Publication Date:
2019-11-13
Description:
Chronic myeloid leukemia (CML) is a typical stem-cell driven malignancy, driven by leukemia stem cells (LSCs). LSCs are resistant to conventional therapies. This resistance is mediated by cell-intrinsic mechanisms and interactions with the microenvironment. LSCs depend on signals from a specialized microenvironment, a so called niche, to maintain their stem cell characteristics. In CML the bone marrow (BM) as a niche is well-investigated and several therapeutic targets, which aim at LSCs by interrupting their interaction with the BM-niche are under investigation. However, even though splenomegaly is a hallmark of CML the contribution of the splenic microenvironment to CML development has not been studied so far. This project aims to investigate the role of the splenic microenvironment as an independent secondary LSC niche and its contribution to disease development. To induce a CML-like disease in mice we retrovirally transduced FACS-sorted Lineage- Sca-1+ cKit+ BM cells with pMSCV-p210BCR/ABL-IRES-GFP and injected the transduced cells into non-irradiated mice. To find out if the spleen contributes to disease development we induced CML in splenectomized and sham operated mice. Splenectomized mice survived significantly longer compared to sham operated controls (median survival 31 vs. 22 days; p=0.0006) with 20% of the splenectomized mice surviving longer than 90 days. Moreover, the number of LSCs in the BM of splenectomized mice was reduced 3.7-fold (p=0.002). Flowcytometric analysis of the spleen and BM compartments of CML bearing mice revealed that the majority of the leukemic stem and progenitor cells (LSPCs) were located in the spleen (19-fold more LSCs in the spleen; p =0.007). Moreover we found the leukemic compartment in the spleen to be enriched for LSPCs compared to the BM (20 % spleen vs. 10 % BM; % LSPCs of total leukemic cells; p=0.01). To confirm this phenotypic observation functionally we performed a limiting dilution transplantation of leukemic cells from spleen and BM. In line with the phenotypic observation we found a higher frequency of LSCs in the spleen compared to the BM (1/41'703 vs. 1/432'594; p=0.02). We next analyzed the gene expression of LSPCs from spleen and BM. We found that the gene expression profile of splenic LSPCs showed higher expression of stemness-related genes and reduced expression of myeloid differentiation genes compared to BM LSPCs, indicating that the spleen is more supportive of primitive LSPCs. Knowing that the spleen contributes to disease development by providing an alternate niche for LSCs we next analyzed the spleens using confocal microscopy. We found that the LSCs resided exclusively in the red pulp. Previous studies have shown that HSCs reside in direct contact with red pulp macrophages (RPMs) during extramedullary hematopoiesis (Dutta et al., JEM, 2015). In addition we found that in spleens from human CML patients CD34+ leukemia cells localized together with macrophages (p=0.001). Furthermore we could show that RPMs are capable of producing both SCF and G-CSF. To test the role of RPMs as a potential niche component in vitro we co-incubated LSCs and RPMs overnight before plating the LSCs in a colony formation assay. We found that the co-incubation with RPMs improved the colony formation capacity of LSCs (CFUs 166 vs. 138; p=0.0356). To test the role of RPMs in vivo we depleted macrophages in CML mice using clodronate liposomes. This resulted in significantly reduced splenomegaly (867mg vs. 249mg; p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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