Cell Population Highly Enriched with LSCs Invades Liver in MPD-Like Myeloid Leukemia.

It is considered that leukemias posses a rare population of leukemia stem cells (LSCs) capable of the limitless self-renewal necessary for cancer initiation and maintenance. These cells also are believed to be responsible for the relapses of leukemias in patients and thus it is important to find the differences between normal HSCs and LSCs to create curative treatment of leukemias. We previously reported a model of transplantable myeloid leukemia in mice. In this model bone marrow (BM) and liver cells of affected mice were fully transplantable; recipients became moribund within 17-32 days since cells injection. Limiting dilutions analysis revealed that the concentration of LSCs in the BM of moribund mice was one LSC per 37000 c-kit+ cells and one per 45 cells from affected liver. Concentration of LSCs in other cell populations was calculated on the base of mice lifespan after transplantation and was shown to be one LSC per 2500000 c-kit-CD45-, 200 c-kit-CD45+, 4500 Ter119+ and 2600 Ter119- cells. Thus LSCs of this disease retain hierarchical organization and are able to differentiate at least among myeloid and erythroid lineages without the loss of self-renewing ability. Extremely high concentration of LSCs in the liver suggests the population of these cells being scaled up during invasion. The aim of this work was to investigate dynamics of liver repopulation by LSCs and to study additional molecular characteristics of leukemia initiating cells.

To trace the development of the disease female mice (C57Bl/6 x CBA) F1 were injected i.v. with 10⁶ BM cells from syngeneic moribund donors. Each day after the injection the liver cells of mice were sorted into CD45+ and CD45- populations. RNA was isolated from both populations and expression of some genes was evaluated using real time PCR and conventional PCR. To assess the role of chemokines secreted by liver in the migration of LSCs the expression of chemokine receptors was analysed by means of PCR in the liver tissue of moribund mice.

Taking into consideration that CD45+ cells comprise 45 percent of total cells in the liver by the terminal stage of the disease the concentration of LSCs among CD45+ cells in the liver may reach 1 per 23 and even higher. It allows isolating relatively homogenious population of LSCs for studying genes that play role in neoplastic transformation. The investigation of the dynamics of the disease showed that the weight of the liver and spleen began to grow on day 10 after cells injection. At the same time the dramatic rise of CD45+ cells occured in the liver and continued till death. Overexpression of genes responsible for self-renewal and proliferation (Bmi-1 and Myc) was revealed since the day 7. The expression of Bmi-1 and Myc in CD45+ cells in leukemic liver from moribund mice was 20-40 and 25-50 times higher than in CD45+ cells of control liver. The expression of Csf1r (M-CSFr) was elevated 100-fold, so this surface antigen may be served as a marker of LSCs in given disease. The analysis of house-keeping genes Rpl13a, Ubc, Hprt1 and Actb expression have shown that none of these genes fits for the normalization of cDNA amount, because the expression of them (by the terminal stage) in CD45+ cells of leukemic liver was elevated 45-, 25-, 98- and 59-fold as compared to CD45+ cells of control liver. It means that LSCs are essentially different from normal hematopoietic precursor cells. The expression analysis of genes coding the receptors of chemokines in the liver of moribund mice has shown that Ccr1 expression appeared and the expression of Ccr2 and Ccr5 increased in comparison with normal liver cells. It allowed to state that the tumor cells in liver express Ccr1 on their surface. This data suggests the role of chemokines secreted by liver and primarily the role of Ccl3 (Mip-1a) and Ccl5 (Rantes) in the migration of tumor cells into the liver. On the other hand this data allowed speculating on the nature of LSCs, because though Ccl3 and Ccl5 are chemoattractants for different subsets of leukocytes, they mainly attract granulocytes and monocytes.

Thus the model used is unique for the study of LSCs properties, the mechanisms of leukemogenesis, the migration and retention of LSCs extranodally in the dependence of specific microenvironment.

No relevant conflicts of interest to declare.