multiparametric in situ messenger rna hybridization analysis to · vol. 1, 1095-1 102, october 1995...

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.

Research. on October 27, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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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.



p�. �‘

�

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

- : : ,‘ .

bFGF .

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mdr-1 � ,.. . ...

EGFR

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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-



EGFR

md r- 1 E-cad herin


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



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.


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.



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.

-‘ .� �A.

,�.,S,

� #{176}:./ .5’

‘I

. . .5‘ ;#{149} S

C

E

‘‘ . �?

D � � ...S

� tr7’$ � �

‘ �S � � Y\ .

S 5��::�H r5��

�:�‘S��

‘-)S,’S � �

“1 � ;:��

�:e1�0. 3(B) 61D) 9�C) 10(B)gg T NT NT NT N kb

EGFR k #{163} � - 10.8- - � -5.6

TypelV � �- . . S -3.1collagenase � ‘ S. #{149} ;�

mdr-1

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.


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.�




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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