Fluorouracil selectively spares acute myeloid leukemia cells with long- term growth abilities in immunodeficient mice and in culture

Abstract

A subset of leukemic cells is assumed to maintain long-term growth of acute myeloid leukemia (AML) in vivo. Characterization of these AML progenitor cells may further define growth properties of human leukemia. In vitro incubations with 5-fluorouracil (5-FU) have been used for enrichment of normal primitive hematopoietic stem cells. By analogy to normal hematopoiesis, it was hypothesized that primitive leukemic stem cells might be kinetically more inactive than colony- forming cells (colony-forming units-AML [CFU-AML]). To examine this hypothesis, conditions were established for incubation with 5-FU that eliminated all CFU-AML. These conditions selected a 5-FU-resistant AML fraction that was evaluated for its capacity for long-term growth by transplantation into mice with severe combined immunodeficiency (SCID) and long-term culture in the quantitative cobblestone area-forming cell (CAFC) assay. Transplantation of the 5-FU-resistant fraction of four cases of AML into SCID mice resulted in growth of AML. Whereas no CFU- AML survived, 31% to 82% of primitive (week-6) CAFC were recovered from the 5-FU-treated cells. Hematopoietic cells proliferating in the CAFC assay were shown to be leukemic by cytologic, cytogenetic, or molecular analysis. The reduction of AML growth as determined by outgrowth of AML in SCID mice was in the same order of magnitude as the primitive (week- 6) CAFC reduction. This indicates that both assays measure closely related cell populations and that the CAFC assay can be used to study long-term growth of AML. These results show a hierarchy of AML cells that includes 5-FU-resistant progenitors. These cells are characterized as primitive (week-6) CAFC and as leukemia-initiating cells in SCID mice.

Reduction of heat‐induced haemotoxicity in a hyperthermic purging protocol of murine acute myeloid leukaemic stem cells by AcSDKP

“…Data from the CAFC assay revealed both short-and long-term growth capacity of leukaemic cells and also differences in proliferative activity between the subsets, confirming the heterogeneity in the clonogenic cell population (Table II). This heterogeneity was also demonstrated in human AML bone marrow using the CAFC-assay (Terpstra et al, 1996a, b). The malignant cell population of AML is maintained by a small fraction of clonogenic cells (Griffin & Lowenberg, 1986).…”

“…Although it has been shown that the expression of CD34 does not necessarily correlate with long-term leukaemia-initiating capacity (Terpstra et al, 1996b), it is generally regarded to be associated with an immature phenotype (Lapidot et al, 1994). Furthermore, it could be shown that CFU-AML subsets were eliminated after a 5-FU treatment without affecting the late-appearing CAs (Terpstra et al, 1996a). The persistent cell population was still capable of initiating leukaemia in SCID mice, indicating a correlation between long-term leukaemia-initiating capacity and late-appearing CAs.…”

Leukaemic stem cells

“…Cell-cycle status of AML progenitor cells AML progenitor cells, like their counterparts in normal haemopoiesis, have been shown to be quiescent and resistant to chemotherapeutic drugs (Terpstra et al, 1997;Guzman et al, 2001). AML progenitor cells have been shown to be resistant to treatment with 5-FU, as the treated cells subsequently engrafted SCID mice and proliferated for over 6 weeks in a cobblestone area forming cell assay, indicating that a population of AML progenitor cells are in a quiescent (G 0 ) state (Terpstra et al, 1996b). Guzman et al (2001) used flow cytometric assays with surface, intracellular and DNA (SID) labelling to assess the cycling status of AML patients and found that 83% of CD34 þ /CD38 -/CD123 þ cells were in G 0 .…”

1 Stem cells: characterization and measurement

“…It is likely that these differences are partly due to a low seeding efficiency of the human HSC in these mice (van Henrick, unpublished data). For example, in SCID mice a 5-10-fold higher engraftment with human AML cells has been reported following pre-treatment of the recipients with macrophage-depleting C12MDP liposomes (Terpstra et al, 1996a). Also, the different growth requirements of these cells may affect the apparent frequencies in NOD/SCID mice.…”