Artigo Acesso aberto Revisado por pares

Carcinoembryonic Antigen Family Members CEACAM6 and CEACAM7 Are Differentially Expressed in Normal Tissues and Oppositely Deregulated in Hyperplastic Colorectal Polyps and Early Adenomas

2000; Elsevier BV; Volume: 156; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)64764-5

ISSN

1525-2191

Autores

Stefan Schölzel, Wolfgang Zimmermann, Georg Schwarzkopf, Fritz Grunert, Brigitta Rogaczewski, John A. Thompson,

Tópico(s)

Monoclonal and Polyclonal Antibodies Research

Resumo

Four members of the carcinoembryonic antigen (CEA) family, CEA, CEACAM1 (BGP), CEACAM6 (NCA-50/90), and CEACAM7 (CGM2), are coexpressed in normal colorectal epithelia but are deregulated in colorectal cancers, where they could play a role in tumorigenesis. As a basis for functional studies, their expression patterns in normal tissues first need to be clarified. This is well documented for CEACAM1 and CEA but not for CEACAM6 or CEACAM7. We have now carried out immunohistochemical expression studies on 35 different organs, using CEACAM6-specific (9A6) and CEACAM7-specific (BAC2) monoclonal antibodies. CEACAM7 was only found on the apical surface of highly differentiated epithelial cells in the colorectal mucosa and on isolated ductal epithelial cells within the pancreas. CEACAM6 was expressed in granulocytes and epithelia from various organs. CEACAM6 and CEACAM7 expression correlated with apoptosis at the table region of the normal colon, and both were absent from highly proliferating cells at the base of colonic crypts. CEACAM6 revealed a broader expression zone in proliferating cells in hyperplastic polyps and adenomas compared with normal mucosa, whereas CEACAM7 was completely absent. Down-regulation of CEACAM7 and up-regulation of CEACAM6 expression in hyperplastic polyps and early adenomas represent some of the earliest observable molecular events leading to colorectal tumors. Four members of the carcinoembryonic antigen (CEA) family, CEA, CEACAM1 (BGP), CEACAM6 (NCA-50/90), and CEACAM7 (CGM2), are coexpressed in normal colorectal epithelia but are deregulated in colorectal cancers, where they could play a role in tumorigenesis. As a basis for functional studies, their expression patterns in normal tissues first need to be clarified. This is well documented for CEACAM1 and CEA but not for CEACAM6 or CEACAM7. We have now carried out immunohistochemical expression studies on 35 different organs, using CEACAM6-specific (9A6) and CEACAM7-specific (BAC2) monoclonal antibodies. CEACAM7 was only found on the apical surface of highly differentiated epithelial cells in the colorectal mucosa and on isolated ductal epithelial cells within the pancreas. CEACAM6 was expressed in granulocytes and epithelia from various organs. CEACAM6 and CEACAM7 expression correlated with apoptosis at the table region of the normal colon, and both were absent from highly proliferating cells at the base of colonic crypts. CEACAM6 revealed a broader expression zone in proliferating cells in hyperplastic polyps and adenomas compared with normal mucosa, whereas CEACAM7 was completely absent. Down-regulation of CEACAM7 and up-regulation of CEACAM6 expression in hyperplastic polyps and early adenomas represent some of the earliest observable molecular events leading to colorectal tumors. The lumen of the normal colon has an enlarged surface area, in the form of crypts whose main functions are the resorption of water and salts as well as mucous secretion. These crypts are coated with a single layer of epithelial cells, which are continuously being regenerated from stem cells located at the base of each crypt.1Potten CS Booth C Pritchard DM The intestinal epithelial stem cell: the mucosal governor.Int J Exp Pathol. 1997; 78: 219-243Crossref PubMed Scopus (407) Google Scholar After division, some daughter cells regenerate stem cells, whereas the rest differentiate into four main epithelial cell types: colonocytes, goblet cells, M-cells, and endocrine cells. During their differentiation, these cells migrate up the crypts to the table region between the crypts and are finally exfoliated into the intestinal lumen. Although the turnover of epithelial cells in the small intestine is relatively similar, one major difference becomes apparent. In humans, colorectal epithelia are much more prone to malignant transformation, and for this reason it is important to understand their normal regulation and to determine any differences that become apparent in transformed cells. Colorectal tumor development is a multistep process that is known to depend on the deregulation or mutation of certain critical genes. It is generally accepted that adenomas represent the first stage of neoplasia, and one of the earliest molecular changes leading to the development of adenomas is mutation in the tumor suppressor gene APC.2Kinzler KW Vogelstein B Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4286) Google Scholar Certain oncogenes are also often mutated in adenomas (eg, K-RAS). Although the mechanism has not yet been elucidated and their role in tumorigenesis is unclear, members of the carcinoembryonic antigen (CEA) gene family also become deregulated in adenomas.3Thompson J Seitz M Chastre E Ditter M Aldrian C Gespach C Zimmermann W Down-regulation of carcinoembryonic antigen-family member 2 expression is an early event in colorectal tumorigenesis.Cancer Res. 1997; 57: 1776-1784PubMed Google Scholar, 4Nollau P Scheller H Kona-Horstmann M Rohde S Hagenmüller F Wagener C Neumaier M Expression of CD66a (human C-CAM) and other members of adhesion molecules in human colorectal adenomas.Cancer Res. 1997; 57: 2354-2357PubMed Google Scholar From a clinical view, it would be of paramount interest to be able to recognize neoplastic growth as early as possible. For example, hyperplastic polyps may be precursors of neoplasia. Indeed, patients with hyperplastic polyps have been reported to have an increased risk of developing adenomas.5Croizet O Moreau J Arany Y Delvaux M Rumeau JL Escourrou J Follow-up of patients with hyperplastic polyps of the large bowel.Gastrointest Endosc. 1997; 46: 119-123Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Among the hyperplastic polyps analyzed from 22 patients, none were found to contain APC mutations, but five carried a K-RAS mutation.6Jen J Powell SM Papadopoulos N Smith KJ Hamilton SR Vogelstein B Kinzler KW Molecular determinants of dysplasia in colorectal lesions.Cancer Res. 1994; 54: 5523-5526PubMed Google Scholar Thus hyperplastic epithelial cells may already be primed for neoplastic transformation. It would be of particular clinical interest to clarify the existence of a neoplastic predisposition in hyperplastic polyps. This could be approached by documenting molecular differences in hyperplastic polyps and normal colorectal epithelial cells. In this context, it would be interesting to see if other markers of neoplasia (eg, members of the CEA family. are deregulated in hyperplastic polyps. CEA is a classical tumor marker for several types of adenocarcinoma, especially those of colorectal origin.7Ballesta AM Molina R Filella X Jo J Gimenez N Carcinoembryonic antigen in staging and follow-up of patients with solid tumors.Tumor Biol. 1995; 16: 32-41Crossref PubMed Scopus (101) Google Scholar Although it is much more prevalent in colorectal tumors compared to the corresponding normal tissue, this may be due to differences in clearance rather than to differential expression.8Kuroki M Arakawa F Yamamoto H Shimura H Ikehara Y Matsuoka Y Active production and membrane anchoring of carcinoembryonic antigen observed in normal colon mucosa.Cancer Lett. 1988; 43: 151-157Abstract Full Text PDF PubMed Scopus (44) Google Scholar CEA is the name-giving member of a family of molecules, three members of which are coexpressed with CEA in the normal colorectal mucosa.9Thompson JA Grunert F Zimmermann W Carcinoembryonic antigen gene family: molecular biology and clinical perspectives.J Clin Lab Anal. 1991; 5: 344-366Crossref PubMed Scopus (593) Google Scholar According to the new official nomenclature system decided at the 9th International CEA/PSG Workshop (Ratzeburg, Germany, September 1998), these three family members are CEACAM1 (formerly BGP), CEACAM6 (formerly NCA-50/90), and CEACAM7 (formerly CGM2).10Beauchemin N Draber P Dveksler G Gold P Gray-Owen S Grunert F Hammarström S Holmes KV Karlson A Kuroki M Lin SH Lucka L Najjar SM Neumaier M Öbrink B Shively JE Skubitz KM Stanners CP Thomas P Thompson JA Virji M von Kleist S Wagener C Watt S Zimmermann W Redefined nomenclature for members of the carcinoembryonic antigen family.Exp Cell Res. 1999; 252: 243-249Crossref PubMed Scopus (328) Google Scholar In colorectal cancers, these family members become deregulated. CEACAM1 is down-regulated,11Neumaier M Paululat S Chan A Matthaes P Wagener C Biliary glycoprotein, a potential human cell adhesion molecule, is down-regulated in colorectal carcinomas.Proc Natl Acad Sci USA. 1993; 90: 10744-10748Crossref PubMed Scopus (230) Google Scholar whereby both mouse (formerly Bgp) and rat CEACAM1 (formerly C-CAM) have been shown to block proliferation after transfection into colorectal and prostatic tumor cell lines.12Kunath T Ordonez-Garcia C Turbide C Beauchemin N Inhibition of colonic tumor cell growth by biliary glycoprotein.Oncogene. 1995; 11: 2375-2382PubMed Google Scholar, 13Hsieh J-T Luo W Song W Wang Y Kleinerman DI Van NT Lin S-H Tumor suppressive role of an androgen-regulated epithelial cell adhesion molecule (C-CAM) in prostate carcinoma cell revealed by sense and antisense approaches.Cancer Res. 1995; 55: 190-197PubMed Google Scholar CEACAM7 reveals an expression pattern similar to that of CEACAM1 in the normal colonic mucosa and is down-regulated in colorectal tumors; however, nothing is known about its function and the protein is not well characterized to date. Interestingly, CEACAM6 expression is up-regulated in colorectal tumors compared to the normal mucosa,11Neumaier M Paululat S Chan A Matthaes P Wagener C Biliary glycoprotein, a potential human cell adhesion molecule, is down-regulated in colorectal carcinomas.Proc Natl Acad Sci USA. 1993; 90: 10744-10748Crossref PubMed Scopus (230) Google Scholar suggesting a role opposite to that of CEACAM1 and/or CEACAM7 during colorectal tumorigenesis. Although it is known that CEACAM6 is expressed in different tissues,14Thompson JA Molecular cloning and expression of carcinoembryonic antigen gene family members.Tumor Biol. 1995; 16: 10-16Crossref PubMed Scopus (70) Google Scholar no systematic analysis has been carried out to date to determine its expression pattern in the human body. Apart from its putative role in colorectal tumorigenesis, CEACAM6, along with other CEA family members, has also been reported to serve as a receptor for mediating adherence and entry of Neisseria bacteria into human tissues.15Bos MP Grunert F Belland RJ Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae.Infect Immun. 1997; 65: 2353-2361Crossref PubMed Google Scholar Thus knowledge of their expression in a given tissue should give an indication of susceptibility to Neisserial infections. In this study we developed and tested a new monoclonal antibody against CEACAM7 (BAC2), as a previously developed CEACAM7 monoclonal antibody named CAC23Thompson J Seitz M Chastre E Ditter M Aldrian C Gespach C Zimmermann W Down-regulation of carcinoembryonic antigen-family member 2 expression is an early event in colorectal tumorigenesis.Cancer Res. 1997; 57: 1776-1784PubMed Google Scholar has since been found to cross-react with CEACAM6. We then used this specific BAC2 monoclonal antibody to analyze CEACAM7 expression and have compared it to that of CEACAM6 in 35 normal organs and during development in the fetal colon by immunohistochemical staining. Their expression patterns were also compared in hyperplastic colorectal polyps and adenomas of various histological types. Finally, CEACAM6 and CEACAM7 expression in the normal colon has been compared to the expression of Ki67 (a marker for cell proliferation) and CD95 (a marker for apoptotically sensitive cells), as well as cells that have undergone apoptosis, which has been assessed by a DNA degradation (TUNEL) test. All adult and fetal organs were from the Institute of Pathology, University Hospital, Freiburg. Adenomas and hyperplastic colorectal epithelia were provided by the Department of Surgery, University Hospital, Freiburg. The tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (10 mmol/L NaH2PO4, 43 mmol/L K2HPO4, and 123 mmol/L NaCl) for 24–48 hours, followed by a 24-hour incubation in 0.5 mol/L sucrose in PBS before being embedded in Jung Freeze medium (Leica Instruments, Nussloch, Germany), which was diluted with an equal volume of water. This was followed by freezing on dry ice or direct freezing in a cryomicrotome at −40°C. Alternatively, unfixed tissues were transported in Dulbecco's modified Eagle's medium (DMEM) and directly embedded and frozen. Frozen blocks were stored at −70°C before cryosectioning. Cryosections of 7 μm thickness were made on a Frigocut 2700 cryotome (Reichert and Jung/Leica Instruments, Nussloch, Germany) and transferred to Superfrost slides (Roth, Karlsruhe, Germany). After drying under a hood at ambient temperature for 30 minutes, the sections were stored at −70°C before immunostaining. The following monoclonal antibodies were used for immunohistochemical staining: 9A6, which has been shown elsewhere to be specific for CEACAM616Grunert F Soubt M Jantscheff P Nagel G Specificity and epitope localization of CD66/CD67 mAb. Leucocyte Typing. V. White Cell Differentiation Antigens, vol 1.in: Schlossman SF Boumsell L Gilks W Harlan JM Kishimoto T Morimoto C Ritz J Shaw S Silverstein R Springer T Tedder TF Todd RF Oxford University Press, New York1995: 907-911Google Scholar; 4/3/17, which recognizes CEACAM1 and CEA17Daniel S Nagel G Johnson JP Lobo FM Hirn M Jantscheff P Kuroki M von Kleist S Grunert F Determination of the specificities of monoclonal antibodies recognizing members of the CEA family using a panel of transfectants.Int J Cancer. 1993; 55: 303-310Crossref PubMed Scopus (46) Google Scholar; 26/3/13, which has specificity for CEA18Grunert F AbuHarfeil N Schwarz K von Kleist S Two CEA, and three NCA species, although distinguishable by monoclonal antibodies, have nearly identical peptide patterns.Int J Cancer. 1985; 36: 357-362Crossref PubMed Scopus (55) Google Scholar; CAC23Thompson J Seitz M Chastre E Ditter M Aldrian C Gespach C Zimmermann W Down-regulation of carcinoembryonic antigen-family member 2 expression is an early event in colorectal tumorigenesis.Cancer Res. 1997; 57: 1776-1784PubMed Google Scholar and BAC2 (see below), which, respectively, recognize and are specific for CEACAM7; Mib-1 (Dako, Hamburg, Germany), which recognizes Ki-67; and Ab-1 (Pharmingen, Hamburg, Germany), which is specific for the human FAS receptor (CD95). In all cases, peroxidase-labeled, rabbit anti-mouse IgG or IgM, with minimal cross-reactivity to human serum proteins (Dianova, Hamburg, Germany), was used as the second antibody. Immunostaining was as described previously.19Thompson J Mössinger S Reichardt V Engels U Beauchemin N Kommoss F von Kleist S Zimmermann W A polymerase chain reaction assay for the specific identification of transcripts encoded by individual carcinoembryonic antigen (CEA) gene family members.Int J Cancer. 1993; 55: 311-319Crossref PubMed Scopus (40) Google Scholar Briefly, the sections were thawed for 15 minutes at ambient temperature under a hood and rehydrated for 3 minutes in PBS. Endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol for 30 minutes, and the sections were washed in PBS. The sections were preincubated in 3% ovalbumin/PBS (Sigma, Deisenhofen, Germany) for 30 minutes in a moist chamber, washed in PBS, and incubated at 4°C for 12–16 hours with the monoclonal antibodies at a concentration of 5–10 μg/ml PBS, or using undiluted hybridoma supernatants for BAC2 (see below). As negative controls, sections were incubated in PBS or cell culture medium. After washing in PBS, the peroxidase-coupled second antibody was used at a concentration of 5–10 μg/ml PBS for 1–2 hours at ambient temperature. The color reaction was achieved using diaminobenzidine as substrate. Counterstaining was omitted or was very brief (1 s) with Mayer's hemalaun. After dehydration, the sections were mounted in VitroClud (Leica Instruments. for microscopy. Apoptotic cells were visualized with the Cell Death Detection Kit POD (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions. The development of monoclonal antibodies that recognize CEACAM7 has already been described.3Thompson J Seitz M Chastre E Ditter M Aldrian C Gespach C Zimmermann W Down-regulation of carcinoembryonic antigen-family member 2 expression is an early event in colorectal tumorigenesis.Cancer Res. 1997; 57: 1776-1784PubMed Google Scholar The same procedure was used to immunize a second CEA-transgenic mouse for the establishment of hybridoma cells that produce antibodies that recognize a CEACAM7/human IgG-Fc fusion protein but not a Ceacam10 (formerly Cea10)/human IgG-Fc fusion protein, proving specificity of the antibodies for the CEACAM7 moiety. One clone (BAC2) was expanded after the first subcloning step. Specificity testing was carried out by FACScan analyses against various HeLa or Chinese hamster ovary (CHO) cell lines that had been stably transfected with cDNA expression vectors for individual members of the CEA family, as described previously.3Thompson J Seitz M Chastre E Ditter M Aldrian C Gespach C Zimmermann W Down-regulation of carcinoembryonic antigen-family member 2 expression is an early event in colorectal tumorigenesis.Cancer Res. 1997; 57: 1776-1784PubMed Google Scholar The PSG1 transfectant and the monoclonal antibody BAP1 have been described elsewhere.20Zhou G-Q Baranov V Zimmermann W Grunert F Erhard B Mincheva-Nilsson L Hammarström S Thompson J Highly specific monoclonal antibody demonstrates that pregnancy-specific glycoprotein (PSG) is limited to syncytio-trophoblast in human early and term placenta.Placenta. 1997; 18: 491-501Abstract Full Text PDF PubMed Scopus (52) Google Scholar A newly developed CHO transfectant was included that expresses CEACAM7 and was recently developed in our laboratory with the use of CEACAM7 cDNA that was described elsewhere.21Thompson J Zimmermann W Nollau P Neumaier M Weber-Arden J Schrewe H Craig I Willcocks T CGM2, a member of the carcinoembryonic antigen gene family is down-regulated in colorectal carcinomas.J Biol Chem. 1994; 269: 32924-32931PubMed Google Scholar This cDNA was subcloned into the pBHE expression vector before transfection using lipofectamine, according to the manufacturer's instructions (Life Technologies, Eggenstein, Germany). Stable transfectants expressing CEACAM7 were identified by FACScan analyses using the CEACAM7/CEACAM6-recognizing monoclonal antibody CAC2.3Thompson J Seitz M Chastre E Ditter M Aldrian C Gespach C Zimmermann W Down-regulation of carcinoembryonic antigen-family member 2 expression is an early event in colorectal tumorigenesis.Cancer Res. 1997; 57: 1776-1784PubMed Google Scholar The specificity of the newly developed BAC2 monoclonal antibody was determined in FACScan analyses against HeLa transfectants that each stably express CEA, CEACAM3 (formerly CGM1), CEACAM6, CEACAM8 (formerly CGM6/NCA-95), and a recombinant membrane-bound PSG1 on their surfaces and against CHO transfectants that each stably express CEACAM1, CEACAM4 (formerly CGM7), and CEACAM7. As a negative control, transfectants that have stably integrated the empty expression vector (HeLa-neo) or the parental cells (CHO-K1) were tested. Positive controls were carried out for each transfectant, using a monoclonal antibody (D14HD11) that recognizes CEACAM1, CEACAM3, CEACAM4, CEACAM6 and CEA, or a monoclonal antibody (80H3) that recognizes CEACAM8, a monoclonal antibody that recognizes PSG (BAP1), as well as the CAC2 monoclonal antibody that recognizes CEACAM7. These results are summarized in Figure 1. It is obvious that BAC2 only reacts with the CEACAM7 transfectant and recognizes no other CEA family transfectants. To rule out any cross-reactivity of BAC2 with CEA, CEACAM1, CEACAM6, or PSGs in immunohistochemical analyses, its staining pattern was compared with that of other CEA family-recognizing monoclonal antibodies with known specificities. These results are summarized in Table 1. CEACAM1 was found in the liver, kidney, submandibulary gland, and placenta, using the 4/3/17 monoclonal antibody, which recognizes CEACAM1 and CEA (the latter is absent from these organs). BAC2 did not stain these organs and thus does not cross-react with CEACAM1. Negativity of BAC2 in the placenta also rules out cross-reactivity with PSG subgroup members of the CEA family. A CEACAM6-specific monoclonal antibody (9A6) and CAC2 stained granulocytes in the bone marrow, whereas BAC2 did not. Finally, cross-reactivity against CEA and CEACAM6 could be ruled out because CEA and CEACAM6-positive colorectal tumors were not stained using BAC2, whereas CAC2 did reveal cross-reactivity. Cross-reactivity of CAC2 was not recognized in previous immunohistological analyses under the conditions used.3Thompson J Seitz M Chastre E Ditter M Aldrian C Gespach C Zimmermann W Down-regulation of carcinoembryonic antigen-family member 2 expression is an early event in colorectal tumorigenesis.Cancer Res. 1997; 57: 1776-1784PubMed Google Scholar Only colonic epithelial cells were stained with BAC2 in these tests, confirming the specificity of this newly developed monoclonal antibody for CEACAM7.Table 1Immunohistochemical Specificity Test for BAC2 Monoclonal AntibodyMonoclonal antibodyBAC2CAC29A626-3-134-3-17Antigen recognizedCEACAM7CEACAM6+ CEACAM7CEACAM6CEACEA+ CEACAM1Colon+++++Colon carcinoma−++++Liver−−−−+Kidney−−−−+Submandibulary gland−++−+Placenta−−−−+Granulocytes−++−++, positively staining cells. Open table in a new tab +, positively staining cells. To gain more insight into the possible functions of CEACAM6 and CEACAM7, their expression was investigated in a variety of normal tissues. For these analyses, 35 different organs and tissues were tested with the antibody 9A6, which has been shown elsewhere to be specific for CEACAM616Grunert F Soubt M Jantscheff P Nagel G Specificity and epitope localization of CD66/CD67 mAb. Leucocyte Typing. V. White Cell Differentiation Antigens, vol 1.in: Schlossman SF Boumsell L Gilks W Harlan JM Kishimoto T Morimoto C Ritz J Shaw S Silverstein R Springer T Tedder TF Todd RF Oxford University Press, New York1995: 907-911Google Scholar and the CEACAM7-specific antibody BAC2. The results of these immunohistochemical analyses are summarized in Table 2, and chosen examples are depicted in Figure 2. Tissues from between two and five individuals were tested in most cases, apart from thyroid, adrenal gland, heart, urether, bladder, and bone marrow, which were only tested from one individual each. Maximum staining intensity was achieved on nonfixed cryosections.Table 2Immunohistochemical Analysis of CEACAM6 and CEACAM7 Expression in Different Normal Organs and TissuesMonoclonal antibody antigen recognized9A6BAC2Expression patternCEACAM6CEACAM7Tongue+−Upper third of squamous epitheliaEsophagus+−Squamous epitheliaStomach body+−Single crypts, luminal cellsDuodenum−−Jejunum−−Ileum−−Appendix++Epithelia, upper fifth of crypt for each antigenColon++Epithelia, upper fifth of crypt for each antigenThyroid gland−−Submandibular salivary gland+−Mucous epithelia most prominentAnterior lingual gland+−Mucous epithelia most prominentPancreas++Small ducts, with BAC2 only individual cellsBreast+−Some breast ductsAdrenal gland−−Liver+−GranulocytesGall bladder+−Apical epitheliaLung+−Pneumocytes, bronchiole epithelia, granulocytesKidney−−Bladder−−Skin+−Eccrine sweat glands, hair follicles, squamous epitheliaBone marrow+−Myeloid cellsTonsils+−Epithelia and granulocytesSpleen+−Myeloid cellsProstate gland+−Single ductsFallopian tube−−Cervix+−Squamous epitheliaUterus−−Ovary−−Testis−−Seminal vesicle−−Epididymus−−Heart−−Urether−−Umbilical cord+−Amnion epitheliaPlacenta+−Amnion epithelia Open table in a new tab CEACAM7 revealed a very narrow expression pattern, being restricted to highly differentiated epithelial cells in the upper fifth of the colorectal crypts (Figures 2a and 3j) and to the apical surface of isolated epithelial cells lining small ducts within the pancreas (Figure 2b). All other tissues and organs tested were negative for CEACAM7 (Table 2). In contrast, CEACAM6 revealed a comparatively broad expression pattern, being found in granulocytes (Figure 2c) and in epithelial cells in a variety of organs (Figure 2, d–l). Positively staining squamous epithelia reveal nonpolarized cell surface localization and cytoplasmic staining (Figure 2d) In simple epithelia, CEACAM6 was present on the apical surface. Organs rich in granulocytes (eg, bone marrow and spleen) revealed CEACAM6 positivity in those cells (data not shown). In addition, the expression of CEACAM6 and CEACAM7 was also investigated during fetal development of the colon and compared to that of CEA. Six fetuses ranging from the 18th to the 35th weeks of pregnancy as well as colon from a 2-day-old child, a 10-day-old child, and an adult were used for this study. All three CEA family members revealed an apical expression throughout development (Figure 3, a–c), which was continued post partum. Whereas CEA was expressed on the apical surface of all epithelial cells down to the base of the crypt, CEACAM6 was only found in the upper half and CEACAM7 in the upper third of the developing crypts. In contrast to CEA and CEACAM6, CEACAM7 also revealed a strong perinuclear staining in the epithelial cells of the upper crypt throughout fetal development (Figure 3a), which became strongly reduced at day 2 and was no longer seen at day 10 post partum. Chromogranin staining was also carried out on a serial section at the 24th week of pregnancy to identify enterochromaffin cells. The CEACAM7 perinuclear staining pattern did not match that of the enterochromaffin cells, which were few in number and were located at various positions throughout the crypts (data not shown). To determine when CEACAM6 and CEACAM7 become deregulated during tumorigenesis, their expression patterns were investigated in 25 colorectal polyps. In five cases, only biopsy specimens were studied, otherwise whole adenomas with adjacent mucosa were analyzed. These results are summarized in Table 3. Eight polyps were hyperplastic, and the rest represented adenomas of various sizes and morphological types (12 tubular, four tubulovillous, and one adenoma with atypia). CEACAM6 was expressed in all polyps (25/25) and often revealed a broader expression zone in the upper crypt epithelia than in the adjacent mucosa (21/25; Figure 3h). In parallel, a loss of polarity was observed, with expression seen not only on the apical surface but throughout the cytoplasm and on the basolateral cell membranes (Figure 3h, inset). In one tubular adenoma, no broader expression zone was found for CEACAM6, but many granulocytes within the polyp stained positive, indicating inflammation (Figure 3e). In comparison, CEA is shown to be located down to the base of the crypts and throughout the same tubular adenoma (Figure 3f). In contrast, CEACAM7 was absent from all 16 polyps that could be analyzed for CEACAM7 expression. The adjacent mucosa was positive for CEACAM7 in all but one case (15/16). The loss of expression of CEACAM7 is shown in a tubular adenoma (Figure 3d) and in a hyperplastic polyp (Figure 3g).Table 3Expression of CEACAM6 and CEACAM7 in Polyps and Adjacent MucosaCEACAM7 expression*Expression of CEACAM7 was found on the apical surface of epithelial cells from the upper fifth of the crypts (+) or was absent(−); it was always absent from polyps.CEACAM6 expression†Expression of CEACAM6 in polyps revealed either a broader expression zone (++), or was comparable (+), or was reduced (+/−) compared to the adjacent mucosa (+).PolypPolyp size (mm)PolypNormal mucosaPolypNormal mucosaHyperplastic polyps1‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for <2 days.0.5 × 1.0−−+++21.5 × 0.5−++/−+3‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for <2 days.1.0 × 1.0−−+++4‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for <2 days.1.5 × 1.0−−+++52.0 × 0.5−++++62.0 × 2.0−+++7‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for <2 days.2.0 × 2.0−−+++8‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for <2 days.2.0 × 2.0−−+++Tubular adenomas9‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for <2 days.1.5 × 1.0−−+++10‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for <2 days.2.0 × 1.5−−+++112.0 × 1.5−+++122.5 × 2.0−++++133.0 × 2.0−++++14‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for <2 days.3.0 × 3.0−−+++154.0 × 2.0−+++16‡These tissue samples were fixed with formaldehyde for more than 4 days; all other samples were unfixed or fixed for 5−++++18<5−++++19<10−++++2014.0 × 12.0−−+++Tubulovillous adenomas214.0 × 4.0−++++22 5−++++2411.0 × 11.0−++++Adenoma with atypia2514.0 × 10.0−++++* Expression of CEACAM7 was found on the apical surface of epithelial cells from the upper fifth of the crypts (+) or was absent(−); it was always absent from polyps.† Expression of CEACAM6 in polyps revealed either a broader expression zone (++), or was comparable (+), or was reduced (+/−) compared to the adjacent mucosa (+).‡ These ti

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