[CANCER RESEARCH 56, 5713-5719,
December 15, 19961
Orthotopic Xenografts of Human Pancreatic Carcinomas Acquire Genetic
Aberrations during Dissemination in Nude Mice'
German Reyes,2 Alberto Villanueva,2 Carmen Garcia, Francisco J. Sancho, Jaume Piulats, Felix LluIs,
and Gabriel Capellá3
Laboratori d'InvestigaciO Gastrointestinal, Institut de Recerca 1G. R., A. V., C. G., G. C.], and Departments ofPathology
i San: Pau, and Laboratori de Bioinvestigació (J. P.], Merck Farina y Qulmica S.A., 08025 Barcelona, Spain
ABSTRACT
Orthotopic transplantation of human tumors in nude mice reproduces
the pattern oflocal growth and distal dissemination. The aim of our study
was to determine the pattern of genetic alterations in human carcinomas
of the exocrine pancreas orthotopically implanted and perpetuated in
nude mice. Eight of the sixteen orthoimplanted human pancreatic carci
nomas
were perpetuated
through
several
passages.
Four
perpetuated
tumors followed distinct patterns of distal dissemination. Point mutations
in the K-rat gene, genetic aberrations in thep53 andpl6 genes, and allelic
losses at retinoblastoma, adenomatous polyposis coli, and deleted in cob
rectal cancer loci were analyzed. Perpetuated tumors maintained the
pattern
ofgenetic
alterations
present in primary
tumors. Five perpetuated
tumors contained K-ras mutations, and all tumors contained p53 and/or
pl6 genetic aberrations. Allelic losses were present in four of the perpet
uated tumors. Additional genetic alterations were detected in 6 of 35
metastases analyzed. Five of 9 peritoneal metastases or malignant ascitic
cells acquired either K-ms or secondpS3 mutations. In contrast, only 1 of
25 liver metastases and none of the lymph node metastases acquired
additional
mutations.
No additional
plt$ gene aberrations
or other allelic
losses were evidenced during tumor dissemination. We conclude that
orthotopically implanted pancreatic carcinomas xenografted In nude mice
show a high degree of genetic stability. Mutations in K-ras and p5.3 genes
can occur in this model system in the more advanced stages of pancreatic
tumor progression,
mainly during peritoneal
dissemination.
INTRODUCTION
Adenocarcinoma of the exocrine pancreas is the fourth leading cause
of cancer death in Western countries (1). Diagnosis of pancreatic cancer
is often difficult, and in most patients tumor is already disseminated when
discovered. Median survival after diagnosis is 6 months, and it has not
improved in the past 30 years (2). Effective treatment is not available, and
in most patients surgical resection is not feasible. Moreover, radio and
chemotherapy have shown limited effectiveness.
The implantation of human tumor cells or fragments in nude mice has
proved useful in the study oftumor growth in vivo. Implantation of tumor
cells in the s.c. tissue of nude mice has been used widely with increasing
success. Recently, a high take rate has been reported for s.c. implantation
of solid fragments of human exocrine pancreas using Matrigel soaking
(3). Tumor cell injection in the corresponding organ of the mice (ortho
topic implantation) has shown a higher uptake rate with preservation of
cell populations with metastatic potential (4). Regarding pancreatic can
ocr, orthotopic implantation of tumor cells has also resulted in the
(F. J. S.] and Surgery (F. L], Hospital de Ia Santa Creu
development of metastatic dissemination (5—7).In a recent study, ortho
topic implantation of human pancreatic tumor solid fragments xc
nografted in nude mice resulted in a 100% take rate and a good repro
ducibility of the tumor metastatic behavior (8).
The majority of carcinomas of the exocrine pancreas (60—100%)
contain mutated K-ras genes (9, 10), and a significant proportion of
tumors (30—70%)harbor mutations in the p53 gene (10—13).The pitS
tumor suppressor gene is inactivated in up to 80% of pancreatic
primary
tumors
(14). Recently,
a novel tumor suppressor
DCC
locus
were
MATERIALS
acquired
during
the metastatic
process.
AND METHODS
Reagents
RPM! 1640, fetal bovine serum, glutamine, penicillin, streptomycin, and
fungizone were purchased from Life Technologies
tamicin
sulfate
from Abbott
was from BioWhittaker
Laboratories
(Madrid,
(Grand Island, NY). Gen
(Walkersville,
MD).
I This
work
was
supported
in
part
by
Comisión
Intetministerial
de
Ciencia
Four 5-week-old male nu/nu Swiss mice weighing 18—22g (Iffa-Credo
Animaux de Laboratoire, L'Abresle, France) were used for tumor implanta
Human Pancreatic Carcinoma Implantation and Perpetuation
Fresh surgical specimens of 16 human pancreatic adenocarcinomas were
Technológico e Industrial Grant 93-0200; FundaciónSALUD 2000; FundaciónCientifica de
in Asociacidn Espafiola Contra ci Cancer of Spain; and Comissionat per Universitats i Recerca
(GRQ93-9501)de Catalunyaand FundacióCatalana de Gastroentemlogia.
exocrine
contributed
equally
to this study.
G. R. was a recipient
cages and water were
autoclaved, and bedding and food was y-ray sterilized.
used
2 Both authors
was
Animals
y TecnologIa
Grant SAL91-0873,FOndOdeInvestigacióne
SanitariasGrant94-0001,and Centro Desan@ollo
Isofluorane
Spain).
tion. Animals were housed in a sterile environment,
Received 7/15/96; accepted 10/16/96.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
gene located
at chromosome 18, the DPC4 gene, has been shown to be altered in
30% of pancreatic carcinomas (15). Other tumor suppressor genes
may also be involved in these tumors (3).
The use of xenografted tumors has facilitated the study of the
allebotype (3), as well as the study of alterations in individual genes
(14) in human pancreatic carcinomas. s.c. xenografts of pancreatic
carcinomas have shown a high degree of genetic integrity compared
with the primary tumor (3).
The aim of this study was to analyze the presence of genetic
alterations in human carcinomas of the exocrine pancreas orthotopi
cally implanted and perpetuated in nude mice and their corresponding
metastasis. We have obtained a library of perpetuated pancreatic
carcinomas with distinct histological, biological, and genetic charac
teristics. A perfect match regarding K-ras mutations, p53 gene aber
rations, and DCC,4 RB, and APC allelic losses has been evidenced
between the human tumor and the pancreatic xenograft. Homozygous
deletions at the pitS gene have been detected in perpetuated tumors.
Moreover, additional genetic aberrations in the K-ras and p53 genes
have been detected, mainly during peritoneal dissemination. No ad
ditional p]6 gene aberrations or allelic losses at the RB, APC, and
(Table
1): 11 primary
tumors,
3 hepatic
metastases,
1 lymph
node
metastasis, and 1 peritoneal metastasis. All tumors were originated in the
pancreas.
None of the patients had received
previous
cytotoxic
of a grant from
the Spanish Ministerio de Educacióny Ciencia.
3 To
whom
requests
for@ta
should
be
addressed,
at
Laboratoti
d'Investigacid
4 The
Gastro
intestinal,Institutde Recerca, Hospital de Ia Santa Creu i Suit Pau, Avgda. Sant Antoni M.
Claret 167,08025 Barcelona,Spain. Phone: 34 3 291 9106; Fax: 34 3 291 9263.
APC,
abbreviations
adenomatous
used
polyposis
are:
DCC,
coli;
deleted
SSCP,
in
single
colorectal
strand
cancer;
RB,
conformation
retinoblastoma;
polymorphism;
LOH, loss of heterozygosity.
5713
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ORTHOTOPIC XENOGRAFI'S OF HUMAN PANCREATIC CARCINOMAS
Table 1 Yieldof implantation. growth characteristics. and dissemination
miceHuman
patterns
of human pancreatic
carcinomas orthoimplanted in nude
mice―Microscopic
elapse
lymph
differentiation―Timebetween passagesDissemination
node metastasiscPeritoneal
seedingPancreatictumorsHistological
patterns in nude
implants
Ascites containing
malignant cellsHematogenous
tumorsI primary
NPI4PoorNo
NP2IModerateNo
——2
——3
NoNo4
local growth——
local growth——
weeksNoNo
NP29Good12—14
NP3OModerateNo
NP3IModerate12—14
——5
PresentNo6
PresentNo7
local growth——
weeksNoPresent
weeksPresentNo
local growth——
weeksNoNo
weeksNoNo
local growth——
weeksNoNo
NP37Moderate7—10
——8
NP38―ModerateNo
NP4OModerate40
NoNo9
NoNo10
NP43Poor28
NP44WellNo
——I
1
NoNoHepatic
metastasis12
NP46Moderate7—10
PresentPresent13 NP18Poor7—10
weeksNoPresent
passages——
local growth——
NP32―na.<4
NP39na.No
——14
——Lymph
node metastasis
——Peritoneal
15
passages——
NP32dna.<4
metastasis16
weeksNoPresent
NP9Poor4
a Perpetuated
tumors
5,
6,
12,
and
16
followed
well-preserved
of
dissemination.
patterns
of
PresentNo
dissemination.
a na., not available.
C _,
not
perpetuated;
No,
absence
d These nonperpetuated pancreatic xenografts showed p53 gene mutation and/or LOH at l7p; p53 status was not assessed in tumors NP3O and NP39; the remaining nonperpetuated
pancreatic xenografts showed no p53 aberration.
therapy. Tumor fragments were obtained in sterile conditions from different
areas of the specimen
and immediately
placed
in RPM!
1640 supplemented
with penicillin, gentamicin, and fungizone. Tissue samples from different areas
were cut into pieces of approximately 2 mm3 that were kept in the medium for
2—4
h until implantation. A total of 10 pieces of each tumor were implanted in
PCR reactionswere carriedout using 500 ng of genomicDNA in the presenceof
radioactivenucleotide([32P]dCFP,2 pCi/PCR tube) in a mixturecontainingPCR
buffer [10mMTris-HCI(pH 8.3),25 ms,iMgCI2,50 mi@i
KCI,and 0.01% gelatin],
100 ma@deoxynucleotide triphosphates, 1 p.M of each primer, and 1 unit of Taq
five nude mice, 2 pieces each. Pieces were implanted in s.c. tissue and in the
DNA polymerase (Life Technologies) in a final volume of 20 s.d. Annealing
temperature,extensiontime, and concentrationof MgC12were optimizedfor each
body-tail of the pancreas (orthotopic implantation). In the last eight tumors
primer set. All reactions
included in the present study, only orthotopic implantation was performed.
Nude mice were anesthetized with isofluorane inhalation. A median or left
lateral laparotomy was performed, and spleen and distal pancreas were mobi
lized. Tumor pieces were anchored to the posterior surface of the pancreas with
a Prolene 6—0suture. Abdominal incision was closed with 4—0Vicryl. After
implantation, mice were inspected twice a week. When no tumor growth was
Five ,.d of the exon 5—6PCR product were digested overnight with restriction
enzyme Hpall to increase sensitivity of the SSCP assay. The remaining PCR
apparent, mice were sacrificed 6 months after implantation. Tumor formation
were performed
in a Hybaid thermal cycler for 35 cycles.
products did not need enzymatic digestion before electrophoresis.Samples were
diluted 1:16 in formamide-dyeloading buffer [95% formamide, 10 mMEDTA,
0.05% bromophenolblue, and 0.05% xylene cyanol] and incubatedfor 3 mm at
95°C.
Tubes were cooled on ice, and 4 p1 of the solutionwere loaded onto a 6%
polyacrylamide/lO%glycerolnondenaturingsequencinggel. Electrophoresiswas
was checked weekly by palpation. The first tumor passage was performed
when an intra-abdominal mass measuring —2cm in diameter was palpated.
Successive passages were performed in two to five animals according to time
elapse for each tumor (Table 1). To assess tumor dissemination pattern, in
every passage at least two mice were kept alive until they were moribund.
carried out at room temperature
After sacrifice, tumors and their metastases were weighed, measured, and
0.1 mai EDTA] for 24 h at 4°C(17). A 5-s.d aliquot of the eluate was subjected to
a new round of PCR amplification. Characterization of mutations was performed
by: (a) cycle sequencing of the amplified product using the AmpliCycle Sequenc
minced into small fragments. At least one fragment was kept in tissue culture
medium and processed to obtain a cell line.5
under S W for 12—15h. Gels were vacuum
dried
at 85°C
and exposedovernightto an X-ray film with intensifierscreen.Only exon
7 primers resulted in coamplificationof a mousep53 gene fragmentthat did not
interfere with mutation analysis. Shifted SSCP bands were excised from the gel
with a razor blade and eluted in 400 @sl
of buffer [0.5 MNH4OAc,0.1% SDS, and
ing kit (Perkin-Elmer,Branchburg,NJ) following the manufacturer's directions;
Detection of K-ras Mutations
and/or (b) cloning of the PCR product using the TA Cloning kit (Invitrogen, San
DNA was extracted from human tumors, pancreatic xenografts, and their
metastases in nude mice following standard protocols. Nude mice tissue (i.e.,
pancreas, liver, and lung) was included in all experiments to rule out contam
ination
by mouse DNA. Mutations
at codons
12 and 13 of the K-ras gene were
detected and characterized by RFLP after PCR amplification of the K-ras first
coding exon using mutant primers as described previously (16). The use of
intronic primers in the first-round amplification of the K-ras gene avoided the
amplification of mouse DNA.
Point Mutations. The wholecodingsequenceof exons4—9
of thep53 gene
was amplified in five PCR reactions using human intronic primers (Table 2). All
Garcia.
A.
Villanueva,
0.
Reyes,
ical, Cleveland,
OH).
Allelic Losses. p53 gene losses were analyzedby an intragenicmicrosat
ellite method as described (18).
Immunohistochemistry
of p53. Two monoclonal antibodies were used
with the avidin-biotin-peroxidase
method: Ab2 Pab 1801 (Oncogene Science,
Uniondale, NY; dilution 1:50) and BP53-12-l (Biogenex, San Ramon, CA;
prediluted), as described previously (19).
Detection ofpl6 Gene Aberrations
Detection ofp53 Gene Aberrations
5 C.
Diego,CA) and sequencingusing Sequenaseversion2.0 (UnitedStatesBiochem
0.
Massd,
R.
M.
Paulés,0. Bachs, G. Capellá, and F. LluIs, Characterization
CaballIn,
A.
Mazo,
A.
Point Mutations. The whole coding sequence of exons 1, 2, and 3 of the
p16 gene was amplified in three independent PCR reactions using human
intronic primers (20). PCR reactions were carried out using 100—200
ng of
genomic DNA in the presence of radioactive nucleotide ([32P]dCTP, 0.1
B.
pCi/PCR tube) in a mixture containing PCR buffer, 100 mi@ideoxynucleotide
of new human pancreatic
cancer cell lines expressing p21 and GADD45 by p53-independent pathways, submitted
for publication.
triphosphates,
0.5 @.LM
of each primer,
5—10% DMSO,
and 1 unit of Taq DNA
polymerase (Life Technologies) in a final volume of 25 [email protected] reactions were
5714
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ORThOTOPIC XENOGRAFTS OF HUMAN PANCREATIC CARCINOMAS
Table 2 Primer sets designedfor
APCGenePrimer
sequences.p531SSCPExonsetAssayPriming
PCR ampl(flcation ofgenes p53 and
regionAmplicon
sizePrimer
4356
bp5'-GGCATTGAAGTCTCATGGA-3'5
5-6408
bp5
7275
8286
bp5'-TCCCCTGCTTGCCACAGGTCT-3'5
,-AGTATGGAAGAAATCGGTAAGAGGTbp5
-3'5
‘
-AATCTGAGGCATAACTGCAC
9225
bp5
-3'2SSCPExon
,-TGGTCCTCTGACTGCTGCTCTT
3'3SSCPExon
-3'5'
‘
-TTCCTCTTCCTGCAGTACTC
-AGTTGCAAACCAGACCTCAG-
3'4SSCPExon
-3'5SSCPExon
‘
-TGGGAGTAGATGGAGCCTGG
3'APC6RFLP
(RsaI)Exon
15 (nucleotide 1458)1
(AspHI)Exon
15 (nucleotide 5037)166
14 bp5
-3'7RFLP
bp5'
‘
-GAGACCAAGGGTGCAGTTAT3'
5‘
-TTGCAGGTAAAACAGTCA3'5
‘
-GTTGAACATCAGATCTGTCCTGCTG‘
-CAAGTTTGTCAAAGCCATTCCAGC
3'5-CCCCTCCAAATGAGTTAGCTGC‘
-CTCTGCTTTATTGTCATCCAATTCA-
performed in a Hybaid thermal cycler for 35 cycles. SSCP analysis was
essentially
performed
as described
above for the p.53 gene, with the only
exception that electrophoresis was carried out at room temperature under 7 W
for 18 h. Exon 2 PCR product was digested with restriction enzyme SmaI to
increase
sensitivity
of the SSCP assay. In this case, coamplification
of a mouse
fragment did not affect SSCP analysis. Characterization of mutations was
performed by cycle sequencing of the amplified product using the AmpliCycle
Sequencing kit (Perkin-Elmer) adding 10% DMSO to the sequencing mixture.
Southern Blot Analysis. PCR-derived probes of exons 1 and 2 were
obtained from normal human pancreas. Sequencing of the cloned PCR product
confirmed the specificity of the reaction. Random primer with [32P]dCTPwas
used for labeling. Filters were hybridized for 12 h and washed under stringent
conditions (65°Cand 0.1% SSC/0.l% SDS). Hybridization with human cDNA
@3-actin probe
was used to test DNA transfer.
Allelic Losses. pitS gene losses were analyzedby meansof two microsat
ellite-length polymorphisms (D9S171 and JFNA)as described (21). The prod
ucts were separated on a 6% polyacrylamide/8
Murea gel, and autoradiography
was performed.
Allelic Losses at the RB, APC, and DCC Loci
The APC locus was studied by intragenic RFLP at nucleotides 1458 and
5037 (Table 2; Ref. 22); the DCClocus
by two intronic MspI polymorphic sites
at D1858 (23); and the RB locus by two intragenic intronic RFLPs (24). PCR
reactions were carried out using 100 ng of genomic DNA in the presence of
radioactive nucleotide (2 @.tCi
of [32PJdCTP each reaction) in a final volume of
20 s.d. Annealing temperature, extension time, and the concentration of MgCI2
were optimized for each primer set. All reactions were performed in a Hybaid
thermal cycler for 35 cycles. After amplification, RFLPIPCR products were
digested overnight with appropriate restriction enzymes and electrophoresed
on 8% polyacrylamide gels.
3'
of tumor cells were occasionally observed in the lymphatic ducts
surrounding mouse pancreas (Fig. 3).
Distal Dissemination Patterns. Distal dissemination was observed
in four of the eight perpetuated tumors (NP9, NP18, NP31, and NP37;
Table 1). The dissemination pattern was tumor specific and stable
throughout several passages (i.e., tumor NP9 is currently at passage 26;
tumor NP18, at passage 22). Malignant cells in the ascitic fluid and/or
peritoneal nodules mainly in the diaphragm were found in all of them
(Table 1). Tumor NP9, obtained from a human peritoneal metastasis,
displayed peritoneal dissemination exclusively (Fig. 4). Tumor NP18,
obtained
from a human hepatic metastasis,
showed a mixed blood-borne
pattern (lung and hepatic metastasis; Fig. 1) along with peritoneal dis
semination. Tumor NP31 showed microscopic peritoneal dissemination
alone. Tumor NP37 produced retroperitoneal and mediastinal lymph
node dissemination as evidenced by the presence of tumor cells@inthe
cortical
sinuses
of lymph
nodes. Finally,
distal dissemination
was not
observed in four perpetuated tumors (NP29, NP4O, NP43, and NP46)
when mice were sacrificed 9 months after implantation (Table 1).
Genetic Alterations in Pancreatic Xenografts and Metastasis.
Mutations at codon 12 of the K-ras gene were present in five of the
eight perpetuated pancreatic xenografts [3 aspartic acid (NP9, NP3I,
and NP37), 1 cysteine (NP29), and 1 valine (NP43) substitution;
Table 3]. No codon 13 mutations were detected. Six of the eight
perpetuated pancreatic xenografts harbored single amino acid substi
tutions in the conserved domains of the p53 gene (Table 3). Two
RESULTS
Yield of Tumor Implantation and Local Growth of Pancreatic
Xenografts. Ten of the 16 human pancreatic carcinomas (62%) grew
as orthotopic implants in nude mice (Table 1). In two of the six initial
cases, s.c. and intrapancreatic growth occurred simultaneously. Time
lapse between passages differed between tumors and remained con
stant through several passages for each tumor (Table 1). Eight tumors
were perpetuated for more than four passages, whereas two tumors
underwent less than four passages (Table I).
For every perpetuated pancreatic xenograft, a big (2—3cm in
diameter) solid mass (Fig. 1) extensively replaced the mouse pancreas
and invaded, in some cases, neighboring organs (i.e., stomach, spleen,
small bowel, and, occasionally, left kidney). A good correlation
between the histological appearance of the primary and the perpetu
ated tumors in mice (Fig. 2) was evidenced. Intrapancreatic perineural
Fig. 1. A big tumor (7) with massive liver dissemination (arrowheads) developed in a
invasion was absent in all cases, in spite of careful searches in a nude mouse 6 months after orthotopic implantation of the human pancreatic tumor NPI8
significant number of serial tissue sections. In contrast, small groups
obtained from a liver metastasis.
5715
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@
@
@
I
@)
I
@s
ORThOTOPIC XENOGRAFTS OF HUMAN PANCREATIC CARCINOMAS
Fig. 2. Microphotograph of primary tumor NP37 (A) and its corresponding pancreatic
implant (B). Note that malignant cells in the implant closely resemble those in the primary
tumor of origin.
animal bearing NP18 tumor differed in K-ras status, with ascitic cells
positive and peritoneal implants K-ras negative. Taken together, all
ascitic malignant cells and the majority of peritoneal implants contained
K-ras aspartic acid substitutions. In contrast, although obtained from a
single perpetuated tumor, only 1 of 21 hepatic metastases was K-ras
positive (Table 3). Because new ras mutations were found during distal
dissemination ofNPl8 xenografts, we analyzed 17 small selected areas of
the NP18 pancreatic xenografts of origin, and no ras mutations were
detected using the same RFLP/PCR technique.
One malignant ascites and one peritoneal metastasis acquired ad
ditional p53 gene mutations at codons 310 and 273, respectively
(Table 3 and Fig. 9). The only lung metastasis analyzed contained an
additional mutation at codon 309 of the p53 gene (Table 3). In
contrast, no additional p53 gene mutations or alleic losses were
detected in hepatic metastasis. Finally, no p16 gene aberrations or
allelic losses at the RB, APC, and DCC loci were acquired during the
metastatic process in the nude mice.
DISCUSSION
In the present study, we have obtained, using orthotopic implantation,
a library of perpetuated human pancreatic carcinomas in nude mice with
distinct histological and metastatic behavior. Histological comparison of
primasy and xenografted tumors revealed that the degree of differentia
tion was stable through several passages. Pancreatic carcinomas are often
associated with a characteristic and prominent desmoplastic host reaction
comprising nonneoplastic stromal cells and leukocytes (14). The enrich
ment for human tumor cells using the xenograft method has been shown
to facilitate genetic analysis (3). The lack of contamination by normal
human cells has been shown to increase the sensitivity of detection of
a, ‘@‘
@
t
‘
.m,.,@..
Fig. 3. Microphotograph
alleic losses and has enabled the identificationof homozygous deletions
,._4
•
showing a cluster of neoplastic cells (vertical arrow) inside
a lymphatic vessel (LV) surrounding the pancreas (P) of a nude mouse bearing an
orthoimplant of the human pancreatic tumor NP37.
tumors (NP9 and NP37) contained a histidine substitution at codon
175 of the p53 gene. Mutations at codons 211, 213, and 246 were
present in tumors NP46, NP43, and NP18, respectively (Table 3). All
tumors harboring p53 gene mutations also displayed p53 protein
accumulation (data not shown). Seven of the eight perpetuated tumors
harbored p16 gene aberrations; pitS microdeletions were evidenced in
tumors NP9 and NP29 (Fig. 5), whereas homozygous deletions were
detected in four tumors (Fig. 6); p16 LOH was present in tumor NP37
(Table 3 and Fig. 7C). Although microdeletions and LOH could be
identified in the corresponding primary tumors, homozygous deletions
could only be detected in human tumors growing in nude mice.
No allelic losses were evidenced at the RB and/or the p5.3 loci (Fig.
7A).
@
@
@
@
In contrast,
allelic
losses
were
detected
at the APC
locus
in two
(i.e., p16 and DPC4 genes) that otherwise would have not been detected
in primary tumors (14, 15, 25). A previous report has shown that both the
allelotype and the presence of point mutations in s.c. xenografts repro
duce the pattern present in their corresponding human pancreatic primazy
tumors of origin (3). In the present study, we have shown that the genetic
status of orthotopically transplanted tumors remains stable through sev
cmi passages in vivo. Because cryostat dissection to enrich for neoplastic
cells in the priinaiy tumors was not used in our study (14), to exclude the
possibility of changes acquired during the xenograft procedure the ge
netic stability was also evaluated by means of the comparisons of K-ras
and p53 status between primary tumors and xenografts in several pas
sages (3).
Using orthotopic implantation, we have obtained a lower take rate
(62%) than the 85% rate reported by Hahn et a!. (3) using Matrigel and
lies
v,-@A
.@
‘-@
;
(NP4O and NP43) of the four informative cases (Fig. 7B); at the DCC
locus in one (NP46) of the three informative cases; and at the pitS
locus in one (NP37) of the eight cases (Table 3 and Fig. 7C).
:‘... .
•
..
:
Acquisition of Genetic Aberrations during Dissemination. Of
@.1
Ak
‘@ “
the 35 metastases analyzed, additional mutations at the K-ras and p53
genes (3 each) were detected in six samples of tumors NP37 and NP18
(Table 3): 3 ascitic fluids, 1 peritoneal metastasis, 1 hepatic metasta
sis, and 1 lung metastasis. Two of the five samples with malignant
cells in the ascitic fluid acquired a K-ras mutation at codon 12 when
compared with their corresponding pancreatic xenograft (Table 3 and Fig.
8). In these cases, sequencing of the PCR product was performed to
Fig. 4. Orthoimplant of human pancreatic tumor NP9 obtained from a peritoneal
confirm the human origin of the amplified fragment. It is interesting that
metastasis produced multiple peritoneal nodules (arrowheads) in the nude mouse model.
SB, small bowel; MV, mesenteric vessel; C, cecum.
ascitic fluid cells and peritoneal metastases obtained from the same
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@
@
—
.
ORTHOTOPICXENOGRAFTSOF HUMAN PANCREATICCARCINOMAS
Table 3 Genetic
aberrations in perpetuated pancreatic xenografts and their ,netastasis according tothe dissemination pattem in
nudemiceXenograft―
gene
gene
genepitS
Metastasis―K-ras
Codon 12bcp53 LOH1st
mutationc2nd
mutation@LOHPeritoneal
Mutation
LOHRB
gene
gene
gene
LOHAPC
LOHDCC
disseminationNP9
——niniPeritoneal
2GAT
——niniAscitic
metastasis
fluid cells
niPeritoneal
NP31
1GAT
Homo'@nininiLymph
metastasis
cellsNP37
node involvement and malignant ascitic
+——niMediastinal
2OAT
+——niLymph
lymph node
+——niAscitic
node
+——niAscitic
fluid cells
+——niHematogenous
fluid cells
(I :0)1
1 bp del'
1 bp del'
1 bp del'
75 CGC—@CAC——1
( I:0)1
75 CGC—I.CAC——1
1
(1:0)
75 CGC—SCAC
GAT (1: 1)1 Exon 7d——
(I :I)Exon
7―ni—
1GAT
2OAT
ni1
—
—
Homcl—
nini
nini
(1:1)1
75 CGC—*CAC——wt
(1:1)1
75 CGC—SCAC——wt
(1: 1)175
COC—SCAC-—wt
75 COC—SCAC——WI
(1:I)1
(1:1)175
COC—SCAC31OAAC—@TAC—wt
2OAT
2OAT
1OAT
1OAT
disseminationNP1
and peritoneal
8
(1:1)246
ATO—SGTO--WI
ATO—SOTO-—WI
ATO—SGTO-—wt
ATG—*GTO273 CGT—SCAT—wt
ATO—*GTG309 CCC—SCGC-WI
(1:1)246
ATO—(ITO——wt
2wt246
--ni-Liver
-—ni—Liver
metastasis
20wt246
1GAT
1WI246
1wi246
——ni-Peritoneal
metastasis
-—ni-Lung
metastasis
--ni-Ascitic
metastasis
——ni—Absence
fluid cells
disseminationNP29
of distal
—ndndndNP4O
2GAT
Homo@-+-NP43
HOmO5—+—NP46
number of samples:
xenografts
(1:I)wt——1
(1 : I)213
2wt21
14
Homo@--+Total
a Pancreatic
1TOT
2WIWI--2011'
were
analyzed
in
bp del'
COA—sTCA———
1 ACT—aA1T---
35
passages
1,
3,
and/or
5.
b Acquired genetic alterations during dissemination are highlighted in italic bold.
C OAT,
aspartic
threonine; All',
acid
substitution;
TOT,
cysteine;
OTT,
valine;
COC,
COT,
and
COA,
arginine;
CAC
and
CAT,
histidine;
ATO.
methionine;
OTO,
valine;
isouleucine; CCC, proline; AAC, asparagine; TAC, tyrosine. wt, wild-type alleles; —
, absence of either mutation or LOH; ni, noninformative;
TCA,
serine;
ACT,
+, presence of LOH;
nd, not done.
d An abnormal SSCP pattern was evidenced in multiple experiments; no mutation was detected by direct sequencing and/or sequencing of the PCR product.
e
@pa deletion
of
1 1 bp at codons
(27—3 1)
)ACO
to OOTACO;
NP29,
deletion
of
1 bp at codon
36 AOT(t)ACO
to AGTACG.
@@Homozygous
deletion at exons 1, 2, and 3.
g Homozygous
deletion
at exon I.
s.c. implantation. The incidence of K-ras mutations in our pancreatic
xenografts (5 of 8) is similar to that detected in Spanish pancreatic tumors
(66%; Refs. 9 and 11). In contrast, p53 gene mutations are overrepre
sented in our pancreatic xenografts. Although six of our eight (75%)
perpetuated tumors contained p53 gene mutations, only 45% of the
primary tumors were p.53 positive,
in good correlation
with data reported
previously (11, 26). It is interesting that although the majority of our
implanted pancreatic tumors that failed to grow lacked p53 gene muta
@
@
A
‘b
‘@b
tions (Table 1), all tumors in the work by Hahn et a!. presented LOH at
l7p (3, 14). Together, these findings suggest that p53 abnormalities
confer growth advantage to tumor cells during tumor implantation in
nude mice. The recent description of a better cell hypoxia endurance of
p53-deficient cells may be related to the higher take rate of p53-positive
tumors perpetuated in nude mice (27). In agreement with previous reports
(14), a high frequency (85%) ofpi6 gene aberrations, mainly homozy
gous deletions, occur in our pancreatic xenografts.
In contrast to the lack of l7p allelic loss, intragenic LOH at the
DCC and APC loci, as well as allelic loss at pitS gene, was detected
in our perpetuated tumors. These findings further support the existing
discrepancy regarding LOH in chromosome 5q in pancreatic carcino
mas (28—30). The APC gene, located at chromosome
B TACG
A TACG
@
NORMAL
A
A
T
A
MUTATED
NP29
Fig. 5. Mutations at exon 1 of the p16 gene in human pancreatic tumor NP29
orthoimplanled and perpetuated in nude mice. A, SSCP/PCR method. Primary tumor and
xenografls in different passages showed an abnormal mobility pattern. Contaminating
nonneoplastic stromal cells contributed to a residual signal in primary tumor (arrowhead)
that disappeared in xenografts. N, normal human tissue; T, primary human tumor; XIII and
X#3, xenografts in passages 1 and 3, respectively.
B, sequencing of the mutated PCR
product showed a T deletion at codon 36 of the p16 gene.
5q2l,
apparently
is not involved in human pancreatic cancer (30). A high percentage of
human pancreatic carcinomas shows allelic loss at chromosome 18q.
Recently, using the xenograft model, the new gene DPC4 has been
identified in neighboring areas of DCC gene located at chromosome
18q (15). Thus, a more detailed analysis of chromosomes 5q and l8q
will be necessary to determine whether the losses detected in our
tumors are restricted to the APC and DCC genes or whether they
affect other neighboring areas within these chromosomes (3, 15).
In contrast to s.c. implantation, in which only local growth occurs,
orthotopic implantation more closely reproduces the metastatic be
havior of the tumor. Dissemination occurred in half (4 of 8) of the
perpetuated tumors, was tumor specific, and was stable through a high
number of passages (i.e., 26 passages in tumor NP9). The use of solid
tumor fragments, in which heterogeneity of tumor cell populations is
likely high, in conjunction with the implantation in a favorable mi
croenvironment, may have contributed to tumor dissemination. Al
though lymphatic, blood-borne, and peritoneal dissemination patterns
were reproducible, limitations in the nude mouse model during dis
semination became apparent. Although all perpetuated tumors caused
5717
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research.
@
@u)@
ORTHOTOPIC XENOORAFTS OF HUMAN PANCREATIC CARCINOMAS
A
NP4O
@
.1L@
NPI8
.I1b@
N43
.k'b
,k'b
_______
.I@4
k's
dL'b@L4
Fig. 6. Homozygous deletions at exon 1 of p16 gene in genomic
DNA obtained from human pancreatic tumors orthoimplanted and
perpetuated in nude mice. A, Southern blot analysis using exon 1
probe. Xenografts NP4O, NP43, and NP46, at different passages,
harbored homozygous deletion; by contrast, deletion was absent in
xenograft NP18. B, Southern blot analysis using human cDNA @3-actin
probe. N, normal human tissue; X#1—X#5,
xenografts at passages 1—5,
B
NP4O
respectively; C, normal mouse tissue.
@
.1i,s
@
NPI8
%llfb%ka,.
.ktb
—
@zI-
NP37
X@[email protected]@-X
118
B
C
NP37
+ 4'
@
NP37
NP4O
114
66
.@
48
Fig. 7. Detection of LOH at p53, APC, and p16 genes in perpetuated human pancreatic
tumors orthoimplantedin nude mice. A, microsatelliteanalysis of p53 gene. No LOH was
detected in NP18 tumor and its metastasis, or in NP37 tumor. B, RFLP/PCR analysis at
nucleotide 1458,exon 15,of the APC gene. No LOH was present in tumor NP37. In contrast,
LOH was found in NP4O,both primary tumor and xenogralt;arrowhead, residual signal due
to contaminationby nonneoplasticcells. C, LOH, as detectedby markerD9S171,whichflanks
thepitS locus,was found in NP37 xenograftand metastasis;LOH was not detectedin primary
tumor due to contaminationby nonneoplasticcells. M, DNA size marker 4X174; N, normal
human tissue; T, primary tumor; X#1, xenograft at passage 1; SM, liver metastasis; AS,
malignant cells in ascites; PM, peritoneal metastasis; NL, lymph node metastasis.
disseminated disease in patients, only half of them metastasized in
mice. Moreover, we did not observe perineural invasion in our model
in spite
of careful
search.
In addition,
lymph
dp@ 1II@
.k43
@‘%
.@
.ktb@L43
-@
sensitivity techniques.
+.c
‘@‘
4. iy
.k'b
_______
majority of peritoneal metastases obtained from mice bearing K-ras
positive or K-ras-negative human pancreatic tumors contained K-ras
mutations. In spite of that, K-ras mutations apparently are not essential
for peritoneal dissemination. In fact, K-ras-positive malignant ascitic
cells coexisted with K-ras-negative peritoneal metastasis in the NP18
K-ras-negative tumor. We do not intend to propose that cells acquire de
novo mutations when they become metastatic. Because we favor the
possibility that metastatic ras-mutated cells are already present in the
pancreatic xenogra.fts, we plan to test these samples using increased
A
NPI8
N43
node
invasion
was
Accumulation of second p53 gene mutations has been occasionally
reported in animal (3 1) and human pancreatic tumor cell lines (1 1),
but not in p53-positive primary tumors in which allelic loss is usually
detected (13). During tumorigenesis, functional copies ofp53 and p16
are presumably rate limiting for tumor growth, and those cells that
have acquired mutation in these genes are then selected for and give
rise to the dominant clones of the neoplasm (14). We have detected
additional mutations in the p.53 gene in two peritoneal metastases and
in one lung metastasis. It is interesting that no additional LOH at Yip
has been evidenced during dissemination in our study. The relatively
low malignant potential of two of the three acquired mutations
(codons 309 and 310) suggests that loss ofp53 function does not play
a significant role in the metastasic process in the nude mouse model.
No additional genetic aberrations in pitS gene were detected during
dissemination. This finding supports the concept that alterations in
pitS gene occur early in pancreatic tumorigenesis (14).
In a separate study, we report that perpetuated tumors and their
metastases have facilitated the establishment of four new human
pancreatic cancer cell lines and a family of eight metastasis-derived
sublines. Derived cell lines from pancreatic xenografts closely resemble
evidenced at a low frequency in our mouse model. Lack of dissemi
nation through these routes is in contrast to their prominent role in
dissemination of human pancreatic carcinoma (31 , 32). This discrep
@
ancy
may
be due
to incompatibility
between
human
and
.
mouse
proteins that participate in the modulation of normal cell-cell and/or
cell-substrate interactions (i.e., nerve cell adhesion molecule and other
members of immunoglobulin gene superfamily; Ref. 33).
K-ras mutations have been shown to occur in early and advanced
65
stages of human carcinomasof the pancreas (34) and have been linked to
49
the metastatic behavior of tumor cells (35, 36). Although the genotype of
pancreatic
xenografts
remained
stable during passages,
additional
genetic
40
alterations in the K-ras and p53 genes were detected during dissemina
lion in our study. It is interesting that additional aspartic acid substitutions
at codon 12 of the K-ras gene were detected, mainly during peritoneal
•S
Fig. 8. Additional mutation at codon 12 of the K-ras gene in malignant cells obtained
dissemination. Moreover, all malignant cells in ascitic fluids and the
from ascites of nude mice bearing tumor NP18. U, uncut PCR product; PC, human DNA
harboring the mutation; MC, mouse DNA. Other abbreviations as in Fig. 7.
5718
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research.
ORThOTOPIC XENOORAFTS OF HUMAN PANCREATIC CARCINOMAS
A
13. Redston, M. S., Caldas, C., Seymour, A. B., Hruban, R. H., da Costa, L. T., Yeo, C. J.,
and Kern, S. E. p53 mutations in pancreatic carcinoma and evidence of common
involvement of homocopolymer tracts in DNA microdeletions. Cancer Res., 54:
3025—3033,
1994.
14. Caldas, C., Hahn, S. A., da Costa, L. T., Redson, M. S., Schutte, M., Seymour, A. B.,
@*‘
4?@ Q
Weinstein, C. L., Hruban, R. H., Yeo, C. J., and Kern, S. E. Frequent somatic
mutations and homozygous deletions of the pitS (MTSI) gene in pancreatic adeno
carcinoma. Nat. Oenet., 8: 27—32,1994.
15. Hahn, S. A., Schutte, M., Shamsul Hoque A. T. M., Moskaluk, C. A., da Costa, L. T.,
Rozemblum, E., Weinstein, C. L., Fisher, A., Yeo, C. J., Hruban, R. H., and Kern,
S. A. DPC4, a candidate tumor suppressor gene at human chromosome l8q21 .I.
B
AG
CT
AG
Science (Washington DC), 271: 350—353, 1996.
16. Villanueva, A., Reyes, 0., Cuatrecasas, M., Martinez, A., Erill, N., Lerma, E., Faire,
CT
@1C@ArgHis@T
@
. ‘I
t.
A
A
C
C
A., LIuls F., and Capellá,0. Diagnostic utility of K-ras mutations in fine needle
aspirates of pancreatic masses. Oaslroenterology, 110: 1587—1594,
1996.
17. Morl, M., and Schmelzer, C. A simple method for isolation of intact RNA from dried
polyacrylamide gels. Nucleic Acids Res., 21: 2016, 1993.
.
18. Kirchweger, R., Zeillinger, R., Schneeberger, C., Speiser, P., Louason, 0., and
Theillet, 0. Patterns of allele losses suggest the existence of five distinct regions of
NORMAL
LOH on chromosome 17 in breast cancer. mt. J. Cancer, 56: 193—199,
1994.
MUTATED
C000N
273
Fig. 9. Second mutations at the p53 gene in periloneal metastasis obtained from mice
bearing tumor NP18. A, SSCP/PCR method of exon 8 showing an abnormal mobility
pattern. Abbreviations as in Fig. 7. B, sequencing of the mutated PCR product showing a
histidine substitution at codon 273 of the p53 gene.
19. Matias-Ouiu, X., Cuatrecasas, M., Musulen, E., and Prat, J. p53 expression in
anaplaslic carcinomas arising from thyroid papillary carcinomas. J. Clin. Pathol., 47:
337—339,
1994.
20. Jen, J., Harper, J. W., Bigner, S. H., Bigner, D. D., Papadopoulus, N., Markowitz, S.,
Willson, J. K., Kinzler, K. W., and Vogelstein, B. Deletion of pitS and p15 genes in
brain tumors. Cancer Res., 54: 6353—6358, 1994.
21. Brenner, A., and Aldaz, C. Chromosome 9p allelic loss and p16/CDKN2 in breast
cancerandevidenceofpitSinactivation
in immortalbreastepithelialcells.Cancer
their corresponding human tumors of origin and show the same K-ras
and p53 status.@In addition, following similar work by Potmesil et al.
(37), we are currently testing the putative antitumoral effect of several
agents, such as gastrointestinal peptide hormones, on the local growth and
distal dissemination of our xenografts of pancreatic cancer.
In conclusion, orthotopic implantation in nude mice facilitated the
perpetuation ofhuman pancreatic carcinomas with distinct biological and
metastatic behavior. Human pancreatic tumors and perpetuated xc
nografts shared the same genotype, which remained stable through sev
cml passages. Neither LOH in genes p13, APC, DCC, and RB nor
additional genetic aberrationsin the pitS gene were detected during distal
dissemination.It is interestingthat additionalgenetic alterationsin K-ras
and p53 genes were detected mainly during peritoneal dissemination.
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5719
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Orthotopic Xenografts of Human Pancreatic Carcinomas
Acquire Genetic Aberrations during Dissemination in Nude Mice
Germán Reyes, Alberto Villanueva, Carmen García, et al.
Cancer Res 1996;56:5713-5719.
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