Figure 6. CXCL12 deletion from MSC sensitizes murine and human CML LSC to TKI treatment.
(A) BCR-ABL expression was induced in SCL-tTA-BCR-ABL mice by tetracycline withdrawal. Whole BM cells were obtained 6–8 weeks after induction and injected into age and sex matched CXCL12f/f (CXCL12f/f-XX-Cre-; Cre-negative), CXCL12f/f-Tek-Cre, CXCL12f/f-Prx1-Cre, (CXCL12f/f-XX-Cre+; XX representing either Tek or Prx1) 6–8-week old littermates irradiated at 8Gy (n=5–6 mice/group). 8 weeks post-transplantation, mice were treated with either vehicle (Veh) or nilotinib (50mg/kg; TKI) for 2 weeks once daily oral gavage after which mice were euthanized and PB, BM, spleen analyzed. Total white blood cells (WBC) (B) and frequency of neutrophils (C) in the PB are displayed. A cohort of mice were followed for their survival after stopping treatment among No Cre and Prx1-Cre mice (E), and No Cre and Tek-Cre mice (F). BM LTHSC (G), STHSC (H), MPP (I) per 1 femur, 1 tibia (2 bones) are shown. FACS sorted LTHSC from primary vehicle or TKI-treated mice (CD45.1/2+) were pooled and transplanted into secondary recipient (CD45.2+) healthy WT 6–8-week-old mice (1000 cells/mouse+250,000 helper WBM CD45.2+ cells) irradiated at 8Gy. Long-term donor engraftment was performed 16 weeks following transplantation. The total WBC levels (J), long-term CML donor engraftment (K), frequency of donor myeloid cells (L), in the PB of mice are shown. Human CML 34+ cells were stained with CFSE. CFSE+ primitive cells (34+ 38-) were FACS sorted and cultured in the presence or absence of FACS sorted tdTomato+ cells from CXCL12+/+-tdTomatof/+-Prx1-Cre+ and CXCL12f/f-tdTomatof/+-Prx1-Cre+ mice for 3 days and treated with or without Nil (1µM). Representative FACS plot (M), proliferation (N) and expansion (O) are shown. Error bars represent mean ± sem. ns (non-significant) P>0.05, *P< 0.05, **P< 0.01, ***P<0.001, ****P<0.0001. See also Figure S7.