Splenic Red Pulp Macrophages Provide a Secondary Stem Cell Niche for Chronic Myeloid Leukemia Stem Cells

Leukemic stem cells (LSCs) are responsible for initiation, progression and relapse of the disease. Chronic myeloid leukemia (CML) is a classical stem-cell driven malignancy and represents an attractive model to study LSCs. Resistance of LSCs to treatment is mediated by cell-intrinsic characteristics but also by the interactions of LSCs with their microenvironment. LSCs depend on signals from their surrounding niche in the bone marrow (BM) and spleen in order to maintain stem cell characteristics such as quiescence and self-renewal. The BM as a niche for LSCs is well-investigated and several therapeutic targets are under consideration.

Splenomegaly is a hallmark of CML. Nevertheless, the role of the spleen on leukemia and LSCs in unknown. Therefore, this project aims to investigate the potential role of the spleen as an independent secondary niche for CML stem cells and its contribution to disease development and progression.

In order to study the role of the spleen in CML development and progression, we retrovirally transduced FACS-purified Lineage^- Sca-1⁺ c-Kit⁺ BM cells (LSKs) with pMSCV-p210BCR/ABL-IRES-GFP and injected transduced LSKs into splenectomized or sham operated mice. Splenectomy prior to CML induction significantly improved survival compared to sham operated controls (median survival 31 vs. 22 days; p=0.0006) with 20 % of splenectomized animals surviving longer than 90 days (endpoint). In addition, splenectomized animals showed a 3.7-fold reduction (p=0.002) in LSC numbers in the BM when analyzed 18 days after CML induction, suggesting that the spleen influences the disease burden in the BM.

We next analyzed the leukemic compartment in the BM and spleen and found a significant enrichment of leukemic stem and progenitor cells in the spleen (20 % spleen vs. 10 % BM; % leukemic stem and progenitor cells of total leukemic cells; p=0.01; 19-fold increase in total amount of LSCs in the spleen; p =0.007). To functionally confirm the phenotypic increase in splenic LSCs, we performed secondary transplantations of total leukemic cells from the BM or spleen at limiting dilution into lethally irradiated secondary recipients. The analysis revealed a significantly higher frequency of LSCs in the spleen compared to the BM (1/41'703 vs. 1/432'594; p=0.02).

In addition, we determined the localization of LSCs in the spleen by confocal microscopy on optically cleared spleens. Splenic LSCs were exclusively localized within the red pulp and in close proximity to macrophages. It was previously shown that hematopoietic stem cells reside in direct contact with macrophages in the red pulp of the spleen during extramedullary hematopoiesis (Dutta et al., JEM, 2015). Therefore, we depleted macrophages in CML mice using clodronate liposomes. Spleen weight (867mg vs. 249mg; p<0.0001) and numbers of splenic LSCs (23-fold; p=0.001) were significantly reduced in clodronate treated animals. Importantly, clodronate treatment did not affect LSC viability, as indicated by comparable amounts of AnnexinV+ cells in both groups.

In summary, we show that the interaction of splenic niche cells (red pulp macrophages) with LSCs results in the generation of a big reservoir of LSCs which contribute to CML progression. Therefore, our study strongly suggest the presence of an independent secondary splenic LSC niche in addition to the well characterized BM LSC niche in CML.