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Ann Thorac Surg 1995;59:835-844
© 1995 The Society of Thoracic Surgeons
Surgery Branch, Thoracic Oncology Section; Naval Medical Oncology Branch; Pediatric Tumor Biology-Ultrastructural Pathology Section and Cytopathology Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
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Abstract |
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Introduction |
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Malignant pleural mesothelioma (MPM) is a neoplasm for which standard treatment regimens, including operation, chemotherapy, and radiation, have had no impact on median survival (approximately 8 to 10 months from diagnosis). Although relatively rare, it is estimated that there will be more than 80,000 new cases of the disease during the next 20 years. The latency period for the disease usually ranges from 15 to 40 years, and its association with asbestos is unquestioned, although there may be other factors that contribute to mesothelial carcinogenesis. Paralleling the rising incidence of mesothelioma, there has been an acceleration of interest in fiber carcinogenesis, autocrine growth pathways, molecular genetics, and altered gene products [1].
As with other thoracic malignancies, it would certainly be to the clinician's advantage to know how these malignancies develop and propagate to formulate treatment strategies precisely. The generation of mesothelioma cell lines has enabled researchers to perform karyotyping analysis and detect specific chromosomal deletions, translocations, and inversions that could give hints regarding changes in genetic material. These changes could down-regulate or completely abolish the action of tumor suppressor genes, leading to uninhibited growth. The development of enzyme-linked assays allow quantitation of abnormal secretory products from the cell lines that may represent autocrine growth factors. Molecular biologic techniques including Northern (RNA) and Southern blot (DNA) analyses, as well as the polymerase chain reaction (which amplifies DNA segments from only a few oligonucleotides) are documenting specific results of chromosomal damage, including base sequence changes. In vitro/in vivo chemotherapy and radiation sensitivity studies have been applied to cell lines and animal models to add preclinical justification for new treatment protocols.
The ability to obtain viable tumor samples of malignant mesothelioma for cell and molecular biologic analyses, and eventual grafting into immunodeficient animals is hindered by the rarity of the disease, and to the time commitment for the development as well as the care and feeding of such cultures. The techniques necessary for deriving the lines are occasionally detailed in the literature; however, as opposed to lung cancer where there are more than 100 established human cell lines, only a few permanent mesothelioma cell lines are available. Moreover, even when the lines are available it is difficult to find any relevant clinical information regarding the patient from whom the line originated.
Since June 1990, July in abstract, 139 patients with malignant pleural mesothelioma have been evaluated for protocols at our institution, of which 77 have been explored for the purpose of maximal debulking with intraoperative and postoperative adjuvant therapy. In addition, another 11 patients on nonsurgical protocols have had thoracenteses from which an attempt was made to generate a cell line. This report describes our methods for establishing MPM cell cultures during this period, and details selected characteristics of the first nine of these mature cultures.
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Material and Methods |
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Patient History
The 9 patients from whom cell lines were derived in this study consisted of 8 men and 1 woman ranging in age from 27 to 69 years. Seven of the 9 patients had definitive asbestos exposure as documented by any of the following: ferruginous bodies in the lung (4 patients), electron microscopic fiber analysis (2), heavy exposure (1 patient). All original pathologic material was analyzed by light microscopic analysis, followed by extensive immunocytochemical analysis using a battery of markers.
Processing of Fresh Material
The initiation and processing of the lines is similar to that described by Versnel [2] and Manning [3] and their colleagues. Briefly, our lines have been derived from primary solid tumor specimens or effusions. Solid tissue was minced into small pieces, 1 to 3 mm2, either with scissors or by cross-cutting with two scalpels. This released tumor aggregates into the medium. Several washes of these tissue pieces usually yielded sufficient tumor cell aggregates for culture. Effusions from small volumes were centrifuged and placed into culture flasks. Large volumes of effusions were centrifuged at 1,500 rpm for 5 minutes, and if there were large amounts of erythrocytes, the cell suspensions were subjected to Ficol gradient separation (Organon Teknika Corporation, Durham, NC). The interface layer of cells was harvested, washed twice with medium, and seeded into flasks. Red blood cells were also removed with ACK lysis (B & B Research, Fiskeville, RI).
Culture Conditions and Isolation Techniques
The effusions or tumor aggregates were cultured in either HITES, ACL-4, Rheinwolds medium for squamous cell carcinoma (SCCRH), RPMI, or DFCI media (Table 1
) Flasks of cells with the varying media were incubated at 37°C in 5% CO2, and the media was changed every 4 to 7 days. When the flasks were confluent, the cells were washed with phosphate-buffered saline, and deplated using a 5- to 7-minute exposure to trypsin--versene (0.05/0.02%). The trypsin was neutralized with serum-containing medium, and the deplated cells were washed twice in complete medium and then replated at high density. All lines were then routinely passaged every 1 to 2 weeks. In general, normal mesothelial cells senesced and failed to grow after 2 months in culture. Normal fibroblasts were eliminated by early, continuous culture in low serum conditions or by the addition of cholera toxin. Cells were grown to confluency, split as necessary, and when colonies of tumor cells appeared, the culture was submitted for immunohistochemical analysis. Natural subcloning (ie, partial trypsinization procedures) and serum deprivation were used for ongoing segregation of normal populations from tumor colonies. Serum was added to the media in increments of 2% for further expansion so that the media was switched to serum- supplemented RPMI. On occasion, however, tumor cells were isolated by scraping the tumor colony with the bent tip of a Pasteur pipet, aspirating it into the pipet and depositing it into the wells of a 24- or 96-well cell culture plate (COSTAR, Cambridge, MA). To verify that the surviving population of cells were abnormal, the final cultures were submitted to cytogenetic analysis. Cultures that showed no growth or only normal cells were discarded after 2 months. All cultures were declared cell lines after 25 passages and/or 1 year in culture.
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Antibodies were run three times on each case: once on air-dried refrigerated cytospins, once on air-dried frozen (-20°C) cytospins, and once on paraffin-embedded cell block sections. The method of fixation for the air-dried cytospins varied according to antibody specifications. For Leu-M1, the slides were fixed in acetone for 5 minutes. For B72.3, CEA, Ber-EP4, and the cytokeratins the slides were fixed in a 1:1 methanol/ethanol mixture for 5 minutes. Two negative control slides (MOPC) were run on each case; one fixed in acetone and one fixed in the alcohol mixture.
The antibody titers were as follows: B72.3, 1:100; CEA, 1:100; Leu-M1, 1:10; Ber-EP4, 1:20; AE1/AE3, 1:100; CAM5.2, undiluted; and MOPC, 1:50.
Electron Microscopic Examination
Seven of the cell lines were evaluated by electron microscopy. Cells were rinsed in phosphate-buffered saline, scraped, and then fixed in 2.5% glutaraldehyde in Sorensen's phosphate buffer (pH 7.4) for 1 hour at room temperature. Subsequently, the cells were washed in phosphate-buffered saline, overlaid onto 1 mL of serum in Eppendorf microfuge tubes and centrifuged for 10 minutes at 1,200 rpm. The cell pellets were fixed with 2.5% glutaraldehyde for another hour, postfixed in OsO4, and embedded in Maraglas 655 (Ladd Research Industries, Burlington, VT). Sections were stained with uranyl acetate lead citrate and examined in a Phillips CM10 electron microscope.
Cytogenetics and Karyotyping
Cytogenetics and karyotyping were performed by the cell culture laboratory of Childrens Hospital of Michigan. All cell lines were reacted with human and murine antiserum to confirm human origin. Isozyme phenotypes using glucose-6-phosphate dehydrogenase, phosphoglucomutase-1 and -3, esterase D, mitochondrial malic enzyme, adenylate, kinase, and glyoxalase-1 were used to calculate the frequency product (ie, the percentage of cultures expected to have the phenotype of the line in question).
A minimum of 100 metaphases were examined for chromosome count and ploidy distribution, and 7 to 10 Giemsa-banded chromosome karyotypes were prepared and photographed from metaphases with varying number of chromosomes.
Growth Properties
Doubling times, cell size, and growth in serum-free media were recorded. For doubling time and serum-free growth data, 1 x 105 cells were plated in T75 or T25 flasks, in either RPMI media with 10% fetal calf serum, ACL4, or HITES media without supplemental fetal calf serum. Cells were harvested by trypsinization, and counted using Coulter Counter model ZM (Coulter Electronics, Hialeah, FL) on days 3 and 6 after plating. Cell size was measured using flow cytometry with beads (Duke Scientific, Palo Alto, CA) as standards.
Cytokine and Growth Factor Production
The elaboration of growth factors using the appropriate enzyme-linked immunoabsorbent assays (ELISA) was measured: epidermal growth factor (EGF); granulocyte macrophage colony-stimulating factor (GM-CSF); platelet-derived growth factor AB (PDGF-AB) (all from R&D Systems, Minneapolis, MN); transforming growth factor-
(TGF-) (Oncogene Science, Uniondale, NY) and TGF-ß (R&D Systems), as well as interleukin-1 (IL-1) (R&D Systems) and IL-6 (Endogen, Boston, MA); and leukemia inhibitory factor (LIF) (R&D Systems). Briefly, 1,000 mesothelioma cells from each cell line were plated into a minimum of six wells of 96-well plates in 200 µL of RPMI supplemented with 10% fetal calf serum. Control wells were included with media alone, and all samples used for generation of the standard curve were diluted with media. The spent media was harvested after a 5-day growth, and tested for the various cytokines or growth factors. Only those levels that were above the minimum detectable level in picograms per milliliter were considered evaluable.
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Results |
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describes the name of the line, its growth medium, whether the specimen was derived from solid tumor, and the original histology of the mesotheliomatab 2. All lines grew as adherent monolayers, and all clearly grew better in the presence of fetal calf serum. Although all tended to have cells ``bud'' off confluent monolayers, line H2595 had a tendency to also form floating spheroids in the media.
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). The cells had abundant dense cytoplasm, frequent mitotic figures, multiple nucleoli, and some were multinucleate. To some extent all cultures had a spindle-shaped look to the cells, but cultures H2596, HP-2, and H2373 were primarily sarcomatoid in appearance with a wild, whorly, fibroblastoid growth pattern. The other cell lines tended toward a more epithelial pattern with thicker, more vacuolated cells, plumper in appearance, grouping together at the beginning of passages as clusters of cells.
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. As predicted for mesothelioma, all cell lines stained positively for cytokeratins, whereas only one line stained positively with an adenocarcinoma marker CEA. Most likely this was a false positive, as the background staining was also high making accurate interpretation difficult. Another cell line stained positively for Leu-M1, and rare mesotheliomas have been reported to show staining with Leu-M1 and CEA. The most predictable immunohistochemical analyses were performed with the paraffin-embedded cell block sections.
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). The degree of complexity and length of the microvillous projections, as well as their numbers varied among the cell lines and were graded in a scale of 1+ (a few short, but slender) to 4+ (numerous, long slender and complex) (Fig 2). The amount of intracytoplasmic filaments also showed slight variation ranging from 1+ (focal) to 2+ (diffuse). Three cell lines showed condensation of the filaments into tonofibrillar bundles (Fig 3). Intercellular junctions were present, but not prominent. Other features included the presence of a well-developed rough endoplasmic reticulum, variable amounts of intracytoplasmic glycogen, and prominent lysosomal activity with myelin figure formation in three cell lines. All cell lines lacked mucin or other secretory material.
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). Seven lines had hypodiploid modal chromosome number, and two lines were hypotetraploid. Table 5 demonstrates the chromosome number determined from 7 to 10 karyotypes on each cell line, as well as the most common copy numbers for selected abnormal chromosomes. Table 6 further subclassifies the specific abnormalities that could be recognized by G-banding of the chromosomes with the marker chromosomestab 6. The most commonly affected chromosomes with deletions, inversions, or translocations were 1, 3, 5, and 9.
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. The doubling times were between 31 and 65 hours and cell diameters ranged from 59 to 81 µm. The majority of the cell lines were able to proliferate for 6 days in serum-free media as seen in Figure 7. Greatest proliferation uniformly occurred in ACL4 and DFCI, both of which contained more essential nutrients than HITES.
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demonstrates the results of growth factor and cytokine production by these nine cell linestab 7. None of the cell lines produced EGF, and only one cell line was noted to produce significant levels of transforming growth factors. All lines were noted to produce significant amounts of GM-CSF, PDGF-AB, and IL-6.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Confirmation that the final population of cells was indeed of mesothelial origin and malignant was based on the morphology of the cells by light and electron microscopy, immunohistochemical profile, and cytogenetic/karyotype analyses. Others have remarked that the morphology of the cells in culture will depend on the type and amount of serum (ie, human versus bovine) in the culture [6]. We did not specifically set up different cultures with varying types of sera. With the addition of serum in the media after exhaustion of the fibroblasts, the remaining population of cells had a spindle-shaped appearance with varying degrees of pleomorphism and an inclination toward colony formation. The cultures would form monolayers and overgrow the flasks with cells ``heaped up'' on each other, characteristic of transformed cells.
All of the cultures stained positively for cytokeratin, supporting the mesothelial nature, and were devoid of other markers, specifically those for adenocarcinoma. This profile was identical to that seen in the primary specimens, all of which were determined to be either epithelial, biphasic, or sarcomatoid mesothelioma. We did not use any of the ``mesothelioma antibodies'' to state definitively whether or not the lines were MPM because these antibodies should be considered investigational and not definitive for the diagnosis. In our opinion, the paraffin-embedded cell block, similar to that used for pathologic determination of surgical specimens was the most reliable, and we believe that future studies should incorporate exclusively this technique for analysis of MPM cell lines by immunohistochemical analysis because of the lower levels of background staining and more reliable staining with our chosen antibody panel.
Cytogenetic analysis of mesothelioma has been reported directly from fresh patient samples, short-term cultures (1 to 5 days), long-term samples (1 to 8 weeks), and established cell lines. Specifically, mesothelioma DNA content has been examined with both classic karyotyping and flow cytometry. Our cytogenetic analysis, performed on all cell lines between the 25th and 50th passages, revealed aneuploidy in all nine lines. The distribution of modal chromosome numbers as well as the individual chromosomal abnormalities confirmed the available data in the literature. Classic karyotypic analysis of mesotheliomas using multiple marker chromosomes demonstrates modal chromosome numbers ranging from 34 to 90. Nine of 12 mesotheliomas classified cytogenetically by Gibas and co-workers [7] demonstrated clonal abnormalities, and the majority were aneuploid (8\9) with modal chromosome numbers between 43 and 85. Nineteen of 30 mesotheliomas had clonal abnormalities in the report by Tiainen and colleagues [8], with chromosomal numbers between 34 and 96. Successful karyotyping was performed in 39 of 46 cases by Hagemeijer and colleagues [9], of which 30 had aneuploid clonal abnormalities with modal chromosome numbers from 38 to 90. Specific cytogenetic studies of mesotheliomas have revealed complex patterns of chromosomal abnormalities with no specific aberrations common to all the tumor samples. However, nonrandom patterns of chromosome aberrations have been reported in many series. Our incidence of translocations, deletions, and inversions specifically in chromosomes 1, 3, 6, and 9 was comparable to that observed by others [10].
The cell lines exhibited varying growth rates, but the growth characteristics were similar to those described by others [2, 3]. It is important, however, to consider that the doubling time variability may affect the results of in vitro drug/radiation/other cytotoxic agent tests, as the susceptibility may depend on cell cycle effects.
The ability of some of the cell lines to proliferate in a serum-free environment will allow investigators to define whether there are specific autocrine or paracrine growth loops that could be targeted for therapy. Such loops, possibly involving PDGF or insulin-like growth factor (IGF), must be defined by demonstrating specific cell surface receptors for the growth factor along with the production of autogenous protein by the cells in serum-free media. Growth in serum-free media will also allow investigations to be performed in which exogenous growth factors can be added to basal media, and proliferative effects recorded. As a prelude to these investigations, we demonstrated that PDGF-AB was produced by our cell lines using ELISA. Gerwin and colleagues [11] were the first to describe elevation of RNA levels for both the and ß chains of PDGF in mesothelioma cell lines and correlated the increase with PDGF-like activity secreted by the cells. Versnel and associates [12] reported elevation of both PDGF chains in malignant mesothelioma cell cultures compared with normal cells, but the elevation was chiefly in the ß chain. Besides growth, factors such as PDGF and IGF, however, there may be a role for cytokines in the pathogenesis of mesothelioma. The association between thrombocytosis and mesothelioma has stimulated investigations searching for increased circulating levels of cytokines such as IL-3, IL-4, GM-CSF, erythropoietin, stem cell factor, IL-11, and IL-6. This report substantiates reports of high levels of IL-6 [13, 14] and GM-CSF [15] from body fluids or cell lines of patients with mesothelioma, and could explain partially the dramatic elevations of platelet counts seen in patients with MPM. Other investigators postulate other growth factors apart from EGF, PDGF, and TGF-ß, which are as yet uncharacterized, that may play a role in autocrine or paracrine growth loops in this disease [16].
Future analysis of our cell lines will include attempts to establish tumor in immunosuppressed mice. Thus far we have been successful with two of the lines in establishing subcutaneous tumors and ascites after tumor inoculation (unpublished data). Such animal models, as detailed by Chahinian and colleagues [17], will prove invaluable in evaluating future treatment strategies.
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Footnotes |
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Address reprint requests to Dr Pass, Thoracic Oncology Section, Surgery Branch, NCI/NIH, Bldg 10, Rm 2B07, Bethesda, MD 20892.
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-receptors. Oncogene 1991;6:2005–11.[Medline]
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