CTC Isolation Using Antibody Cocktails

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SUMMARY

This document is intended to guide the user through a method for using multiple antibody types conjugated to magnetic beads to recover circulating tumor cells (CTCs). This protocol provides specific instructions for the use of EpCAM, EGFR, and HER-2 antibodies, and guidelines for protocol optimization if other surface markers are to be used. The protocol incorporates use of the IsoFlux System and the IsoFlux Rare Cell Enrichment Kit.

This protocol has been validated by Fluxion using tumor cell line spike-in experiments. Please consult Fluxion for any updates to this protocol.

BACKGROUND

Immunomagnetic cell separation in the IsoFlux System is based on the binding of magnetic particles to surface antigens that are present in CTCs but absent in surrounding leukocytes. The types and numbers of target cells recovered with the IsoFlux System will depend on the capture markers used. Certain markers, such as EpCAM, are generally understood to be present in most carcinomas (cancers of epithelial origin), but the expression of single markers can be heterogeneous in CTCs.

Given the heterogeneity of the CTC population and likely variations between different cancer types, it is advantageous to be able to separate CTCs based on multiple surface markers. The ability to select the surface marker of interest can also aid in selecting specific CTC sub- populations.

In addition to CTCs, many other rare cell types of scientific and clinical interest can exist in the peripheral blood circulation. These include cancer stem cells, immune cells, and circulating endothelial cells. In many cases, these cells can also be enriched from the background cells using one or more surface-expressed antigens.

PRINCIPLES OF THE PROCEDURE

The IsoFlux RCE Kit contains immunomagnetic capture beads, microfluidic cartridges, and additional reagents required for performing CTC enrichment. The magnetic beads consist of micron-scale particles with a magnetic core surrounded by a polymeric layer coated with a monoclonal human anti-mouse IgG. The user can identify one or more capture antibodies of their own choosing and bind to the beads (direct method) or add to the sample (indirect method).

A biological sample (e.g. whole or fractionated blood) is pre-enriched and mixed with the immunomagnetic particles. The sample/bead mixture is loaded into the microfluidic cartridge by the user. The cartridge is then loaded into the IsoFlux System instrument where the sample passes through the fluidic channel on the cartridge. Midway through the fluidic channel is a cell isolation zone that is exposed to an external magnetic field inside the instrument. The target cells each have magnetic beads attached to them that are attracted towards the magnetic field. The target cells collect on a removable disk that forms the roof of the isolation zone. After the sample is processed, the enriched cells are transferred inside the instrument to a microfuge 

tube or low volume recovery holder. The cartridge is then ejected from the instrument and the enriched CTCs are ready for further analysis.

REAGENT REQUIREMENTS

  • IsoFluxTM Rare Cell Enrichment Kit (Fluxion Cat#910-0092)

  • Anti-EpCAM mouse monoclonal antibody [Ber-EP4] (Abcam ab7504)

  • Anti-EGFR mouse monoclonal antibody (Millipore GR01-100UG)

  • Anti-HER2 mouse monoclonal antibody (R & D Systems MAB1129)

INSTRUMENT REQUIREMENTS

  • IsoFlux Instrument (Catalog #950-0100)

  • Swing bucket centrifuge capable of 800 x g

  • Test tube racks

  • Calibrated micro-pipettors and tips

  • Vortex mixer

MATERIALS REQUIRED, NOT PROVIDED

  • IsoFlux Instrument (Catalog No. 950-0100)

  • Swing bucket centrifuge capable of 1500xg (with brake settings)

  • Test tube racks

  • Calibrated micro-pipettors and tips

  • Serological pipettes and pipettor

  • 1.5 or 2mL microfuge tubes (preferably low retention)

  • Microfuge tube rotator (capable of 10 to 20 RPM)

  • 50mL Leucosep® tubes (with frit) (Greiner, Catalog No. 227290)

  • Phosphate Buffer Saline without Ca2+ Mg2+ (PBS-CMF)

  • 50mL conical tubes

  • Ficoll-PaqueTM Plus (GE Healthcare, Catalog No. 17-1440-02)

  • Permanent magnets (accessory parts included with IsoFlux instrument, large round and

    small cylindrical magnet)

  • CTL-WashTM Supplement (CTL, Catalog No. CTLW-010)

  • RPMI tissue culture media

  • Optional: Benzonase® Nuclease (Sigma, Catalog No. E8263)

  • Optional: Nylon Mesh Cell Strainer, 40 Micron (BD, Catalog No. 352340)

CELL CULTURE SUPPLIES
(NEEDED IF DOING SPIKING STUDIES)*

  • Tumor cell line that is positive for one of the surface markers to be used with clinical samples

  • Celltracker Green (Life Technologies, Catalog No. C7025)

  • Dimethyl sulfoxide (DMSO)

  • Serum Free media

  • Complete growth media

  • TrypLE Express (Life Technologies, Catalog No. 12605010)

  • Cell culture incubator (37°C, 5% CO2, >95% humidity)

    * Fluxion has recommended use of certain reagents and supplies from third-party vendors. While these kits have been tested for suitability in the protocol, Fluxion does not have any affiliation with the respective vendors of these kits. All issues with performance of these kits should be addressed directly to the supplier.

WARNINGS AND PRECAUTIONS

  1. For Research Use Only

  2. Please read the entire contents of this protocol before processing samples.

  3. Caution: Care should be taken to collect and transfer blood samples before processing.

    Cells are fragile and can be damaged or lost if not handled properly.

  4. Caution: All personnel should follow universal precautions for biological sample

    handling and use laboratory safety equipment (i.e., safety glasses, laboratory coat,

    gloves).

  5. Caution: Microbial contamination of reagents can cause erroneous results and should be

    avoided.

  6. Warning: All biological specimens, cartridges and other materials coming into contact with the specimen(s) are considered biohazardous. Handle as if capable of transmitting infection. Treat and dispose of waste using proper precautions and in accordance with local, state, and federal regulations. Never pipette by mouth.

  7. Warning: Some of the reagents contain sodium azide as a preservative. If swallowed, seek medical advice immediately. Keep out of reach of children. Keep away from food and drink. Wear suitable protective clothing. Contact with acids liberates very toxic gas. Azide compounds should be flushed with large volumes of water during disposal to avoid deposits in lead or copper plumbing where explosive conditions can develop.

  8. Operator training is required to perform the test procedure.

REAGENT STORAGE AND HANDLING

    • Reagents are supplied ready for use. Store unopened at 2 to 8°C.

    • After opening, reagents should be stored for no longer than 60 days at 2° to 8°C.

    • Protect reagents from heat in excess of 35°C. Do not freeze.

    • Bring to room temperature (15 to 30°C) before use.

    • Protect reagents from exposure to sunlight.

    • When properly stored, reagents are stable until the expiration date printed on the

      reagent container or kit box. Do not use expired reagents.

    • Do not mix and match reagents from different kits.

ENRICHMENT PROCEDURE

Specimen collection and preparation

  1. Collect biological samples aseptically into an appropriate sample collection tube.

  2. If samples are being shipped or transported, take care to pack the samples

    accordingly to control exposure to excessive temperatures or agitation.

  3. Depending on the sample type, a pre-enrichment step might be required such

    as density centrifugation (e.g.Ficoll) or red blood cell lysis.

  4. The final sample should be spun down in a centrifuge to create a pellet.

Reagent preparation

  1. Binding Buffer (BB): Make 50mL of 0.5% BSA 2mM ETDA in PBS without Ca2+ Mg2+. Filter through a 0.22μm filter. Keep on ice. For each coupling reaction to be performed add 1mL of BB to a 2mL maximum recovery tube and rotate at 4°C until ready.

  2. Beads Suspension (BS): This preparation suffices for 8 samples. Scale up or down as appropriate. Resuspend the beads stock with a micropipette; avoid frosting the suspension (do not vortex). Dispense 400μL of beads stock into a microfuge tube. Pull down the beads with a large magnet. Aspirate off the buffer while holding the beads in place on the magnet. Wash the beads with 1mL of BB. Repeat the pulling down of the beads on a large magnet and aspirate off the buffer while holding the beads in place on the magnet. Resupend the beads in 400μL of BB. Store on ice.

Antibody-Bead Coupling Reaction – Direct Method

Coupling antibodies to beads may be done before the experiment (i.e. coupling over night at 4 ̊C). If kept sterile and stored at 4 ̊C, the coupled beads are stable for at least 2 weeks. It is also recommended to perform the coupling in batch. Refer to Section 3.1 in the Rare Cell Enrichment Kit Instruction for Use Coupling reaction direct method (coat RCE beads with mouse antibody) for detailed instructions. Below are the suggested antibody concentrations per 8 samples. Scale up or down as appropriate.

  1. Add the following antibody contents to each tube containing 400μL of washed beads:

  2. Active mix 20 times with magnet by rotating the magnetic field 180 ̊ each time so that beads are brought directly across the microfuge tube to collect on the opposite wall.

  3. Incubate the coupling reaction on a rotator at room temperature for at least 1 hour (or overnight at 4 ̊C).

  4. Place each tube on a permanent magnet for 1 min and discard the supernatant. Remove tubes from the magnet and wash the beads with 1 mL Binding Buffer.

  5. Place each tube on a permanent magnet for 1 min and discard the supernatant.
  6. Repeat previous two washing steps (e and f) once more to remove excess antibody.
  7. Resuspend the beads in 400uL BB. Keep beads on ice and resuspend with a
    micropipette before use.

Cell Coupling Reaction – Cells and Bead Cocktail

  1. Prepare PBMC fraction from whole blood sample. Please refer to the

    Enrichment procedure Section 1 and 2 in the Rare Cell Enrichment Kit

    Instruction For Use for detailed instructions.

  2. Ensure that the pellet is resuspened in about 500μL with BB. Add CTL Wash

    Supplement to improve viability.

  3. Add 40μL FcR blocker to cells and keep on ice for 5 minutes.

  4. Add 30μL each of the antibody-coupled beads to the cell preparation (total 90μL

    of coupled beads). Note: keep the coupling PBMC/beads coupling volume between 800μL to 1mL. Maximum sample volume to be loaded onto the cartridge is 1.1mL. Do not exceed this volume.

  5. Active mix 20 times using a magnet.

  6. Incubate at 4 ̊C on a rotator for 1 to 2 hours.

  7. Proceed with enrichment on IsoFlux System.

Cell enrichment

  1. Refer to the IsoFlux System Instructions for Use and on-screen commands for full instructions to process samples for cell enrichment.

  2. Prepare for cell collection in a Low-Volume Recovery Holder. Remove the microfuge tube from the sample retrieval well (well No. 5) and insert the Low Volume Recovery Holder into the collection tube holder.

  3. Carefully open the cartridge lid, add 3 mL Binding Buffer to the buffer reservoir (well No. 2) of each cartridge. Carefully close the cartridge lid.

  4. Load cartridge(s) onto the carriage(s). Press Prime. Cartridge loading carriage(s) will slide in automatically. Machine will prime for about 6 minutes.

  5. After priming is completed, touch screen will show Ready to Load Sample. Press Ready to Load Sample. Left carriage will slide out automatically. Carefully open cartridge lids. Add beads coupled cell samples to Sample well (well No. 1), avoid  forming bubbles. Carefully snap close the cartridge lid and load onto the carriage. 

  6. After all cell samples are loaded for the left carriage, press Load. Left carriage will slide in automatically. If running one or two samples, cell isolation will start at this point. If running more than two samples, right carriage will slide out automatically. Load the rest of samples and press Load, right carriage will slide in automatically. Cell isolation will start. Cell isolation typically takes about 45 minutes but it may vary for different samples.
  7. After cell isolation is completed, touch screen will show Extract Sample. Press
    Extract Sample. Carriage(s) will slide out automatically.

Cell retrieval

  1. If using the Microfuge Tube – cells will be suspended from the cap of the tube. Gently invert the tube 2-3 times until all the cells are suspended in the Binding Buffer at the bottom of the tube.

  2. If using the Low Volume Recovery Holder – flip the holder upside down such that the enriched cells are facing up on the bench. Gently aspirate the cells into a pipette by first pushing up to 10μL of Binding Buffer onto the sample, then aspirating back into the pipette. Dispense the collected cells into a new microfuge tube.

  3. Cells are now ready for further testing

ANALYTICAL VALIDATION

The breast cancer cell line BT549 was chosen in order to validate improved recovery of tumor cells exhibiting low or no EpCAM expression. This cell line was reported to have no detectable EpCAM expression and no recovery using the CellSearch system (Fig. 1). Low EpCAM expressing cell lines, such as MDA-MB-231, were also reported to have low (12%) recovery using CellSearch. Fluxion has previously reported significantly improved CTC recovery using the IsoFlux System (approx. 70%) due to higher antigen sensitivity in the microfluidic capture region.

Surface antigen expression for three model cell lines was assessed by microscopy in order to identify some possible alternative markers to use as immunomagnetic bead targets (Fig. 2). The BT549 data indicates extremely low to no EpCAM expression, low to mid EGFR expression, and low to no HER2 expression. As a result, we chose to compare recovery using only EpCAM or only EGFR. Finally, data was collected using all three capture markers as a cocktail in order to maximize recovery.



Figure 1: Test cell line selection. The selected cell line, BT549, was previously reported to have undetectable EpCAM expression and no cells recovered using the EpCAM based CellSearch system (data reproduced from "Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells", Sieuwerts et al.) Note that, by cytometry, MDA-MB-231 cells are very close to background, and BT549 cells are indistinguishable from background.

Figure 2: Immunofluorescence data for different model cell lines. We confirmed expected
protein expression profiles via immunofluorescence imaging for three different cell lines.
While SKBR3 is a very high EpCAM expressor, both MDA-MB-231 and BT549 exhibit no
visible EpCAM expression and medium EGFR expression.

lines were pre-stained, spiked into healthy blood samples, isolated using the IsoFlux protocol described above, and counted after mounting on microscope slides. The results shown in Fig. 3 demonstrate that, for BT549, a subset of cells (40-50%) can still be recovered using EpCAM alone, likely due to the higher sensitivity of the IsoFlux System as compared to literature data. The recovery is similar for the pre-conjugated CTC bead preparation and the user conjugated custom gG bead when EpCAM is the selected marker. EGFR alone is a similarly effective marker, with 50% recovery for this cell line. The multiple antibody bead cocktail was most effective, with 76% of cells recovered on average.
Figure 3: Recovery using individual antibodies and cocktails. The CTC beads (pre-
conjugated with EpCAM), IgG Beads with EpCAM, and IgG beads with EGFR all displayed
recovery of BT549 cells in the 45-50% range when used alone. The antibody cocktail using
EGFR + EpCAM + HER2 increased the total recovery to 76%.
 

In conclusion, this protocol makes possible tumor cell isolation from whole blood using a combination of surface markers. A specific antibody cocktail and protocol is provided for three different markers (EpCAM, EGFR, and HER2) that enable efficient recovery of cells that appear EpCAM negative by both cytometry and microscopy.