Purging of contaminating breast cancer cells from hematopoietic stem cell grafts by adenoviral GAL-TEK gene therapy and magnetic antibody cell separation.
The presence of contaminating tumor cells in autologous bone marrow or peripheral blood stem cell (PB-SC) preparations increase the likelihood of relapse in women receiving transplants for metastatic breast cancer. We describe a new technique for purging breast cancer cells (BCCs) that combines two independent strategies: (a) the specific enrichment of CD34+ progenitor stem cells by magnetic antibody cell separation (MACS), and then (b) infection of the contaminating BCCs with a recombinant adGAL-TEK marker/suicide gene adenovirus (ad-v), followed by the addition of ganciclovir (GCV). Infection with this ad-v results in three to four times greater expression of ad-v-delivered reporter gene in BCCs than in CD34+ cells. In addition -2 h, -low multiplicity of infection (50:1) adGAL-TEK infections of BCC lines (MCF-7 and BT474) eradicated >99% of BCCs after 72 h of exposure to 20 microM GCV. However, exposure to both adenovirus and GCV at the MOIs and doses used had little effect on hematopoietic stem cells to form colonies in colony-forming unit assays. adGAL-TEK infection in our model system (10(3)-10(5) BCCs added into 10(7) HSCs) also resulted in the 3 to 5 log eradication of clonogenic BCCs after the addition of GCV. MACS enrichment/purification of CD34+ cells from PB-SC contaminated with 2 x 10(6) to 5 x 10(7) BCCs followed by adGAL-TEK infection and GCV addition resulted in 5-7-log depletion of clonogenic BCCs as well as enrichment of CD34+ progenitor cells to >98%, with the recovery of >70% of hematopoietic stem cells. This adenoviral purging system is so robust that poor MACS purification, resulting in 1.5-log depletion of BCCs, still permits excellent ad-v infection and BCC killing.
Marini
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Department of Molecular Hematology and Therapy, University of Texas, M.D. Anderson Cancer Center, Houston 77030, USA.
Snell
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Yu
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Zhang
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Singletary
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Champlin
R
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Andreeff
M
M
D000256:Adenoviridae / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D002470:Cell Survival / Q000187:drug effects; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D018189:Immunomagnetic Separation; D014162:Transfection; D014407:Tumor Cells, Cultured
D015774:Ganciclovir