Sucrase-isomaltase expression and enterocytic ultrastructure of human colorectal tumors

Int. J . Cancer: 44, 238-244 (1989) 0 1989 Alan R. Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer

SUCRASE-ISOMALTASE EXPRESSION AND ENTEROCYTIC ULTRASTRUCTURE OF HUMAN COLORECTAL TUMORS Bella CZERNICHOW', Patricia SIMON-ASSMANN' , Michkle KEDINGER', Christiane ARNOLD', Michel PAR ACHE^, Jacques MAREsCnUx3, Alain ZWEIBAUM' and Katy H A ~ N ' " 'Unit& 61 INSERM 3, avenue Molikre, 67200 Strasbourg; 2Centre Alexis Vautrin, 54511 Nancy; 3Pavillon Chirurgical A, Hospices Civils, 67000 Strasbourg; and 'Unit6 178 INSERM, Bat. INSERM, 16, avenue P . Vaillant Couturier, 94807 Villejuif, France.

We report the relative frequency of sucrase-isomaltase (Sl) antigen expression in human colonic adenocarcinoma (22/57), in peritumoral mucosa taken next to the tumor (31141) or distant from it (29/42) as well as in 21/23 polyps. Our results are based on indirect immunofluorescence with a monoclonal antibody (MAb) specific for human intestinal SI. A regular and intense expression of SI occurred only in 6 tumor specimens. In the remaining 16 SI-positive tumor samples, labelling was heterogeneous, Le., scattered over more or less extensive areas. A similar irregular staining pattern was also found in polyps and in peritumoral mucosa, irrespective of i ts distance from the tumor. Electron microscopic examination of 19 car- cinomas mostly revealed altered brush-border membrane features, irrespective of histological SI staining pattern. Brush-border enzyme activities of sucrase, alkaline phosphatase and maltase showed no difference between tumor specimens and peritumoral mucosa, but aminopeptidase was depressed in the former. Sucrase activity was extremely low (mean values I. I to I .8 mU/mg protein) and rose only excep- tionally to 17.5 mU/mg prot.

The first evidence of enterocytic differentiation character- ized by apical brush-border membrane and brush-border- associated hydrolases specific to the small intestine in 2 human colon carcinoma cell lines was provided by Pinto et al. (1982, 1983). These 2 cell lines, Caco-2 and HT-29, were established by Fogh et al. (1977). Caco-2 cells exhibit these differentiation characteristics spontaneously under standard culture conditions (Pinto et al., 1983) whereas HT-29 cells are differentiated only when adapted to growth in glucose-free medium (Pinto et al., 1982; Zweibaum et al., 1985; Wice et al., 1985).

The assumption that expression of brush-border digestive hydrolases in colonic cancer cells corresponds to fetal resur- gence was supported by the following observations: (a) the same hydrolases are transiently present in the fetal colon between 12 and 30 weeks (Zweibaum et al., 1983; Lacroix et al., 1984); (b) the molecular form of sucrase-isomaltase synthe- sized by HT-29 and Caco-2 cells corresponds to the high- molecular-weight form of the enzyme (precursor of sucrase and isomaltase subunits; Hauri et al., 1979) and is found in fetal small and large bowel (Zweibaum et al., 1983, 1984; Triadou and Zweibaum, 1985); (c) fetal sucrase-isomaltase, like that of the cell lines, has a faster mobility in SDS PAGE than the adult small-intestinal high-molecular-weight form of the enzyme (Triadou and Zweibaum, 1985; Zweibaum et al., 1989).

Further investigations performed with MAbs produced against human small-intestinal hydrolases were extended to tumors developed in nude mice from injected HT29 and Caco- 2 cell lines and to primary colorectal tumors from patients (Zweibaum et al., 1984). This approach consisted of the immunohistological detection of the brush-border enzymes, sucrase-isomaltase (SI), aminopeptidase N (APN) and dipepti- dylpeptidase IV (DPPIV). A pattern of positive staining for SI, APN and DPPIV similar to that found in tumor xenografts of HT-29 and Caco-2 cells and in 16-week fetal colon was observed in 7 out of 27 tumor specimens. The reaction was con- fined to the apical luminal borders of moderately differentiated adenocarcinoma. Of particular interest was the presence of SI

at the surface epithelium of juxta-tumoral mucosa in 9 out of 1 1 tested samples, corresponding in 4 cases to SI-negative tumors.

In this preliminary study, however, SI expression was not analyzed in the mucosa distant from the carcinoma, although structural alterations have been emphasized (Dawson and Fi- lipe, 1976; Shamsuddin et al . , 1982), nor in polyps. In addition, immunohistochemical findings have never been correlated with ultrastructural brush-border membrane features from which quite consistent abnormalities have been described in various primary colorectal tumors (Imai and Stein, 1963; Car- doso et al., 1971; Dawson and Filipe, 1976; Riddell and Levine, 1977; Mughal et al., 1981; Hickey and Seiler, 1981; Balazs and Kovacs, 1981; Ahnen et al., 1982; Shamsuddin et al., 1982).

Our present study was conducted in order to screen immunohistological expression of sucrase-isomaltase in colonic tumors and in mucosa adjacent or distant from tumors, in parallel with ultrastructural brush-border differentiation characteristics and/or with quantitative determination of enzyme activities. We also report preliminary results concerning the incidence of SI immunoreactivity in polyps.

MATERIAL AND METHODS

Tissue specimens Primary colonic carcinoma tissue samples were obtained

from 57 patients undergoing surgical resection at the Centre Alexis, Vautrin, Nancy, and at the Chirurgie A Department, Hospices Civils, Strasbourg. Of these, 20 were from the prox- imal colon (1 cecum, 15 right colon, 4 transverse colon) and 37 from the distal colon (26 sigmoid and 11 rectum); 18 were well differentiated, 30 moderately and 8 poorly differentiated; 1 was a colloid-type adenocarcinoma.

In 45 surgical specimens, mucosa adjacent to the tumor or 5-30 cm distant from the surgically removed tumor was also evaluated.

Samples of 23 colorectal polyps were included in our study. Of these, 21 corresponded to adenomatous polyps of tubulo- villous type (Morson, 1962; Morson et al., 1976). They were associated with adenocarcinoma (12 cases) or were removed from patients with familial polyposis (9 cases). The 2 remaining polyps were diagnosed as glandular dysplasia and as large hyperplastic polyp, respectively.

Control colonic mucosal biopsies without histological abnormalities were obtained by endoscopy at the Clinique MC- dicale B Department, Hospices Civils, Strasbourg, from 6 patients whose primary symptoms were abdominal pain or con- stipation.

5To whom reprint requests should be addressed.

Received: March 3, 1989.

SI EXPRESSION IN HUMAN COLON CANCER 239

Preparation of tissues All specimens were received immediately after excision and

rapidly sampled under the initial direction of a diagnostic pa- thologist. Small pieces of tissue from the resected specimens were cleaned in Ham F10 culture medium. Unless otherwise stated, the samples were processed in parallel for immunocy- tochemistry, enzyme determination and electron microscopy. For diagnosis, the tumor samples and the polyps were processed using conventional procedures.

Antibody The HBB2/614/88 MAb specific for human small-intestinal

sucrase-isomaltase (Hauri et al., 19856) was obtained from Dr. H.P. Hauri (Biocenter, Base1 University, Switzerland). This antibody has already been used in a study of SI expression in human adenocarcinoma (Zweibaum et al., 1984), in studies concerned with SI deficiency in children (Hauri et al., 1985a) and with biosynthesis of SI in Caco-2 cells (Hauri et al . , 19856) and HT-29 cells (Trugnan et al., 1987).

Indirect immunojluorescence of sucrase-isomaltase For localization of SI, the samples were fixed with 2%

paraformaldehyde in piperazine 1,4-bis (2-ethanesulfonic acid) (PIPES) PH 7.0 for 1 hr at +4"C and stored in PIPES buffer enriched with sucrose (10%) at 4°C until use. The fixed samples were then embedded in Tissue-Tek 11 (Miles, Naperville, IN), frozen in freon cooled in liquid N, and kept at -70°C until processing. Frozen 5-6 pm sections were collected on gelatin-coated slides and incubated with the HBB2/614/88 MAb (ascites fluid:1/75) for 2 hr. After washing, sections were further incubated for 2 hr with fluorescein-coupled sheep anti- mouse IgG antibodies (1/200, Institut Pasteur, Paris). For the controls, sections were incubated without MAbs. Preparations were mounted under coverslips in phosphate-buffered glycerol supplemented with 0.1 % paraphenylenediamine. The sections were observed with a Leitz epifluorescent microscope. In sev- eral cases, after observation the cryosections were stained with periodic-acid Schiff for light microscopy.

Enzymatic analysis Enzymatic analysis was performed on membrane fractions

prepared from the tissue specimens according to the protocol used for the preparation of brush-border-enriched fractions (Schmitz et al., 1973). Sucrase activity was assayed according to Messer and Dahlqvist (1966), lactase according to Kol- dovsky et al. (1969), alkaline phosphatase according to Garen and Levinthal (1960), and aminopeptidase according to Maroux et al. (1973). Proteins were assayed by the method of Lowry et al. (1951). All enzyme activities are expressed as milliunits per milligram of protein: one unit is defined as the activity that hydrolyzes 1 pmole of substrate/min under our experimental conditions.

Electron microscopy For transmission electron microscopy, specimens were fixed

in 0.2 M cacodylate-buffered 2% glutaraldehyde (PH 7.4) for 2 hr at 4"C, post-fixed in cacodylate-buffered 1 % osmium tetrox- ide (PH 7.4) for 30 rnin at 4°C and embedded in araldite. Thin sections (1 pm) were stained with toluidine blue. Ultra-thin sections were stained with uranyl acetate and lead citrate before observation using a Philips 300 electron microscope.

RESULTS

Immunohistological detection of sucrase-isomaltase Colorectal neoplasms. As listed in Table I, 22 out of 57

tumors examined were positive for SI. However, only 6 of them showed intense and continuous staining lining the apical border of tumor cells (Fig. la ) . In the remaining 16 positive

tumors, staining was uneven within single tumor samples; label was located over more or less extensive regions (Fig. 16,c). In all specimens, SI staining appeared as a narrow band lining cell apices either along surface epithelium, crypts or tumor tubules. Extent of staining could not be correlated to tumor differentiation.

Mucosaji-om adjacent and remote regions of the colon cancer. As listed in Table I, positive but heterogeneous staining was detected in 31/41 mucosal specimens adjacent to the tumor and in 29/42 samples taken 5-30 cm from the surgically removed tumor. In almost all of these specimens, the staining pattern corresponded to a scattered or patchy topc?graphy, partly due to the presence of goblet cells (Fig. 2a,b). This labelling was observed at the surface epithelium (Fig. 2a), in groups of neighboring glands (Fig. 2a) or in limited areas inside a single gland (Fig. 26). In juxta-tumoral specimens the staining pattern appeared comparable to that seen in remote regions. It was, however, more extensive in certain cases, due to elongation of crypts. This can be related to thickening of the mucosa in the vicinity of tumors, which is considered as an anomaly by Riddell and Levine (1 977).

Colorectal polyps. Immunofluorescence of sucrase-isomaltase was analyzed in 23 polyps. Intense and regular expression of SI lining the epithelium was observed in 2 cases, corresponding respectively to one hyperplastic polyp (Fig. 3a) and to 1 polyp exhibiting glandular dysplasia (not shown). Among 21 adenomatous polyps of the tubulo-villous type, irregular distribution, closely similar to that found in pen-tumoral specimens, was observed in 18 samples. The presence of SI could not be correlated to the morphology of the specimens. SI staining was almost negative in 3 cases (Fig. 36).

Control colonic mucosa. Biopsy specimens originating from 6 different patients were examined for the presence of SI. One biopsy was completely negative. In the 5 remaining cases a scattered distribution of SI-positive colonocytes could be observed (Fig. 4a). This staining pattern was independent of the anatomic region of biopsy sampling (right, transverse colon, or sigmoid) . Ultrastructural study

This study was performed in 19 tumor fragments, 6 peritumoral specimens and 3 polyps. From a general point of view, ultrastructural alterations affected most, if not all, of the cells in the tumor samples examined. Special attention was directed towards brush-border membrane features in tumors, whether or not these expressed SI. Various pictures could be observed. For example, in a specimen showing regular and intense SI- expression microvilli were either sparse and short (Fig. Id), or hypertrophied with long rootlets of microfilaments extending deep into the cytoplasm (Fig. ljj, or completely distorted (Fig. 1 6 , ~ ) . In other tumor specimens (Fig. 3), in which small SI- positive areas were obvious, most cells displayed subnormal

1ABLE I - PRESENCE OF SI-BlMU?JOSTAINING IN COLORECTAL ADENOCARCINOMA AND PERI? UMOKAL COLONIC .ZIUCOSA

Pen-tumoral mucosa'

Differentiation Adenocarcinomal Immediate vicinity Distant of the tumors (<2 cm) (5-30 cm)

nz + + + - n + + + - n + + + -

Poor 8 1 1 6 5 0 3 2 7 0 4 3 Moderate 30 4 9 17 22 1 16 5 21 0 14 7 Good 18 1 6 11 13 0 10 3 14 0 11 3 Colloid 1 1 1 1 Total 57 6 16 35 41 1 30 10 42 - 29 13 'Staining pattern: + + regular and intense; + heterogeneous; - negative.-*n:

number of subjects.

240 CZERNICHOW ET AL.

FIGURE 1 - SI-immunostaining (a-c) illustrating the uneven distribution of SI between colorectal tumor specimens. The staining produced by SI-MAb is either intense and regular (a), restricted to a minute area (b) or located over more or less extensive areas (c). Electron micrographs (d-fl show various ultrastructural brush border membrane features which are encountered within a single tumor sample. The surface epithelial cells of the tumor illustrated in la ) exhibit microvilli which are either short and sparse (d), clustered together (P) or hypertrophied and extending long rootlets of their filamentous core into the apical cytoplasm If?. Arrow indicates glycocalyceal bodies. Bars: 30 p (a<); 2 p (d-f). Moderately differentiated adenocarcinoma of the right colon, patients JT (a,d-fl and ML (b). Well-differentiated adenocarcinoma of the rectum, Dukes’ stage B, patient AT (c).

apical microvilli similar to those described in Figure ld-f. In the highly SI-positive hyperplastic polyp illustrated in Figure 3a, microvilli appeared barely recognizable in some areas of

the section (Fig. 3c) , while in others they were intermingled with edematous apical blebs (Fig. 3 4 or exhibited prominent intracytoplasmic rootlets (Fig. 3e) .

FIGURE 2 - SI-immunostaining of colonic mucosa adjacent to the tumor (a) of patient AT, illustrated in Figure lc, and of colonic mucosa taken at 20 cm from the tumor (b) of patient RP (moderately differentiated adenocarcinoma of the right colon, Dukes’ stage B). Note more or less patchy labelling of cells located at the surface epithelium (a) and lining cryptal lumen (a,b). Electron micrograph of a juxta-tumoral specimen (c ) shows well-organized brush border at the apex of vacuolated epithelial cells. Bars: 30 p (ah); 2 p (c).

SI EXPRESSION IN HUMAN COLON CANCER 24 1

FIGURE 3 - SI-immunostaining and electron microscopy of polyps. Hyperplastic polyp displaying regular expression of ST lining epithelial cells (a). Almost SI-negative adenomatous polyp (b). Hyperplastic polyp exhibiting at the electron microscopic level various brush-border membrane features (c-e). Microvilli barely recognizable (c), intermingled with edematous apical blebs (d) or extending long rootlets into the apical cytoplasm (e ) . Bars: 30 p (a,6); 2 (c-el.

FIGURE 4 - SI-immunostaining and electron microscopy of control colonic mucosa. Focal apical labelling of SI (a) and normal ultrastructure of brush border membrane (b). Bars: 30 (u); 2 p (6).

242 CZERNICHOW ET AL.

Examples of brush-border membrane features in tumor- adjacent mucosa and in control colon are illustrated in Figure 2c and 4b, respectively. Due to patchy or focal occurrence of SI-positive cells, no actual correlation with the ultrastructurally more or less well-differentiated microvilli could be established. Highly vesiculated cells present in juxta-tumoral mucosa (Fig. 2c) are not necessarily artefactual. Their occurrence has already been mentioned as an atypical feature of transitional mucosa, in addition to other changes such as those observed in the composition of mucin produced by goblet cells (Dawson and Filipe, 1976). Activity of brush-border hydrolases

Quantitative determination of brush-border enzymes was performed in adenocarcinoma and in corresponding peritumoral mucosa (Table 11). The mean average values of sucrase, maltase and alkaline phosphatase did not vary between tumor specimens and mucosal samples taken close to or distant from the tumor. Aminopeptidase activity was reduced by about 50% in the tumors as compared to peri-tumoral mucosa.

As regards sucrase activity, individual values (Fig. 5) indi- cate that in 68% of the adenocarcinomas, there was no activity at all (values below 1 mU/mg prot.); in the remaining cases values ranged between 1.0 and 7.8 mU/mg prot. Distribution of sucrase activity in pen-tumoral and in tumor samples showed only minor differences, although one fragment of the former showed 17.5 mU/mg. In addition, sucrase activity measured in the hyperplastic polyp (Fig. 3a) corresponded to 13.8 mU/mg prot. (not depicted in Fig. 5).

DISCUSSION

This study c o n f i i s some previously reported observations (Zweibaum et al., 1984) about the occurrence of sucrase- isomaltase in human colonic adenocarcinoma, and provides new information about the relationship between tissular SI antigen expression, ultrastructure and quantitative brush-border enzyme activities.

Immunoreactivity of SI could be detected in 22/57 primary colonic tumors as well as in 31/41 juxta-tumoral mucosa and in 29/42 mucosa taken at various distances from the tumor. Marked heterogeneity appeared in the staining pattern of SI. Intense and regular SI labelling of tumors similar to that seen in fetal colon at mid-gestation (Zweibaum et al., 1983) was observed in only 6 tumors. In the 16 other SI-positive tumors, the staining pattern was uneven. The remaining 35 tumor samples were negative. Uneven SI expression was also a charac- teristic of most peri-tumoral specimens, whatever their distance from the tumor, and of colorectal polyps. However, intense SI staining lining the surface epithelium was seen in 1 dysplastic and in 1 hyperplastic polyp. Further screening of SI in hyperplastic polyps would be of interest in relation to other fundamental biological differences occurring between hyperplastic and adenomatous polyps (Kaye et al., 1973; Bara et al., 1983; Bedossa et al., 1987).

I; @ 7

I

“1 5

17j @ :I 5 I

.*

. . *+AT

0 n J 0 n=10 n = 7 n = 8 n = 9 n z l

5 I@ 4

3

2 -

RP .? .

*. : %

. - .. * .. : : * . I

n-12 n=13 n = 1 0 n = 6 n = 4 n - 4

FIGURE 5 - Histogram of sucrase-specific activities in 56 specimens of adenocarcinoma, in 45 mucosal samples taken in the immediate vicinity (<2 cm) of the tumor and in 49 samples taken 5-30 cm away from it. JT, AT, RP and ML correspond to specimens from which tissular SI expression is illustrated in Figures In-c and 2n,b.

TABLE 11 - SPbCItIC BRLSIi-BORDLR ENLYMt ACTIVITIFS (rnLirng PROT r SIMJ IK COLONIC AD1 NOCARCINOMA AND PFRI-TIMORAI MIICOSA

Sucrase Maltase Alkaline ohosohatase Aminopeptidase

Adenocarcinoma 1 . 1 f 0.3, 10.5 k 1.9 55.2 -C 3.6 35.3 f 2.7 (n = 57) (n = 54) (n = 53) (n = 54)

Peritumoral mucosa 1.8 f 0.3 9.8 f 0.7 52.2 k 5.2 61.0 * 12.3 (Vicinity <2cm) (n = 46) (n = 40) (n = 43) (n = 45)

Peritumoral mucosa 1.5 ? 0.2 9.3 f 0.8 52.4 f 4.4 78.4 f 10.9 (remote: 5-30 cm) (n = 49) (n = 46) (n = 49) (n = 451

‘(n) = number of individual cases

ST EXPRESSION IN HUMAN COLON CANCER 243

An unexpected observation was the occurrence of spot-like apical immunofluorescence in glands of 516 control colonic mucosal biopsies. Such a staining pattern was similar to that of some mucosal specimens distant from the tumor but was not observed in our previous study (Zweibaum et al., 1984) where the normal colonic mucosa analyzed came from kidney donors having undergone irreversible brain damage. Thus, it is not known whether the control patients used in this study were tumor-bearers or not.

Concerning the relationship between ultrastructural brush- border membrane features and immunofluorescence of SI in colonic adenocarcinoma, completely abnormal microvilli could correspond to intense immunohistological staining of SI, whereas more normal-appearing brush borders could be observed within tumor specimens displaying focal SI labelling. We cannot exclude the possibility that such differences might be inherent to differences in parallel but not identical fields examined within a single tumor. Altered brush-border membrane features have been considered as markers of colorectal adenocarcinoma (Hickey and Seiler, 1981). This is in contrast with the well-organized brush borders of differentiated CaCo-2 and HT-29 cells, but can be explained by the fact that culture conditions may suppress the deleterious environmental influ- ences of inflammation, necrosis or infection which prevail in primary tumors.

Overall quantitative data on brush-border enzyme activities showed no difference between tumor specimens and peritumoral colonic mucosal samples except in the case of aminopeptidase which was depressed in the former. This remains unexplained. Concerning sucrase activity, the mean levels detected in the tumor and the peri-tumoral specimens were re- markably low (1.1-1.8 mU/mg prot.). Individual data show more elevated sucrase activity in I tumor specimen (7.8 mu/ mg prot.), in 1 mucosal sample next to a colloid-type adenocarcinoma (17.5 mU/mg prot.) and in the single hyperplastic polyp (1 3.8 mU/mg prot.). Such amounts remained far below those measured in CaCo-2 cells (1,565 mUimg prot.) and even in HT-29 cells grown in sugar-deprived medium (18-32 mu/ mg prot.) where 50-60% of the population was labelled with SI-antibody (Zweibaum et al., 1985; Trugnan et al., 1987; Chantret et al., 1988). No clear-cut relationship could be es-

tablished between intense tissular distribution of SI and brush- border enzyme activities. This should be related to difficulties in the procedure of isolating altered apical plasma membranes. Indeed, the procedure of Schmitz et al. (1973), which is well adapted for small-intestinal enterocytes and cancer cell lines, is less efficient for colonocytes (Hauri et al., 1986) and could be even less efficient for solid tumor specimens.

Taken together, our results suggest that heterogeneous SI expression is frequent in human colorectal tumors and even more so in peri-tumoral mucosa, although regular and intense staining of tumors appears rather uncommon. This conclusion is in agreement with screening experiments of SI activity and immunoreactive expression in 20 established human colon carcinoma cell lines showing that only Caco-2 and HT-29 cells exhibit this particular marker, although the presence of villin is consistent (Chantret et al., 1988). This latter actin-binding protein present in intestinal brush-border microvilli is produced in all cancer cell lines tested, independently of polarization and brush-border membrane differentiation (Chantret et al., 1988; Dudouet et aE., 1987). In contrast to SI, its regularity and intensity in colonic adenocarcinomas is impressive (Moll er al., 1987; Carboni et al., 1987; West et al., 1988) although not restricted to them (Moll et al., 1987).

Finally, low incidence of SI expression within human colorectal tumors and established cell lines derived from them also reflects the cellular heterogeneity of adenocarcinomas which contain multiple populations of cells with different properties (Arends et al., 1985). It also strengthens the idea that the established SI-positive cell lines Caco-2 and HT-29 correspond either to cell types which were predominant in the original tumor or to a subpopulation of cells which was selected from the tumor during the initial phase of culture.

ACKNOWLEDGEMENTS

We are very grateful to Mrs. E. Alexandre and Mrs. C. Leberquier for skillful technical assistance and to Mrs. C. Haf- fen for photographic processing. We thank Drs. M. Doffoel, S. Evrard and R. Paleau for their help in providing biopsies, surgical specimens and histopathological diagnosis. Financial support was given by INSERM, ARC and the FRMF.

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Sucrase-isomaltase expression and enterocytic ultrastructure of human colorectal tumors

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