Comparison of CD45+ Depletion Methods for Enrichment of Disseminated Tumor Cells in Bone Marrow Samples
Poster Dec 07, 2017
Birte Möhlendick, Imke Hoffmann, Swetlana Seidschner, Sarah Schumacher, Alexander Smyczek, Carina Vaerst, Wolfram Trudo Knoefel, Nikolas Hendrik Stoecklein
Disseminated tumor cells (DTC) in bone marrow (BM) samples are, with a frequency of only one DTC per 1x106 cells, quite rare. DTCs are usually detected utilizing immunofluorescence staining of 1-2x106 mononuclear cells (MNC) and consecutive microscopic analysis. To increase the detection rate of DTCs a higher number of cells should be analyzed. Since microscopic detection of these cells is very laborious DTC enrichment strategies are needed. Here, we investigated the efficiency of three different depletion strategies of CD45+ cells for DTC enrichment in BM samples.
CD45+ cell depletion was performed on BM samples from cancer patients with different tumor entities. Immunomagnetic depletion (DYN) of CD45+ cells was performed after Ficoll density gradient centrifugation on 1x107 MNCs utilizing CD45 Dynabeads® (Invitrogen) with i) 4x107 beads/ml cell suspension (DYNLO) and ii) 1x108 beads/ml cells suspension (DYNHI). Antibody-based depletion (ROS) of CD45+/CD66b+ cells was performed on 1x108 BM cells with the RosetteSep® Human CD45 Depletion Cocktail (Stemcell Technologies). For the different strategies at least 60 samples were analyzed, the depletion efficiency was calculated and DTC numbers were extrapolated to compare the respective methods.
We were able to detect DTCs in 32.7% (DYNLO), 12.5% (DYNHI) and 66% (ROS) of the patient samples, respectively. CD45+ cells could be depleted most efficiently up to 440-fold using ROS, followed by 44-fold with DYNHI and 20-fold using DYNLO. DTC numbers were significantly higher (p<0.0001) in the ROS samples (mean=3.69) with up to 21 DTCs per 1x106 cells. With the immunomagnetic procedures DYNLO samples (mean=0.48) had slightly higher DTC counts (8.33 vs 6.35 DTCs per 1x106 cells) compared to DYNHI (mean=0.39).
In our hand the ROS procedure showed the best results for DTC enrichment from BM samples. This method enables the analysis of a high number of BM cells since it depletes unwanted CD45+ cells most efficiently and thus increases DTC detection rates in the patient samples.
Multiplexing cell-based assays is possible using 3D culture models that are larger and more complex than monolayers
Real-time detection methods to measure live or dead cells provide much flexibility for multiplexing
All multiplexed assay combinations should be verified using appropriate controls for each 3D cell culture model.