Journal: Cell reports
Article Title: TNF-α-induced alterations in stromal progenitors enhance leukemic stem cell growth via CXCR2 signaling
doi: 10.1016/j.celrep.2021.109386
Figure Lengend Snippet: (A) Experimental schema. BCR-ABL expression was induced in SCL-tTA-BCR-ABL mice (CML) by tetracycline withdrawal. WBM cells obtained 8 weeks after leukemia induction from CML mice and from WT healthy mice (normal) were injected into WT mice irradiated at 8 Gy (n = 6–8 mice/group). Mice were treated 8 weeks after transplantation with Veh, CXCR2i (5 mg/kg), Nil (50 mg/kg), or the combination once daily by oral gavage for 2 weeks, after which they were euthanized and PB and BM were analyzed by flow cytometry. (B–D) WBC numbers (B) and neutrophil frequency (C) in the PB and LSC numbers in (D) in the BM (2 femora + 2 tibiae) of CML mice. (E) A cohort of mice was followed for survival after stopping treatment (n = 7–8 mice/group). After primary transplantation of CML WBM cells from (A), LTHSCs were FACS-purified and transplanted into secondary healthy WT mice irradiated at 800 cGy. (F) Serial blood draw was performed every 4 weeks until 16 weeks. (G) WBM cells from Cxcr2 +/+ -SCL-tTA-BCR-ABL or Cxcr2 −/− -SCL-tTA-BCR-ABL double-mutant mice (2 × 10 6 cells/mouse, n = 6–8 mice/group) were injected into WT mice irradiated at 8 Gy (n = 6–8 mice/group). Transplanted mice were maintained off doxycycline to induce BCR-ABL expression and generate leukemia. Mice were treated 8 weeks after transplantation with Veh or Nil (50 mg/kg) once daily by oral gavage for 2 weeks and euthanized, and PB and BM were analyzed by flow cytometry. (H–K) Total WBC (H) and frequency of multi-lineage reconstitution of donor cells (I) in the PB and numbers of donor LSCs (J) and GMP (K) in the BM (2 femora + 2 tibiae). (L) Primary human CML CD34+ cells were labeled with CFSE, and CFSE max CD34+CD38− primitive LSCs were FACS purified and co-cultured with or without primary human BM MSC (hMSCs) with nilotinib (Nil; 1 μM), the CXCR2i (10 μM), or both or were left untreated for 72 h (n = 4 biological replicates). The proliferation index was calculated on the basis of reduction in CFSE levels. (M and N) The effects of treatment on the proliferation index in the presence of hMSC cells (M) and the percentage of apoptosis, calculated based on Annexin V+ labeling, of CML cells in the presence of hMSCs (N) are shown. (O) Annexin V+ labeling of murine LSCs cocultured in the presence of CML 6C3+ cells and treated with various drugs for 48 h. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Article Snippet: Nilotinib (TKI) supplied by Novartis Pharmaceuticals, and CXCR2 inhibitor (SB225002) purchased from Selleckchem (cat no. S7651, Houston, TX) were stored in 10mM dimethylsulfoxide (DMSO) at 20°C.
Techniques: Expressing, Injection, Irradiation, Transplantation Assay, Flow Cytometry, Purification, Mutagenesis, Labeling, Cell Culture
