Collagen type XVIII/endostatin is differentially expressed in primary and metastatic colorectal cancers and ovarian carcinomas

Collagen type XVIII (C18) is a nonfibrillar collagen of basement membranes. Its C-terminal fragment, endostatin, has been identified as an inhibitor of angiogenesis. C18 is predominantly expressed by hepatocytes of normal, cirrhotic and neoplastic liver. We compared the patterns of C18 RNA-expression in colonic adenocarcinoma metastases, which represent the most frequently occurring liver tumours, to normal colon mucosa, to primary colon cancers and to ovarian cancers which are often morphologically similar to colonic cancer or metastasis. Two C18-specific RNA-probes were generated to perform in situ hybridization combined with immunohistochemistry for cytokeratin, vimentin and the endothelial marker CD31, in order to characterize the C18-expressing cells. C18/endostatin protein was localized by immunohistology. In colorectal carcinomas and their liver metastases high levels of C18 transcripts were observed in endothelial cells and fibroblasts/myofibroblasts, whereas C18 RNA was virtually absent from carcinoma cells. Ovarian carcinomas displayed high C18 RNA expression both in carcinoma and stromal cells, indicating that induction of C18 transcription in tumour stromal cells is independent of the ability of carcinoma cells to express C18. While the role of tumour cell derived C18 in cancer growth regulation remains unknown, stimulation of proteolysis of the locally strongly expressed C18 to endostatin could offer an attractive approach for a targeted antineoplastic therapy. © 2001 Cancer Research Campaign   http://www.bjcancer.com

cells in cirrhosis and hepatocellular carcinoma. The high expression by neoplastic hepatocytes in vivo supports a unique role of this collagen not only in the formation of the sinusoidal basement membrane but also in the regulation of angiogenesis and neoplastic transformation (Schuppan et al, 1998).
Here we extended these studies analysing the origin and distribution of C18 in colonic adenocarcinoma metastases as the most frequently occurring secondary liver tumours, compared to normal colonic mucosa and to primary colon cancer. In addition, we studied primary ovarian carcinomas which are difficult to distinguish from colon carcinoma metastases to the ovaries.

Tissues
Tissue samples were obtained from patients undergoing resection for colorectal tumours (n = 16) or partial liver resection for solitary colorectal liver metastases (n = 5). Tissue samples were snapfrozen in liquid nitrogen immediately after removal and submitted for histopathologic diagnosis. The patient data and the histopathologic characteristics are listed in Table 1. As controls, we used colonic tissues with regular histology (at a distance of >10 cm from the focal lesions, n = 6) as well as resectates of ovarian carcinomas (n = 10) and one normal ovary.

Tissue sectioning and fixation
Frozen sections (7 µm) were collected onto 3-aminopropyl triethoxysilane-coated slides, dried briefly on a hot plate at 80˚C, and fixed

Immunohistology
The immunohistochemical detection of endostatin was performed with polyclonal rabbit antibodies specific for murine endostatin (kind gift of Dr U Eberspaecher and Dr A Menrad, Schering-AG, Berlin, Germany) with cross-reactivity to human endostatin. The alkaline phosphatase anti-alkaline phosphatase (APAAP) method was used for immunohistology, using affinity-purified mouse anti-rabbit immunoglobulin (Dianova, Hamburg, Germany), affinity-purified rabbit anti-mouse immunoglobulin (Dako, Glostrup, Denmark), and the APAAP complex (1:20 dilution, Dako). Alkaline phosphatase was developed with new fuchsin.

In situ hybridization
Pre-hybridization, hybridization, washing procedures including removal of non-specifically bound probe by ribonuclease A digestion and autoradiography of slides were as described before (Milani et al, 1990;Herbst et al, 1997). Controls included hybridization with C18 sense probes as well as with a procollagen α1(I) probe (Milani et al, 1990).

Normal colon
Some C18 RNA-expressing cells were found within the lamina muscularis propria, particularly in interspersing stromal tissue.
Only very few positive cells were seen in the lamina propria. Epithelial cells did not display C18 RNA at detectable levels ( Figure 1A).

Colorectal carcinomas and colorectal liver metastases
A highly increased C18 RNA-expression was observed in peritumourous stromal cells, both of primary tumours and metastases. The phenotype of these C18-positive cells was consistent with stromal fibroblasts or endothelial cells of small vessels and capillaries. C18 RNA was only marginally detectable in the carcinoma cells. Only a few tumour cells in 5 of 16 cases of colorectal carcinomas and 2 of 5 cases of colorectal liver metastases displayed a weak signal slightly above the background ( Figure 1B-D). In colorectal liver metastases the C18 RNA-expression in the adjacent liver tissue showed a homogeneous labelling of all hepatocytes as previously described (Schuppan et al, 1998). In situ hybridizations performed with both of the C18-specific RNAprobes, encoding either the endostatin-domain or part of the collagenous region, revealed an identical expression pattern. By combined immunohistochemical staining and in situ hybridization the C18 RNA-expressing cells in the tumour stroma were identified as vimentin-positive mesenchymal cells. Many of the C18 RNA-positive cells also expressed smooth muscle α-actin, a marker of a myofibroblastic differentiation, or the endothelial CD31 antigen (Figure 2A), but cells were consistently negative for cytokeratin ( Figure 2B).
By immunostaining, the C18/endostatin protein was found within the tumour stroma, with prominent labelling of endothelial cells of small vessels and capillaries, well in agreement with the results of in situ hybridization. Carcinoma cells did not display endostain immunoreactivity, whereas the sinusoidal basement membranes of neighbouring liver parenchyma showed a linear but weaker deposition of C18/endostatin ( Figure 2E, F).

Ovarian carcinomas
In contrast to colon carcinomas, ovarian carcinoma cells showed highly elevated C18 RNA transcript levels in 8 of 10 cases. However, as in colorectal carcinomas and colorectal liver metastases, C18 mRNA expression was high in the tumour stroma ( Figure 1E, F). C18 transcripts were almost homogeneously distributed among the groups of tumour cells without variation along morphologically distinct sites such as the front of invasion vs. more central tumour regions. There was no relationship of C18 transcript levels with cellular or nuclear characteristics of the tumour cells. Double-labelling experiments documented that the vast majority of the cytokeratin-positive carcinoma cells were expressing C18 RNA ( Figure 2C). In the 2 cases that displayed a transitional or endometrioid-type morphology only a weak signal for C18 RNA was detected over the carcinoma cells (not shown). As in colon carcinomas, combined immunohistochemical staining and in situ hybridization allowed identification of the C18 RNA-expressing cells in the tumour stroma as vimentin (α-actin)positive fibroblasts/myofibroblasts or anti-CD31-positive endothelial cells ( Figure 2D).
In one case of normal ovary no C18 RNA transcripts were detectable, whereas the neighbouring epithelial lining of the Fallopian tube displayed a weak signal. In situ hybridizations with sense (noncomplementary control) probes revealed a weak, nonspecific background labelling (not shown). High procollagen α1(I) transcript levels were detectable over fibroblastic cells of all specimens as proof for integrity of the examined tissue samples (not shown).

DISCUSSION
As a component of specialized basement membranes, C18 merits particular attention, since its proteolysis by proteases such as cathepsin L and elastase generates the potent angiogenesis inhibitor endostatin (Wen et al, 1999;Felbor et al, 2000). Previous quantitative and in situ hybridization studies revealed a prominent expression of C18 mRNA and protein in epithelia of liver and to a minor degree in kidney and lung (Oh et al, 1994b;Muragaki et al, 1995;Rehn and Pihlajaniemi, 1995;Musso et al, 1998;Schuppan et al, 1998). However, the patterns of C18 RNA expression in primary colonic adenocarcinoma and its metastases as the most frequently occurring secondary liver tumours, compared to normal colon as well as to often morphologically similar ovarian carcinomas is largely unknown.
Expression patterns of C18 in the liver tissue adjacent to the metastases displayed high transcript levels in hepatocytes, and lower levels in epithelial cells of small bile ducts and in endothelial cells as described previously (Schuppan et al, 1998). However, epithelia of colon carcinoma did not express C18 mRNA, while high C18 RNA transcript levels were present in the tumour stroma of both primary neoplasms and liver metastases. Here, fibroblasts and endothelial cells were identified as prominent sources of C18.
In contrast, ovarian carcinomas showed a strong C18 RNA expression in tumour cells in 8 out of 10 cases. Also, C18 expression was highly elevated in tumour endothelial cells and fibroblasts in these tumours. The finding of a high epithelial expression of C18 in ovarian cancer is of practical interest, since ovarian carcinoma may spread to the intestinal wall and may then be present in biopsy materials. Vice versa, gastrointestinal carcinomas may spread to the ovary. However, on microscopic examination ovarian endometrioid and mucinous carcinomas are often indistinguishable from colon colorectal carcinoma metastatic to or infiltrating the ovary, even when using the tumour markers CEA and CA125. Therefore, C18 may serve as a novel marker to distinguish carcinoma of intestinal (absent C18 expression) vs. ovarian (high C18 expression) origin.
Our study demonstrates that epithelial cells of different origin differ widely in their ability to express C18. While the low C18 expression of normal colonic epithelium is largely maintained in its neoplastic counterparts, and a high C18 expression is found both in normal and neoplastic hepatocytes (Schuppan et al, 1998), C18 is clearly up-regulated in the epithelium of ovarian carcinomas. The finding of variable C18 expression in adenocarcinomas depending on their site of origin argues against a pivotal function of C18 in early neoplastic transformation, but does not exclude a role of C18 in progression of ovarian carcinoma. In all cases, however, induction of peritumoural C18 expression by stromal endothelial cells and fibroblasts appears to be relevant for tumour progression and metastatic behaviour.
Whereas intact C18 may promote endothelial cell survival and angiogenesis, its C-terminal proteolytic fragment, endostatin, has been shown to be a potent inhibitor of angiogenesis O'Reilly et al, 1997). Cathepsin L, one of the proteases implicated in proteolytic cleavage of endostatin from its precursor C18, is widely expressed in colorectal carcinomas as well as in the colorectal cancer cell line HT-29 (Sheahan et al, 1989;Chauhan et al, 1991;Shuja et al, 1991;De Stefanis et al, 1997). Thus, epithelial tumours potentially modulate their growth by generating the proteases liberating endostatin from C18. The balance between C18 expression and cathepsin L, elastase or other proteases possibly involved in C18 processing may, therefore, be central for the regulation of angiogenesis and the dynamics of tumour growth. Importantly, the local production of C18 by the tumour cells opens