autologous blood and marrow transplantation - Blog Science ...

Mapara et al. 533
Hematopoietic colony-forming assay and tumor cell clonogenic assay
CD34 +
cells (3Xl0 5
/well) or MT-1 cells (lxl0 5
/well) were seeded as
triplicates into a 24-well plate. Cells were infected with AdCMV.CD (20
multiplicity of infection [m.o.i.]), followed by the addition of 1 mM 5-FC (Sigma
Chemie GmbH, Germany). Controls were treated as indicated. After 48 hours of
incubation, 2X10 3
CD34 +
cells or 1X10 3
MT-1 cells were plated into 1 ml of
standard methylcellulose (MethoCult GF H4434, Stem Cell Technologies,
Vancouver, Canada) in a 35-mm dish. Colonies were scored after 8 (MT-1 cells)
and 14 (CD34 +
cells) days of culture.
Recombinant adenovirus vector
The recombinant adenovirus vector AdCMV.CD (generously provided by Dr.
Ronald Crystal, New York) carries the cDNA of the E. coli cytosine deaminase
driven by the cytomegalovirus (CMV) major immediate promoter. 20
The Ad vector
AdCMV.nlsft-gal carries the reporter gene -ft-galactosidase combined with a
nuclear localization signal driven by the CMV promoter. 21
The Ad vector
AdCMV.p21 (generously provided by Dr. Michael Strauss, Berlin, Germany)
carries the cDNA p21 driven by the CMV promoter. All vectors were propagated
on 293 cells and purified and stored at -70°C, as previously described. 22
RESULTS
Combined positive and negative selection
We have conducted a phase I/II study in breast cancer patients evaluating the
feasibility and purging efficiency of a combined positive/negative purging
approach using the ISOLEX 300 device (Baxter, Munich, Germany). Positive
selection of CD34 cells was followed by an immunomagnetic tumor cell depletion
using a cocktail of monoclonal antibodies directed against epithelial antigens.
Preclinical experimentation had suggested that this combination of CD34
selection and subsequent immunomagnetic purging leads to an improved purging
efficiency, resulting in an overall tumor cell depletion of 4.9 log (Table 1). These
results could be confirmed in our trial. Thus, after double purging, tumor cells
could only be detected in 6.3% of the autografts (1/16) compared with 50% before
purging (8/16). A median of >4 log of tumor cell depletion could be achieved
(Table 2). Positive/negative purging resulted in excellent results concerning CD34
purity and CD34 recovery leading to a median CD34 purity of 97.2% and a median
cell loss of 48.9% after both purging procedures. 23
Furthermore, these cells led to
rapid and sustained engraftment after high-dose chemotherapy. Thus, it can be