Leukaemia exposure alters the transcriptional profile and function of BCR::ABL1 negative macrophages in the bone marrow niche

Abstract

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages.

Fig. 1. CML results in an increase in Ph⁻ progenitor cells and myeloid skew at the expense of lymphocytes.

a Schematic outline of experimental design to generate CML chimeric mice. 7.5 × 10⁵ bone marrow (BM) cells from either CD45.2 SCLtTA⁺/BCR::ABL1⁻ (Control) or SCLtTA⁺/BCR::ABL1⁺ (CML) mice mixed with 7.5 × 10⁵ CD45.1 BM cells from wild-type (WT) mice was transplanted into WT mice. CML-exposed cells refer to CD45.1 WT BM cells exposed to CD45.2 SCLtTA⁺/BCR::ABL1⁺ (CML) BM cells. CML-exposed cells have been compared to CD45.1 WT BM cells exposed to CD45.2 SCLtTA⁺/BCR::ABL1⁻ (Control). Created with BioRender.com (Agreement number: XA268HUG33). b Quantification of myeloproliferation in BM (b) and blood (c) at experimental endpoint (n = 4 mice per experimental arm) d–i Flow cytometry analysis of CD45.1 and CD45.2 arms of BM of chimeric mice after 15 days off TET. Percentage of CD19⁺ B cells (n = 3 mice for CD45.2⁺CD19⁺ control, n = 4 mice for CD45.2⁺CD19⁺ CML, CD45.1⁺CD19⁺ control and CD45.1⁺CD19⁺ CML exposed) (d), CD8⁺ T cells (n = 4 mice per experimental arm) (e), CD4⁺ T cells (n = 3 mice for CD45.2⁺CD4⁺ control, n = 4 mice for CD45.2⁺CD4⁺ CML, CD45.1⁺CD4⁺ control and CD45.1⁺CD4⁺ CML exposed) (f), CD11b⁺Ly6G⁺ cells (n = 3 mice for CD45.2⁺CD11b⁺Ly6G⁺ control, n = 4 mice for CD45.2⁺CD11b⁺Ly6G⁺ CML, CD45.1⁺CD11b⁺Ly6G⁺ control and CD45.1⁺ CD11b⁺Ly6G⁺ CML exposed) (g), CD11b⁺F4/80⁺ cells (n = 3 mice for CD45.2⁺CD11b⁺F4/80⁺ control, n = 4 mice for CD45.2⁺CD11b⁺F4/80⁺ CML, CD45.1⁺CD11b⁺F4/80⁺ control and CD45.1⁺CD11b⁺F4/80⁺ CML exposed) (h), and CD11b⁺Ly6G⁻F4/80⁻ cells (n = 3 mice for CD45.2⁺CD11b⁺Ly6G⁻F4/80⁻ control, n = 4 mice for CD45.2⁺CD11b⁺Ly6G⁻F4/80⁻ CML, CD45.1⁺CD11b⁺Ly6G⁻F4/80⁻ control and CD45.1⁺CD11b⁺Ly6G⁻F4/80⁻ CML exposed) (i). Data are shown as the mean ± s.e.m. P-values were calculated using unpaired two-tailed t-test (b–i). Source data are provided as a Source Data file.

Fig. 2. Macrophage depletion promotes leukaemogenesis.

a Schematic overview of macrophage depletion following CSF1R blockage in CML chimeric mice. Mice were treated with anti-CSF1R antibody or IgG2a isotope control one day prior to tetracycline withdrawal. Anti-CSF1R was then administered 3 times per week. Schematic created with BioRender.com (Agreement number: FA268HUUTO). b Percentage of CD11b⁺F4/80⁺ cells in CD45.1 or CD45.2 BM following treatment with anti-CSF1R (n = 3 mice) or IgG2a isotope control (n = 4 mice). c–e Overall survival (c) and myeloproliferation in blood (d) and BM (e) at experimental endpoint following treatment with anti-CSF1R (n = 5 mice) or IgG2a isotope control (n = 4 mice). The control (CTR) experimental arm (n = 3 mice) refers to irradiated control mice. Data are shown as the mean ± s.e.m. P-values were calculated using unpaired two-tailed t-test (b, d, e), or log-rank (Mantel–Cox) test for survival analysis (c). Source data are provided as a Source Data file.

Fig. 3. CML-exposed macrophages have altered gene expression and form unique subpopulations.

a, b Schematic outline of experimental design (a) and heatmap (b) of Fluidigm gene expression analysis of CD11b⁺F4/80⁺ macrophages sorted from CD45.1 (Control/CML exposed) BM (n = 7 mice for CD11b⁺F4/80⁺ macrophages exposed to control; n = 4 mice for CD11b⁺F4/80⁺ CML-exposed macrophages). Schematic created with BioRender.com (Agreement number: MH268HV0JI). c t-Distributed Stochastic Neighbour Embedding (t-SNE) visualisation of CD45.1⁺ CD11b⁺F4/80⁺ cells from control and CML BM scRNA-seq. d Unsupervised clustering of top 20 marker gene expression heatmap. e Volcano plot of differentially expressed genes in cluster 1 (C1) compared to cluster 2 (C2) and 6 (C6). Upregulated genes with a C1 versus C2 and C6 log2 (fold change) of at least 0.7 are shown in red, while downregulated gene with a C1 versus C2 and C6 log2 (fold change) of at least −0.7 are shown in blue. Differential expression (DE) testing was carried out using the default wrapper function present inside the scran package in R (DeSeq2), which combines pairwise two-tailed t-test into a single rank list markers. The Benjamini–Hochberg method was used to correct for multiple comparisons (e). Source data are provided as a Source Data file.

Fig. 4. CML exposure suppresses clearance of apoptotic cells through downregulation of CD36.

a CTV⁺CD11b⁺F4/80⁺ flow cytometry analysis following Control/CML CTV⁺ c-Kit⁺ cells co-culture with BMDM for 48 h (n = 4 independent experiments). b Percentage of CTV/CD11b^high BMDM conditioned with medium from control (CM) or CML c-Kit+ enriched BM (LCM) for 24 h in the presence of CML apoptotic cells for the final two hours (n = 3 independent experiments). c Quantification of phagocytosis of latex beads measured as percentage of PE^high in BMDM following exposure to CM or LCM for 24 h (n = 4 independent experiments). d Surface marker expression of CD36 in BMDM measured as mean fluorescent intensity (MFI) following exposure to CM or LCM (n = 3 independent experiments). e Quantification of efferocytosis in BMDM conditioned with CM, CM supplemented with 50 μM sulfo-N-succinimidyl oleate (SSO), or LCM for 24 h (n = 4 independent experiments). f Quantification of phagocytosis of latex beads in BMDM following exposure to CM in the presence or absence of 50 μM SSO or LCM for 24 h (n = 4 independent experiments). g–i Surface marker expression of CD86 (g), CD301 (h) and CD206 (i) in BMDM exposed to CM with or without 50 μM SSO addition or LCM for 24 h (n = 3 independent experiments). Data are presented as the mean ± s.e.m. P-values were calculated using unpaired two-tailed t-test (a–d), or ordinary one-way ANOVA with Dunnet’s multiple comparisons test (e–i). Source data are provided as a Source Data file.

Fig. 5. CML stem/progenitor cells have a dysregulated protein secretome.

a, b Schematic diagram of BM LT-HSC RNA sequencing experimental setup and gene ontogeny (GO) enrichment analysis of significant differentially expressed genes (FDR < 0.05) in CML LT-HSCs (n = 4 mice per experimental arm). Schematic created with BioRender.com (Agreement number: QN268HV791). c Schematic diagram of c-kit isolation and conditioned medium generation for secretory proteomics (MS). created with BioRender.com (Agreement number: HF268HUP38). d Volcano plot representing log₂ fold change between CML and WT mice against log₂ p-value for secreted proteins (N = 3 mice per experimental arm). e Log₂ fold change (CML/Control) of significantly changed proteins (FDR < 0.05) in CML vs WT. f Lactotransferrin (LTF) ELISA in c-KIT⁺ conditioned medium (48 h) from SCLtTA⁺/BCR-ABL⁻ (n = 9 conditioned medium samples) or SCLtTA⁺/BCR-ABL⁺ (n = 7 conditioned medium samples). Data are presented as the mean ± s.e.m. P-values were calculated using DESeq2 pairwise two-tailed t-test (fold change < −1 or > 1; p < 0.05) and the Benjamini–Hochberg method to correct for multiple comparisons (b), unpaired two-tailed t-test (f), and paired two-tailed t-test (e). Source data are provided as a Source Data file.

Fig. 6. LTF exposure suppresses clearance function and CD36 expression in mouse macrophages.

a Quantification of uptake of apoptotic cells in BMDM conditioned with CM, CM in the presence of 50 μg/mL LTF, or LCM for 24 h (n = 3 independent experiments). b Quantification of phagocytosis of latex beads in BMDM following culture with CM in the presence or absence of 50 μg/mL LTF or LCM for 24 h (n = 4 independent experiments). c–f Surface marker expression of CD86 (c), CD301 (d), CD206 (e) and MHC II (f) in CM-exposed BMDM with or without 50 μg/mL LTF or LCM exposed BMDM following 24 h incubation (n = 3 independent experiments). g Relative log fold change in mRNA expression levels in BMDM exposed to CM with or without the addition of 50 μg/mL LTF for 24 h (n = 4 independent experiments). h Surface marker expression of CD36 in BMDM following exposure to CM in the presence or absence of 50 μg/mL LTF for 24 h (n = 3 independent experiments). Data are presented as the mean ± s.e.m. P-values were calculated using ordinary one-way ANOVA with Dunnet’s multiple comparisons test (a–f), and unpaired two-tailed t-test (g, h). Source data are provided as a Source Data file.

Fig. 7. LTF exposure alters the clearance function of human macrophages.

a Quantification of clearance of apoptotic cells in human BMDM exposed to CM, CM in the presence of 50 μM SSO or 50 μg/mL LTF, or LCM for 24 h (n = 4 independent experiments). b Percentage of CTV/CD11b^high THP-1-derived macrophages following a 24 h exposure to 50 μM SSO, 50 μg/mL LTF, or a combination of both (n = 6 independent experiments). c Quantification of phagocytosis of latex beads in THP-1 macrophages treated with 50 μM SSO, 50 μg/mL LTF, or a combination of both (n = 5 independent experiments). d Surface marker expression of CD36 in CM or LCM exposed THP-1-derived macrophages for 24 h (n = 4 independent experiments). e Expression of CD36 in untreated (UT) THP-1 macrophages or exposed to 50 μg/mL LTF (n = 4 independent experiments). f Percentage of CTV/CD11b^high THP-1 control (CTR) or CD36 KO macrophages treated with 50 μM SSO or 50 μg/mL LTF for 24 h (n = 4 independent experiments). g Expression of CD11b in THP-1-derived macrophages treated with 50 μM SSO or 50 μg/mL LTF, or a combination of both for 24 h (n = 4 independent experiments). h Quantification of CD11b MFI in THP-1 CTR or CD36 KO macrophages incubated with 50 μM SSO or 50 μg/mL LTF for 24 h (n = 4 independent experiments). Data are shown as the mean ± s.e.m. P-values were calculated using ordinary one-way ANOVA with Dunnet’s multiple comparisons test (a–c, f–h), and unpaired two-tailed t-test (d, e). Source data are provided as a Source Data file.