Desmoplastic reaction of gastric carcinoma: A light- and electron-microscopic immunohistochemical analysis using collagen type-specific antibodies

Desmoplastic Reaction of Gastric Carcinoma: A Light- and Electron-Microscopic Immunohistochemical Analysis Using Collagen Type-Specific Antibodies TOSHINARI MINAMOTO, MD,* AKISHIOOI, MD,?YOSHIKATSUOKADA,MD,* MASAYOSHIMAI, MD,t YUTAKANAGAI, PHD,*AND ISAO NAKANISHI,MD* The desmoplastic reaction in ten cases of gastric carcinoma was investigated light and electron immunohistochemically by using monospecifie antibodies to collagen types. In addition to type I and III collagens, type V collagen was constantly recognized in the fibrous stroma, increasingly of the scirrhous carcinoma. Type IV collagen delineated the basement membranes of carcinoma nests linearly with occasional discontinuity, whereas in the scirrhous carcinoma, it was present along the thick bundles of collagenous fibers. Immunoelectron microscopic studies revealed that type I and III collagens were distributed on the collagen fibers, and type V collagen was stained in the margin of these fibers. These antibodies also reacted in the rough endoplasmic reticulum of fibroblasts or myofibroblasts in a few cases. Type IV collagen was localized in the periphery of smooth muscle cells, endothelial cells of collapsed capillaries, and myofibroblasts scattered in the stroma of scirrhous carcinoma. Carcinoma cells were not reactive with any antibodies examined. These findings suggest that type V collagen, as well as type I and III collagens, is involved in the formation of desmoplastic stroma, and that these collagens are reactively synthesized by fibroblasts and myofibroblasts in some interaction with invading carcinoma cells. HuM PATHOL 19:815--821, 1988. 9 1988 by W.B. Saunders Com-

pany.

Gastric carcinoma of scirrhous type, which has long been termed "linitis plastica," affects young women with relatively high incidence and rapidly progresses with a miserable prognosis. 1 The scirrhous carcinoma is characterized by excessive deposition of collagens in the stroma of invasive poorly differentiated carcinoma cells. In terms of the formation of the desmoplatic stroma, several mechanisms have been proposed. They include the seeming increase caused by the collapse of preexisting matrix, 2 synthesis by neoplastic cells themselves, 3-6 excessive production by host stroma cells, 7-~~ and some combination o f them; whereas there have been a few reports 7'8 concerning the collagen types and their distribution in the gastric scirrhous carcinoma, both of which have to be elucidated to understand the pathogenesis of tumor desmoplasia. In the present study, we comparatively examine the localization of collagen

From the *Department of Pathology, School of Medicine and the ?Department of Surgery, Cancer Research Institute, Kanazawa University, lshikawa; and the :~Department of Tissue Physiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo. Revision accepted for publication October 7, 1987. Address correspondence and reprint requests to Dr. Minamoto: Department of Pathology, School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa-shi, Ishikawa 920, Japan. Key words: gastric carcinoma, desmoplasia, collagen. 9 1988 by W.B. Saunders Company. 0046-8177/88/1907-002355.00/0

815

types in the gastric carcinomas with mild to marked stromal proliferation by light and electron immunohistochemical techniques, for the purpose of analyzing the possible pathogenesis of desmoplastic reaction. MATERIALSAND METHODS

Preparation of Collagens Collagen was extracted from human placenta by pepsin digestion, then type I, III, IV, and V collagens were isolated essentially according to the differential salt precipitation procedures, 11 with partial modifications. Type III collagen was further isolated by dif9 procedure. I2 For ferentialdenaturation-renaturatton removal of contaminated noncollagenous proteins, each type of collagen isolated above was passed through diethylaminoethyl (DEAE)-cellulose column, a3 Purity of each type of collagen was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 14 and by SDS-PAGE combined with digestion by pure bacterial collagenase derived from Clostridium histolyticum (Advance Biofactures Co, Form III; Lynbrook, NY). 15

Preparation of Type-Specific Anticollagen Antibodies Spraque-Dawley rats were immunized with human type I, III, IV, or V collagen with several booster injections. The antisera against each type of collagen were obtained i week to 10 days after the last booster injection. The antibody titer of each antiserum was monitored by enzyme-linked immunosorbent assay (ELISA) 16 during immunization. Type-specific anticollagen antibodies were purified from crude antisera by cross-adsorption on affinity columns consisting of each of the other types of collagen coupled with activated CH-Sepharose 4B (Pharmacia Fine Chemicals; Uppsala, Sweden), followed by immunoadsorption on affinity columns prepared from the same type of collagen immunized and by elution. 17 The specificity of each antibody was analyzed by the inhibition test, using ELISA, 18 according to the experinaental conditions described in table 1.

The Cases of Gastric Carcinoma Ten cases of gastric carcinoma were examined histologically and immunohistochemically. Gross appearance, histologic type, and amount of stroma of these cases were classified according to the classifica-

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Volume 19, No. 7 [July 1988]

TABLE 1. Experimental Conditions of ELISAInhibition Tests

shown in figure 1. Compositional oLchains of collagen types were identical with those of previous report. 24 Since it is known that pepsin-sensitive sites are present in type IV collagen molecules, 25 several fragments of a chains were found in prepared type IV collagen fraction besides oL1 (IV) and a2 (IV) chains. All of the bands recognized in figure 1 were completely cleaved on analysis of SDS-PAGE after digestion of purified collagen types with bacterial collagenase (data not shown). On analysis by ELISA, affinity purified anticollagen antibodies were selectively reactive with corresponding types of collagen (data not shown). ELISA inhibition tests, as shown in figure 2, indicated that the binding activity of anticollagen antibodies to each type of collagen were exclusively inhibited by the same type of collagen used for immunization, but not by another type of collagen above the concentration of 1,000 ng/mL. T h e s e data c o n f i r m the typespecificity of anticollagen antibodies.

Concentration of antigen* coated in well: 10 p,g/mL Dilution of 1st antibodies?: types I, III, and V, x400; type IV, x 100 Inhibition: 6.25 to 3,200 ng/mL of antigen was preincubated with 1st antibody (24 hr at 4~ Dilution of ALP-labeled 2nd antibody$: x 1,000 Substrate for enzyme (ALP): paranitrophenyl phosphate disolved in diethanolamine buffer (5 mg/mL) ABBREVIATIONS:hr, hour; ALP, alkaline phosphatase. * Antigens: Type I, III, IV, and V collagens. t 1st Antibodies: Rat anticollagen antibodies. $ ALP-labeled 2nd antibody: Alkaline phosphatase-labeled rabbit antirat IgG (Cappel Laboratories; Chochranville, PA).

tion o f the Japanese Research Society for Gastric Cancer 19'2~ (table 2). Histologically, 4 cases were classified poorly differentiated adenocarcinoma with scirrhous stroma, and two of five cases with intermediate stroma showed partially scirrhous reaction. Immunohistochemical Studies

For light microscopic observatin of collagen type distribution, freshly prepared frozen sections were stained by avidin-biotin-complex (ABC) method 2~ and counterstained with methyl green. As to observe the electron microscopic localization of collagen types, frozen sections after fixation with 2% periodate-lysine-paraformaldehyde (PLP) were stained by indirect peroxidase-labeled antibody method'22 '23 and observed under a Hitachi H-500 electron microscope operating at 75 kV without additional staining or stained with uranyl acetate and lead citrate. Working dilutions of the respective anticollagen antibodies were 1:100 for ABC method and 1:10 for immunoelectron microscopic technique. As a negative control for immunohistochemical stainings, normal rat IgG with appropriate dilution was used instead of typespecific anticollagen antibodies. RESULTS Purity of Collagen Types and Specificity of Anticollagen Antibodies

SDS-PAGE pattern of type I, III, IV, and V collagens isolated and purified from human placenta is

Immunohistochemical Findings

Light microscopic observation by ABC method revealed that, in gastric carcinoma with intermediate stroma, type I and III collagens were diffusely distributed in the fibrous stroma (fig. 3A and B), and in addition, type V collagen was also present in the stroma (fig. 3D). Type IV collagen delineated the basement membrane region of carcinoma cell nests linearly with occasional discontinuity (fig. 3C). Type IV collagen was also localized in vascular basement membrane but not in the fibrous stroma. In gastric scirrhous carcinoma (fig. 4A), dense fibrous tissue was mainly composed of type I and III collagens. Type IV collagen, in contrast to carcinoma with intermediate stroma, was irregularly present along the thick bundles of collagen fibers besides carcinoma cells and blood vessels (fig. 4B). Type V collagen was m o r e intensely stained in the fibrous stroma of scirrhous carcinoma than in that of carcinoma with intermediate stroma (fig. 4C). Such an unusual distribution of type IV collagen and increased immunoreactivity of type V collagen were found in the partial scirrhous portion of carcinoma with intermediate stroma.

TABLE 2. Cases of Gastric Carcinoma Examined

Case

Age (years)

Sex

1 2 3 4 5 6 7 8 9 10

54 68 72 51 51 57 69 74 52 61

F F M M M M M M F M

Gross Appearance Borrmann 4 Borrmann 4 Borrmann 4 IIc-advanced Borrmann 3 Borrmann 3 Borrmann 3 Borrmann 3 Borrmann 2 Borrmann 3

Histology and Condition of Stroma por, scirrhous por, scirrhous por, scirrhous por, scirrhous tub2, intermediate (partially scirrhous) tub2, intermediate (partially scirrhous) tub1, intermediate tub 2, mtermediate por, intermediate pap, medullary

ABBREVIATIONS:F, female; M, male; por, poorly differentiated adenocarcinoma; tUbl, well differentiated tubular adenocarcinoma; tub2, moderately differentiated tubular adenocarcinoma; pap, papillary adenocarcinoma

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DESMOPLASTICREACTIONOF GASTRICCARCINOMA [Minamoto et all

12 FI@UI~| t. SDS-PAGEpattern of type I, III, IV, and V collagens isolated from human placenta. Each type of collagen was dissolved in loading buffer and applied on 3% stacking gel and 5% running gel, without reduction or with reduction by 0A mol/L dithiothreitol. Lanes '1 and 2, type I collagen; lanes 3 and 4, type III collagen; lanes 5 and 6, type IV collagen; lanes 7 and 8, type V collagen. Lanes %3, 5, and 7 were electrophoresed without reduction; lanes 2, 4, 6, and 8 were electrophoresed under reduced condition. The electrophoretic position of c o m p o s i t i o n a l chains of coIFagen types are illustrated.

34

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FIGURE 2. ELISAinhibition tests of the antibodies to {A} type 1, {B} type Ill, (C} type IV, and (D} type V collagens. The experimental condition is referred to Table 1. The vertical axis shows percent of optimal density in comparison with that of uninhibited condition, and the transverse axis shows the concentration of collagen [antigen] used for inhibition. 9 type I; O, type III; rq, type IV; I , type V.

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FIGURE 3. Gastric carcinoma with intermediate stroma immunostained by an ABC method using the antibodies to (A) type I, (B) type Ill, (C) type IV, and (D) type V collagens, Approximate magnifications: [A] • [B] • [C] and [D) •

I m m u n o e l e c t r o n microscopic studies showed that, as to either cases with intermediate or scirrhous stroma, type I and III collagens were distributed on the collagen fibers (fig. 5A) and type V collagen was stained in the margin of these fibers. In addition, these antibodies were reactive in the rough endoplasmic reticulum of fibroblast and myofibroblast in some cases (fig. 5A and D). Type IV collagen was exclusively localized in the basal lamina in the cases with intermediate stroma (fig. 5B), while it was localized in the periphery, corresponding to basal lamina, of smooth muscle cells, endothelial cells of collapsed capillaries, and myofibroblasts scattered in the fibrous stroma of scirrhous carcinoma (fig. 5C). Carcinoma cells were not reactive with the antibodies examined either by ABC method or immunoelectron microscopic technique in any of the cases presented here. Normal rat IgG, used as a control instead of anticollagen antibodies, showed no positive reaction with the specimens of any cases.

carcinoma is mainly composed of type I and III collagens, and that, on light microscopic observation, an increased immunoreactivity of type V collagen in the stroma is one of the characteristics of the scirrhous carcinoma. The same stromal features have been demonstrated in breast carcinoma 1~ and pulmonary scar carcinoma. 26-28 Therefore, it is suggested that type V collagen, as well as type I and III collagens, is involved in the formation of desmoplastic stroma. Similarly, an increased content of type V collagen has been shown both biochemically and immunohistoc h e m i c a l l y in benilgn f i b r o t i c lesions such as a t h e r o s c l e r o s i s , 2 9 ' 3 ~ p u l m o n a r y fibrosis, 31 hypertrophic s c a r Y and postburn wound tissueY In vitro study demonstrates that type V collagen forms fine fibrils with a D-periodic banding pattern and directly interacts with type I collagen in forming hybrid fibrils. 34 Based on these data, type V collagen appears to be increased not only in desmoplastic t u m o r stroma but also in the process of progressive fibrosis. Another stromal characteristic of scirrhous carcinoma was an unusual localization of type IV collagen at the light microscopic level. An unusual localization means that, though it has been confirmed that type IV collagen is one of the intrinsic components of and exclusively localized in basement membrane, 35 in

DISCUSSION The distribution pattern of collagen types in the present study indicates that fibrous stroma of gastric

818

DESMOPLASTICREACTIONOF GASTRICCARCINOMA [Minamoto et al]

stroma of scirrhous carcinoma 9 These components reactive with the antibody to type IV collagen seem to correspond to an unusual locallization of type IV collagen on a light microscopic observation 9 There have been several arguments about the origin of desmoplastic tumor stroma, and it is not yet elucidated whether desmoplastic stroma is produced by mesenchymal cells or invading tumor cells themselves, or is caused by an interaction between these two kinds of cells. Recent immunohistochemical studies using anticollagen antibody suggest that the desmoplastic matrix components are derived from reactive proliferation of host stroma. 7,s'l~ However, to date there is no evidence indicating that mesenchymal cells such as fibroblasts in the tumor tissue excessively synthesize collagen or other matrix proteins in vivo, either on autoradiography or immunohistochemical techniques. In the present study, immunoelectron microscopic observation showed that antibodies to type I, III, and V collagens reacted in the rough endoplasmic reticulum of fibroblast and myofibroblast presented in the fibrous stroma. These findings favor the presumption that matrix collagen of desmoplastic carcinoma is mainly produced by fibroblasts and myofibroblasts. This study may be the first immunohistochemical representation suggestive of mesenchymal cells in desmoplastic tumor synthesizing collagen in vivo. The cell, which we called myofibroblast, is known to have combining ultrastructural characteristics of fibroblast and smooth muscle cell; that is, the nuclear indentation, well developed rough endoplasmic reticulum, microfilaments with dense bodies, and focal basal lamina-like structure. 9'36 Myofibroblasts are frequently found in desmoplastic tumors, including scirrhous carcinoma, and thought to play an active role in desmoplastic reaction.9,'ao, a6-39 The majority of cell culture studies of the mechanism of tumor desmoplastic reaction have demonstrated that conditioned medium or preformed matrix of cultured carcinoma cells stimulates the cell growth rate and collagen and elastin synthesis of mesnchymal cells.S'4~ has been shown that carcinoma cells in culture synthesize little or only trace amounts of collagen or elastin, s'41'42 Our immu" nohistochemical study also showed that carcinoma cells in any cases examined were not reactive with anticollagen antibodies 9 Considering the data of the present study and previous investigations, it may be concluded that desmoplastic stroma of scirrhous carcinoma is formed by reactive stromal proliferation and that the major part of matrix collagens are excessively synthesized by fibroblasts and myofibroblasts in some interaction with invading carcinoma cells9 Since several properties of the generation of tumor stroma have been thought to be similar to the wound-healing response, 43 tumor cells may constantly secrete some factors such as plasminogen activator and tumorderived growth factors, which directly or indirectly stimulate the migration, growth, and collagen biosynthesis of mesenchymal cells9

? 9 t "~

~J r

7 .

-

j,

-

f

FIGURE 4. Gastric scirrhous carcinoma stained (A) with hematoxylin-eosin and immunostained with the antibodies to (B) type IV and (C) type V collagens. Approximate magnifications: [A], [B], and [(2] x125

scirrhous carcinoma, type IV collagen was unexpectedly localized in the fibrous stroma, particularly in the margin of thick collagen bundles other than basement membrane region. Sakakibara 4 and Nagai et al. 8 have also observed the same findings on light microscopic immunohistochemical study of gastric scirrhous carcinoma, although they couldn't adequately explain it. Immunoelectron microscopic observations in this study revealed that type IV collagen delineated the basal laminae of smooth muscle cells, collapsed capillaries undetectable by light microscopy, and myofibroblasts scattered in the fibrous 819

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FIGURE 5. Immunoelectron microscopic localization of type Ill, IV, and V collagens in gastric carcinoma. (A) Type III collagen is diffusely distributed on the stromal collagen fibers and its immunoreactivity is also found in the rough endoplasmic reticulum of a fibrobtast in case 7. (B) Type IV collagen exclusively delineates the basal lamina of a carcinoma cell nest with occasional discontinuity in case 7. (C) Type IV collagen is localized around the smooth muscle cell and myofibroblast scattered in the fibrous stroma of scirrhous carcinoma of case 2. (D) Type V collagen is stained along the collagen fibers, and its immunoreactivity is also recognized in the rough endoplasmic reticulum of the myofibroblast in case 2. [C] and [D] are counterstained with uranyl acetate and lead citrate. Approximate magnifications: [A] x8,100, [B] x7,200, [C] x4,500, and [D] x8,200. Ca, carcinoma cell; Fb, fibroblast; Mf, myofibroblast; Sm, smooth muscle cell.

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DESMOPLASTICREACTIONOF GASTRICCARCINOMA [Minamoto et al]

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