ORIGINAL
ARTICLE
JOP. J Pancreas
(Online) 2000; 1(4):183-190.
Subcutaneously
Inoculated Cells and Implanted Pancreatic Cancer Tissue Show Different Patterns
of Metastases in Syrian Golden Hamsters
Cintia Yoko Morioka1, Seiji Saito1, Kouji Ohzawa1, Shinji Asano2, Yasuhide Hibino3, Yuji Nakada1, Kei-ichiro Kita1, Akiharu
Watanabe1
1Third Department of
Internal Medicine, 2Molecular Genetics Research Center, and 3First
Department of Biochemistry, Toyama Medical and Pharmaceutical University.
Toyama, Japan
ABSTRACT
Context We studied behavior
of the subcutaneously implanted pancreatic tumors and the process of metastasis
using syngeneic Syrian golden hamsters. Design HaP-T1, a cell line
derived from nitrosamine-induced pancreatic cancer in Syrian golden hamsters
was used for this experiment. Thirty-five animals were divided into two groups:
subcutaneous cell inoculation and subcutaneous tissue implantation. The tumor
tissue was obtained from subcutaneously implanted cancer cells. One month after
implantation, the tumors were resected and studied histopathologically.
The animals were followed-up weekly by palpation of the peripheral lymph nodes
in order to identify local recurrence. After death, necropsy was performed.
Liver, lungs and pancreas specimens were taken for histopathogical
study and detection of K-ras point mutation
using the PCR/RFLP method. Results The mean survival time in the
subcutaneous cell inoculation group was 151±17.5 days, and in the subcutaneous
tissue implantation group was 137±12.9 days. During the follow-up, 13
subcutaneously cell inoculated hamsters (86.7%) had right axillary lymph node
metastasis while subcutaneously tissue implanted hamsters did not show any
palpable lymph nodes. After necropsy, 10 of the 20 subcutaneously tissue
implanted animals (50%) showed metastases in the lungs at the histopathological level. However, 16 of the 20
subcutaneously tissue implantated animals (80%)
showed K-ras point mutation in the lung
specimens. The lungs of the animals of the subcutaneous cell inoculation group
did not show any metastases. No metastases were found in the liver or the
pancreas in either group. Conclusion This study suggests that homologous
subcutaneous cell inoculation and subcutaneous tissue implantation models
showed completely different patterns of metastasis. These models may aid
further research to clarify the mechanisms of metastasis in pancreatic cancer.
INTRODUCTION
The
process of metastasis in pancreatic cancer is not clear. Subcutaneously
implanted nude mice models have been widely used in in-vivo research.
However, the response to the therapy may sometimes be a false positive. The
immunological rejection response to the graft may give a false impression that
the drug works [1]. The homologous implantation model appears to be suitable
for in-vivo experiments because of the low rejection rate.
Nitrosamine-induced
pancreatic cancer in Syrian golden hamsters resembles that of humans
immunologically, biologically and morphologically [2]. Thus, subcutaneously
implanted tumor models in syngeneic golden hamsters appear suitable for use in in-vivo
experiments for the biological study of pancreatic cancer. However, there have
been no studies done to see if cell implantation and tissue implantation show
different metastatic behaviors in this model.
The
purpose of the present study was to clarify the behavioral differences of
subcutaneously inoculated pancreatic cancer cells versus subcutaneously
implanted pancreatic cancer tissue with respect to the process of metastasis
depending on the method of tumor implantation.
MATERIALS
AND METHODS
Cells
A cultured
cell line derived from a pancreatic cancer induced by N-nitrosobis (2-hydroxypropyl) amine (BHP) in Syrian golden
hamsters, HaP-T1, established by Saito et al. [3], was used in this
study. The cell culture was maintained in Eagle's Minimum Essential Medium,
containing glutamine, non-essential aminoacids and
NaHCO3, as described previously [4], through serial passages. This
cell line shows a mutation from GGT to GAT in codon 12 of the K-ras gene [4].
Animals
Thirty-five
Syrian golden hamsters of both sexes from 8 to 26 weeks of age were used.
Preparation
of Suspension of Cell Lines
Subconfluent cultures were washed once with PBS, and harvested with trypsin 0.25%
and EDTA 0.02%. After checking cell viability with the trypan
blue-dye exclusion test, the cells were counted and adjusted to 2x106
cells/mL, using cold serum-free culture medium. They were kept cold until use.
Preparation
of Tumor Grafts
0.1 ml of
tumor cell suspensions was injected into the subcutis
of 4 animals. After one month, the tumors were resected aseptically, and cut
into pieces of approximately 1 mm3. They were maintained in a cold
serum-free medium until use. Part of the resected tumors was taken for histopathological study.
Experimental
Design
The
animals were divided into two randomized groups, subcutaneous cell inoculation
(SCI), and subcutaneous tissue implantation (STI). All implantations in both
groups were performed on the back of the animal. The growth of the tumor and
the body weight were monitored weekly. After one month, tumorectomy
was performed to avoid death of the hamsters due to tumor necrosis or invasion
of the deep tissues. Thus, we could study the process of metastasis during the
early stage of tumor growth. The resected tumor specimens were studied histopathologically. The animals were followed-up until
death. After death, necropsy was performed.
Subcutaneous
Implantation
SCI (n=15)
animals were anesthetized with diethyl-ether inhalation. After asepsy, 0.1 mL of cell suspension was inoculated once,
subcutaneously, using a 29-Gauge needle.
STI (n=20)
hamsters were anesthetized with diethyl-ether inhalation. After asepsy, a hole was opened in the cutis using a scalpel, and
then one piece of the tumor was implanted in the subcutis.
The hole was closed using a 4.0 nylon suture (Keisei Co., Tokyo, Japan).
Resection
of the Implanted Tumor
After one
month, hamsters were anesthetized with diethyl-ether inhalation and sodium
pentobarbital 5 mg/kg body weight, intraperitoneally.
They were placed in a sterile field. Asepsy was
carried out. The skin was opened with a scalpel 2 mm from the developed tumor
to avoid rupture of the capsule, and possible spreading of the tumor cells. The
dissection was made "circumferencially"
around the tumor. Thick vessels were linked using 4.0 nylon sutures. After the
linkage, the tumor was isolated in the lower part using Kelly, and was
sectioned. Therefore, the resected specimen consisted of tumor and the upper
skin. Next, the hole was closed with simple sutures using 4.0 nylon.
Follow-up
of the Animals
The
hamsters were followed-up weekly, in order to observe local recurrence of the
tumor, palpable lymph nodes and general condition, i.e. if they become moribund
or not. After death, the animals were necropsied in order to confirm the
presence or absence of metastasis. The liver, lungs and pancreas were removed
and fixed in formalin and some parts were frozen in liquid nitrogen for DNA analysis.
Histopathological Examinations
The
resected tumor and necropsied tissues were stained with hematoxilin-eosin
and alcian-blue/periodic acid Schiff.
Detection
of K-ras Point Mutation at Codon 12
DNA
extraction and detection of the K-ras gene
were made according to the previous described PCR/RFLP method [5]. When
mutation is present, the sample shows two bands, a mutant and a wild type. DNA
extracted from HaP-T1, and from the liver of a 12-week-old hamster without
tumor, were used as positive and negative controls, respectively.
ETHICS
The Syrian
golden hamsters (GN strain), used in the present study, were purchased from the
Nippon Institute for Biological Science (Oume,
Japan), and they were maintained in the Laboratory Animal Center of our
University, in a 12h/12h light/dark cycle, fed standard rations and water ad
libitum. The use of these animals was approved by the Animal Studies
Committee of our University.
STATISTICAL
ANALYSIS
Results
were shown as mean values ± SD. Survival time was compared between the groups
by means of the Mann-Whitney test, while the Fisher exact test was used to
compare the incidence of metastasis. Histopathology and PCR/RFLP analysis for
the detection of lung metastases were compared by means of the Mc-Nemar test. Statistical evaluations were performed by means
of the SPSS/PC+ package running on a personal computer. A two-tailed P value
less than 0.05 was accepted as statistically significant [5].
RESULTS
Success
rate of Implantation, Appearance of Resected Tumors and Survival Time
All 35 animals developed tumors at
the site of implantation, palpable after one week. At the time of resection,
the diameter of the tumor ranged between 18 and 22 mm. Five hamsters in the STI
group, but none in the SCI group, showed adhesion to deep tissue. The skin
covering the tumor was adhered in all animals of both groups. All tumors showed
a well-circumscribed surrounding capsule. Some necrosis was found in the center
of all tumors in the SCI group, and in the center and periphery of all tumors
in the STI group. All resected specimens were confirmed histologically as
moderately differentiated adenocarcinoma. However, tumors of the STI group
consisted of more connective tissue, as shown in Figure 1. Thus, the success
rate of implantation was 100%. The mean survival time in the SCI group was 151±
17.5 days (range: 120-156), and in the STI group was 137± 12.9 days (range:
130-170); the survival time was not significantly different between the two
groups (P=0.200).
Table 1. Comparison
between the subcutaneous cell inoculation (SCI) and subcutaneous tissue
implantation (STI) groups. |
|||
|
SCI |
STI |
P values |
Survival time (days)a |
151± 17.5 |
137± 12.9 |
P=0.200b |
Success of implantation |
15 (100.0%) |
20 (100.0%) |
ND |
Local tumoral recurrence |
0 |
0 |
ND |
Regional metastasis |
13 (86.7%)c |
0 |
P<0.001c |
Distant metastasis |
|
|
|
ND: not done; NS: not
significant |
Figure 1. A subcutaneously
implanted hamster. A. Appearance before tumor resection. B.
Panoramic view after resection. C. Resected specimen without the
covering skin. D. Histopathologic view
showing a moderately differentiated adenocarcinoma (H&E, 200x). |
Follow-up,
Local Recurrence and Findings in the Necropsy
In the follow-up, neither the SCI nor the STI group showed local recurrence at the site of implantation. Thirteen SCI hamsters (86.7%) developed right axillary lymph node metastasis (Table 1), which was palpable between 4 and 12 weeks after the resection. This lymph node in the early stage was movable. After approximately 2 weeks, it became adhered to deep tissue and grew to 6 cm in diameter. When the lymph node reached this size, the animals died. In the necropsy, there was no local recurrence, or macroscopic internal-organ metastases. The metastatic axillary lymph node showed central necrosis (Figure 2). All lymph nodes were confirmed as metastasis at the histopathological and molecular level. The lungs, liver and pancreas did not show any metastases. The remaining two animals of this group without axillary lymph node metastasis did not have any metastases in their internal organs. The cause of death was unknown.
Figure 2. A case from the
SCI group. A. Appearance of the right lymph node. B. Necropsied
lymph node specimen cut sagitally. C. Histopathologic view of the lymph node (H&E, 100x). |
The STI animals did not show any
palpable lymph nodes. They became moribund about 17 to 21 weeks after tumorectomy, and quickly lost weight (about 10 g/day during
the last 3 days of life). The occurrence of axillary lymph node metastases was
significantly lower than that observed in the SCI group (P<0.001). After
death, when necropsy was performed, 10 of 20 animals (50.0%) showed metastases
in the lungs at the histopathological level, which
was significantly higher when compared with SCI group (P=0.002). On the other
hand, the incidence of lung metastases found by PCR/RFLP analysis (Figure 3)
was 80.0%. This figure was significantly higher in comparison with the SCI
group as well as in comparison with the histopathological
findings observed in the same animals (P=0.031). No metastases were found in
the liver and pancreas. The remaining 4 animals in this group did not show any
macroscopic or microscopic metastatic sites.
Figure 3. An ethidium-bromide stained agarose gel electrophoresis of
PCR/RFLP analysis. (1: mutant band; 2: wild band; mk:
marker; pc: positive control (HaP-T1 cell line); li: liver; lu: lung; pa: pancreas; rt: subcutaneously-resected
tumor (STI); nc: normal liver tissue. |
DISCUSSION
With the
purpose of studying the response to the drugs in-vivo, many studies
prefer to use the subcutaneous transplantable tumor models because the method
is simple and monitoring of the results is easy. Since the majority of these
experiments use human cell lines, nude mice have been widely used. However, in
most of these studies, the animals have not been studied over a long period
because of the immunological response which increases as the animal gets older
[6]. Klein and Bevan [7] stated that it could be due to age-related
reconstitution of the nude mouse immune system by endogenous production of
interleukin-2. Thus, monitoring of the local response to the therapy obscures
the validity of long-term treatment studies since tumor reduction may be due to
histocompatibility of the tumor rejection rather than specific anti-tumor
activity. Therefore, we used a homologous model of subcutaneous implantation.
Kyriaziz et
al. [8] subcutaneously injected human carcinoma of the larynx and human
colon carcinoma cell lines into nude mice. After observing for 6 months, they
noted regional lymph node metastases, capsule infiltration and invasion of
lymphatic vessels. However, they did not resect the local tumor, as was done in
the present study. In our preliminary findings, when the local tumor was
resected within 21 days, no metastases were found in either group at 6 months
follow-up (data not shown).
Kyriaziz et
al. [9] implanted pieces of various human tumors, including pancreatic
cancer, in the anterior part of the lateral thoracic region in nude mice. Most
of the implanted tumors metastasized to regional and mediastinal lymph nodes
and the lungs. The authors stated that the lung metastases occurred through
lymphatic and hematogenous routes.
There
appear to be two patterns of arrest of tumor cells at target sites; one is
based on anatomic and physiologic factors, and the other is based on
selectivity [1]. In the pathogenesis of the metastasis cascade, the process
consists of a long series of sequential interrelated steps. Thin-walled venules, like lymphatic channels, offer very little
resistance to penetration by tumor cells and provide the most common pathways
for tumor cell entrance into circulation [1, 10]. Thus, cells entering the lymphatics could have been sequestered via the draining
lymph node in the SCI group. Metastatic cells of STI group could have entered
the venous circulation, via the metastatic cascade, and encountered their first
barrier in the lung, as Nicholson and Poste reported [1]. This could be
explained by the non-specific trapping of metastatic tumor cells, which often
occurs in the first organ encountered by the circulating cells. Greene and
Harvey [11] stated that the localization of a metastatic colony to a particular
organ might depend on the formation of an initial bond between the tumor cells
and the adhesive molecules on the luminal side of the vascular endothelium of
that organ. However, the reasons why lymphatic dissemination was absent in the
STI group should be clarified in the future.
Growth
rates, invasiveness and metastatic behavior of the transplanted tumors differ
depending on the route of implantation [2, 12]. Although the subcutaneous route
of implantation was similar in the present experiments, the histology of the
STI resected tumors showed more developed architecture of the interstitial
tissue. Therefore, the tumoral architecture,
including vascularization, may be an important factor in the process of the
spread of metastatic cells. Moreover, in the STI model, it should be taken into
account that the implanted tumor tissue was derived from the tumor cell line
inoculated subcutaneously in Syrian golden hamsters. The different pattern of
metastasis might be due to the fact that a second step of implantation could
modify the cell biology, thus favoring hematogenous
spread.
In human
pancreatic cancer, the first target organ of hematogeneous
metastases is the liver because the tumoral cells
usually spread via the portal vein [13]. We reported a high liver metastatic
rate when orthotopic implantation of pancreatic tumor
tissue was performed [4]. Thus, other routes of tumor implantation, i.e. intrapancreatic or intraperitoneal, could favor tumor
spread to the liver, more closely resembling to the human pattern. Lung
metastases found in the present experiments may be a consequence of
non-specific trapping of metastatic tumor cells, as stated before.
Ductal
adenocarcinoma, the most common form of pancreatic cancer in humans, is
associated with activation of the K-ras
oncogene in approximately 90% of cases [14]. This has been shown in other
nitrosamine pancreatic cancer induced models to take place an elevated high
rate [9, 15, 16, 17]. The present cell line has had the point mutation
confirmed [4]. Moreover, the early diagnosis of pancreatic cancer in humans may
be carried out by the detection of the K-ras
point mutation. In the present study, the incidence of lung metastases found by
PCR/RFLP analysis was greater than the histopathological
findings (80% vs. 50%) and was statistically significant as well. Thus,
detection of the K-ras point mutation could
identify the micrometastasis, which could not be
found using histopathologic examinations alone.
Therefore, the sensitivity of the detection of the K-ras
point mutation might be greater than that of histology.
The cause
of death of the animals not showing any metastases was unknown. However, we
believe that it was related to the metastasis phenomena, considering that
healthy animals have a two to three year life-span. We think that these animals
could have had metastases in the brain, bone or bone marrow, sites, which were
not observed at necropsy.
In conclusion, homologous SCI and
STI models showed different patterns of metastasis. The event of metastasis
appeared to occur between 21 and 30 days after subcutaneous implantation. The
SCI model combined with tumorectomy may be used as a
lymphatic metastasis model, (simple to monitor) and the STI model combined with
tumorectomy may be used as a hematogenic
metastatic model of the lung. However, these findings are preliminary and
further studies are required to clarify the mechanisms of metastasis. In the
future, the different patterns of metastases after implantation of tumor cells
and tumor tissue should be evaluated using other cell lines.
Received
August 30th, 2000 – Accepted October 4th, 2000
Key
words Animal;
Genes, ras; Injections, Subcutaneous; Mesocricetus; Neoplasm Metastasis; Neoplastic Processes;
Pancreatic Neoplasms; Transplantation, Homologous
Abbreviations SCI: subcutaneous cell
implantation; STI: subcutaneous tissue implantation
Acknowledgements
The authors would
like to thank Mr. Yoshihiro Kuwabara, the Laboratory
Animal Center, and the Molecular Research Genetics Center of our University,
for their support.
Correspondence
Seiji Saito
Third Department of Internal Medicine
Toyama Medical and Pharmaceutical University
2630 Sugitani
Toyama 930-0194
Japan
Phone: +81-76-434.2281
Fax: +81-76-434.5027
E-mail address: sights@ms.toyama-mpu.ac.jp
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