Journal: F1000Research

Article Title: General anaesthetics reduce acute lymphoblastic leukaemia malignancies in vitro and in vivo via CXCR4 and osteopontin mediated mechanisms

doi: 10.12688/f1000research.125877.2

Figure Lengend Snippet: a) NALM-6 cells were initially treated with intralipid (VC), 10 μg/ml propofol and 3.6% sevoflurane for six hours. Then, treated cells were loaded into the migration chamber with 1 nM SDF-1 in the lower chamber as the chemoattractant. Cells were allowed to pass through migration chambers for six hours. Cells in the lower chamber were collected and counted in a flow cytometer. Data are shown as mean±sd (n=4). NC: naïve control, VC: intralipid, P10: 10 μg/ml propofol, and Sevo: 3.6% sevoflurane. *p<0.05, **p<0.01, b) Reh cells were initially treated with intralipid (VC), 10 μg/ml propofol and 3.6% sevoflurane for six hours. Then, treated cells were loaded into the migration chamber with 1 nM SDF-1 in the lower chamber as the chemoattractant. Cells were allowed to pass through migration chambers for six hours. Cells in the lower chamber were collected and counted in a flow cytometer. Data are shown as mean±sd (n=4). NC: naïve control, VC: intralipid, P10: 10 μg/ml propofol, and Sevo: 3.6% sevoflurane. *p<0.05, **p<0.01 c) Intravital confocal calvarium imaging of GFP-transduced NLAM-6 cells (red) was performed after injection of GFP-NALM-6 cells into C57BL/6 mice IV. GFP-NALM-6 cells were pre-treated with either 10 μg/ml of propofol or 3.6% sevoflurane overnight in vitro prior to injections. Respective time-lapses were shown following IV injection of tritc-dextran to identify blood vessels (green); non leukaemia cells with high autofluorescent signals (blue) were recorded as well to improve the identification of target cells. Scale bar: 50 μm. Each position was imaged at three-minute intervals for 66 minutes. We only investigated the migration of cells in bone marrow. Cells in blood vessels were excluded from our investigations. Top row: Mouse injected with untreated cells (red). NC: naïve control. Middle row: Cells were pre-treated with 10 μg/ml of propofol overnight in vitro prior to injection. Bottom row: Cells were pre-treated with 3.6% of sevoflurane prior to injection. Displacement of each cell captured in all time-lapses was recorded. Data are pooled from 3 naïve control mice (N=3, 113 cells), 4 mice injected with propofol treated cells (n=4, 86 cells) and 4 mice injected with sevoflurane treated cells (N=4, 88 cells). Data are illustrated as mean±sd. For displacement data, *p<0.05, ***p<0.001. Data are analysed by one-way non-parametric ANOVA test (Kruskal-Wallis test). Propofol: 10 μg/ml of propofol and Sevoflurane: 3.6% of sevoflurane. Mean speed of each cell captured in time-lapses was recorded. Data is pooled from 3 naïve control mice (N=3, 113 cells), 4 propofol pre-treated mice (N=4, 86 cells) and 4 sevoflurane treated mice (N=4, 88 cells). Data are illustrated as mean±sd. *p<0.05, **p<0.01. Data is analysed by one-way non-parametric ANOVA test (Kruskal-Wallis test). Propofol: cells were pre-treated with 10 μg/ml of propofol for six hours and Sevoflurane: cells were pre-treated with 3.6% of sevoflurane for six hours.

Article Snippet: Multiple group comparisons were performed using ANOVA with a Kruskal-Wallis nonparametric or Bonferroni’s post hoc test for comparisons GraphPad Prism 7.0, RRID:SCR_002798.

Techniques: Migration, Flow Cytometry, Control, Imaging, Injection, In Vitro, IV Injection

Journal: F1000Research

Article Title: General anaesthetics reduce acute lymphoblastic leukaemia malignancies in vitro and in vivo via CXCR4 and osteopontin mediated mechanisms

doi: 10.12688/f1000research.125877.2

Figure Lengend Snippet: a) Upper left: diagram showing strategy for calvarium imaging. Intravital confocal calvarium imaging of GFP-transduced NLAM-6 cells (red) was performed after injection of GFP-NALM-6 cells into C57BL/6 mice IV. GFP-NALM-6 cells were pre-treated with either 10 μg/ml of propofol or 3.6% of sevoflurane prior to injection. Representative maximum projection tile scans and corresponding high-magnification inserts were shown following IV injection of tritc-dextran to identify blood vessels (green); non leukaemia cells with high autofluorescent signals (green, spotty signal) were recorded as well to improve the identification of target cells. Higher magnification images show portions of low magnification tile scans. Scale bar: 500 μm for low magnification tile scan. 50 μm for high magnification images. b) The distance between the nearest bone surface (not shown) and each GFP-NALM-6 cell was measured. Data illustrated as mean±sd. Data are pooled from 4 naïve control mice (N=4, 279 cells), 4 mice injected with propofol treated cells (n=4, 134 cells) and 6 mice injected with sevoflurane treated cells (n=6, 366 cells). *p<0.05. Data are analysed by one-way non-parametric ANOVA test (Kruskal-Wallis test). c) We calculated the number of cells enter bone marrow space from IVM images for each treatment group. Data are illustrated as mean±sd. Data are pooled from nine naïve control mice, eight mice injected with propofol pre-treated cells and six mice injected with sevoflurane pre-treated cells. *p<0.05. Data are analysed by one-way non-parametric ANOVA test (Kruskal-Wallis test). Propofol: cells were pre-treated with 10 μg/ml of propofol for six hours, Sevoflurane: cells were pre-treated with 3.6% of sevoflurane for six hours.

Article Snippet: Multiple group comparisons were performed using ANOVA with a Kruskal-Wallis nonparametric or Bonferroni’s post hoc test for comparisons GraphPad Prism 7.0, RRID:SCR_002798.

Techniques: Imaging, Injection, IV Injection, Control

Journal: F1000Research

Article Title: General anaesthetics reduce acute lymphoblastic leukaemia malignancies in vitro and in vivo via CXCR4 and osteopontin mediated mechanisms

doi: 10.12688/f1000research.125877.2

Figure Lengend Snippet: a) NALM-6 cells were treated by intralipid (VC), 5 μg/ml and 10 μg/ml of propofol for six hours and 24 hours of recovery time is given for samples treated with 10 μg/ml of propofol. Or cells were treated by 3.6% of sevoflurane for two, four, and six hours, and 24 hours of recovery time is given to samples treated by initial 2 hours of exposure of sevoflurane. Following the general anaesthetics treatment and recovery time, treated cells were stained with a CXCR4 antibody and analysed by flow cytometer for mean fluorescence intensity. Data are illustrated as mean±sd (N=4). Data are analysed by one-way ANOVA followed by Bonferroni’s post hoc test. For propofol statistics, *p<0.05, **p<0.01, ***p<0.001. VC: intralipid, P5: 5 μg/ml of propofol, P10: 10 μg/ml of propofol, Sevo 2: two hours sevoflurane, Sevo 4: four hours sevoflurane and Sevo 6: six hours sevoflurane. Grey histogram: isotype control; Red histogram: CXCR4. b) NALM-6 cells were either untreated or treated with 3.6% sevoflurane for six hours initially. Then they were allowed to adhere to plates pre-coated with thrombin cleaved osteopontin for two hours. Data are illustrated as mean±sd (N=4). *p<0.05. Data are analysed by one-way ANOVA followed by Bonferroni’s post hoc test. c) NALM-6 cells were either untreated or treated with 10 μg/ml of propofol for six hours initially. Then they were allowed to adhere to plates pre-coated with thrombin-cleaved osteopontin for two hours. Data are illustrated as mean±sd (N=4). *p<0.05. Data are analysed by one-way ANOVA followed by Bonferroni’s post hoc test. VC: intralipid. c) Reh cells were treated by intralipid (VC), 5 μg/ml and 10 μg/ml of propofol for six hours. Or cells were treated by 3.6% of sevoflurane for two, four, and six hours. Following the general anaesthetics treatment, treated cells were stained with a CXCR4 antibody and analysed by flow cytometer for mean fluorescence intensity. Data are illustrated as mean±sd (N=4). Data are analysed by one-way ANOVA followed by Bonferroni’s post hoc test. **p<0.01. ****p<0.0001. VC: intralipid, P5: 5 μg/ml of propofol, P10: 10 μg/ml of propofol, Sevo 2: two hours sevoflurane, Sevo 4: four hours sevoflurane and Sevo 6: six hours sevoflurane. d) Reh cells were either untreated or treated with 3.6% sevoflurane for six hours initially. Then they were allowed to adhere to plates pre-coated with thrombin cleaved osteopontin for two hours. Data are illustrated as mean±sd (N=4). **p<0.01. Data are analysed by one-way ANOVA followed by Bonferroni’s post hoc test. d) Reh cells were either untreated or treated with 10 μg/ml of propofol for six hours initially. Then they were allowed to adhere to plates pre-coated with thrombin-cleaved osteopontin for two hours. Data are illustrated as mean±sd (N=4). *p<0.05, **p<0.01. Data are analysed by one-way ANOVA followed by Bonferroni’s post hoc test. VC: intralipid.

Article Snippet: Multiple group comparisons were performed using ANOVA with a Kruskal-Wallis nonparametric or Bonferroni’s post hoc test for comparisons GraphPad Prism 7.0, RRID:SCR_002798.

Techniques: Staining, Flow Cytometry, Fluorescence, Control

Journal: F1000Research

Article Title: General anaesthetics reduce acute lymphoblastic leukaemia malignancies in vitro and in vivo via CXCR4 and osteopontin mediated mechanisms

doi: 10.12688/f1000research.125877.2

Figure Lengend Snippet: a and c) NALM-6 and Reh cells were treated with 10 μg/ml propofol or intralipid and Ara-C (0.05 to 50μM) for 4 hours. Cell viability analysis was carried out by CCK-8. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Data is shown as mean±sd (n=4). Data is analysed by two-way ANOVA followed by Bonferroni’s post-hoc test. VC: Vehicle control, P10: 10μg/ml. b and d) NALM-6 and Reh cells were treated with 3.6% sevoflurane and Ara-C (0.05 to 50μM) for 4 hours. Cell viability analysis was carried out by CCK-8. Data is shown as mean±sd (n=4). *p<0.05, ****p<0.0001. Data is analysed by two-way ANOVA followed by Bonferroni’s post-hoc test. e-f) NALM-6 and Reh cells were treated with 0.1 μM Ara-C, Ara-C plus 10 μg/ml propofol or intralipid and Ara-C plus 3.6% sevoflurane for 4 hours followed by flow cytometry analysis of autophagy influx by Cyto ID autophagy detection kit. Data is illustrated as mean±sd (n=4). *p<0.05, **p<0.01. Data is analysed by one-way ANOVA followed by Bonferroni’s post-hoc test. Grey histogram: Isotype control, Red histogram: Cyto-ID.

Article Snippet: Multiple group comparisons were performed using ANOVA with a Kruskal-Wallis nonparametric or Bonferroni’s post hoc test for comparisons GraphPad Prism 7.0, RRID:SCR_002798.

Techniques: CCK-8 Assay, Control, Flow Cytometry