In vitro chemoseparation of leukemic cells from murine bone marrow using VP16-213: importance of stem cell assays.
The use of chemopurified autologous bone marrow (BM) is being explored as a transplant source for patients with leukemia who do not have a HLA-matched donor. Because stem cell assays have not previously been found to predict engraftment after transplantation, the optimal drug(s) and drug concentrations have not been determined. To determine the effectiveness of the podophyllotoxin derivative, VP16, and the value of stem cell assays in chemopurification studies, a murine model using the C57B1/6 mouse and its syngeneic leukemia EL-4 was developed. Kill of committed (CFU-C) and pluripotent (CFU-S) hematopoietic stem cells and tumor (tCFU) stem cells after a 1-h exposure to VP16 was first determined. A marked kill differential of tCFU compared to that of the CFU-C/S populations was found, with no tCFU surviving at VP16 concentrations greater than 30 micrograms/ml. No kill differential of CFU-C versus CFU-S was seen at VP16 doses greater than 10 micrograms/ml. All mice transplanted with a mixture of 25 X 10(6) BM cells and 1 X 10(7) EL-4 cells treated in vitro with 40 micrograms/ml of VP16 died of their tumors. However by reducing the tumor burden to 1 X 10(6) EL-4 and 5 X 10(6) EL-4, nine of ten and four of six of the mice, respectively, survived 90+ days tumor free. On the basis of survival data, it was found that engraftment of VP16-treated BM was directly proportional to the product of the degree of CFU-S inhibition and BM cell inoculum, i.e., the number of viable CFU-S transplanted. The maximum VP16 concentration that led to predictable engraftment at BM doses less than 25 X 10(6) cells was 55 micrograms/ml. Thus, 15 mice were transplanted with 1 X 10(7) BM cells and 1 X 10(7) EL-4 cells, incubated with 55 micrograms/ml of VP16; 13 out of 15 survived tumor free for 90+ days. When VP16 was used as a BM chemopurification agent, up to 50% of contaminating tumor cells were eliminated from BM suspensions without affecting engraftment after transplantation. Because stem cell inhibition predicted engraftment, drug concentrations that maximized tumor cell kill could be chosen.
D000818:Animals; D001853:Bone Marrow / Q000187:drug effects; D001854:Bone Marrow Cells; D016026:Bone Marrow Transplantation; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D003114:Colony-Forming Units Assay; D004305:Dose-Response Relationship, Drug; D005047:Etoposide; D007942:Leukemia, Experimental / Q000473:pathology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains
D005047:Etoposide