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Non-myeloablative busulfan chimeric mouse models are less pro-inflammatory than head-shielded irradiation for studying immune cell interactions in brain tumours

Youshani, A. Saam, Rowlston, Samuel, O’Leary, Claire, Forte, Gabriella, Parker, Helen, Liao, Aiyin, Telfer, Brian, Williams, Kaye, Kamaly-Asl, Ian D., Bigger, Brian W.·2019·Journal of Neuroinflammation
DOI 10.1186/s12974-019-1410-yPMC PMC6362590
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Fig. 1 Peripheral chimerism analysis of transplanted mice after different pre-conditioning regimens. a Four groups received different pre-conditioning regimens: (i) non-myeloablative conditioning (NMC) using a single dose of busulfan at 25 mg/kg (n = 6/group), (ii) full conditioning (FC) busulfan 125 mg/kg given over five consecutive days (n = 6/group), (iii) head-shielded irradiation (HIR) at a divided dose of 11 Gy given 3 h apart (n = 6/group) and (iv) untransplanted controls (UnTx) (n = 6/group). b Peripheral blood chimerism at 2 weeks post-transplant, c 4 weeks post-transplant, d 8 weeks post-transplant and e 12 weeks post-transplant. f Bone marrow chimerism measured at 12 weeks post-transplant and g spleen chimerism at 12 weeks. One-way ANOVA with Tukey’s post hoc correction for multiple comparisons was used. Error bars represent the SEM. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns = non-significant
Fig. 1 Peripheral chimerism analysis of transplanted mice after different pre-conditioning regimens. a Four groups received different pre-conditioning regimens: (i) non-myeloablative conditioning (NMC) using a single dose of busulfan at 25 mg/kg (n = 6/group), (ii) full conditioning (FC) busulfan 125 mg/kg given over five consecutive days (n = 6/group), (iii) head-shielded irradiation (HIR) at a divided dose of 11 Gy given 3 h apart (n = 6/group) and (iv) untransplanted controls (UnTx) (n = 6/group). b Peripheral blood chimerism at 2 weeks post-transplant, c 4 weeks post-transplant, d 8 weeks post-transplant and e 12 weeks post-transplant. f Bone marrow chimerism measured at 12 weeks post-transplant and g spleen chimerism at 12 weeks. One-way ANOVA with Tukey’s post hoc correction for multiple comparisons was used. Error bars represent the SEM. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns = non-significantCC BY · PubMed Central
Fig. 2 Gene expression of peripheral blood at 2 weeks and peripheral blood, spleen and brain at 12 weeks. a Gene expression of peripheral blood samples taken at 2 weeks post-transplant and tested for Il1b, Ccl2, Il6 and Tnfa (n = 6/group). b Gene expression of peripheral blood samples taken at 12 weeks post-transplant and tested for Il1b, Ccl2, Il6, and Tnfa (n = 6/group). c Spleen sample gene expression profiles were measured for the cytokines Il1b, Ccl2, Il6, Tnfa and Ifng. d Brain samples without a tumour were analysed for the cytokines Il1b, Ccl2, Il6 and Tnfa. Ccl2, Tnfa and Il6 expression in blood at 2 weeks, Il1b, Ccl2, Tnfa and Il6 expression in the blood at 12 weeks, Il6 expression in the spleen and Ccl2 expression in the brain failed Shapiro-Wilk normality test and were analysed using Kruskal-Wallis test with Dunn’s post hoc correction for multiple comparisons. All other samples were analysed using one-way ANOVA with Tukey’s post hoc correction for multiple comparisons. Error bar represents the SEM. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. Samples with ≥ 5-fold difference in gene expression and unequal variance were log transformed (spleen; Tnfa, brain; Il1b, Ccl2 and Tnfa). Error bar represents the SEM. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05
Fig. 2 Gene expression of peripheral blood at 2 weeks and peripheral blood, spleen and brain at 12 weeks. a Gene expression of peripheral blood samples taken at 2 weeks post-transplant and tested for Il1b, Ccl2, Il6 and Tnfa (n = 6/group). b Gene expression of peripheral blood samples taken at 12 weeks post-transplant and tested for Il1b, Ccl2, Il6, and Tnfa (n = 6/group). c Spleen sample gene expression profiles were measured for the cytokines Il1b, Ccl2, Il6, Tnfa and Ifng. d Brain samples without a tumour were analysed for the cytokines Il1b, Ccl2, Il6 and Tnfa. Ccl2, Tnfa and Il6 expression in blood at 2 weeks, Il1b, Ccl2, Tnfa and Il6 expression in the blood at 12 weeks, Il6 expression in the spleen and Ccl2 expression in the brain failed Shapiro-Wilk normality test and were analysed using Kruskal-Wallis test with Dunn’s post hoc correction for multiple comparisons. All other samples were analysed using one-way ANOVA with Tukey’s post hoc correction for multiple comparisons. Error bar represents the SEM. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. Samples with ≥ 5-fold difference in gene expression and unequal variance were log transformed (spleen; Tnfa, brain; Il1b, Ccl2 and Tnfa). Error bar represents the SEM. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05CC BY · PubMed Central
Fig. 3 Brain immunophenotyping of different transplant groups with and without GBM. Brain immune cells were identified and quantified using flow cytometry. a Schematic diagram demonstrating how mouse brain samples in different groups were processed by brain digestion and flow cytometry with (n = 3/group) or without (n = 3/group) a brain tumour. b Naïve brain chimerism at 12 weeks, measured by calculating total CD45.2 cell engraftment. c Brain chimerism of mice implanted with GBM at 14 weeks, measured by calculating total CD45.2 engraftment. One-way ANOVA analysis with Tukey’s post hoc correction for multiple comparisons was used for all groups. Error bars represent the SEM. ****p < 0.0001; ***p < 0.001; *p < 0.05. d Gating strategy used to identify immune cell populations within the brain. e Immune cell profile, within the brain of different chimeric mice 12 weeks post-transplant (n = 3/group) without GBM. f Tumour-tropic immune cells found within the tumour-bearing hemisphere of different chimeric mice implanted with GBM (n = 3/group). Two-way ANOVA analysis with Tukey’s post hoc correction for multiple comparisons was used throughout. Error bar represents the SEM. **** or ####p < 0.0001; *** or ###p < 0.001; ** or ##p < 0.01; * or #p < 0.05; ns = non-significant. *Differences between the same type of immune cell amongst different transplant groups. #Differences between different immune cells within the same transplant group. One mouse from the NMC group was excluded due to reflux of tumour cells during surgery, thus providing unreliable downstream analysis
Fig. 3 Brain immunophenotyping of different transplant groups with and without GBM. Brain immune cells were identified and quantified using flow cytometry. a Schematic diagram demonstrating how mouse brain samples in different groups were processed by brain digestion and flow cytometry with (n = 3/group) or without (n = 3/group) a brain tumour. b Naïve brain chimerism at 12 weeks, measured by calculating total CD45.2 cell engraftment. c Brain chimerism of mice implanted with GBM at 14 weeks, measured by calculating total CD45.2 engraftment. One-way ANOVA analysis with Tukey’s post hoc correction for multiple comparisons was used for all groups. Error bars represent the SEM. ****p < 0.0001; ***p < 0.001; *p < 0.05. d Gating strategy used to identify immune cell populations within the brain. e Immune cell profile, within the brain of different chimeric mice 12 weeks post-transplant (n = 3/group) without GBM. f Tumour-tropic immune cells found within the tumour-bearing hemisphere of different chimeric mice implanted with GBM (n = 3/group). Two-way ANOVA analysis with Tukey’s post hoc correction for multiple comparisons was used throughout. Error bar represents the SEM. **** or ####p < 0.0001; *** or ###p < 0.001; ** or ##p < 0.01; * or #p < 0.05; ns = non-significant. *Differences between the same type of immune cell amongst different transplant groups. #Differences between different immune cells within the same transplant group. One mouse from the NMC group was excluded due to reflux of tumour cells during surgery, thus providing unreliable downstream analysisCC BY · PubMed Central
Fig. 4 Monocytes, macrophages and microglia subpopulation analysis in the brain prior to and after GBM implantation. a Following the cytometry gating used in Fig. 2d, the remaining myeloid subpopulations of monocytes, macrophages (Mɸ) and microglia were further separated using Ly6C/MHCII/MerTK/CD64 into monocytes, undifferentiated Mɸ and a combination of differentiated Mɸ and microglia. The combined group of differentiated Mɸ and microglia were separated using CD11b/CD45. All brain samples were analysed using flow cytometry (n = 3/group). b Bar graph showing the relative glioma-associated macrophages and microglia (GAMM) subpopulations in tumour-bearing hemisphere of GBM-implanted mice. All samples were analysed using a two-way ANOVA with Tukey’s post hoc correction for multiple comparisons. Error bar represents the SEM. **** or ####p < 0.0001; ***or ###p < 0.001; **or ##p < 0.01. One mouse from the NMC group was excluded due to reflux of tumour cells during surgery, thus providing unreliable downstream analysis
Fig. 4 Monocytes, macrophages and microglia subpopulation analysis in the brain prior to and after GBM implantation. a Following the cytometry gating used in Fig. 2d, the remaining myeloid subpopulations of monocytes, macrophages (Mɸ) and microglia were further separated using Ly6C/MHCII/MerTK/CD64 into monocytes, undifferentiated Mɸ and a combination of differentiated Mɸ and microglia. The combined group of differentiated Mɸ and microglia were separated using CD11b/CD45. All brain samples were analysed using flow cytometry (n = 3/group). b Bar graph showing the relative glioma-associated macrophages and microglia (GAMM) subpopulations in tumour-bearing hemisphere of GBM-implanted mice. All samples were analysed using a two-way ANOVA with Tukey’s post hoc correction for multiple comparisons. Error bar represents the SEM. **** or ####p < 0.0001; ***or ###p < 0.001; **or ##p < 0.01. One mouse from the NMC group was excluded due to reflux of tumour cells during surgery, thus providing unreliable downstream analysisCC BY · PubMed Central

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Metadata

Authors
Youshani, A. S., Rowlston, S., O’Leary, C., Forte, G., Parker, H., Liao, A., Telfer, B., Williams, K., Kamaly-Asl, I. D., & Bigger, B. W.
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2019-02-05
Volume
16
Issue
1
ISSN
1742-2094
DOI
10.1186/s12974-019-1410-y
PMC
PMC6362590
Cited by
16
References
0
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (tdm)
Funders
Royal College of Surgeons of England (Fellowship)

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CoChat. (2026). Citation check: Non-myeloablative busulfan chimeric mouse models are less pro-inflammatory than head-shielded irradiation for studying immune cell interactions in brain tumours. https://app.cochat.ai/papers/10.1186/s12974-019-1410-y
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Youshani, A. S., Rowlston, S., O’Leary, C., Forte, G., Parker, H., Liao, A., Telfer, B., Williams, K., Kamaly-Asl, I. D., & Bigger, B. W. (2019). Non-myeloablative busulfan chimeric mouse models are less pro-inflammatory than head-shielded irradiation for studying immune cell interactions in brain tumours. Journal of Neuroinflammation. https://doi.org/10.1186/s12974-019-1410-y