Rapid BCP-ALL Engraftment in NOD/SCID Mice Is Characterized by a High Leukemia Initiating Cell (LIC) Frequency.

Abstract 2985

Recently, we identified in a NOD/SCID human (hu) B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) mouse model two different engraftment phenotypes that we named time to leukemia (TTL) short (TTL^short) and TTL long (TTL^long) and that reflected the rapid or late onset of leukemia in the mice. We showed that the rapid and late leukemia engraftments in the mice highly correlated with the relapse free survival of patients initially stratified as standard risk at diagnosis.

In order to analyze if the two distinct phenotypes are characterized by different frequency of leukemia initiating cells (LICs), we investigated the NOD/SCID repopulating activity of 4 individual BCP ALL samples.

We performed a limiting dilution transplantation assay using freshly isolated spleen cells form 2 TTL^short and 2 TTL^long xenografted mice with full-blown leukemia. We created 5 groups of 8 mice each and transplanted intravenously 10⁵, 10⁴, 10³, 10², 10¹ cells per mouse/group. Leukemia engraftment in the peripheral blood (PB) was routinely evaluated by flowcytometry and the mice were sacrificed at the onset of disease manifestation. We defined a mouse as engrafted when we could detect ≥ 1% CD19 positive cells in the PB. In 3 out of 4 samples, 100% of the mice transplanted with 10⁵ and 10⁴ cells engrafted; only in one sample (TTL^long) we had a 75% and 12,5% successful engraftment using 10⁵ and 10⁴ cells respectively. The percentage of engrafted mice using 10³ cells was 87,5% and 75% for the 2 TTL^short samples and 37,5% and 0% for the 2 TTL^long specimens. No mice transplanted with the 10² cells isolated from the 2 TTL^long samples engrafted whereas 50% and 12,5% of the mice transplanted with the 10² cells isolated from the 2 TTL^short engrafted successfully. No one of the mice transplanted with 10¹ cells engrafted. The LIC frequency was calculated using the Poisson single-hit model; a higher LIC frequency was calculated in the 2 TTL^short (1/329 and 1/739) compared to the 2 TTL^long (1/2159 and 1/74028).

As cells in the G0 phase of the cell cycle are considered as the reservoir for the new cycling cells and putative LICs, we analyzed by flowcytometry the percentage of G0 cells, defined as cells staining negative for the Ki-67 antigen, within TTL^short (N=8) and TTL^long (N=7). No statistical difference was found between the analyzed samples. Nevertheless, we observed that in both TTL^short and TTL^long almost all cells were actively progressing through the cell cycle.

Aiming to further characterize distinctive biological features related to the different LIC frequency, we analyzed by flowcytometry the cell cycle profiles of 9 TTL^short and 8 TTL^long mice. We used a cell cycle analysis based on the simultaneous labeling of DNA (7-AAD) and RNA (Pyronin Y) that allows to distinguish between cells that are in G1 but have not yet actively entered the S phase of the cell cycle (named G1a cells) from cells that are actively progressing form G1 to S (named G1b cells). A higher percentage of G1a cells was detected in the TTL^short compared to TTL^long (p = 0.004 Mann-Whitney Test) suggesting the presence of a stand-by-cell fraction in the TTL^short almost ready to proceed through the cell cycle. On the other hand, a higher percentage of G1b cells was found in the TTL^long compared to TTL^short (p = 0.001, Mann-Whitney Test). No significant difference was found in the proportion of cells in the “S/G2/M subgroup”.

We also analyzed by Western Blot CYCLIN B1 expression in TTL^short (N=5) and TTL^long (N=5): a statistically different CYCLIN B1 expression was found between the two groups (p = 0.009 Mann-Whitney Test). As this molecule is involved in the progression of cells from the G2 to M phase of the cell cycle, we analyzed by flowcytometry the mitotic cells fraction defined as the cells positive for the phosphorilation of the Ser10 on the histone H3 in 6 TTL^short and 5 TTL^long specimens. A higher proportion of P-H3(Ser10) positive cells was detected in the TTL^short compared to the TTL^long (p = 0.045, Mann-Whitney Test).

All together these data indicate that the two NOD/SCID engraftment phenotypes are characterized by different frequency of LICs. Furthermore, our functional analyses reveal a distinctive progression of the leukemic cells through the cell cycle showing a higher number of cells (G1a cells) ready to progress through the G1-S phases in the TTL^short/poor prognosis leukemia.