Malignant cell-specific gelatinase activity in human endometrial carcinoma.

BACKGROUND. The protease activity leading to degradation of the extracellular matrix was compared between human endometrial cancer and normal uterine endometrium. METHODS. Conditioned medium from tumor cells and normal endometrial cells was subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gel containing gelatin as a substrate. After electrophoresis, the gel was stained with Coomassie blue, and then the enzyme activity, expressed as the zone of dye clearing, was analyzed by densitometry. RESULTS. Densitometric analysis showed that all the endometrial cancers expressed a very high molecular weight enzyme activity (Mr 220,000), which was not detected in medium from normal endometrial cells. The analysis also showed that in endometrial cancer the activity of a Mr 92,000 enzyme was always superior to that of a Mr 64,000 enzyme, which was in contrast to the situation for normal endometrium. CONCLUSIONS. These results indicate that the expression of Mr 220,000 enzyme activity and the higher activity of the Mr 92,000 enzyme than the Mr 64,000 enzyme are involved in the malignant phenotype of native endometrial cancer.     

Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer

Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P<0.05), 31 in grade 2 (P<0.05) and 25 in grade 3 (P<0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.     

Disulfide bonding of a human prostate tumor-associated membrane antigen recognized by monoclonal antibody D83.21

Previous reports from our laboratory have described the binding specificity of a monoclonal antibody, D83.21, prepared by fusing P3X63/Ag8 mouse myeloma cells with mouse lymphocytes immunized against the DU145 human prostate adenocarcinoma cell line. The D83.21 monoclonal antibody displayed preferential binding to human prostate and bladder carcinoma cell lines and tissues. This antibody was not reactive with a variety of other normal and malignant human cell lines or tissues. Immunofluorescence analysis indicated that the D83.21 antigen was located on the plasma membrane. Biochemical characterization of the target antigen was performed by subjecting detergent-soluble extracts of [3H]glucosamine-labeled cells to D83.21 monoclonal antibody affinity chromatography. The radioactive material that was specifically bound and eluted from the affinity column was analyzed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis and fluorography. In the absence of 2-mercaptoethanol, the antigen displayed two prominent bands with molecular weights of 180 and 110 kilodaltons. In the reduced form, the antigen is composed of an Mr 60,000 heavy chain and an Mr 28,000 light chain. The antigen was further resolved using two-dimensional (intact/reduced) sodium dodecyl sulfate: polyacrylamide gel electrophoresis. These analyses indicated that the Mr 180,000 chain was composed entirely of Mr 60,000 subunits, whereas the Mr 110,000 band consisted only of Mr 28,000 subunits. The antigen recognized by the D83.21 monoclonal antibody is therefore a membrane glycoprotein with a subunit structure cross-linked together through disulfide bonds.     

Carcinoembryonic antigen has a different molecular weight in normal colon and in cancer cells due to N-glycosylation differences.

Carcinoembryonic antigen, an apical membrane glycoprotein expressed in normal human colonic epithelial cells, colonic polyps, tumor, and tissue culture cell lines originating from colonic adenocarcinomas, is generally considered to have a molecular weight of 180,000. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis associated with immunoprecipitation or immunoblotting with both monoclonal (Mab 517 and Mab 601) and polyclonal antibodies, we observed that carcinoembryonic antigen was actually expressed as two discrete apparent molecular weight forms in normal tissues: a broad band averaging at Mr 200,000 and a sharp band at Mr 130,000. This constituted the phenotype of the normal colon. In cancer cells we detected a single band at Mr 170,000 or lower. This variation was mainly the consequence of a modification of the glycosylation pattern of the molecule since deglycosylation by N-glycanase or biosynthesis in the presence of tunicamycin always produced a single molecular weight form, whether or not the source of tissue was normal or cancerous. By close inspection of benign, moderately transformed, and carcinomatous human colonic polyps we noticed that this shift in the molecular weight of carcinoembryonic antigen preceded the detection of other cancer markers such as nonspecific cross-reacting antigen at Mr 95,000 or the histological modifications leading to malignant diagnosis. Carcinoembryonic antigen constitutes, therefore, an important model with which to study the modifications of the glycosylation pattern induced during cancer biogenesis.     

Multiplicity of transforming growth factors in human malignant effusions

Human malignant effusions were found to contain transforming growth factor (TGF) activity capable of stimulating anchorage independent growth of nontransformed rodent fibroblasts. Bio-Gel P-60 chromatography of acid-ethanol extracts demonstrated the presence of three populations of TGF activities in 57% of malignant effusions. Two activities were similar to those of TGF alpha and TGF beta as judged by their size (Mr approximately equal to 6,000 and approximately equal to 25,000, respectively), biological activity (ability to stimulate anchorage independent growth of NRK fibroblasts in the absence or presence of epidermal growth factor, respectively), and capacity to competitively inhibit binding of 125I-labeled epidermal growth factor to A-431 membranes and 125I-TGF beta to baby hamster kidney fibroblasts, respectively. In addition a third factor which stimulated anchorage independent growth of nontransformed rodent fibroblast and human colonic epithelial cells was also recovered following Bio-Gel P-60 chromatography of extracts from several cytology positive human malignant effusions of patients with colonic and breast carcinoma as well as other malignancies. The latter malignant effusion related transforming growth factor was not present in benign or cytology negative effusions. Malignant effusion related TGF factor was inactivated by sulfhydryl reducing agents, heat, and trypsin treatment but was stable in 1% acetic acid and ethanol. Partial purification was accomplished by chromatography of an acid-ethanol extract on Bio-Gel P-60 followed by high performance liquid chromatography with C18-mu Bondapak to yield a nearly pure protein with apparent molecular weights of 64,000 by sodium dodecyl sulfate-polacrylamide gel electrophoresis when run in nonreducing conditions and 32,000 when run in reducing conditions. Malignant effusion related TGF was able to stimulate anchorage independent growth of nontransformed fibroblasts in the absence of other growth factors. It did not competitively inhibit binding of 125I-labeled epidermal growth factor, 125I-TGF beta, or 125I-labeled platelet derived growth factor. Therefore, this factor isolated from human malignant effusions may be distinct from previously described transforming growth factors. Collectively these observations indicate that human malignant effusions contain a multiplicity of transforming growth factors. It is possible that the malignant effusion related transforming growth factors play a role or reflect the metastatic growth properties of various tumors.     

Use of a monoclonal antibody (B72.3) as an immunocytochemical adjunct to diagnosis of adenocarcinoma in human effusions

A monoclonal antibody (MAb), designated B72.3, has been generated using membrane-enriched fractions of a metastatic human breast carcinoma as the immunogen. Previous studies have demonstrated that the reactive antigen, a novel Mr 220,000 to 400,000 glycoprotein complex, can be detected in formalin-fixed, paraffin-embedded tissue sections of human breast and colon carcinomas, and not in a variety of normal adult human tissues. In this preliminary study, we report that MAb B72.3 may be used as an adjunct for diagnosis of adenocarcinoma in cytological preparations of human effusions. Using the avidin-biotin complex method of immunoperoxidase staining and formalin-fixed, paraffin-embedded cell suspensions, MAb B72.3 detected adenocarcinoma cells in effusions from all of 21 patients with adenocarcinoma of the breast. No reactivity was demonstrated in any cell type in benign effusions from 24 patients without cancer, or 13 patients with prior or extant cancer in other body sites; moreover, B72.3 showed no reactivity to leukemic or lymphomatous effusions, or apparent mesothelial cells from malignant effusions. MAb B72.3 also detected adenocarcinoma cells in cytological effusion specimens from 12 of 12 patients with adenocarcinoma of the lung and 16 of 16 patients with adenocarcinoma of the ovary. Thus, these data suggest that the immunocytochemical application of MAb B72.3 should now be considered as an adjunct in the discrimination of adenocarcinoma cells from reactive mesothelial cells in the cytological diagnosis of malignant effusions.     

Cross-reactivity of a monoclonal antibody directed against an estrogen-induced protein in MCF-7 human breast cancer cells with murine Leydig cell tumor proteins

An estrogen-responsive murine Leydig cell tumor (M5480A) was examined for the presence of cross-reactive proteins to a monoclonal antibody directed against a Mr 24,000 estrogen-regulated protein in human breast cancer cells. Human breast tumor biopsies were used as controls for the cytosol preparations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot conditions used in these experiments. The estrogen-regulated Mr 24,000 protein was detected in sodium dodecyl sulfate-polyacrylamide gels of cytosols from four human breast tumor biopsies examined. Larger amounts of the Mr 24,000 protein were present in the two estrogen-progesterone receptor-positive tumor biopsies in comparison to the two estrogen-progesterone receptor-negative samples. In addition, the two receptor-positive samples demonstrated an additional, less intense immunoreactive band at Mr 21,000. Under identical conditions, the same monoclonal antibody bound to two major protein bands from sodium dodecyl sulfate-polyacrylamide gels of Leydig cell tumor cytosols at Mr 56,000 and Mr 86,000. Antibodies prepared from BALB/c mouse ascites fluid of animals bearing the parent myeloma cell line (P3X63NS1) exhibited no immunoreactivity against the human breast or Leydig cell tumor proteins. In light of the high degree of specificity which this monoclonal antibody exhibits, our results suggest that similar antigenic determinants may exist in these proteins from two distinct tumors.     

Biosynthesis and secretion of laminin and laminin-associated glycoproteins by nonmalignant and malignant human keratinocytes: comparison of cell lines from primary and secondary tumors in the same patient

Laminin biosynthesis was compared in four pairs of human squamous cell carcinoma cultures derived from primary and recurrent or metastatic tumors in four patients with cancer of the larynx and hypopharynx to determine if changes in laminin production accompany tumor progression. Laminin profiles of the malignant cells were compared with laminin biosynthesized by nonmalignant human keratinocytes. Pulse-chase biosynthetic labeling of the cultures with [35S]methionine established that all of the squamous carcinoma cell lines synthesize immunoreactive A (Mr 400,000), B1 (Mr 205,000), and B2 (Mr 200,000) laminin subunits; assemble them to form the intact laminin molecule (Mr 950,000); and secrete a portion of the laminin they produce into the culture media. One aspect of laminin expression unique to keratinocytes, both malignant and nonmalignant, was the occurrence of three additional glycoprotein forms (Mr 195,000, 170,000, and 160,000) in the laminin immunoprecipitates. In contrast to the laminin subunits, these glycoproteins were not immunoreactive with the anti-laminin antiserum on Western blots. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis without and with reduction of disulfide bonds revealed that the laminin immunoprecipitates contained a family of oligomeric molecules. These ranged in apparent molecular weight from 370,000 to 950,000 and were composed of laminin subunits and the glycoprotein forms linked by interchain disulfide bonds. The malignant keratinocyte cell lines from different patients were distinguishable in terms of the array of laminin and glycoprotein forms displayed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the rate of [35S]methionine incorporation into laminin during the pulse-labeling, the fraction of [35S]methionine-labeled laminin secreted into the medium during the chase incubation, and the absolute amount of laminin secreted into the culture medium as determined by enzyme-linked immunosorbent assay. However, cell lines established from primary and metastatic or recurrent cancer in the same patient were indistinguishable in their profile of laminin biosynthesis and secretion. In comparison to primary cultures of nonmalignant foreskin basal keratinocytes, the malignant cells secreted into the culture medium a larger fraction of the laminin that they produce. This is an indication that the malignant keratinocytes in culture deposited a less stable basal lamina-like extracellular matrix than their malignant counterparts. The diminished integrity of the basal lamina matrix may be an important factor in the invasive growth of human epithelial cancer.     

Purification and biochemical characterisation of a novel breast carcinoma associated mucin-like glycoprotein defined by antibody 3E1.2

A member of the high molecular weight glycoproteins of human milk and breast cancer was isolated from the sera, ascites and breast carcinoma tissue of patients with breast cancer using monoclonal antibody 3E1.2. The 3E1.2 defined antigen, termed mammary serum antigen (MSA) was obtained by immunoaffinity chromatography and a solid phase immuno-precipitation technique (SPIT) from serum of patients with metastatic breast cancer. MSA was found to be a high molecular weight glycoprotein with a Mr greater than 300,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a native Mr approximately 1 x 10(6) by gel filtration chromatography; in accord with the published Mr of other high molecular weight glycoproteins obtained from human milk and breast cancer. A high degree of glycosylation of MSA molecule was shown by its poor staining with Coomassie blue but good staining in a PAS-silver stain. In addition, MSA contained N-acetyl neuraminic acid and N-acetyl glucosamine as indicated by its binding to wheat-germ agglutinin. The epitope defined by antibody 3E1.2 is sensitive to treatment by sodium periodate and neuraminidase, implying that both carbohydrate and sialic acid are required for binding of antibody 3E1.2. Sandwich immunoassays demonstrated that MSA+ molecules are likely to express repeated 3E1.2 defined epitopes. Furthermore, MSA was susceptible to degradation by pronase, subtilisin and proteinase K and gave a different peptide profile from that of the PAS-O glycoprotein of human milk. MSA+ molecules were found to carry epitopes for a number of other monoclonal antibodies which were reactive with the PAS-O glycoprotein. It is suggested that MSA has the same core protein as is recognised by antibody DF3 which has been used to clone the same cDNA as was cloned with antibodies HMFG-1, HMFG-2 and SM-3. However, the epitope detected by the 3E1.2 antibody is either absent or weakly expressed on human milk, human milk-fat globule membrane (HMFGM) or deglycosylated HMFGM--all of which react strongly with various anti-HMFG antibodies. The antibody 3E1.2 thus recognises a unique epitope of the high molecular weight glycoproteins of human milk and breast cancer, being found in cancer tissue, serum and ascitic fluid of patients with breast cancer but weakly expressed or absent in human milk.     

A novel ribonucleoprotein complex defined by monoclonal antibodies to NIH 3T3 cells transfected with human pancreatic adenocarcinoma DNA

Three monoclonal antibodies elicited to NIH 3T3 cells transfected with DNA from a human pancreatic adenocarcinoma cell line recognized a novel ribonucleoprotein complex. Minimally, this ribonucleoprotein complex contained a Mr 240,000 protein (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and two RNA species with apparent sizes of 1.5 and 3.0 kilobases (by formaldehyde agarose gel electrophoresis). In addition to a cytoplasmic and nuclear subcellular localization, the RNA antigen was secreted from human tumor cell lines and NIH 3T3 cells transfected with pancreatic tumor DNA (inhibitable by monensin) and was apparently not a viral or Mycoplasma contaminant. The ribonucleoprotein antigen was detected in some normal tissues by immunoperoxidase but was not found in or secreted from in vitro cultured normal human fibroblasts, nontransfected or spontaneously transformed NIH 3T3 cells, or normal peripheral blood leukocytes.     

Mouse monoclonal antibodies which recognize a human (beta 1-4)galactosyl-transferase associated with tumor in body fluids.

Mouse monoclonal antibodies against human (beta 1-4)galactosyl-transferase (GalT) purified from human ovarian tumor effusion fluids were prepared and characterized. GalT purified from normal human plasma showed a single diffused band in nondenaturing polyacrylamide gel electrophoresis, but GalT purified from human ovarian tumor effusion fluids showed several oligomeric bands and a monomeric band in nondenaturing polyacrylamide gel electrophoresis. These oligomeric bands were dissociated into monomer by urea treatment and polymerized by a 2-mercaptoethanol treatment. Nine monoclonal antibodies (MAb) were prepared by immunization of purified GalT from human ovarian tumor effusion fluids and classified into three groups. Type I MAbs (MAb8611, MAb8913, and MAb8919) reacted only to the GalT monomer. Type II MAbs (MAb4880, MAb8507, and MAb8628) reacted to both the GalT monomer and the GalT polymer. Type III MAbs (MAb7907, MAb8513, and MAb8677) reacted only to the GalT polymer. These MAbs except MAb7907 could recover GalT enzyme activity from effusion fluids by immunoprecipitation. A fraction passed through MAb8513 affinity chromatography still showed reactivity to MAb8919, demonstrating that an epitope of MAb8513 resides on a minor part of GalT. A sandwich immunoassay (MAb8513-MAb8628HRP) was developed, and serum samples from ovarian cancer patients and benign ovarian patients were tested. The levels of sandwich immunoassay of serum samples from cancer were elevated significantly compared to those from benign and did not necessarily correlate to total GalT enzyme activity in serum samples. These results suggested that MAb8513 (Type III) might recognize a unique GalT associated with tumor (GAT).     

Generation of monoclonal antibodies to human milk-fat globule membrane antigens, with special reference to a precipitable secretory product of breast and ovarian carcinomas

Monoclonal antibodies were generated against protein antigens extracted from human milk-fat globule (HMFG) membranes. Of more than 500 hybridoma clones, 16 secreted antibodies reacting with breast and ovarian carcinoma cells, but not with lymphocytes or macrophages. Five clones were further expanded and characterized; four reacted with plasma membranes of all breast and ovarian carcinomas rested, but also with normal breast. One of the monoclonal antibodies, III D 5, reacted mainly against the cytoplasmic components of breast cancer cells, whereas the reaction with normal or mastopathic breast was against the luminal plasma membranes. The antigen recognized by the antibody was secreted in ascites fluid or pleural effusions of patients with widely spread ovarian or breast carcinomas, and could be demonstrated by immunodiffusion. It is suggested that the antigen is composed of several glycoprotein subunits bearing the same repetitive epitope. This might be the physicochemical basis for its precipitability with a monoclonal antibody.     

Cadherin switching in ovarian cancer progression

The roles of the cadherins in the progression of ovarian cancer to the late stages of the disease state when malignant cells have disseminated within the peritoneal cavity remain poorly understood. In view of these observations, we have undertaken a comprehensive survey of the cadherin subtypes present in normal ovarian surface epithelium and peritoneum and in the tumors and peritoneal effusions of women diagnosed with Stage I or Stage II primary ovarian cancer using a degenerate cloning strategy for sequences highly conserved among this family of cell adhesion molecules. On the basis of the nucleotide sequences of the resultant PCR products, multiple cadherin subtypes (E-, N-, P-cadherin, and cadherin-4, -6, and -11) were found to be present in these normal and malignant tissues and cells. P-cadherin was determined to be the predominant cadherin subtype in normal peritoneum, peritoneal effusions and Stage II tumor masses. An increase in P-cadherin mRNA and protein expression levels in ovarian tumor masses with progression to later stages of the disease state was confirmed by Northern and Western blot analysis, respectively. In addition, we have determined that the cadherin-associated protein, known as beta-catenin, is expressed in normal peritoneum, ovarian tumors and malignant cell effusions obtained from women with Stage I or Stage II cancer. Immunoprecipitation studies demonstrated that P-cadherin was capable of interacting with beta-catenin in these normal and malignant tissues and cells. Collectively, these findings suggest that the regulated expression of P-cadherin/beta-catenin complexes in ovarian tumor cells may represent a key step in disease progression.     

Ligand-induced phosphorylation of a murine tumor surface protein (TSP-180) associated with metastatic phenotype

A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to Mr 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (Mr 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 32PO4, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the Mr 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.     

Isolation of a lipolytic factor (toxohormone-L) from ascites fluid of patients with hepatoma and its effect on feeding behavior

The ascites fluids from patients with hepatoma or ovarian tumor and the pleural fluid from patients with malignant lymphoma elicited fatty acid release in slices of rat adipose tissue in vitro. The lipolytic factor, named toxohormone-L, was isolated from the ascites fluid of patients with hepatoma. The isolated preparation gave a single band on both disc gel electrophoresis and sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis in the presence of β-mercaptoethanol. Its molecular weight was determined to be 70,000–75,000 and 65,200 by SDS-acrylamide gel electrophoresis and analytical ultracentrifugation respectively. Injection of toxohormone-L into the lateral ventricle of rats significantly suppressed food and water intakes. There was at least a 5-hr delay between its injection and the appearance of its suppressive effect.     

Characterization of a malignant phenotype-associated cell surface glycoprotein common to various human tumor cells and preferentially expressed on adenocarcinoma of the lung

Since both the liver and lung are derived from the endoderm, common antigens may appear on both tissues during malignant transformation. In an attempt to delineate cell surface alterations associated with the neoplastic transformation of these tissues, we have produced a library of monoclonal antibodies against a human hepatoma cell line termed FOCUS. One of these monoclonal antibodies, designated AF-10, recognized an antigen preferentially expressed on human lung adenocarcinoma cells, both in vitro and in vivo. This antigen has been characterized using Western immunoblot analysis and immunoprecipitation from surface-iodinated or metabolically labeled cells. The mature antigen is a cell surface glycoprotein with a core polypeptide with a molecular weight of 75,000 bearing N-glycosylation units. This protein migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 100,000-115,000 in reducing conditions and Mr 115,000-130,000 in nonreducing conditions. The epitope recognized by monoclonal antibody AF-10 is borne by the core protein. This antigen is shed from the cell surface and was identified in the culture supernatant from lung adenocarcinoma cell lines. Studies of the biodistribution of the AF-10 antigen showed that it was also expressed at low levels in normal human small intestine and kidney. The AF-10 monoclonal antibody may be useful for the study of the antigen expression between normal lung and the transformed phenotype.     

Monoclonal antibodies to human squamous cell carcinoma of the lung and their application to tumor diagnosis

Three immunoglobulin G1 monoclonal antibodies, LuCa2, LuCa3, and LuCa4, were produced by fusing murine myeloma NS1 cells with splenocytes obtained from a BALB/c mouse immunized with SK-MES1 cells derived from human squamous cell carcinoma of the lung. These three monoclonal antibodies were shown to recognize different protein antigens on SK-MES1 cells by indirect immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. While the pattern of cell line distribution of antigens recognized by these antibodies was not tumor type specific, their reactivity with tissue and pleural effusion was much more informative than with cell lines. The presence of target antigens in vivo was analyzed by immunoperoxidase staining of frozen tissue sections and immunofluorescence staining of tumor cells in pleural effusions. LuCa2 antibody was reactive with lung squamous carcinoma and adenocarcinoma tumor tissues and pleural effusions, but only infrequently with those of small cell carcinoma. This antibody was also reactive with many tumor tissues from other organs as well as with various normal tissues, including alveoli and bronchus. LuCa3 and LuCa4 antibodies reacted with lung squamous carcinoma in tissues and pleural effusions, but not with lung adenocarcinoma nor with small cell carcinoma. These two antibodies reacted only weakly with normal squamous tissues of the esophagus, skin, and cervix uteri, but not with various other normal tissues. Moreover, LuCa3 had weak reactivity with squamous cell carcinoma tissue of tongue and esophagus, whereas LuCa4 had no reactivity with nonpulmonary tumor tissues. LuCa3 and LuCa4 antibodies should be of clinical interest, because our data suggest that these antibodies may be potentially useful for the diagnosis of the histological type of lung tumor cells in both cancer tissue and pleural effusions.     

Characterization of the shed form of the human tumor-associated glycoprotein (TAG-72) from serous effusions of patients with different types of carcinomas

Monoclonal antibody (MAb) B72.3 binds a high molecular weight tumor-associated glycoprotein designated TAG-72. This study reports the isolation and characterization of secreted TAG-72 directly from effusions of ovarian, colorectal, pancreatic, and endometrial carcinoma patients and compares them to TAG-72 derived from the LS-174T colon carcinoma xenograft. The B72.3-reactive antigen, TAG-72, was used as immunogen to produce second generation anti-TAG-72 MAbs. One of these second generation MAbs, CC49, had a higher affinity than that of B72.3 and was utilized as an affinity reagent in a procedure to purify the TAG-72 present in the serous effusions of carcinoma patients. A three-step purification procedure, utilizing heat extraction, CC49 antibody affinity chromatography, and gel filtration chromatography, resulted in 1000- to 4400-fold purifications of the TAG-72 derived from effusions, as analyzed using a double-determinant radioimmunoassay. Radiolabeled TAG-72 from each of the effusions demonstrated similar high molecular weight bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similar results from the various effusions were also obtained in Western blotting analyses. Analyses of TAG-72 from the different effusions in radioimmunoassay using five different anti-TAG-72 MAbs revealed similar binding patterns. The results of these studies thus indicate that TAG-72 obtained directly from patients with ovarian, colorectal, endometrial, and pancreatic carcinomas and the LS-174T xenograft are highly similar in terms of immunochemical properties and antigenic profile.     

Monoclonal antibody DS6 detects a tumor-associated sialoglycotope expressed on human serous ovarian carcinomas

A newly developed murine monoclonal antibody, DS6, immunohistochemically reacts with an antigen, CA6, that is expressed by human serous ovarian carcinomas but not by normal ovarian surface epithelium or mesothelium. CA6 has a limited distribution in normal adult tissues and is most characteristically detected in fallopian tube epithelium, inner urothelium and type 2 pneumocytes. Pre-treatment of tissue sections with either periodic acid or neuraminidase from Vibrio cholerae abolishes immunoreactivity with DS6, indicating that CA6 is a neuraminidase-sensitive and periodic acid-sensitive sialic acid glycoconjugate ("sialoglycotope"). SDS-PAGE of OVCAR5 cell lysates has revealed that the CA6 epitope is expressed on an 80 kDa non-disulfide-linked glycoprotein containing N-linked oligosaccharides. Two-dimensional non-equilibrium pH gradient gel electrophoresis indicates an isoelectric point of approximately 6.2 to 6.5. Comparison of the immunohistochemical distribution of CA6 in human serous ovarian adenocarcinomas has revealed similarities to that of CA125; however, distinct differences and some complementarity of antigen expression were revealed by double-label, 2-color immunohistochemical studies. The DS6-detected CA6 antigen appears to be distinct from other well-characterized tumor-associated antigens, including MUC1, CA125 and the histo-blood group-related antigens sLea, sLex and sTn.     

Regulatory T cells and cytokines in malignant pleural effusions secondary to mesothelioma and carcinoma

Immunotherapy against a variety of malignancies, including pleural-based malignancies, has shown promise in animal models and early human clinical trials, but successful efforts will need to address immunosuppressive factors of the tumor and host, particularly certain cytokines and CD4(+) CD25(+) regulatory T cells (Treg). Here, we evaluated the cellular and cytokine components of malignant pleural effusions from 44 patients with previously diagnosed mesothelioma, non-small cell lung cancer (NSCLC), or breast cancer and found significant differences in the immune profile of pleural effusions secondary to mesothelioma vs. carcinoma. Although a high prevalence of functionally suppressive CD4(+) CD25(+) T cells was found in carcinomatous pleural effusions, mesothelioma pleural effusions contained significantly fewer CD4(+) CD25(+) T cells. Activated CD8(+) T cells in pleural fluid were significantly more prevalent in mesothelioma than carcinoma. However, there is clear patient-to-patient variability and occasional mesothelioma patients with high percentages of CD4(+) CD25(+) pleural effusion T cells and low percentages of CD8(+) CD25(+) pleural effusion T cells can be identified. Mesothelioma pleural effusions contained the highest concentrations of the immunosuppressive cytokine transforming growth factor (TGF)-beta. Thus, the contribution of cellular and cytokine components of immunosuppression associated with malignant pleural effusions varies by tumor histology and by the individual patient. These results have implications for the development of immunotherapy directed to the malignant pleural space, and suggest the need to tailor immunotherapy to overcome immunosuppressive mechanisms in tumor environments.