Immunomagnetic separation, in which the target cells are identified using monoclonal antibodies (Mabs) and separated by attachment to paramagnetic particles and passage through a magnetic field, is widely used for both negative and positive cell
selection. This report describes an immunomagnetic method for the selective purging and enrichment of tumor cells from bone marrow using two different panels of Mabs and polystyrene microspheres. In model experiments, cultured LA-N-5
neuroblastoma
cells
that were premarked with the fluorescent DNA stain (Hoechst 33342) were seeded into normal marrow. Mixture of normal marrow and tumor cells were sensitized with anti-neuroblastoma antibodies (positive selection) or anti-hematopoietic antibodies
(negative selection), and then magnetic immunobeads coated with goat anti-mouse immunoglobulin (GAM) to form bead-target cell conjugate, and removed from the mixture using magnets. All the anti-hematopoietic Mabs such as L243 (anti-Ia), Gap8.3
(anti-myeloid), BBM1(anti-β2 microglobulin), and W6/32 (anti-HLA-A,B,C) showed strong reactivity to bone marrow cells and negativity to LA-N-5 neuroblastoma cell, while Mab390 (anti-fetal brain) and Mab459 (anti-neuroblastoma) showed strong
reactivity
to LA-N-5 and negativity to marrow cells. Positive selection with a combination of four anti-neuroblastoma antibodies (Mab390, Mab549, HSAN 1.2, 126-4) produced significant enrichment (e.g. 1% to 10% or 5% to 45% after Ficoll/Hypaque
sedimentation).
Higher enrichments were obtained from low percentage (1%, 5%) of tumor cells in marrow by positive selection and from high percentage (10%, 20%) of tumor cells in marrow by negative selection.
Depletion of tumor cells from marrow was affected by concentrations of GAM and bead:tumor cell ratios. With 50μg GAM/mg and 100 beads/tumor cell ratio, maximum depletion was obtained (2.2 log depletion after one step and 3.7 log after two). With
clinical marrows, variable degree of enrichments was achieved according to tumor percentages before enrichment and two samples turned out to be positive in marrow metastases after enrichment.
We conclude that tumor cell depletion and enrichment by this system can be extended to any type of tumor cell by changing panels of Mabs and that this method could be applied to autologous BMT and to increase the sensitivity in tumor cell
detection
in
addition to preparing tumor samples from marrow for molecular biological studies.
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