multiparametric in situ messenger rna hybridization analysis to · vol. 1, 1095-1 102, october 1995...
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Vol. 1, 1095-1 102, October 1995 Clinical Cancer Research 1095
Advances in Brief
Multiparametric in Situ Messenger RNA Hybridization Analysis to
Detect Metastasis-related Genes in Surgical Specimens of Human
Colon Carcinomas’
Yasuhiko Kitadai, Lee M. Ellis,
Yutaka Takahashi, Corazon D. Bucana,
Haruyuki Anzai, Eiichi Tahara,
and Isaiah J. Fidler2
Departments of Cell Biology [Y. K., C. D. B., I. J. F.] and Surgery
[L. M. E., Y. I.], The University of Texas M. D. Anderson CancerCenter, Houston, Texas 77030; Department of Surgery, Saitama350-02 Medical School, Saitama, Japan [H. Al; and Department of
Pathology, Hiroshima University School of Medicine, Hiroshima 734,
Japan [Y. K., E. T.]
Abstract
We examined the expression ofseveral genes that regulate
different steps of metastasis in surgical specimens of human
colon carcinomas. The expression of epidermal growth factor
receptor (growth), basic fibroblast growth factor [(bFGF), an-giogenesis], type IV collagenase (invasion), E-cadberin (adhe-
sion), and multidnig-resistant (mdr)-1 (drug resistance) mRNAwas examined using an in situ mRNA hybridization (ISH)
technique and Northern blot analysis. Dukes’ stage C and Dtumors exhibited a higher level of expression (P < 0.05) for
bFGF, type IV collagenase, and mdr-1 mRNA than Dukes’
stage B tumors. The expression level of epidermal growth
factor receptor and E-cadherin did not correlate with the stage
of the disease. The ISH technique revealed intertumoral het-erogeneity for expression of several genes among Dukes’ stage
B neoplasms. In some Dukes’ stage B tumors, we also found
intratumoral heterogeneous staining for bFGF and type IV
collagenase, with the highest expression level at their invasive
edge. In Dukes’ stage C and D tumors, the expression of these
genes was more uniform. These results recommend the suit-
abifity of the multiparametric ISH analysis for metastasis-
related genes to identify individual colon cancers with meta-
static potential.
Introduction
Most deaths from cancer are due to metastases that are
resistant to therapy (1). Therefore, the most urgent question
facing the clinician is whether metastasis has already occurred at
the time of initial diagnosis. The prognosis and choice of ther-
Received 4/14/95; revised 6/12/95; accepted 6/29/95.
1 This work was supported in part by Cancer Center Support Core Grant
CA 16672 and Grant R35-CA 42107 (1. J. F.) from the National CancerInstitute, National Institutes of Health, by American Cancer SocietyCareer Development Award 94-21 (L. M. E.), and by the Josef SteinerFoundation.
2 To whom requests for reprints should be addressed, at Department ofCell Biology, Box 173, The University ofTexas M. D. Anderson CancerCenter, 1515 Holcombe Boulevard, Houston, TX 77030.
apy for most colon cancer patients are based on the stage of their
disease and the metastatic potential of their cancer (2). Tradi-
tionally, these parameters have been determined by microscopic
examination of tissue sections from the primary tumor (3).
However, advances in molecular biology and in our understand-
ing of the process of cancer metastasis have provided new tools
with which to predict the malignant potential of human cancers.
To produce clinically relevant metastases, tumor cells must
complete a series of sequential and selective steps that include
growth, vascularization, invasion, survival in the circulation,
adhesion, and extravasation (1). There have been numerous
reports that show that the metastatic potential of tumors directly
correlates with the expression level of several genes, which
include (among many others) EGFR3 (4-9), bFGF (10-12),
interleukin 8 (13), type IV collagenase (14-20), mdr-i (21), and
carcinoembryonic antigen (22, 23), and inversely correlates with
expression of E-cadherin (24, 25). Most of these studies, how-
ever, focused on single factors to conclude that the expression of
these genes is necessary but in itself insufficient to explain the
metastatic potential of neoplasms. Because discrete steps in the
pathogenesis of metastasis are regulated by independent genes
(26), the identification of cells capable of metastasis requires
multiparametric analysis.
We have recently developed a rapid ISH technique for
detecting the activity of genes that regulate discrete steps of
metastasis (27, 28). The colorimetric ISH technique can be used
on frozen or formalin-fixed, paraffin-embedded tissues to mea-
sure the specific mRNA activity of a particular gene. Examining
tumor tissues for concurrent expression of several genes that
regulate different steps in metastasis should permit identifica-
tion and quantitation of cells with metastatic potential among
those comprising an individual patient’s tumor. This study used
ISH and Northern blot analyses to detect mRNA transcripts of
EGFR (growth), bFGF (angiogenesis), type IV collagenase (in-
vasion), E-cadherin (adhesion), and mdr-1 (drug resistance) in
surgical specimens of HCC. The results suggest that the mul-
tiparametric ISH technique can be used to identify HCCs with
metastatic potential.
Materials and Methods
Surgical Specimens. Immediately after resection, surgi-
cal specimens (tumor and normal mucosa) were divided into
two parts. One was snap frozen in liquid nitrogen (for Northern
blotting), and the other was placed in 10% buffered formalin and
processed for routine histopathology. The frozen samples and
the parallel tissue blocks were received from the Department of
3 The abbreviations used are: EGFR, epidermal growth factor receptor;bFGF, basic fibroblast growth factor; mdr, multidrug resistant; ISH, in
situ mRNA hybridization; HCC, human colon carcinoma.
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1096 ISH Analyses for Metastasis-related Genes
Table I Sequence of oligonucleotide probes
WorkingProbe Sequence 5-3’ (GC content) dilution Reference
EGFR GGA’GCG’CTG’CCC’CGG’CCG’TCC’CGG (87.5%) 1 :800 27,29
bFGF CGG’GAA’GGC’GCC’GCT’GCC’GCC (85.7%) 1:200 30,31Type IV collagenase TGG’GCG’ACG’GCG’CGG’CGG’CGG’CGT’GGC’ (85.2%) 1:500 32
E-cadherin (mixture) TGG’AGC’GGG’CTG’GAG’TCT’GAA’CFG (62.5%)GAC’GCC’GGC’GGC’CCC’lTC’ACA’GTC (75.0%)
1:200 33
mdr-1 (mixture) CAG’ACA’GCA’GCT’GAC’AGT’CCA’AGA’ACA’GGA’CT (53. 1%)
GCATFC’TGG’ATG’GTG’GAC’AGG’CGG’TGA’G (60.7%)1 :200 34
Poly (dT)20 1:1(X)0
Pathology (M. D. Anderson Cancer Center). The specimens
studied were staged according to Astler-Coller modification of
Dukes’ classification. Six Dukes’ stage B, two Dukes’ stage C,
and three Dukes’ stage D surgical specimens of primary colon
carcinomas were evaluated.
Oligonucleotide Probes. Specific antisense oligonucleo-
tide DNA probes were designed complementary to the mRNA
transcripts based on published reports of the eDNA sequences
(Table 1; Refs. 27 and 29-34). The specificity of the oligonu-
cleotide sequences was initially determined by a GenEMBL
data base search using the Genetics Computer Group sequence
analysis program (Genetics Computer Group, Madison, WI)
based on the FastA algorithm (35) showing 100% homology
with the target gene and minimal homology with nonspecific
mammalian gene sequences. The specificity of each of the
sequences was also confirmed by Northern blot analysis. A
poly(dT)2() oligonucleotide was used to verify the integrity and
lack of degradation of mRNA in each sample. All DNA probes
were synthesized with six biotin molecules (hyperbiotinylated)
at the 3’ end via direct coupling using standard phosphoramidile
chemistry (Research Genetics, Huntsville, AL; Ref. 36). The
lyophilized probes were reconstituted to a stock solution at 1
jig/jil in 10 mM Tris (pH 7.6) and 1 msi EDTA. The stock
solution was diluted with Probe Diluent (Research Genetics)
immediately before use (Table 1).
ISH. ISH was performed as described previously (27, 28)
with a minor modification. ISH was carried out using the Mi-
croprobe Manual Staining System (Fisher Scientific; Ref. 37).
Tissue sections (4 jim) of formalin-fixed, paraffin-embedded
specimens were mounted on silane-treated ProbeOn slides
(Fisher Scientific). The slides were placed in the Microprobe
slide holder, dewaxed, and rehydrated with Autodewaxer and
Autoalcohol (Research Genetics), followed by enzymatic diges-
tion with pepsin (38). Hybridization of the probe was carried out
for 45 mm at 45#{176}C,and the samples were then washed three
times with 2X SSC for 2 mm at 45#{176}C. The samples were
incubated with alkaline phosphatase-labeled avidin for 30 mm at
45#{176}C,rinsed in 50 mM Tris buffer (pH 7.6), rinsed with alkaline
phosphatase enhancer for 1 mm, and incubated with a chromo-
gen substrate for 15 mm at 45#{176}C.Additional incubation with
fresh chromogen substrate was done if it was necessary to
enhance a weak reaction. Positive reaction in this assay stained
red. Control for endogenous alkaline phosphatase included treat-
ment of the sample in the absence of the biotinylated probe and
use of chromogen alone.
To check the specificity of the hybridization signal, the
following controls were used: (a) RNase pretreatment of tissue
sections; (b) substitution of the antisense probe with a biotin-
labeled sense probe; and (c) competition assay with unlabeled
antisense probes. Markedly decreased or no signal was obtained
after all of these treatments.
Intensity Quantitation. The intensity of the cytoplasmic
staining was quantified in three different areas of each sample
by an image analyzer using the Optimas software program
(Bioscan, Edmonds, WA). Three different areas in each sample
were quantified to yield an average measurement.
Northern Blot Analysis. Polyadenylated mRNA was ex-
tracted from cultured cells or tumor tissues using the FastTrack
mRNA isolation kit (Invitrogen Co., San Diego, CA). mRNA
was electrophoresed on a 1% denaturing agarose gel contain-
ing formaldehyde, electrotransferred at 0.6 A to a GeneScreen
nylon membrane (DuPont Co., Boston, MA), and UV cross-
linked with 120,000 jiJ/cm2 using a UV Stratalinker 1800
(Stratagene, La Jolla, CA). Hybridizations were performed as
described previously (39). Nylon filters were washed at 65#{176}C
with 30 mM NaCI, 3 msi sodium citrate (pH 7.2), and 0.1%
SDS (w/v).
cDNA Probes. The eDNA probes used in this analysis
were: (a) a 1.4-kb EcoRI eDNA fragment of bovine bFGF (30),
(b) a 3.8-kb XhoI eDNA fragment of human EGFR (kindly
provided by Dr. F. Kern, Washington, DC; Ref. 29), (c) a
1.1-kb EcoRI eDNA fragment of human type IV collagenase
(kindly provided by Dr. W. G. Stetler-Stevenson, Bethesda,
MD), (‘0 a 1.4-kb EcoRI eDNA fragment of human mdr-1
(34), and (e) a 0.6-kb EcoRI-AvaI eDNA fragment of mouse
E-cadherin (kindly provided by Dr. M. Takeichi, Kyoto,
Japan; Ref. 40). The �3-actin eDNA probe was purchased
from Oncor, Inc. (Gaithersburg, MD). Each eDNA fragment
was purified by agarose gel electrophoresis recovered using
GeneClean (BIO 101, Inc., La Jolla, CA) and radiolabeled
with the random primer technique using [a-32P]deoxyribo-
nucleotide triphosphate (41).
Densitometric Quantitation. Expression of the mRNA
was quantified by densitometry of autoradiograms using the
Image Quant software program (Molecular Dynamics, Sunny-
vale, CA), with each sample measurement calculated from the
ratio of the average areas between the specific mRNA transcripts
and �3-actin mRNA transcript in the linear range of the film.
Statistical Analysis. The significance of the ISH data
was determined using Student’s t test.
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p�. �‘
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Clinical Cancer Research 1097
Fig. I ISH of Dukes’ stage B car-cinoma (case 10). Tumor cells (7)expressed a high level of EGFRmRNA as compared with adjacentnormal mucosa (W). The expressionof bFGF, type IV collagenase, and
mdr-1 mRNA was low. These datawere confirmed by Northern blot
analysis (see Table 3 and Fig. 4).HE., hematoxylin and eosin. X275.
Case No. 10
Dukes’ stage B
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E-cadherin
Results
Expression of Metastasis-related Genes in HCC: ISH
Technique. Paraffin blocks of each specimen were cut into
4-p.m sections and stained with hematoxylin and eosin for
histopathological evaluation or processed for ISH analysis. The
integrity of the mRNA in each sample was verified by using a
poly(dT)20 probe. All samples had an intense histochemical
reaction, indicating that the mRNA was not degraded. Next, we
examined the expression level of EGFR, bFGF, type IV colla-
genase, E-cadherin, and mdr-1 genes. For each probe, the in-
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EGFR
md r- 1 E-cad herin
1098 ISH Analyses for Metastasis-related Genes
Fig. 2 ISH of Dukes’ stage D car-cinoma (case 6). Tumor cells con-
currently expressed high levels of
EGFR, bFGF, type IV collagenase,and ,ndr- 1 mRNA. These data wereconfirmed by Northern blot analy-sis (see Table 3 and Fig. 4). HE.,
hematoxylin and eosin. X275.
Case No. 6Dukes’ stage D
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Type IV collacienase
tensity of cytoplasmic staining was quantified by an image
analyzer and compared to the intensity of staining with the
poly(dT)2() probe taken to be the maximum reaction and as-
signed a numerical value of 5.0. Representative examples for the
ISH are shown in Fig. 1 (Dukes’ stage B neoplasm) and Fig. 2
(Dukes’ stage D neoplasm), and the results of all analyses are
summarized in Table 2.
Colon epithelial cells (not tumor cells) expressed detect-
able levels of EGFR, E-cadherin, and mdr-1 mRNA but not
bFGF and type IV collagenase (Fig. 1). Collagenase type IV and
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Table 3 Statistical analysis for expression level of metastasis-related genes: ISH technique
Stage”
mRNA expression index (mean ± SD)
EGFR bFGF Collagenase ,ndr-1
1.3! ± 0.50” i.08 ± 0.82’ 1.53 ± 0.44’
2.20 ± o�34h 2.04 ± 0.27’ 2.38 ± 0.47’
E-cadherin
1.65 ± 0.55
2.16 ± 0.33
B 2.73 ± 0.71
C+D 3.04±1.42a Astler-Coller modificaiton of Dukes’ classification.hp < 0.01.
C p < 0.05.
Clinical Cancer Research 1099
Table 2 Expression level o f metastasis-rel ated genes in HCCs: ISH technique
Case Stage” Histology/differentiation
mRNA expression index’�
EGFR bFGF Collagenase mdr-l E-cadherin
1
3
4
5
1011
8
9
2
67
B
B
B
B
BB
C
C
D
DD
Moderate
WellModerate
Moderate
ModerateModerate
Moderate
Moderate
WellWellModerate
1.9
3.61.9
2.7
3.33.0
1.2
3.3
4.14.6
2.0
2.ic
1.6’1.5
0.9
0.90.9
1.8
2.5
1.92.6
2.2
2.0’2.2’0.9
0.2
0.50.7
1.8
2.0
1.92.5
2.0
1.8
1.41.2
1.2
1.32.3
1.9
2.3
2.02.7
3.0
2.1
2.4
1.5
1.0
1.11.8
1.6
2.2
2.22.5
2.3
(1 Astler-Coller modification of Dukes’ classification.
b The intensity of cytoplasmic staining was quantified by an image analyzer and compared to the intensity of poly(dT)2() staining defined as 5.0.C The value is of the highest staining zone in heterogeneously staining tumors.
bFGF mRNA were also detected in macrophages infiltrating the
interstitial tissue (data not shown). In general, the level of bFGF
(P < 0.01), collagenase type IV (P < 0.05), and mdr-1 (P <
0.05) mRNA was higher in the Dukes’ stage C or D tumors as
compared with Dukes’ stage B tumors (Tables 2 and 3). The
level of mRNA for EGFR and E-cadherin varied among differ-
ent Dukes’ stage B tumors. In contrast, Dukes’ stage C and D
neoplasms exhibited a uniform and intense reactivity with all
probes, including E-cadherin (Table 2).
The present observations agree with the conclusion that
early stage HCCS are heterogeneous for invasion and metastasis,
whereas advanced stage HCCs are more homogeneous for these
properties (42, 43). In addition to intertumoral heterogeneity,
using the ISH technique, we found intratumoral heterogeneity
for expression of bFGF and collagenase type IV within Dukes’
stage B neoplasms. Specifically, the expression of collage-
nase type IV and bFGF was higher in the less-differentiated
lesions and highest at the invasive edge (Fig. 3). Expression
of E-cadherin was detected in both cancer cells and normal
colon epithelium and did not correlate with the Dukes’ clas-
sification.
Expression of Metastasis-related Genes in HCC:
Northern Blot Analysis. To confirm the ISH data, we exam-
med the level of EGFR, bFGF, type IV collagenase, mdr-l, and
E-cadherin mRNA transcripts using Northern blot analysis in 7
of the 1 1 HCC surgical specimens. The data of four represen-
tative samples are shown in Fig. 4, and the data of all seven
specimens are summarized in Table 4. In each sample, we
compared the mRNA level in the carcinomas with that found in
adjacent normal mucosa. Dukes’ stage C and D tumors ex-
pressed higher levels of EGFR, type IV collagenase, bFGF, and
mdr-1 mRNA compared with adjacent normal mucosa. For
example, Dukes’ stage D tumors (cases 6 and 9) expressed
higher levels of EGFR, type IV collagenase, and mdr-1 mRNA
transcripts when compared with Dukes’ stage B lesions (Fig. 4),
closely agreeing with the results obtained using the ISH tech-
nique. The results with Dukes’ stage B neoplasms were more
complicated. For example, in case 3, the ISH technique revealed
heterogeneous staining for type IV collagenase and bFGF,
which was not detected by Northern blot analysis. Northern
blotting also showed that the mRNA level for E-cadherin was
higher in the carcinoma tissue than in the normal mucosa and
that this level did not correlate with staging.
Discussion
We examined the expression of several genes that control
angiogenesis (bFGF), invasion (collagenase type IV), adhesion
(carcinoembryonic antigen), growth (EGFR), and resistance to
chemotherapy (mdr-1) in surgical specimens of human colon
carcinomas. We hypothesized that the metastatic profile of
human colon carcinomas can be identified by multiparametric
ISH analyses. We were especially interested in determining
whether this analysis could differentiate between metastatic
and nonmetastatic Dukes’ stage B tumors. To confirm the
results of the ISH technique, we carried out parallel Northern
blot analyses. The comparison revealed several unexpected
findings.
Research. on October 27, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Fig. 3 Intratumoral heterogeneity for
mRNA expression of bFGF and type IVcollagenase in Dukes’ stage B carcinomas
(A-D, case 1 ; E and F, case 3). Case 1 was
composed of well- and moderately differ-
entiated carcinoma cells (A). Moderately
differentiated carcinoma cells (C) ex-pressed higher levels of bFGF mRNA than
did well-differentiated carcinoma cells (D).The expression of type IV collagenase
mRNA was higher at the invasive edge (F)
compared with the center of the tumor (F).Hybridization of cells with a hyperbiotiny-
lated poly(dT)20 oligonucleotide probe
confirmed mRNA integrity and lack of
degradation (B). X275.
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E-cadherin � � I I I I V� -
�-actin � ..Fig. 4 Representative autoradiographs for Table 2. Surgical specimenswere frozen, and mRNA was then extracted. Polyadenylated mRNA (3jig/lane) was used to detect the mRNA expression for EGFR, type IVcollagenase, mdr-1, and E-cadherin. A �3-actin probe was used as aninternal control. T, tumor; N, normal.
1100 ISH Analyses for Metastasis-related Genes
Tumor tissues consist of multiple cell types including tu-
mor cells, normal stromal cells, endothelial cells, and infiltrating
host cells. Northern blot analysis represents the average level of
mRNA of all of the cells in the sample. In contrast, the ISH
technique can identify the cellular source of the mRNA, as well
as intratumoral heterogeneity for gene expression. Dukes’ stage
C and D neoplasms expressed higher levels of bFGF, type IV
collagenase, and mdr-1 mRNA compared with the majority of
the Dukes’ B lesions. The ISH technique, however, revealed
heterogeneous intratumoral expression for type IV collagenase
and bFGF. In some Dukes’ stage B neoplasms, the invasive
edge as well as satellite lesions exhibited an intense reaction,
whereas the center of the tumor did not. Increased expression of
bFGF mRNA was also found in the less-differentiated zones of
the neoplasms. Northern blotting failed to demonstrate either
pattern of reactive cells.
At the time of diagnosis, malignant neoplasms are hetero-
geneous and contain cells with different biological properties
that include invasion and metastasis (1, 44). Metastases can
originate from preexisting subpopulations of cells (45), which
comprise < 1 % of the tumor (1). The presence of these few
metastasis-competent cells could dramatically alter the prog-
nosis. The present studies were carried out with fresh surgical
II... -4.3.�
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Clinical Cancer Research 1101
Table 4 Expres sion level of metastasis-related genes in human coloncarcinomas: Northern blot analysis
Case Stage”
mRNA expression (TIN ratio)”
EGFR bFGF Collagenase mdr-1 E-cadherin
3
4
10
8
967
B
B
B
C
CDD
1.0 -‘ 1.2 1.1 3.8
1.0 - 1.0 1.0 3.31.7 - 1.0 1.2 2.01.0 - 1.8 1.1 2.0
2.4 - 2.7 3.5 2.5
2.0 - 2.6 2.7 2.4
2.0 - 2.2 2.6 3.3
“ Astler-Coller modification of Dukes’ classification.I, The ratio of densitometric measurements of autoradiographic
signals from Northern blot analysis in colon carcinoma tissues (T) and
corresponding normal tissues (N).
C _� no mRNA transcripts were detected.
specimens of HCC, and, therefore, the data cannot be corre-
lated with clinical course of the disease. Of the seven cases of
Dukes’ stage B tumors, case 1 and case 3 tumors showed a
high level of mRNA expression for bFGF (angiogenesis) and
collagenase type IV (invasion), primarily at their invasive
edge. Although it is highly speculative, our analysis suggests
that these two cases may be Dukes’ stage B tumors with
metastatic potential.
The ISH technique described here can examine the con-
current expression of metastasis-related genes in surgical spec-
imens of HCC. Since this analysis can be performed on forma-
lin-fixed, paraffin-embedded tissues, a large study of archival
material should determine whether the results correlate with
disease recurrence and patient survival. These studies are now
under way.
Acknowledgments
We thank Ricardo Sanchez, Norma Llansa, and Kenneth Dunner,
Jr., for technical assistance, and Lola Lopez for expert assistance in thepreparation of the manuscript.
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1995;1:1095-1102. Clin Cancer Res Y Kitadai, L M Ellis, Y Takahashi, et al. human colon carcinomas.detect metastasis-related genes in surgical specimens of Multiparametric in situ messenger RNA hybridization analysis to
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