Contribution of Leukemia-Induced Alterations in Mesenchymal Cell Subpopulations to Altered Regulation of Leukemic and Normal Stem Cells in the CML BM Microenvironment

Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem cell (HSC) maintenance. The function of microenvironmental niches in regulating leukemia stem cell (LSC) growth is not well understood. We have reported that alterations in chemokine and cytokine expression in the CML BM microenvironment, including reduction in expression of the critical hematopoietic chemokine CXCL12, may differentially alter CML and normal stem cell localization and growth, and provide a competitive growth advantage to CML LSC. Here we evaluated the contribution of leukemia-induced alterations in BM mesenchymal cell subpopulations to altered BM microenvironmental function in CML. Our group has used cell surface markers to characterize mesenchymal cells in the BM of adult mice. These studies have identified three distinct osteoprogenitor subpopulations [CD166+CD146- (CD166+);CD166-CD146+ (CD146+); and CD166-CD146-Sca1+ (Sca1+) cells] as well as a phenotypically defined population of CXCL12-adundant reticular (CAR) cells (Sca1-CD44+CD51+CD106+CD140a+) within CD45-Ter119-CD31- BM mesenchymal cells. We have shown that Sca1+ cells represent primitive mesenchymal progenitors that can generate the other mesenchymal populations, whereas CD146+ cells may represent an intermediate population between Sca1+ cells and CD166+ osteoblasts. Using the SCL-tTA-BCR/ABL mouse model of CML, we examined leukemia-induced alterations in distribution and LTHSC supportive function of mesenchymal cells in CML BM. We observed that the frequency of Sca1+ (p=0.001) and CD146+ (p=0.02) progenitors were significantly increased, and of CD166+ osteoblastic cells (p<0.05) and CAR (p<0.02) cells were significantly reduced, in CML compared to normal BM. CD146+ and CD166+ cells, but not Sca1+ cells, from CML BM showed reduced proliferation in vitro compared to their normal counterparts. To evaluate hematopoietic supportive function, selected normal and CML mesenchymal subpopulations were cocultured with CD45.1 normal and CML LTHSC for 3 days at a 1:1 ratio. LTHSC were then transplanted into CD45.2 recipient mice (200 normal LTHSC/mouse; 1000 CML LTHSC/mouse). Normal LTHSC cocultured with normal Sca1+ (p<0.03), CD146+ (p<0.02), CD166+ (p<0.02), and CAR (p<0.03) cells demonstrated significantly increased hematopoietic engraftment in recipient mice at 16 weeks compared with control LTHSC cultured without mesenchymal cells. In contrast engraftment of normal LTHSC co-cultured with CML compared to normal BM mesenchymal subsets was significantly reduced [Sca1+ (p<0.03), CD146+ (p<0.02), CD166+ (p=0.06), and CAR (p<0.02) cells]. CML LTHSC cultured with normal Sca1+ (p<0.002), CD146+ (p<0.001), CD166+ (p<0.001), and CAR (p<0.03) cells showed significantly increased engraftment in recipient mice after transplantation and increased leukemia development compared with CML LTHSC cultured without stromal cells. CML LTHSC showed similar engraftment in recipient mice after coculture with CML and normal Sca1+ and CD146+ cells, but significantly reduced engraftment after coculture with CML compared to normal CD166+ (p<0.01) and CAR (p<0.02) cells. In conclusion, our studies have identified leukemia associated alterations in the distribution of mesenchymal subpopulations in the CML BM microenvironment, with decrease in more differentiated CD166+ osteoblastic cells and CAR cells and increase in primitive Sca-1+ and CD146+ cells. The reduction in CXCL12 producing CAR cells, and the reduced ability of CML Sca1+ and CD146+ cells to support normal compared to CML LTHSC, may provide a competitive growth advantage to the leukemic clone within the CML BM microenvironment.