Cell lines and antibodies
A2058 metastatic melanoma cells and human embryonic kidney 293 (HEK293) cells were cultured in DMEM supplemented with 10% foetal bovine serum (FBS).
Mouse anti-human monoclonal antibodies to MCAM (clone P1H12), MCSP (clone 9.2.27), CD271 (clone C40-1457) and CD45 (clone HI30) were purchased from BD Biosciences (San Jose, CA, USA). Rabbit anti-human polyclonal antibody to ABCB5 (clone RB16781) was purchased from Abgent (San Diego, CA, USA). Secondary antibodies were anti-mouse and anti-rabbit conjugated to Alexa-Fluor 488 (A488) and biotinylated anti-mouse, which was used with streptavidin-A488.
Patient blood samples
Patients, recruited from 3 clinics in Perth, Australia, were diagnosed and staged according to guidelines of the American Joint Committee on Cancer (AJCC). All patients recruited between August 2011 and March 2012 were included in the study. Peripheral blood samples were obtained from 10 non-metastatic (stage I and II) and 13 metastatic (stage III and IV) patients. Additionally, 15 control blood samples were obtained from healthy volunteers. Blood was drawn by phlebotomists into EDTA tubes, after the first few millilitres was discarded to avoid epithelial contamination, and refrigerated until use. For 21 of 23 patients, blood was collected into five EDTA tubes; only one tube was collected for the remaining two patients and all controls. Patient samples ranged from 4 to 10 ml and control samples were 9 ml. All results were therefore calculated per ml of whole blood. Participants signed informed consent with the clinician in accordance with protocols safeguarding patient rights. All procedures have been accepted by the Human Research Ethics Committees at ECU (No. 2932) and SCGH (No. 2007–123).
Expression of MCAM, MCSP, ABCB5, CD271 and CD45 was tested in A2058 and HEK293 cells by immunofluorescence. Approximately 60,000 cells were seeded per coverslip and allowed to adhere overnight. Attached cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and stained with primary antibody, diluted in PBS with 3% BSA for one hour at room temperature. MCAM, MCSP and CD45 antibodies were diluted 1/500, ABCB5 1/250 and CD271 1/750. Cells were then stained with secondary antibody, diluted 1/500, for 20 minutes at room temperature. Cells were washed three times with PBS between each step. Coverslips were mounted onto microscope slides with ProLong Gold anti-fade mounting medium containing DAPI for nuclear staining (Invitrogen, Carlsbad, CA, USA) before analysis with an Olympus BX51 microscope equipped with an Olympus DP71 camera.
MCSP, MCAM, ABCB5 and CD271 antibodies were covalently bound to magnetic beads using the Dynabeads Antibody Coupling Kit (Invitrogen), as per the manufacturer’s instructions. For efficient antibody-bead coupling, 10 μg of antibody was used per mg of Dynabeads.
Binding capacity of antibody coupled beads
A2058 cells were harvested in DMEM containing 5 mM EDTA and analysed with the Vi-Cell-XR Cell Viability Analyser (Beckman Coulter, Brea, CA, USA). 0.5 × 106 A2058 cells were added to six tubes. 0.5 μl of MCSP beads was added to three tubes and 0.5 μl of MCAM beads was added to the remaining three tubes. Tubes were incubated for one hour at 4°C with rotation and subsequently placed on a DynaMag-2 magnet (Invitrogen). Enriched cells were washed three times with PBS, resuspended in 500 μl of PBS and counted with the Vi-Cell-XR Cell Viability Analyser. 0.5 μl of MCSP beads bound a mean of 1.42 × 105 cells and 0.5 μl of MCAM beads bound a mean of 0.98 × 105 cells.
Quantification of spiked melanoma cells and patient CMCs
A2058 cells were harvested as previously described. Only cells with greater than 90% viability were used. Cells were spiked into DMEM/10% FBS or white blood cells (WBCs) resuspended in 1 ml incubation buffer (0.5% BSA, 2 mM EDTA in PBS, pH 7.2), following lysis of control blood with lysis buffer (140 mM NH4Cl, 17 mM Tris, pH 7.65). Spikes of 1, 10 and 20 cells were performed by pippetting under a microscope, while 50 and 100 cell spikes were performed by dilution. Cells were incubated with 0.5 μl of MCSP beads or a combination of MCSP, MCAM, ABCB5 and CD271 beads (with a combined volume of 0.5 μl) for one hour at 4°C with rotation. Enriched cells were placed on a DynaMag-2 magnet (Invitrogen), washed three times with PBS and mounted on microscope slides as previously described. Before mounting, cells enriched from control blood were fixed with 4% paraformaldehyde for 10 minutes at room temperature, stained with CD45 antibody (diluted 1/500 in PBS containing 3% BSA) for one hour at room temperature, followed by biotinylated anti-mouse and streptavidin-A488, both for 10 minutes at room temperature. Two PBS washes were performed between each antibody incubation. Enriched cells were quantified by microscopy where WBCs were defined as CD45 positive and melanoma cells as CD45 negative.
Control and patient blood was processed within 24 hours of collection. Blood was lysed as previously described. For controls and patients, one blood sample was incubated with a combination of MCSP, MCAM, ABCB5 and CD271 beads (combined volume 0.5 μl). The four remaining patient samples were incubated with 0.5 μl of one of the four individual bead types. Samples were incubated, washed and stained as previously described and quantified by microscopy.
DNA amplification and detection of B-raf V600E mutation
Enriched cells attached to magnetic beads were lysed for whole genome amplification using the Repli-g kit (Qiagen, Germantown, MD, USA). In summary, after two PBS washes, all buffer was removed and cells attached to the magnetic beads were resuspended in 3.5 μl of cell lysis buffer containing DTT, incubated 10 minutes at room temperature then stop solution was added. Whole genome amplification was carried out as per the manufacturer’s instructions.
The V600E mutation was detected using the qBiomarker Somatic Mutation PCR Assay (c.1799 T > A) relative to a reference PCR for the B-raf gene (SABiosciences, Qiagen). Quantitative PCR was performed in a ViiA 7 Real-Time System (Applied Biosystems, Foster City, CA, USA).
Statistical tests were performed using SPSS Statistics 19 (IBM). The Mann–Whitney U Test was used to compare the number of CMCs between patients and controls and between metastatic and non-metastatic patients. The Wilcoxon Signed Rank test was used to compare the number of CMCs enriched with a combination of beads versus each individual bead type. P < 0.05 was considered statistically significant. The two patients with missing data were excluded from the appropriate analyses.