327 @ 1990 The Japanese Society of Pathology
Immunoelectron Microscopic Localization of Human Type 1 1 1 Collagen in Human Ga st ro intest ina I Ca rcinoma s
Haruo Ohtani' and Akira Ooshima2
Ultrastructural immunolocalization of human type 111 collagen/procollagen was investigated in 28 cases of human gastrointestinal carcinoma by the pre-embedding method using a monoclonal antibody. In addition to immunoreactivity on collagen fibers, amorphous or finely fibrillar immunoreactive substances were observed in the extracellular matrix adjacent to the plasma membrane and in the cisternae of rough endoplasmic reticulum (rER). These substances were considered to represent type 111 procollagen and/or collagen before fiber formation. Cytoplasmic recesses containing immunoreactive fibers were also observed. These findings related to collagen production were detected in stromal fibroblasts in 16 of 28 cases and in carcinoma cells in six of 28 cases. The positivity rate of these findings was lower in diffuse-type gastric carcinoma. The present study thus clarified further details of collagen production and secretion, and revealed that carcinoma cells may also be involved i n the production of stromal collagen in some cases as well as stromal fibroblasts. Acta Pathol Jpn 40: 327-334, 1990. Key words : Cancer stroma, Collagen, Fibroblast,
lmmunoelectron microscopy
INTRODUCTION Interaction between cancer cells and the surrounding stroma is one of the key aspects in the mechanism of tumor cell invasion (1, 2). Deposition of increased amounts of stromal collagen (desmoplastic reaction) is Received September 7, 1989. Accepted for publication January 24, 1990. '2nd Department of Pathology, Tohoku University School of Medicine, Sendai. '1 st Department of Pathology, Wakayama Prefectural Medical Collage, Wakayama. Mailing address : Haruo Ohtani, M.D. (AgSJA), 2nd Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Preliminary results of this study were presented at the 47th Annual Meeting of the Japanese Cancer Association in Tokyo, 1988 (No. 1285).
one of the major host reactions to carcinoma growth. Ultrastructurally, fibroblasts in the cancer stroma are usually endowed with well developed rough endoplasmic reticulum (rER) and other cell organelles (3, 4) showing the features of active protein secretion. These findings suggest that stromal collagen originates from fibroblasts. In recent years, however, there has been some debate on the origin of stromal collagen, i.e. whether it is produced by carcinoma cells (5, 6), stromal fibroblasts (7-9) or both(l0). Such debate has been focused particularly on scirrhous carcinoma of the stomach, and no conclusion seems to have been reached. The major components of stromal collagen in human carcinomas are types I and Ill (9, 11, 12). However, immunoelectron microscopic studies on these components are limited in number (9) and no thorough investigation of the intracellular immunolocalization of collagen has yet been performed in cancer stroma. Immunoelectron microscopy is useful for the study of intracellular localization of a substance in relation to its production. In the present study we attempted to clarify the intracellular immunolocalization of type Ill collagen/ procollagen using a monoclonal antibody (MoAb). We use the term fibroblasts in a wider sense to include rnyofibroblasts because of their morphological continuity and their similar phenotypic features (4, 13, 14) despite the presence of some minor differences (15, 16). Most of the stromal fibroblasts investigated in this study had various amounts of microfilament bundles in their peripheral cytoplasm.
MATERIALS AND METHODS Antibody: The method of generation and the properties of the mouse monoclonal antibody (MoAb) against human type Ill collagen have already been described elsewhere (17). This MoAb reacts with the helical determinant of human type Ill collagen and not with types I,
328
lmmunoelectron Microscopy of Collagen (Ohtani and Ooshima)
11, IV, V, or VI collagens, and thus theoretically it reacts with human type Ill procollagen (Ooshima et a/., manuscript in preparation). Tissues : We studied 9 colorectal carcinomas and 19 gastric carcinomas surgically resected at Tohoku Rosai Hospital and Tohoku University Hospital in Sendai. All the patients were Japanese. The gastric carcinomas were classified into intestinal (differentiated) type (six cases) and diffuse (undifferentiated) type (13 cases) (18, 19). All diffuse-type carcinomas had a scirrhous stroma. All the colorectal carcinomas were classified as well or moderately differentiated adenocarcinomas (20). Histologically normal gastric (one case) and colonic mucosa (two cases) and gastric mucosa with intestinal metaplasia (one case) were obtained from carcinoma cases. Carcinoma tissues were taken from invasive growth areas beyond the muscularis mucosae. lmrnunostaining method : Details of our method have already been described (21). Immediately after resection, specimens (measuring 6 x 6 x 2 mm) were fixed with perioda te-lysine-pa raformaldehyde fixative (PLP) (22) for 6 - 8 h at 4°C. In seven cases, specimens were fixed with microwave radiation in 4% paraformaldehyde-0.1% glutaraldehyde followed by postfixation as described by us previously (23, 24). After immersion in 10% nonirnmunized goat serum, 6pm-thick cryostat sections were incubated with MoAb (undiluted culture medium) for 48-72 h at 4°C and then overnight with peroxidase-conjugated F(ab)’, fragment of goat anti-mouse IgG antibody (Cappel, Malvern, PA ; diluted 1 : 60). The reaction products formed with 3,3’diaminobenzidine tetrahydrochloride were visualized by osmification, and the specimens were embedded in Epon 812. Ultrathin sections were stained with lead citrate for 1 min and observed with a JEOL lOOB electron microscope in all cases. Specimens with massive necrosis or severe neutrophilic infiltration were excluded. For the negative control, the adjacent sections were processed by replacing the MoAb with phosphatebuffered saline (PBS) or MoAb preabsorbed with human type Ill collagen (Sigma, St. Louis, MO). In some cases, the specimens were pretreated with 0.03-0.3% Triton X100 in PBS, or saponin was added to the MoAb solutions (0.03%) to facilitate antibody penetration (25). For light microscopy, the neighboring sections were processed in the same way and counterstained with 1% methyl green in all cases. Ceff identification : In diffuse-type carcinoma, stromal cells and carcinoma cells are mixed. However, differentiation between the two was made with certainty
by the following features. Stromal fibroblasts are spindle-shaped cells with abundant rER stacks, and they never have mucin granules or microvilli. Dissociated-. type carcinoma cells are oval-shaped cells with nuclear atypia. They may have mucin granules, microvilli and intracytoplasmic lumina, and may be joined t o neighboring cells by desmosomes with convergence of intermediate filaments. Light microscopic morphometry : Hematoxylin-eosinstained sections ( 3 p m thick) were prepared from paraffin blocks of five intestinal-type gastric, seven diffuse-type gastric, and six colonic carcinomas from among the cases examined by immunoelectron microscopy. The distribution density of stromal fibroblasts was measured by counting fibroblasts in a randomly placed square, 100 x 100 p m in size, using a x 2 0 0 microscopic field. Twenty squares were counted in all cases. The nuclear area of stromal fibroblasts was measured with a semi-automatic image-analyzer (Canon BX-1, Tokyo) using 5 4 randomly taken photographs (three per case, x 1,000 final magnification). The data were summed in each group and statistical analysis was carried out among the three groups by student’s t test.
RESULTS Light and electron microscopic immunohistochemistry Microwave fixation gave better preservation of subcellular structures without significant changes in the results (23, 24). Light microscopically, the stromal area was positively stained in all cases (Figs. l a , 2a). Diffuse cytoplasmic immunoreactivity was not observed in carcinoma cells in any cases. The negative controls uniformly showed no reaction (Figs. 1b, 2b). UItrast ruct ura IIy, the immuno react io n products with the MoAb were observed on collagen fibers, measuring 50-90 nm in diameter, in all cases (Figs. 3, 5, 6b). The immunoreactivity was also observed on amorphous or finely fibrillar substances in the extracellular matrix near the surface of the stromal fibroblasts (Fig. 3) in 16 of 2 8 cases. Collagen fibers were admixed with them (Fig. 3, inset). Cisternae of the rER were positive in seven cases, mostly very sparsely (Fig. 3) and rarely abundantly (Fig. 4). Membranes of the Golgi apparatus were positive in one case. These reactivities were thought to represent the localization sites of human type Ill procollagen, thus indicating the cells producing it (see Discussion for details). Stromal fibroblasts were found to have cytoplasmic recesses containing im m uno react ive col-
Acta Pathologica Japonica 40 (5) : 1990
Figure 1. Diffuse-type gastric carcinoma stained with MoAb against human type Ill collagen (a) and a control section (b, preabsorbed with human type Ill collagen). Counterstained with methyl green.
329
Figure 2. Intestinal-type gastric carcinoma stained with MoAb against type 111 collagen (a) and a control section (b, preabsorbed with human type Ill collagen). Counterstained with methyl green.
Figure3. A stromal fibroblast in colonic carcinoma (stained with MoAb against human type Ill collagen). lmmunoreactive fibers and amorphous substance are co-localized in the extracellular matrix adjacent t o the plasma membrane (arrows). Surface of some of the rER is positive (small arrow). Asterisk indicates cytoplasmic reactivity beneath the plasma membrane. Fixed by microwave radiation. Scale b a r y 0 . 2 p m . Inset shows higher magnification of amorphous immunoreactivity admixed with collagen fibers near the cell surface. ~ 5 7 , 0 0 0 .
330
lmrnunoelectron Microscopy of Collagen (Ohtani and Ooshima)
Figure 4. A stromal cell in colonic carcinoma. Dilated cisternae of rER are imrnunoreactive. Fixed by microwave radiation. Scale bar= 1 um.
Figure 5. A stromal cell in intestinal-type gastric carcinoma showing two cytoplasmic recesses containing immunoreactive collagen fibers (arrows). Fixed by conventional method. Scale b a r = l urn.
lagen fibers in three cases (Fig. 5). In five cases, the amorphous substances were also observed in the cytoplasm between the cytoplasmic membrane and the rER (Fig. 3, asterisk). The number of cases showing the amorphous or finely fibrillar substances was larger in intestinal-type gastric carcinoma and colorectal carcinoma (5/6 and 7/9,respectively) than in diffuse-type gastric carcinoma (4/13)(Table 1). lmmunoreactive amorphous or finely fibrillar s u b stances were also observed around or within carcinoma cells in one case of intestinal- and three cases of diffuse-
type gastric carcinoma, and two cases of colorectal carcinoma (six cases in totaI)(Figs. 6a, b). The distribution pattern of the substances was the same as that in fibroblasts, and the substances were not observed in the extracellular matrix remote from carcinoma cells. In one case each of intestinal- and diffuse-type gastric carcinoma among these six cases, carcinoma cells possessed cytoplasmic recesses containing immunoreact ive collagen fibers and rER showing immunoreactivity on its membranes (Fig. 7). In non-neoplastic mucosa, fibroblasts in the lamina
Table 1. Occurrence Rate of lmmunoreactivity Related to Production and Secretion of Type Ill Collagen/Procollagen (number of positive caseslnumber of total cases)
Site of immunoreactivity Amorphous substances in extracellular matrix near cell membrane
Gastric carcinoma diffuse type intestinal type 4/13 (F) 5 / 6 (F) 3/13 (Ca) 1 / 6 (Ca)
Colorectal carcinoma 7 / 9 (F) 219 (Ca)
Total 16/28 (F) 6/28 (Ca)
rER
Cytoplasmic recess with collagen fibers
0113 (F) 1/13 (Ca)
1 / 6 (F) 1 / 6 (Ca)
0/9 (Ca)
2/9 (F)
3/28 (F) 2/28 (Ca)
Total
4/13 (F) 3/13 (Ca)
5/6 (F) 1 / 6 (Ca)
7/9 (F) 2 i 9 (Ca)
16/28 (F) 6/28 (Ca)
F, fibroblast; Ca, carcinoma cell. Total means cases in which at least one of the three immunoreactive sites was positive.
331
Acta Pathologica Japonica 40 (5): 1990
Figure 6. An isolated carcinoma cell in diffuse-type gastric carcinoma. a. Low magnification. Scale b a r = l pm. b. Higher magnification of the area indicated by an arrow in Fig. 7a showing co-localization of extracellular amorphous/finely fibrillar substances and collagen fibers. Fixed by conventional method. Scale bar=0.2 pm.
propria generally possessed less rER than those in the cancer stroma (4),and immunoreactive rER was sporadically detected in two of four cases. Pretreatment of the specimens with Triton X-100 or saponin did not augment the intracellular immunoreactivity. Light microscopic morphometry The data are summarized in Table 2. Both the distribution density and nuclear size of stromal fibroblasts were significantly lower in diff use-type gastric carcinoma (approximately half) than those in intestinal-type gastric and colorectal carcinomas. There were no significant differences in the results between the last two groups.
DISCUSSION
Figure 7. Part of another carcinoma cell in the same case as in Fig. 6 showing immunoreactivity on the surface of the rER (small arrows), which is similar to that in Fig. 3. Large arrow indicates by con. immunoreactive collagen fibers. ~ u = n u c l e ~ Fixed ~. ventional method. Scale bar=0.2 pm.
On the basis of a previous ultrastructural autoradiographic study (26),the biosynthetic and secretory pathway of procollagen has been postulated to proceed from the rER t o the Golgi apparatus, and then via secretory granules to the ground substance. lmmunoelectron microscopic studies on collagen/procollagen in experimental animals have shown positive staining in the cisternae of the rER, dilated Golgi saccules, and vacuoles (or granules) (27-32). 1” human cancer stroma, Minamoto et a/. (9)reported similar results in stromal fibroblasts of gastric carcinoma but not in the cancer
332
lmmunoelectron Microscopy of Collagen (Ohtani and Ooshima) Table 2. Summary of Results of Light Microscopic Morphometry (expressed as meanflSD) Gastric carcinomas Diffuse type Intestinal type
Colorectal carcinoma
Distribution density (cell count per 10,000 pm2)
10.8+ - 3.4a
22.0k 5.5
22.7k 5.6
Nuclear area (pm’) (n=number of fibroblasts measured)
27.8+ 17.2a (n=595)
43.9k22.4 (n=393)
44.4k25.9 (n=642)
Fibroblasts
a
smaller than those in the other two groups (p
Immunoelectron Microscopic Localization of Human Type 1 1 1 Collagen in Human Ga st ro intest ina I Ca rcinoma s
Haruo Ohtani' and Akira Ooshima2
Ultrastructural immunolocalization of human type 111 collagen/procollagen was investigated in 28 cases of human gastrointestinal carcinoma by the pre-embedding method using a monoclonal antibody. In addition to immunoreactivity on collagen fibers, amorphous or finely fibrillar immunoreactive substances were observed in the extracellular matrix adjacent to the plasma membrane and in the cisternae of rough endoplasmic reticulum (rER). These substances were considered to represent type 111 procollagen and/or collagen before fiber formation. Cytoplasmic recesses containing immunoreactive fibers were also observed. These findings related to collagen production were detected in stromal fibroblasts in 16 of 28 cases and in carcinoma cells in six of 28 cases. The positivity rate of these findings was lower in diffuse-type gastric carcinoma. The present study thus clarified further details of collagen production and secretion, and revealed that carcinoma cells may also be involved i n the production of stromal collagen in some cases as well as stromal fibroblasts. Acta Pathol Jpn 40: 327-334, 1990. Key words : Cancer stroma, Collagen, Fibroblast,
lmmunoelectron microscopy
INTRODUCTION Interaction between cancer cells and the surrounding stroma is one of the key aspects in the mechanism of tumor cell invasion (1, 2). Deposition of increased amounts of stromal collagen (desmoplastic reaction) is Received September 7, 1989. Accepted for publication January 24, 1990. '2nd Department of Pathology, Tohoku University School of Medicine, Sendai. '1 st Department of Pathology, Wakayama Prefectural Medical Collage, Wakayama. Mailing address : Haruo Ohtani, M.D. (AgSJA), 2nd Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Preliminary results of this study were presented at the 47th Annual Meeting of the Japanese Cancer Association in Tokyo, 1988 (No. 1285).
one of the major host reactions to carcinoma growth. Ultrastructurally, fibroblasts in the cancer stroma are usually endowed with well developed rough endoplasmic reticulum (rER) and other cell organelles (3, 4) showing the features of active protein secretion. These findings suggest that stromal collagen originates from fibroblasts. In recent years, however, there has been some debate on the origin of stromal collagen, i.e. whether it is produced by carcinoma cells (5, 6), stromal fibroblasts (7-9) or both(l0). Such debate has been focused particularly on scirrhous carcinoma of the stomach, and no conclusion seems to have been reached. The major components of stromal collagen in human carcinomas are types I and Ill (9, 11, 12). However, immunoelectron microscopic studies on these components are limited in number (9) and no thorough investigation of the intracellular immunolocalization of collagen has yet been performed in cancer stroma. Immunoelectron microscopy is useful for the study of intracellular localization of a substance in relation to its production. In the present study we attempted to clarify the intracellular immunolocalization of type Ill collagen/ procollagen using a monoclonal antibody (MoAb). We use the term fibroblasts in a wider sense to include rnyofibroblasts because of their morphological continuity and their similar phenotypic features (4, 13, 14) despite the presence of some minor differences (15, 16). Most of the stromal fibroblasts investigated in this study had various amounts of microfilament bundles in their peripheral cytoplasm.
MATERIALS AND METHODS Antibody: The method of generation and the properties of the mouse monoclonal antibody (MoAb) against human type Ill collagen have already been described elsewhere (17). This MoAb reacts with the helical determinant of human type Ill collagen and not with types I,
328
lmmunoelectron Microscopy of Collagen (Ohtani and Ooshima)
11, IV, V, or VI collagens, and thus theoretically it reacts with human type Ill procollagen (Ooshima et a/., manuscript in preparation). Tissues : We studied 9 colorectal carcinomas and 19 gastric carcinomas surgically resected at Tohoku Rosai Hospital and Tohoku University Hospital in Sendai. All the patients were Japanese. The gastric carcinomas were classified into intestinal (differentiated) type (six cases) and diffuse (undifferentiated) type (13 cases) (18, 19). All diffuse-type carcinomas had a scirrhous stroma. All the colorectal carcinomas were classified as well or moderately differentiated adenocarcinomas (20). Histologically normal gastric (one case) and colonic mucosa (two cases) and gastric mucosa with intestinal metaplasia (one case) were obtained from carcinoma cases. Carcinoma tissues were taken from invasive growth areas beyond the muscularis mucosae. lmrnunostaining method : Details of our method have already been described (21). Immediately after resection, specimens (measuring 6 x 6 x 2 mm) were fixed with perioda te-lysine-pa raformaldehyde fixative (PLP) (22) for 6 - 8 h at 4°C. In seven cases, specimens were fixed with microwave radiation in 4% paraformaldehyde-0.1% glutaraldehyde followed by postfixation as described by us previously (23, 24). After immersion in 10% nonirnmunized goat serum, 6pm-thick cryostat sections were incubated with MoAb (undiluted culture medium) for 48-72 h at 4°C and then overnight with peroxidase-conjugated F(ab)’, fragment of goat anti-mouse IgG antibody (Cappel, Malvern, PA ; diluted 1 : 60). The reaction products formed with 3,3’diaminobenzidine tetrahydrochloride were visualized by osmification, and the specimens were embedded in Epon 812. Ultrathin sections were stained with lead citrate for 1 min and observed with a JEOL lOOB electron microscope in all cases. Specimens with massive necrosis or severe neutrophilic infiltration were excluded. For the negative control, the adjacent sections were processed by replacing the MoAb with phosphatebuffered saline (PBS) or MoAb preabsorbed with human type Ill collagen (Sigma, St. Louis, MO). In some cases, the specimens were pretreated with 0.03-0.3% Triton X100 in PBS, or saponin was added to the MoAb solutions (0.03%) to facilitate antibody penetration (25). For light microscopy, the neighboring sections were processed in the same way and counterstained with 1% methyl green in all cases. Ceff identification : In diffuse-type carcinoma, stromal cells and carcinoma cells are mixed. However, differentiation between the two was made with certainty
by the following features. Stromal fibroblasts are spindle-shaped cells with abundant rER stacks, and they never have mucin granules or microvilli. Dissociated-. type carcinoma cells are oval-shaped cells with nuclear atypia. They may have mucin granules, microvilli and intracytoplasmic lumina, and may be joined t o neighboring cells by desmosomes with convergence of intermediate filaments. Light microscopic morphometry : Hematoxylin-eosinstained sections ( 3 p m thick) were prepared from paraffin blocks of five intestinal-type gastric, seven diffuse-type gastric, and six colonic carcinomas from among the cases examined by immunoelectron microscopy. The distribution density of stromal fibroblasts was measured by counting fibroblasts in a randomly placed square, 100 x 100 p m in size, using a x 2 0 0 microscopic field. Twenty squares were counted in all cases. The nuclear area of stromal fibroblasts was measured with a semi-automatic image-analyzer (Canon BX-1, Tokyo) using 5 4 randomly taken photographs (three per case, x 1,000 final magnification). The data were summed in each group and statistical analysis was carried out among the three groups by student’s t test.
RESULTS Light and electron microscopic immunohistochemistry Microwave fixation gave better preservation of subcellular structures without significant changes in the results (23, 24). Light microscopically, the stromal area was positively stained in all cases (Figs. l a , 2a). Diffuse cytoplasmic immunoreactivity was not observed in carcinoma cells in any cases. The negative controls uniformly showed no reaction (Figs. 1b, 2b). UItrast ruct ura IIy, the immuno react io n products with the MoAb were observed on collagen fibers, measuring 50-90 nm in diameter, in all cases (Figs. 3, 5, 6b). The immunoreactivity was also observed on amorphous or finely fibrillar substances in the extracellular matrix near the surface of the stromal fibroblasts (Fig. 3) in 16 of 2 8 cases. Collagen fibers were admixed with them (Fig. 3, inset). Cisternae of the rER were positive in seven cases, mostly very sparsely (Fig. 3) and rarely abundantly (Fig. 4). Membranes of the Golgi apparatus were positive in one case. These reactivities were thought to represent the localization sites of human type Ill procollagen, thus indicating the cells producing it (see Discussion for details). Stromal fibroblasts were found to have cytoplasmic recesses containing im m uno react ive col-
Acta Pathologica Japonica 40 (5) : 1990
Figure 1. Diffuse-type gastric carcinoma stained with MoAb against human type Ill collagen (a) and a control section (b, preabsorbed with human type Ill collagen). Counterstained with methyl green.
329
Figure 2. Intestinal-type gastric carcinoma stained with MoAb against type 111 collagen (a) and a control section (b, preabsorbed with human type Ill collagen). Counterstained with methyl green.
Figure3. A stromal fibroblast in colonic carcinoma (stained with MoAb against human type Ill collagen). lmmunoreactive fibers and amorphous substance are co-localized in the extracellular matrix adjacent t o the plasma membrane (arrows). Surface of some of the rER is positive (small arrow). Asterisk indicates cytoplasmic reactivity beneath the plasma membrane. Fixed by microwave radiation. Scale b a r y 0 . 2 p m . Inset shows higher magnification of amorphous immunoreactivity admixed with collagen fibers near the cell surface. ~ 5 7 , 0 0 0 .
330
lmrnunoelectron Microscopy of Collagen (Ohtani and Ooshima)
Figure 4. A stromal cell in colonic carcinoma. Dilated cisternae of rER are imrnunoreactive. Fixed by microwave radiation. Scale bar= 1 um.
Figure 5. A stromal cell in intestinal-type gastric carcinoma showing two cytoplasmic recesses containing immunoreactive collagen fibers (arrows). Fixed by conventional method. Scale b a r = l urn.
lagen fibers in three cases (Fig. 5). In five cases, the amorphous substances were also observed in the cytoplasm between the cytoplasmic membrane and the rER (Fig. 3, asterisk). The number of cases showing the amorphous or finely fibrillar substances was larger in intestinal-type gastric carcinoma and colorectal carcinoma (5/6 and 7/9,respectively) than in diffuse-type gastric carcinoma (4/13)(Table 1). lmmunoreactive amorphous or finely fibrillar s u b stances were also observed around or within carcinoma cells in one case of intestinal- and three cases of diffuse-
type gastric carcinoma, and two cases of colorectal carcinoma (six cases in totaI)(Figs. 6a, b). The distribution pattern of the substances was the same as that in fibroblasts, and the substances were not observed in the extracellular matrix remote from carcinoma cells. In one case each of intestinal- and diffuse-type gastric carcinoma among these six cases, carcinoma cells possessed cytoplasmic recesses containing immunoreact ive collagen fibers and rER showing immunoreactivity on its membranes (Fig. 7). In non-neoplastic mucosa, fibroblasts in the lamina
Table 1. Occurrence Rate of lmmunoreactivity Related to Production and Secretion of Type Ill Collagen/Procollagen (number of positive caseslnumber of total cases)
Site of immunoreactivity Amorphous substances in extracellular matrix near cell membrane
Gastric carcinoma diffuse type intestinal type 4/13 (F) 5 / 6 (F) 3/13 (Ca) 1 / 6 (Ca)
Colorectal carcinoma 7 / 9 (F) 219 (Ca)
Total 16/28 (F) 6/28 (Ca)
rER
Cytoplasmic recess with collagen fibers
0113 (F) 1/13 (Ca)
1 / 6 (F) 1 / 6 (Ca)
0/9 (Ca)
2/9 (F)
3/28 (F) 2/28 (Ca)
Total
4/13 (F) 3/13 (Ca)
5/6 (F) 1 / 6 (Ca)
7/9 (F) 2 i 9 (Ca)
16/28 (F) 6/28 (Ca)
F, fibroblast; Ca, carcinoma cell. Total means cases in which at least one of the three immunoreactive sites was positive.
331
Acta Pathologica Japonica 40 (5): 1990
Figure 6. An isolated carcinoma cell in diffuse-type gastric carcinoma. a. Low magnification. Scale b a r = l pm. b. Higher magnification of the area indicated by an arrow in Fig. 7a showing co-localization of extracellular amorphous/finely fibrillar substances and collagen fibers. Fixed by conventional method. Scale bar=0.2 pm.
propria generally possessed less rER than those in the cancer stroma (4),and immunoreactive rER was sporadically detected in two of four cases. Pretreatment of the specimens with Triton X-100 or saponin did not augment the intracellular immunoreactivity. Light microscopic morphometry The data are summarized in Table 2. Both the distribution density and nuclear size of stromal fibroblasts were significantly lower in diff use-type gastric carcinoma (approximately half) than those in intestinal-type gastric and colorectal carcinomas. There were no significant differences in the results between the last two groups.
DISCUSSION
Figure 7. Part of another carcinoma cell in the same case as in Fig. 6 showing immunoreactivity on the surface of the rER (small arrows), which is similar to that in Fig. 3. Large arrow indicates by con. immunoreactive collagen fibers. ~ u = n u c l e ~ Fixed ~. ventional method. Scale bar=0.2 pm.
On the basis of a previous ultrastructural autoradiographic study (26),the biosynthetic and secretory pathway of procollagen has been postulated to proceed from the rER t o the Golgi apparatus, and then via secretory granules to the ground substance. lmmunoelectron microscopic studies on collagen/procollagen in experimental animals have shown positive staining in the cisternae of the rER, dilated Golgi saccules, and vacuoles (or granules) (27-32). 1” human cancer stroma, Minamoto et a/. (9)reported similar results in stromal fibroblasts of gastric carcinoma but not in the cancer
332
lmmunoelectron Microscopy of Collagen (Ohtani and Ooshima) Table 2. Summary of Results of Light Microscopic Morphometry (expressed as meanflSD) Gastric carcinomas Diffuse type Intestinal type
Colorectal carcinoma
Distribution density (cell count per 10,000 pm2)
10.8+ - 3.4a
22.0k 5.5
22.7k 5.6
Nuclear area (pm’) (n=number of fibroblasts measured)
27.8+ 17.2a (n=595)
43.9k22.4 (n=393)
44.4k25.9 (n=642)
Fibroblasts
a
smaller than those in the other two groups (p
