A chordoma is a rare low-grade malignant tumor thought to arise from vestigial notochordal tissue. The classic site is in the axial skeleton. Studies vary on the prevalence of the sites; however, approximately one-half are located in the sacrococcygeal region, with the others distributed mainly to the remainder of the spine and the spheno-occipital region1,2.
The tumor is histologically composed of variably sized myxoid lobules, with multivacuolated, large epithelioid or so-called physaliferous cells3. When a tumor with histologically similar features is encountered in the extra-axial skeleton, then the differential diagnosis broadens to include other neoplasms such as parachordoma, malignant myoepithelioma, extraskeletal myxoid chondrosarcoma, and extra-axial chordoma. The staining patterns and cytogenetic characteristics are variable, so arriving at an accurate diagnosis can be challenging4-6. Brachyury is thought to be a specific marker for notochord-derived tumors, and a positive stain on a peripheral musculoskeletal tumor provides strong support for the diagnosis of an extra-axial chordoma7,8. Wide excision is the treatment of choice of all chordomas. They have a high risk of local recurrence, and they also metastasize9,10.
The lack of a good in vitro model on which to perform more extensive characterization of this tumor led us to explore the possibility of obtaining a sustainable cell line to aid with further research. We present these data to describe the case of a patient with an extra-axial chordoma, emphasizing the diagnostic challenges with the hope that the development of our cell line will help to further evaluate this tumor in future studies. The patient was informed that data concerning the case would be submitted for publication, and he consented.
Case Report
A twenty-eight-year-old man presented for evaluation of a mass in the right shoulder. He had a one-year history of pain in the right shoulder, both at rest and with activity, with no history of trauma. There was no other relevant clinical history. Plain radiographs showed a subtle destructive pattern within the scapular body (Fig. 1-A). The magnetic resonance imaging showed a partially enhancing solid mass within the belly of the right supraspinatus muscle. It was oval shaped and well circumscribed. The mass measured 6.6 × 4.7 × 3.4 cm (Fig. 1-B), exhibited markedly high intensity on the short tau inversion recovery (STIR) sequence, and was relatively isointense to muscle on the T1-weighted sequences. There was destruction of the posterior scapular spine, suggesting invasion of the adjacent cortex. Computed tomography scans of the chest, abdomen, and pelvis showed no abnormalities.
An initial computed tomography-guided needle biopsy was performed because of the concern of malignancy and the difficulty in accessing the mass. It revealed nests of small round cells, many with vacuoles. The cells had uniform, oval-shaped nuclei containing small amounts of eosinophilic cytoplasm (Fig. 2-A). The tumor was strongly positive for vimentin, focally positive for S-100, moderately positive for cytokeratin AE1/AE3 (Figs. 2-B, 2-C, and 2-D), and negative for p63. A preliminary diagnosis was a parachordoma, or malignant myoepithelioma of the soft tissues.
A wide resection of the tumor was then performed through a posterior approach. Care was taken to excise a cuff of normal tissue surrounding the mass in order to prevent local recurrence. It was also necessary to osteotomize a portion of the scapula to remove the reactive bone. Histological examination revealed clear margins. The resected tumor consisted of a cellular neoplasm composed of physaliferous-like cells with uniform nuclei. It was multinodular and composed of cords of large, partially vacuolated, epithelioid cells in a myxoid matrix (Figs. 2-E and 2-F). Further immunohistochemistry stains were identical to the frozen section with the addition of a weakly positive epithelial membrane antigen (Fig. 3-A) and a negative CK-7. A brachyury antibody stain (polyclonal brachyury antibody, diluted at a ratio of 1:50; Santa Cruz Biotechnology, Santa Cruz, California) was diffusely and strongly positive (Fig. 3-B). This confirmed the diagnosis of an extra-axial chordoma. The patient was doing well clinically with no recurrence or metastasis at two years of follow-up.
The tumor sample was mechanically dissociated with a scalpel into 1-mm pieces and plated in 20 mL of Dulbecco modified Eagle medium (DMEM; Gibco Invitrogen, Grand Island, New York) with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, Georgia) and 1% penicillin-streptomycin (Gibco Invitrogen). The media was changed every two to three days. After ten days, some adherent cells grew from the tumor pieces in a monolayer. Once cells reached 70% to 80% confluency, they were lifted with use of 0.25% trypsin-EDTA (Gibco Invitrogen) and analyzed by flow cytometry. The high expression of cell surface markers CD73a, CD105, and CD166, and the lack of expression of CD11b, CD34, CD45, and CD79a (Table I) confirmed their derivation from a mesenchymal lineage11.
One million cells were injected subcutaneously into the posterior abdominal flank of a nude (nu/nu) mouse according to an approved protocol from the Tulane University Institutional Animal Care and Use Committee. After ten weeks, a growing tumor was detected at the site of injection, and it was allowed to grow until it reached 5 mm in diameter. The tumor was removed from the mouse after it was killed twelve weeks after injection, dissected in 1-mm pieces, and placed in culture medium from which we derived the new extra-axial chordoma cell line named EACH-1 (extra-axial chordoma-1).
Flow Cytometry
A volume of 3 × 105 cells was concentrated by centrifugation at 500 g for five minutes and suspended in 50 µL of phosphate-buffered saline solution containing 1% (w/v) bovine serum albumin and the fluorescent-labeled antibodies (BD Pharmingen, San Jose, California) indicated in Table I. The samples were incubated for thirty minutes at room temperature and then were washed three times in phosphate-buffered saline solution by centrifugation. The samples were analyzed by a Cytomics FC-500 flow cytometer (Beckman Coulter, Miami, Florida). The percentage of positive cells for each marker was determined by the ratio of positive events compared with the negative control (CXP software; Beckman Coulter).
Growth Curve Assay
Extra-axial chordoma-derived cells were seeded into two twelve-well plates with 1 mL of DMEM with 10% fetal bovine serum media (500 cells per well). Three wells were trypsinized each day, and cells were counted for total cell number with use of a hemocytometer for eight days.
Tumor Growth Assay
Extra-axial chordoma-derived cells were grown to 70% to 80% confluency and then were lifted with trypsin and counted with use of a hemocytometer. A volume of 10 ×106 cells was suspended in 200 µL of Hanks balanced salt solution (Gibco Invitrogen). Subcutaneous injections of cells into the posterior abdominal flank of six to eight-week-old nu/nu mice were carried out in triplicate. Perpendicular tumor diameters were measured biweekly with use of digital calipers, and the volume calculated from the formula: 4/3 × p × L × M2, where L is the larger radius and M is the smaller radius.
Western Blotting
Cells were prepared and lysed in buffer (RIPA lysis buffer; Santa Cruz Biotechnology) supplemented with protease inhibitor cocktail, and protein concentration was determined (BCA protein assay kit; Pierce Biotechnology, Rockford, Illinois). The cell lysate (20 µg of total protein) was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NuPAGE Novex 4-12% Bis-Tris gels; Invitrogen, Carlsbad, California). The sample was transferred to a filter (Immobilon-P PVDF membrane; Millipore, Billerica, Massachusetts) by electroblotting (XCell II Blot Module; Invitrogen). The filter was blocked for two hours with Tris-buffered saline solution (TBS [Sigma, St. Louis, Missouri]; phosphate-buffered saline solution containing 0.05% Tween 20) added with 5% nonfat dry milk and then incubated overnight at 4°C with a rabbit polyclonal brachyury antibody (Bry, clone H-210, number sc-20109; Santa Cruz Biotechnology) diluted at a ratio of 1:200 in Tris-buffered saline solution with 1% nonfat dry milk. The membrane was washed three times for ten minutes each with Tris-buffered saline solution. Bound primary antibody was detected by incubating for one hour with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (Chemicon Millipore, Billerica, Massachusetts). The filter was washed and developed with use of a chemiluminescence assay (Visualizer Spray and Glow ECL detection system; Upstate Biotechnology, Lake Placid, New York).
Immunohistochemistry
Slide sections were deparaffinized and rehydrated. Antigen retrieval was performed by using a vegetable steamer containing a boiling solution of sodium citrate buffer (10 mM sodium citrate and 0.05% Tween 20; pH 6.0) for thirty minutes, then slides were washed twice for five minutes in Tris-buffered saline solution with 0.025% Triton X-100. Sections were blocked in 10% normal goat serum with 1% bovine serum albumin in Tris-buffered saline solution overnight at 4°C and incubated for twenty-four hours with a rabbit polyclonal Bry antibody (clone H-210, number sc-20109; Santa Cruz Biotechnology) diluted at a ratio of 1:50 in Tris-buffered saline solution with 1% bovine serum albumin. Negative control slides were obtained by incubating with isotype rabbit IgG. Slides were rinsed twice for five minutes in Tris-buffered saline solution with 0.025% Triton and then incubated in 0.3% H2O2 in Tris-buffered saline solution for fifteen minutes. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (number AP132P; Chemicon Millipore) diluted at a ratio of 1:5000 in Tris-buffered saline solution with 1% bovine serum albumin was applied for one hour at room temperature, then slides were rinsed twice for five minutes in Tris-buffered saline solution and developed with a diaminobenzidine (DAB) chromogen kit (ImmPACT DAB; Vector Laboratories, Burlingame, California) for fifteen minutes at room temperature. Slides were counterstained with hematoxylin, dehydrated, cleared, and mounted.
Nonadherent Clonogenicity Assay
Single cell suspensions of each cell line were collected, and 2 × 103 cells were plated in each well of a six-well Nunc low-cell-binding plate (Nunc, Rochester, New York). Cells were incubated for twelve days before media with colonies was transferred to adherent plates for twenty-four hours, after which colonies were stained with crystal violet. Formation of colonies with >200 cells was quantified. Clonal density was used as described by Patrawala et al.12, and nonadherent plates were used as substitutes for agar plating.
Karyotyping
A 150-mm cell culture dish containing a 50% confluent cell population was trypsinized. The pellet was resuspended in a 75-mM KCl solution and was fixed in methanol and acetic acid (in a 3:1 solution). After two washes in fixative, cells were spread on a slide and air-dried, then treated with RNase (0.1 mg/mL) for one hour at 37°C, washed in 2X SSC (saline-sodium citrate buffer), and incubated for five minutes at 37°C in 0.005% pepsin. Slides were then washed in phosphate-buffered saline solution, fixed in 1% formaldehyde, and dehydrated. After the slides were air-dried, they were aged for at least one week at room temperature before denaturation. Some slides were stained in Giemsa (4% in phosphate-buffered saline solution) for scoring dicentric chromosomes. At least 100 Giemsa-stained cells were analyzed for each point, with use of a bright-field microscope.
Source of Funding
This research was supported by the Louisiana Cancer Research Consortium; HCA—The Healthcare Company; and the Louisiana Gene Therapy Research Consortium. None of these agencies played a role in the investigation.
Note: The authors thank pathologists Barry F. Faust, MD, of the Ochsner Health System, New Orleans, Louisiana; Jorge E. Ruiz, MD, of the Mayo Medical Laboratories, Jacksonville, Florida; John Reith, MD, of the University of Florida, Gainesville, Florida; and Adrienne Flanagan, MD, PhD, of University College of London, United Kingdom, for the diagnostics of the tumor; Alan Tucker of the FACS analysis core of the Gene Therapy Center, New Orleans, Louisiana, for the cell surface marker analysis; and Marilyn Li, MD, of the Cytogenetics Core of Tulane Medical School, New Orleans, Louisiana, for the karyotyping.