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(Chest. 2003;124:682-687.)
© 2003 American College of Chest Physicians

Defensive Role of Pleural Mesothelial Cell Sialomucins in Tumor Metastasis*

Ramit K. Sharma, MD; Kamal A. Mohammed, PhD; Najmunnisa Nasreen, PhD; Joyce Hardwick, BA; Robert D. Van Horn, BS; Carlos Ramirez-Icaza, MD and Veena B. Antony, MD, FCCP

* From the Division of Pulmonary and Critical Care Medicine, Department of Medicine, Veterans Affairs Medical Center, Indiana University School of Medicine, Indianapolis, IN.

Correspondence to: Veena B. Antony, MD, FCCP, Veterans’ Affairs Medical Center, 1481 West Tenth St, 111-P, Indianapolis, IN 46202; e-mail: vantony{at}.iupui.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Sialomucin complex (SMC) is a heterodimeric glycoprotein, and is found on the surfaces of the mesothelia of the pleura, pericardium, and peritoneum. Sialomucins play a significant role in adhesion as well as in defense. In this study, we hypothesized that pleural mesothelial cells (PMCs) express SMC and thus prevent the adherence of ovarian cancer cells (HTB-77) to the pleura.

Methods: PMCs were plated, and the adherence of HTB-77 cells was observed using a cytofluor. The PMC monolayer was pretreated with sialidase, and HTB-77 adherence was observed using cytofluor. In another set of HTB-77 cells, adherence was observed when the PMC monolayer was pretreated with supernatants of HTB-77 cells. Last, supernatants of HTB-77 cells were assayed for sialidase activity.

Results: The removal of SMC by sialidase greatly increased the adherence of HTB-77 cells to the PMC monolayer, which was statistically significant (p < 0.05). Similar results were obtained when the PMC monolayer was pretreated with the supernatants of HTB-77 cells. Supernatants of HTB-77 cells showed the presence of sialidase.

Conclusions: The presence of SMC on the PMC acts as a defense layer, and its removal by sialidase increases the susceptibility of the PMC layer to the adherence of malignant cells and to increased metastasis. HTB-77 cells also express sialidase, which by its action on the monolayer aids in the adherence of tumor cells to the pleural surface.

Key Words: metastasis • ovarian cancer • pleura • sialidase • sialomucins


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The parietal and visceral pleura are lined with mesothelial cells, which are cells flattened on a basal lamina over a layer of reticular fibers and collagen. These cells are adhered to one another near their free surfaces by a zonula adherens, and by the interdigitations of cell membranes and one or more desmosomes.1 Mesothelial cells have extensive interlacing microvilli on their free surfaces and appear to be embedded in a layer of protein.2 The free surfaces of mesothelial cells also have been shown to have mucins, which are negatively charged.3

Mucins are best known as the major constituent of mucus. They form a distinct group of highly glycosylated glycoproteins that contain numerous glycans, which are predominantly linked to Ser or Thr residues. Glycans are tightly packed and cover a larger part of the protein backbone of these molecules, and their molecular masses far exceed 200 kd. Mucins protect the underlying epithelial cells and other cells, and also act as lubricants.4 Large mucin-like domains also are found on certain cell membrane-associated molecules such as leukosialin and episialin. These membrane-associated mucins differ from mucus mucins. Among these mucin-like proteins, one of the most unique is the sialomucin complex (SMC), a cell-surface glycoprotein complex. It was first isolated from highly proliferative and metastatic ascitic sublines of 13762 rat mammary adenocarcinoma.5 SMC is a large heterodimeric glycoprotein complex that is composed of mucin subunit ascitic sialoglycoprotein (ASGP)-1 and a transmembrane subunit ASGP-2, which is N-glycosylated. ASGP-2 anchors the SMC to the plasma membrane.6 The sialomucin molecule shares many properties with episialin with respect to its structure, biosynthesis, and processing. Like episialin, SMC is continuously recycled and resialylated.7

The free surface of the peritoneal mesothelium has been shown to have a coat with glycoconjugates containing anionic sites. This has been demonstrated using cationic probes, such as colloidal iron.8 These strong anionic sites coat the surface with a negative charge and are considered to act repulsively in order to maintain the peritoneal cavity.8 9 10 Light and electron microscopic studies have shown that the pleura also has strong anionic sites that are associated with a free surface of mesothelium. These strong anionic sites ionized even at pH 1.5, suggesting that the latter sites are derived from sialic acid.9 10 11 Neuraminidase treatment before colloidal iron staining erases the Prussian blue reaction for colloidal iron stain on the free surface of the pleural mesothelium.10 11 12 Enzyme digestion with hyaluronidase, chondroitinase ABC, heparitinase, and keratinase have no effect on colloidal iron staining. These findings suggest that mesothelial surface anionic sites are derived from sialic acid. Although the free mesothelial surface of the parietal and visceral pleura abut each other and come into contact, the anionic charge present on the surface may act by electrostatic repulsion to avoid serosal adhesions and to reduce friction during organ movement.

Earlier studies performed on various malignancies, especially adenocarcinoma of the colon and of the ovary, have demonstrated that these malignant cells express sialomucins on their surfaces, which has been postulated as aiding in their distant metastasis. These tumors also invade the pleura preferentially, and the question arises about why this relatively selective metastasis has not been explored previously. Understanding this important mechanism could have great clinical significance and implications in patient management.

The present study demonstrates that sialomucins play a significant role, both in adhesion and in defense. The removal of the SMC present on the free surface of pleural mesothelial cells (PMCs) makes them more prone to later adhesion by malignant cells.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Neuraminidase (sialidase) EC 3.2.1.18 was obtained from Sigma Chemical Co (St. Louis, MO), colloidal iron was obtained from Waco Pure Chemical Industries (Tokyo, Japan), ovarian cancer cells (HTB-77) and Met 5-A cells were obtained from the American Type Culture Collection (Manassas, VA), and the neuraminidase (sialidase) assay kit was purchased from Molecular Probes (Eugene, OR).

Colloidal Iron Staining
Briefly, PMCs were plated on a four-chamber slide and were grown to confluence. The cells were fixed and subsequently stained with colloidal iron using a modified Hale and Mowry method. Initially, cells were hydrated and placed in acetic acid for 8 min. Cells then were stained with colloidal iron for 10 min. After washing with distilled water until the slides appeared colorless, the slides were stained with potassium ferrocyanate and 1% HCl. Finally, the slides were counterstained with nuclear fast red and rinsed with 95% ethyl alcohol. Last, these slides were mounted in xylene-soluble medium and studied under microscopy.

In another set of similar experiments, PMCs were plated on a four-chamber slide, and the cells were grown to confluence. Two of the chambers were treated with neuraminidase (sialidase) at a concentration of 2 mmol/L for a period of 60 min. Excess sialidase was washed off using Hanks balanced salt solution (HBSS). The slides were stained with colloidal iron, as described above. Finally, these slides were mounted in xylene-soluble medium and studied under microscopy.

Determination of Adherence of HTB-77 Cells to PMC Monolayer
The adherence of the HTB-77 cells to the PMC monolayer was studied using a 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF) adherence assay. Briefly, Met 5-A cells were plated in a 24-well microplate and were brought to confluence. HTB-77 cells then were counted and palletted. This pallet was brought into 500 µL HBSS. These were mixed with 20 µL BCECF, which had been prepared earlier by dissolving 50 µL BCECF into 40.3 µL dimethyl sulfoxide.

The cell pellet was kept on ice for a period of 30 min, and then was washed and rinsed three times in HBSS. After the last spin, enough cells were brought into the HBSS for a cell monolayer to form, and they were placed in different wells for different times periods (ie, 30, 60, 90, and 120 min). At appropriate time intervals, these were rinsed gently with HBSS solution three times and studied under cytofluor. The cytofluor settings were set for an excitation range of 480 to 510 nm and an emission range of 510 to 540 nm.

Determination of Adherence of HTB-77 Cells to PMC Monolayer After Pretreating With Sialidase
PMCs were plated in a 24-well microplate and were brought to confluence. Twelve wells were treated with sialidase at a concentration of 2 µm/µL for periods of 30 and 60 min. Then the wells were gently rinsed with HBSS. Subsequently, an ovarian cancer cell adherence assay was performed as described above, and the microplate was read for cytofluor studies.

Adherence of HTB-77 Cells to PMC Monolayer After Pretreating With Supernatants of the HTB-77 Cells
PMCs were plated in a 24-well microplate and were brought to a confluent monolayer. From the HTB-77 cells growing in the culture flask, a supernatant was aspirated and placed onto the PMC monolayers (500 µL in each well) for periods of 30 and 60 min. Subsequently, the microplate was gently rinsed, and the excess supernatant was washed off with HBSS. The BCECF adherence assay then was performed as described above, and the cytofluor studies were performed using different time intervals.

Determination of Sialidase in Supernatants of HTB-77 Cells
Supernatants of HTB-77 cells were assayed for sialidase activity using the neuraminidase assay kit according to manufacturers instructions. In brief, supernatants of HTB-77 cells were obtained and sialidase activity was assessed. The assay kit utilizes the Amplex Red to detect H2O2 generated by galactose oxidase oxidation of desialidated galactose, which is the end result of the sialidase reaction. H2O2 in the presence of horseradish peroxidase reacted with Amplex Red stain to generate the red fluorescence oxidation product resorufin, which was measured by the cytofluor.

Statistical Analysis
Results are expressed as the mean ± SE using the Student unpaired t test. All experiments were performed at least three times unless otherwise indicated. Statistical significance was considered significant if p values were < 0.05.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PMCs Express SMC
A monolayer of PMCs was prepared and stained with colloidal iron. SMC on pleural cells showed a purple coloration when stained with colloidal iron (Fig 1 , middle, B), confirming the presence of SMC on the free mesothelial surface. The results of PMC monolayer staining after pretreatment with sialidase showed no purple coloration, confirming the removal of SMC from the free surface of the PMC monolayer by sialidase (Fig 1 , bottom, C) when compared to the control (Fig 1 , top, A). A similar result was obtained when the PMC monolayer was treated with a supernatant of the HTB-77 cells and then was stained with colloidal iron (data not shown).



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  		Figure 1.. PMCs express SMC. Top, A: PMC only. Middle, B: PMCs stained with colloidal iron. Bottom, C: PMC stained with colloidal iron after pretreatment with sialidase (2 mmol). This is representative of three independent experiments.

 
Adherence of HTB-77 Cells to PMC
The PMC monolayer was grown to confluence, and the HTB-77 cell adherence studies were performed. The HTB-77 cells were left on top of the PMC monolayer for adherence, after labeling with BCECF for periods of 30, 60, 90, and 120 min. The adherence of HTB-77 was noticed to be highest at 60 min (Fig 2 . ) However, the adherence of HTB-77 cells plateaued after 90 min of incubation.



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  		Figure 2.. Adherence of ovarian cancer cells to PMC. The adherence of ovarian cancer cells (HTB-77) to PMC was measured using a cytofluor, as described in the "Materials and Methods" section. The data represent the mean ± SE of four independent experiments. * = p < 0.05 (ie, considered to be significant).

 
HTB-77 Cells Show an Increasing Detachment to PMC When Treated With Sialidase
The PMC monolayer wells were pretreated with sialidase for a period of 60 min, and HTB-77 cells were placed on the PMC monolayer for varying time intervals, with wells treated with sialidase showing an increase in the adherence of the HTB-77 cells compared to controls (Fig 3 ). Cells showed maximum adherence at the 60-min interval and then leveled off.



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  		Figure 3.. PMC pretreatment with sialidase/ovarian cancer cell supernatants increases ovarian cancer cell adherence. The adherence of ovarian cancer cell (HTB-77) to the PMC was measured using a cytofluor, as described in the "Materials and Methods" section. The data represent the mean ± SE of six independent experiments. * = p < 0.05 when compared to the control.

 
Supernatant of HTB-77 Induce Sialidase-Like Activity in Increasing Detachment of the HTB-77 Cells to PMC Monolayer
The PMC monolayers were pretreated with supernatants of HTB-77 cells for 60 min each. Subsequently, the adherence studies for the HTB-77 cells were performed (Fig 3) , and there was a significantly increased adherence of the HTB-77 cells in the wells that had been pretreated with supernatants compared to the control. However, when comparing the sialidase group with the HTB-77 culture supernatant group, no significant difference was noticed.

Evidence of Sialidase in the Supernatants of HTB-77 Cells
HTB-77 cells were cultured, and supernatants were obtained after 60 min. The sialidase levels were assessed in the supernatants using a cytofluor. The levels of sialidase were significantly higher in the supernatants of HTB-77 cells when compared to the supernatants of the PMC (Fig 4) .



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  		Figure 4.. Ovarian cancer cells (HTB-77) release sialidase in vitro. The data represent the mean ± SE of four independent experiments. * = p < 0.05 when compared to PMCs.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The free surface of mesothelial cells have SMCs, which are negatively charged,3 on their surface, and these act as repulsive forces to maintain pleural cavity. These also act as a part of a defense mechanism by preventing the adhesion of noxious substances to its free surfaces.11 12 13 The findings in our study confirm the presence of SMCs on the free surface of PMCs, which also had been reported earlier by others.8 9 Treating PMC monolayer with sialidase removes these anionic sites, thus removing the negative charge. As a result of this, the free surface of the PMCs no longer acts as a repulsive barrier, forming rather a nidus for the adherence of noxious cells. It was shown in our study that there was an increased adherence of ovarian cancer cells (HTB-77) to the free mesothelial surface, which has been pretreated with sialidase when compared to controls. This difference was statistically significant (p < 0.05). Interestingly, a similar result was obtained when PMCs were pretreated with a supernatant of ovarian cancer cells. This set of experiments also showed an increased adherence of ovarian cancer cells, and this increased adherence was statistically significant (p < 0.05). Also, the supernatants of ovarian cancer cells showed the presence of sialidase, which is released by HTB-77 cells.

The findings of our study are very interesting because of the insight given into the preferential metastasis of certain malignancies to the pleural surface. It is already known that certain tumors have predilection for metastasis to pleural surfaces. There have been studies12 13 that have reported that few malignant tumors (ie, ovarian cancer and adenocarcinoma of colon) express sialomucins on their free surface, and that these mucins play a role in their metastasis to distant sites. A more likely explanation is that, in addition to the presence of sialomucins, ovarian cancer cells also release a sialidase or sialidase-like substance, which aids in removing the SMCs that are present on the free surface of PMCs, thus increasing the adherence of the malignant cells. The pleural mesothelium was originally thought of as a passive membrane in the process of the development of infections and malignant metastasis. The pleural mesothelium is now emerging as a coordinator of concerted response to both microbial and malignant invasion.13 In this study, we have demonstrated that PMCs express SMCs, which may in part decrease the seeding of invading malignant cells onto the pleura. Depending on the types of cells that express sialomucins and the type of glycosylations, SMCs may serve different functions.

As reported earlier, there have been previous studies that have demonstrated the possible role of sialomucins that are present on malignant cells in the promotion of distant metastasis, especially those on ovarian and colonic tumors. However, there has been no previous study that has shown a possible role for sialidase or for a similar sialidase-like substance in these tumors to facilitate tumor adhesion to the pleura. Our study is the first of its kind to look in this direction and to demonstrate a sialidase-like activity by the ovarian cancer cells. Strong expressions of increased adhesions by ovarian malignant cells suggest for the first time that sialomucin and the sialidase-like activity expressed by these cancer cells serve in transporting metastases and adhesions to distant sites. The expression of sialidase by HTB-77 goes a step further to show that the former is likely to be involved in the mechanism of metastasis of cancers. Further studies are needed to look into these novel paths of the mechanism of metastasis and of preferential adhesion. Further studies may enable us to understand these mechanisms of metastasis better and may help us to develop strategies to break the sequence of events, to help in the reduction of metastasis, and, thus, to take a step toward improving the quality of life of these patients. These studies may include looking into the secretion and/or the production of sialidase by HTB-77 cells, their migration to the surface, and their mechanisms of adherence to SMCs that are present on the pleural surface.

In summary, we have reported that PMCs have SMCs on their surface. We also have shown that when this SMC is removed by sialidase or by a substance that has sialidase-like activity, as shown by the supernatants of HTB-77 ovarian cancer cells, there is an increased adherence of malignant cells to the pleural surface. We further speculate that the sialomucin present on mesothelial cells might be protective against adhesions of various malignant cells and other noxious cells.


   Footnotes
 
Abbreviations: ASGP = ascitic sialoglycoprotein; BCECF = 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; HBSS = Hanks balanced salt solution; PMC = pleural mesothelial cell; SMC = sialomucin complex

This research was supported in part by grants NIH RO1 AI45338-02 and NIH RO1 AI41877-04 from the National Institutes of Health.

Received for publication August 12, 2002. Accepted for publication January 9, 2003.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  8. Ohtsuka, A, Murakami, T Anionic sites on the free surface of the peritoneal mesothelium: light and electron microscopic detection using cationic colloidal iron. Arch Histol Cytol 1994;57,307-315[ISI][Medline]
  9. Miller, RL, Collawn, JF, Jr, Fish, WW Purification and macromolecular properties of a sialic acid-specific lectin from the slug Limax flavus. J Biol Chem 1982;257,7574-7580[Abstract/Free Full Text]
  10. Ohtsuka, A, Yamana, S, Murakami, T Localization of membrane-associated sialomucin on the free surface of mesothelial cells of the pleura, pericardium, and peritoneum. Histochem Cell Biol 1997;107,441-447[CrossRef][ISI][Medline]
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