acknowledgments
 
Supported by National Institute of Health National Cancer Institute grant 5RO1 CA60479 and SPORE grant P50CA72712, the National Cancer Institute Laboratory Cancer Research Center support grant CA367127, and the American Cancer Society Special Institutional Grant.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Flanagan,S.P. (1966) 'Nude', a new hairless gene with pleiotropic effects in the mouse. Genet. Res., 8, 295–309.[ISI][Medline]
  2. Marincola,F.M., Drucker,B.J., Siao,D.Y., Hough,K.L. and Holder,W.D. Jr. (1989) The nude mouse as a model for the study of human pancreatic cancer. J. Surg. Res., 47, 520–529.[ISI][Medline]
  3. Dexter,D.L., Matook,G.M., Meitner,P.A., Bogaars,H.A., Jolly,G.A., Turner,M.D. and Calabresi,P. (1982) Establishment and characterization of two human pancreatic cancer cell lines tumorigenic in athymic mice. Cancer Res., 42, 2705–2714.[Abstract]
  4. Kyriazis,A.P., McCombs,W.B.D., Sandberg,A.A., Kyriazis,A.A., Sloane,N.H. and Lepera,R. (1983) Establishment and characterization of human pancreatic adenocarcinoma cell line SW-1990 in tissue culture and the nude mouse. Cancer Res., 43, 4393–4401.[Abstract]
  5. Tomikawa,M., Kubota,T., Takahashi,S., Matsuzaki,S.W. and Kitajima,M. (1998) Chemosensitivity of human pancreatic cancer cell lines serially transplanted in nude mouse. Anticancer Res., 18, 1059–1062.[ISI][Medline]
  6. Kokkinakis,D.M., Schold,S.C. Jr., Hori,H. and Nobori,T. (1997) Effect of long-term depletion of plasma methionine on the growth and survival of human brain tumor xenografts in athymic mice. Nutr. Cancer, 29, 195–204.[ISI][Medline]
  7. Kyriazis,A.P., Yagoda,A., Kyriazis,A.A., Kereiakes,J.G. and McCombs,W.B.D. (1985) Response to ionizing radiation of human bladder transitional cell carcinomas grown in the nude mouse. Exp. Cell Biol., 53, 281–286.[ISI][Medline]
  8. Chahinian,A.P., Mandeli,J.P., Gluck,H., Naim,H., Teirstein,A.S. and Holland,J.F. (1998) Effectiveness of cisplatin, paclitaxel, and suramin against human malignant mesothelioma xenografts in athymic nude mice. J. Surg. Oncol., 67, 104–111.[ISI][Medline]
  9. Marincola,F., Taylor-Edwards,C., Drucker,B. and Holder,W.D. Jr. (1987) Orthotopic and heterotopic xenotransplantation of human pancreatic cancer in nude mice. Curr. Surg., 44, 294–297.[Medline]
  10. Bettan-Renaud,L., Bayle,C., Teyssier,J.R. and Benard,J. (1989) Stability of phenotypic and genotypic traits during the establishment of a human neuroblastoma cell line, IGR-N-835. Int. J. Cancer, 44, 460–466.[ISI][Medline]
  11. Sharkey,F.E. and Fogh,J. (1984) Considerations in the use of nude mice for cancer research. Cancer Metastasis Rev., 3, 341–360.[ISI][Medline]
  12. Satyaswaroop,P.G., Zaino,R., Clarke,C.L. and Mortel,R. (1987) Nude mouse system in the study of tumor biology, treatment strategies and progesterone receptor physiology in human endometrial carcinoma. J. Steroid Biochem., 27, 431–438.[ISI][Medline]
  13. Maguire,H.Jr., Outzen,H.C., Custer,R.P. and Prehn,R.T. (1976) Invasion and metastasis of a xenogeneic tumor in nude mice. J. Natl Cancer Inst., 57, 439–442.[ISI][Medline]
  14. Hollande,E., Trocheris,de St-Front,V., Louet-Hermitte,P., Bara,J., Pequignot,J., Estival,A. and Clemente,F. (1984) Differentiation features of human pancreatic tumor cells maintained in nude mice and in culture: immunocytochemical and ultrastructural studies. Int. J. Cancer, 34, 177–185.[ISI][Medline]
  15. Goji,J., Sano,K., Nakamura,H. and Ito,H. (1992) Chondrocytic differentiation of peripheral neuroectodermal tumor cell line in nude mouse xenograft. Cancer Res., 52, 4214–4220.[Abstract]
  16. Schmied,B., Liu,G., Moyer,M.P., Hernberg,I.S., Sanger,W., Batra,S. and Pour,P.M. (1999) Induction of adenocarcinoma from hamster pancreatic islet cells treated with N-nitrosobis(2-oxopropyl)amine in vitro. Carcinogenesis, 20, 317–324.[Abstract/Free Full Text]
  17. Pour,P.M. and Stepan,K. (1987) Induction of prostatic carcinomas and lower urinary tract neoplasms by combined treatment of intact and castrated rats with testosterone propionate and N-nitrosobis(2-oxopropyl)amine. Cancer Res., 47, 5699–5706.[Abstract]
  18. Inagaki,T., Matsuwari,S., Shimada,K., Abe,N., Takahashi,R. and Maeda,S. (1993) Effective removal of the contaminating host fibroblasts for establishment of human-tumor cultured lines. Hum. Cell, 6, 137–142.[Medline]
  19. Ikematsu,Y., Liu,G., Fienhold,M.A., Cano,M., Adrian,T.E., Hollingsworth,M.A., Williamson,J.E., Sanger,W., Tomioka,T. and Pour,P.M. (1997) In vitro pancreatic ductal cell carcinogenesis. Int. J. Cancer, 72, 1095–1103.[ISI][Medline]
  20. Takahashi,T., Moyer,M.P., Cano,M., Wang,Q.J., Mountjoy,C.P., Sanger,W., Adrian,T.E., Sugiura,H., Katoh,H. and Pour,P.M. (1995) Differences in molecular biological, biological and growth characteristics between the immortal and malignant hamster pancreatic cells [Erratum in Carcinogenesis (1995) 16, 1257]. Carcinogenesis, 16, 931–939.[Abstract]
  21. Telford,W.G., King,L.E. and Fraker,P.J. (1991) Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell. Prolif., 24, 447–459.[ISI][Medline]
  22. Takahashi,T., Moyer,M.P., Cano,M., Wang,Q.J., Adrian,T.E., Mountjoy,C.P., Sanger,W., Sugiura,H., Katoh,H. and Pour,P.M. (1995) Establishment and characterization of a new, spontaneously immortalized, pancreatic ductal cell line from the Syrian golden hamster. Cell Tissue Res., 282, 163–174.[ISI][Medline]
  23. Toshkov,I., Schmied,B., Adrian,T.E., Murphy,L., Haay,W. and Pour,P.M. (1998) Establishment of tumor cell culture (ILA) derived from hamster pancreatic islets treated with BOP. Int. J. Cancer, 78, 636–641.[ISI][Medline]
  24. El-Metwally,T.H. and Adrian,T.E. (1999) Optimization of treatment conditions for studying the anticancer effects of retinoids using pancreatic adenocarcinoma as a model. Biochem. Biophys. Res. Commun., 257, 596–603.[ISI][Medline]
  25. Hornbeck,P. (1994) Enzyme-linked immunosorbent assays. In Coico,R. (ed.) Current Protocols in Immunology. John Wiley & Sons, New York, Vol. 1, pp. Unit 2.1.
  26. Schaffert,C., Pour,P.M. and Chaney,W.G. (1998) Localization of galectin-3 in normal and diseased pancreatic tissue. Int. J. Pancreatol., 23, 1–9.[ISI][Medline]
  27. Armstrong,J.M., Myers,D.V., Verpoorte,J.A. and Edsall,J.T. (1966) Purification and properties of human erythrocyte carbonic anhydrases. J. Biol. Chem., 241, 5137–5149.[Abstract/Free Full Text]
  28. Rygaard,J. and Povlsen,C.O. (1969) Heterotransplantation of a human malignant tumour to `Nude' mice. Acta Pathol. Microbiol. Scand., 77, 758–760.[Medline]
  29. Hahn,S.A., Seymour,A.B., Hoque,A.T., Schutte,M., da Costa,L.T., Redston,M.S., Caldas,C., Weinstein,C.L., Fischer,A., Yeo,C.J. et al. (1995) Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res., 55, 4670–4675.[Abstract]
  30. Bepler,G. and Neumann,K. (1990) Nude mouse xenografts as in vivo models for lung carcinomas. In Vivo, 4, 309–315.[Medline]
  31. Kitamura,H., Cho,M., Lee,B.H., Gum,J.R., Siddiki,B.B., Ho,S.B., Toribara,N.W., Lesuffleur,T., Zweibaum,A., Kitamura,Y., Yonezawa,S. and Kim,Y.S. (1996) Alteration in mucin gene expression and biological properties of HT29 colon cancer cell subpopulations. Eur. J. Cancer, 32A, 1788–1796.
  32. Sordat,B., Fritsche,R., Mach,J.P., Carrel,S., Ozzello,L. and Cerottini,J.C. (1974) Morphological and functional evaluation of human solid tumours serially transplanted in nude mice. In Rygaard,J, Povlsen,CO, (eds). Proceedings of the First International Workshop on Nude Mice. Verlag, Stuttgart, pp. 269–278.
  33. Gershwin,M.E., Ikeda,R.M., Kawakami,T.G. and Owens,R.B. (1977) Immunobiology of heterotransplanted human tumors in nude mice. J. Natl Cancer Inst., 58, 1455–1461.[ISI][Medline]
  34. Fabra,A., Nakajima,M., Bucana,C.D. and Fidler,I.J. (1992) Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice. Differentiation, 52, 101–110.[ISI][Medline]
  35. DiBerardino,M.A., Hoffner,N.J. and Etkin,L.D. (1984) Activation of dormant genes in specialized cells. Science, 224, 946–952.[ISI][Medline]
  36. Hui,M.Z., Sukhu,B. and Tenenbaum,H.C. (1996) Expression of tissue non-specific alkaline phosphatase stimulates differentiated behaviour in specific transformed cell populations. Anat. Rec., 244, 423–436.[ISI][Medline]
  37. Razin,A. and Riggs,A.D. (1980) DNA methylation and gene function. Science, 210, 604–610.[ISI][Medline]
  38. Friess,H., Berberat,P., Schilling,M., Kunz,J., Korc,M. and Buchler,M.W. (1996) Pancreatic cancer: the potential clinical relevance of alterations in growth factors and their receptors. J. Mol. Med., 74, 35–42.[ISI][Medline]
  39. Irimura,T., Matsushita,Y., Sutton,R.C., Carralero,D., Ohannesian,D.W., Cleary,K.R., Ota,D.M., Nicolson,G.L. and Lotan,R. (1991) Increased content of an endogenous lactose-binding lectin in human colorectal carcinoma progressed to metastatic stages. Cancer Res., 51, 387–393.[Abstract]
  40. Schoeppner,H.L., Raz,A., Ho,S.B. and Bresalier,R.S. (1995) Expression of an endogenous galactose-binding lectin correlates with neoplastic progression in the colon. Cancer, 75, 2818–2826.[ISI][Medline]
  41. Kim,Y.W., Kern,H.F., Mullins,T.D., Koriwchak,M.J. and Metzgar,R.S. (1989) Characterization of clones of a human pancreatic adenocarcinoma cell line representing different stages of differentiation. Pancreas, 4, 353–362.[ISI][Medline]
  42. Hirota,M., Egami,H., Mogaki,M., Kazakoff,K., Chaney,W.G. and Pour,P.M. (1993) Relationship between blood group-A antigen expression and malignant potential in hamster pancreatic cancers. Teratogen. Carcinogen. Mutagen., 13, 217–224.[ISI][Medline]
  43. Juhl,B.R., Hartzen,S.H. and Hainau,B. (1986) Lewis a antigen in transitional cell tumors of the urinary bladder. Cancer, 58, 222–228.[ISI][Medline]
  44. Davidsohn,I., Ni,L.Y. and Stejskal,R. (1971) Tissue isoantigens A, B, and H in carcinoma of the pancreas. Cancer Res., 31, 1244–1250.[ISI][Medline]
  45. Weiss,T., Bernhardt,G., Buschauer,A., Jauch,K.W. and Zirngibl,H. (1997) High-resolution reversed-phase high-performance liquid chromatography analysis of polyamines and their monoacetyl conjugates by fluorescence detection after derivatization with N-hydroxysuccinimidyl 6-quinolinyl carbamate. Anal. Biochem., 247, 294–304.[ISI][Medline]
  46. Rosewicz,S., Stier,U., Brembeck,F., Kaiser,A., Papadimitriou,C.A., Berdel,W.E., Wiedenmann,B. and Riecken,E.O. (1995) Retinoids: effects on growth, differentiation, and nuclear receptor expression in human pancreatic carcinoma cell lines. Gastroenterology, 109, 1646–1660.[ISI][Medline]
  47. Chaudhry,A., Oberg,K., Gobl,A., Heldin,C.H. and Funa,K. (1994) Expression of transforming growth factors beta 1, beta 2, beta 3 in neuroendocrine tumors of the digestive system. Anticancer Res., 14, 2085–2091.[ISI][Medline]
  48. Houghton,J.A. and Taylor,D.M. (1978) Maintenance of biological and biochemical characteristics of human colorectal tumours during serial passage in immune-deprived mice. Br. J. Cancer, 37, 199–212.[ISI][Medline]
  49. Heerema,N.A., Sather,H.N., Sensel,M.G., Kraft,P., Nachman,J.B., Steinherz,P.G., Lange,B.J., Hutchinson,R.S., Reaman,G.H., Trigg,M.E., Arthur,D.C., Gaynon,P.S. and Uckun,F.M. (1998) Frequency and clinical significance of cytogenetic abnormalities in pediatric T-lineage acute lymphoblastic leukemia: a report from the Children's Cancer Group. J. Clin. Oncol., 16, 1270–1278.[Abstract]
  50. Alimena,G., Dallapiccola,B., De Cuia,M.R., Gallo,E., Gastaldi,R., Nanni,M., Franchi,A. and Mandelli,F. (1984) Cytogenetic findings in acute promyelocytic leukaemia. A report of 25 cases. Scand. J. Haematol., 33, 135–143.[ISI][Medline]
  51. Dergham,S.T., Dugan,M.C., Kucway,R., Du,W., Kamarauskiene,D.S., Vaitkevicius,V.K., Crissman,J.D. and Sarkar,F.H. (1997) Prevalence and clinical significance of combined K-ras mutation and p53 aberration in pancreatic adenocarcinoma. Int. J. Pancreatol., 21, 127–143.[ISI][Medline]
Received November 3, 1999; accepted December 28, 1999.