Technical Notes Method to deplete carryover white blood cells from CELLSEARCH Epithelial Cell Profile Kit samples Introduction This paper details a method to deplete carryover white blood cells from samples following CTC enrichment when using the CELLSEARCH Epithelial Cell Profile Kit. Circulating tumor cells (CTCs) of epithelial origin are present in the blood of cancer patients at very low frequency (< 10 / ml blood). In order to characterize CTCs, they need to be enriched from large volumes of blood (7.5 mls). The CELLSEARCH Circulating Tumor Cell (Epithelial) assay uses magnetic particles conjugated to antiepithelial cell adhesion molecule (EpCAM) to selectively capture CTCs. After enrichment, the sample contains CTCs and a number (1000 5000) of white blood cells (WBCs). The presence of WBCs in the CTC enriched sample may interfere with the molecular analysis of CTC biomarkers, especially if it is coexpressed by WBCs. We have improved the ability to perform molecular characterization of CTCs by selectively depleting WBCs carried over during CTC enrichment. The CELLSEARCH Epithelial Cell Profile Kit enriches CTCs without permeabilization or staining of cells. Investigators must confirm the utility of these methods for their intended application. Principle of Operation Samples after processing on the CELLTRACKS AUTOPREP SYSTEM using the CELLSEARCH Epithelial Cell Profile Kit are labeled with antibody to the leukocyte marker CD45. The anti-cd45 antibody used is conjugated to fluorescein isothiocyanate (FITC). The anti-cd45-fitc binds to the WBCs carried over into the CTC enriched cell population. Samples are incubated for 1 hour in microtiter plates coated with anti-fitc antibody. The anti-fitc binds the FITC present on the anti-cd45-fitc and causes the CD45 positive WBCs to adhere to the plate. The CD45 negative CTCs remain unbound, and are subsequently harvested with the supernatant fluids. Figure 1 shows the principle of white blood cell separation from tumor cells after tumor cells enrichment in the CELLSEARCH Circulating Epithelial Cell Kit. Page 1 of 8
Figure 1: Principle of WBC depletion EpCAM FF EpCAM FF WBC CD45-FITC Tumor Cell FITC WBC Tumor Cell Anti FITC Materials Required 1. Anti FITC from Novus (catalog # NB110-7898) 2. CD45-FITC (Becton-Dickinson #340664 @50ug/ml, or Biolegend #304006 @200ug/ml) 3. R&D Systems Cellect Magnet #MAG997 (Magnetic separator) 4. 6 well plate from VWR (Falcon #353046) 5. PBS/1%BSA (BSA from Seracare # AP-4510-01 or equivalent BSA from other vendor) 6. 12x75mm 5ml polystyrene round bottom tube BD#352008 Method A. Coating of 6 well plate with anti FITC 1. Prepare 50mM bicarbonate buffer ph 8.5. Prepare buffer fresh same day before use. 2. Dilute anti FITC from Novus in 50mM bicarbonate buffer (step 1) to 50ug/ml. Coating each well requires 800ul of anti FITC solution. Prepare sufficient anti FITC solution to coat at least one well per sample to be WBC depleted. Page 2 of 8
3. Add 800ul of anti FITC to each well and make sure the entire surface of the well is completely covered with no bubbles or empty spots. 4. Incubate at Room Temperature (RT) for 3 hours, followed by overnight incubation at 2-8 o C (Cover the plate with the lid during all the incubations). 5. After overnight incubation, warm the plate to RT for 30minutes and aspirate all the supernatant. 6. Rinse each well with PBS twice using 1ml for each rinse and aspirate the supernatant after the 2 nd rinse. 7. Block each well with 1ml of PBS/1%BSA to prevent any non-specific binding of CD45-FITC or cells to the plate. 8. Incubate the plate at RT for 4 hours. 9. Aspirate all the supernatant and rinse the plate twice with 2ml/rinse of PBS. 10. Aspirate all the buffer and air dry the plate at RT for one hour. 11. Cover the plate with the lid and store the plate at 2-8 o C until use. 12. Plate may be stored at 2-8 o C for up to 30 days before use. Other storage conditions or times must be validated by the investigator. B. Labeling of cells with CD45-FITC 1. Prepare the samples using CELLSEARCH Epithelial Cell Profile kits 2. Take the sample out of the CELLTRACKS AUTOPREP SYSTEM and transfer the entire sample (~ 900ul) to a 12x75mm polystyrene tube. 3. Add CD45-FITC to the sample to final concentration of 2ug/ml and mix by gently vortexing. 4. Incubate the sample in the dark for 15 minutes at RT. 5. Remove excess unbound CD45-FITC by washing as follows. 6. Add 3ml of PBS/1%BSA to the sample, vortex gently and place the sample in the magnetic separator. 7. After 15 minutes, and with the sample tube still in the magnetic separator, aspirate the supernatant using a glass Pasteur pipette or plastic transfer pipettes. Discard the supernatant fluid. 8. Remove the tube from the magnet and wash the sample again by repeating steps 6 and 7 above. 9. Remove the tube from the magnet, add 900ul of PBS/1% BSA and vortex gently. Page 3 of 8
C. Application of sample to 6 well plate coated with anti FITC 1. Bring the anti FITC coated plate to RT (minimum of 30 minutes) on the day of use. 2. Add 2ml of PBS/1% BSA to the wells and incubate for 15minutes. 3. Aspirate the buffer and rinse the well once more with PBS/1%BSA 4. Aspirate all of the buffer before sample is added to the well. 5. Add the entire sample from the step B-9 to one of the wells of 6 well plate. 6. Incubate the sample for 1 hour in the dark at RT. Every 15 minutes, gently rock the plate forward and back 3x (no more than a 45 degree angle) to mix the sample. 7. After 1 hour incubation, gently rock the plate forward and back 3x (no more than a 45 degree angle) 8. Keep the plate tilted at a 45 degree angle, aspirate the sample using 1ml pipette and dispense it at the top of the well to get all the unbound cells into the sample. Repeat this aspiration and dispension step 2 more times and then transfer the entire sample (~ 900ul) from each well to a desired tube for downstream molecular analysis. Preliminary results: The method described above was tested with normal EDTA blood spiked with tissue cultured tumor cells. 7.5ml of EDTA blood spiked with SKBR3 cells was processed on the CELLTRACKS AUTOPREP SYSTEM using a CELLSEARCH Epithelial Cell Profile Kit. An enriched sample (900ul) from the CELLTRACKS AUTOPREP SYSTEM using a CELLSEARCH sample tube was transferred to a 12x75mm tube. After labeling the cells with CD45-FITC, the excess CD45-FITC was removed by washing the sample 2-times with 3ml of PBS/1%BSA by magnetic separation for 15minutes. The final sample was resuspended in 900ul of PBS/1%BSA and applied to the wells coated with anti-fitc. The samples were incubated for 1 hour with gentle mixing every 15 minutes. After one hour, the supernatant was removed from the wells and the number of white blood cells and SKBR3 cells were determined by flow cytometry. The cells were labeled with anti-her2neu conjugated to allophycocyanin dye (APC) to detect SKBR3 cells by flow cytometry. The white blood cells were detected in the FITC channel since they were labeled with anti-cd45-fitc. The number of white blood cells and SKBR3 cells present in the samples was calculated before and after depletion step. The cell count present before depletion was normalized to 100% to calculate percent cell depletion.the results from this study are shown in the Figure 2. Page 4 of 8
Figure 2: Percent Removal of white blood and tumor cells by anti-fitc coated plate 120 100 97 96 95 95 95 97 96 Cell Depletion (%) 80 60 40 20 10 14 19 18 13 8 14 WBC Dep (%) SKBR3 Dep (%) 0 1 2 3 4 5 6 Ave. Number of Samples Tested Flow cytometry results show that greater than 85% of the CTCs were recovered in the supernatant fluids, while greater than 90% of WBCs had been depleted. These results were then confirmed by molecular analysis. Molecular measurements of WBC depletion in CELLSEARCH Epithelial Cell Profile kit enriched samples Duplicate 7.5ml of EDTA blood from 6 healthy donors was processed on the CELLTRACKS AUTOPREP SYSTEM using a CELLSEARCH Epithelial Cell Profile Kit. Following CELLTRACKS AUTOPREP SYSTEM enrichment, one tube from each donor was subjected to either the Depleted method which removed the WBC from the sample or the Non-depleted method which serves as a control to measure the WBC contamination in the sample without the depletion process. Non-Depleted method Samples are removed from the CELLTRACKS AUTOPREP SYSTEM and placed in a magnet for 15 minutes. The buffer is aspirated and the cells are lysed in RLT buffer (Qiagen). Depleted method Following CELLTRACKS AUTOPREP SYSTEM enrichment, the excess CD45-FITC was removed by washing the sample 2-times with 3ml of PBS/1%BSA by magnetic separation for 15minutes. The final sample was resuspended in 900ul of PBS/1%BSA and then applied to the wells coated with anti-fitc. Page 5 of 8
The samples were incubated for 1 hour with gentle mixing every 15minutes. After one hour, the supernatant was removed from the wells and the cells were placed in a magnet for 15 minutes. The buffer was removed and the cells were lysed in RLT buffer (Qiagen). For both the depleted and non depleted samples, the RNA was purified using the Qiagen AllPrep Kit and reverse transcribed using the High Capacity cdna Reverse Transcription Kits kit (Life Technologies). Complimentary DNA was amplified using TaqMan PreAmp Master Mix Kit (Life Technologies) and primer sets as for genes listed in the figures below. Quantitative PCR was carried out on the amplified samples for two genes known to be expressed in white blood cells (CD45 (aka PTPRC) and BST1) as well as a housekeeping gene expressed (B-Actin). Efficacy of WBC depletion was measured by the loss of WBC and housekeeping specific gene signals in the depleted sample relative to the non-depleted sample. Figure 3 - Molecular measurement of WBC removal. Circles and squares represent individual measurements for the non-depleted and depleted samples respectively. Lines represent the mean measurement for each sample type. P values represent the results of student s T-tests performed between the depleted and non-depleted samples for each gene measured. Page 6 of 8
T-tests between the depleted and non-deleted samples showed a significant loss of WBC specific and housekeeping genes as a result of CD45 depletion. Mean delta Ct measurements of >2 cycles indicate a >75% reduction of the WBC signals in the CD45 depleted samples. Improvement of CTC detection following white blood cell depletion One potential advantage of the CD45 depletion method is an improved ability to detect CTC or characterize CTC gene expression when there is some level of background gene expression contributed by the white blood cells. Reducing the number of white blood cells should reduce the background gene expression and improve the ability to detect transcripts in lower numbers of CTC. To demonstrate this principle four samples were prepared for each of six donors; two samples were spiked with 10 VCaP cells and two samples were unspiked. One spiked and unspiked sample was prepared with both the Depleted and Non-depleted protocol as described above. RNA extraction, Reverse Transcription and Pre-Amplification kit were carried out as above. Public databases were mined to identify a panel of potential CTC markers that could be useful for measuring utility of the WBC depletion method. Genes were selected that had moderate to high expression in VCaP cells and lower expression in WBC. p = 0.09 p <0.001 p = 0.24 p =0.01 Figure 4 Improved detection of CTC markers following CD45 depletion. RT-PCR results for potential CTC markers S100A13 and AKR1C3. Without WBC depletion (non-depleted samples) both genes show no significant difference in expression between unspiked and 10-cell spiked sample sets. In the depleted set the background contributed by WBC is reduced and there is a significant difference between the unspiked and 10-cell spike sample sets. Page 7 of 8
Conclusions: The white blood cell depletion method demonstrates that the cell population following WBC removal is significantly depleted of WBC with minimal loss of cancer cells. Thus, this method has a potential to improve the detection of tumor-specific markers in clinical samples enriched for the presence of circulating tumor cells. The decision on whether to implement the WBC depletion protocol will depend on the background expression level of the specific genes of interest. The depletion method may not be necessary for genes that have no background expression in WBC. For genes that have high levels of expression in WBC, it is likely that signals from WBC will mask signals from CTC despite WBC depletion method. It is likely that the method will be most useful in improving the detectability of markers that have a low level of background expression in WBC. It is recommended that the depletion method be tested on healthy donors for each gene of interest to determine the WBC background level and benefit of the procedure. References: 1. Chandra Rao, Brad Foulk, Karl Nielsen, Bhavesh Vaidya, Jaymala Patel, Dana Gaffney, Denis Smirnov, Jayaprakash Karkera and Mark Connelly Janssen Research & Development, LLC, Huntingdon Valley, PA 19006 Improved Molecular Characterization of Circulating Tumor Cells AACR 2013 Abstract #1443 Page 8 of 8