Brain tumors induce widespread disruption of calvarial bone and alteration of skull marrow immune landscape

Abstract

The skull marrow niche has recently been identified as a reservoir that supplies the brain with monocytes and neutrophils in the context of disease and injury, but its role in brain cancers remains unknown. Here we show that glioblastoma, the most malignant type of brain tumor, induces calvarial bone abnormalities in murine models and patients with glioblastoma, altering osteoclast activities and increasing the number of skull channels in mice. Single-cell RNA sequencing revealed glioblastoma-mediated alterations in the immune landscape of skull marrow and femoral bone marrow, including expansion of neutrophils and deterioration of various B cell subsets. In vivo inhibition of bone resorption reduced bone abnormalities, but promoted tumor progression in mesenchymal subtype tumors. This also abolished the survival benefit of the checkpoint inhibitor anti-PD-L1, by reducing activated T cell and increasing inflammatory neutrophil numbers. Together, these data provide insight into how brain tumors affect skull bone and the immune environment.

Fig. 1. GBM induces calvarial bone abnormality and OC activity in several anatomical regions of the calvarium delineating osteogenic edges adjacent to the skull sutures.

a, microCT imaging showed that GBM induced bone erosion in the skull mainly at the osteogenic edges adjacent to the skull sutures. Tumor was sterotactically injected in frontal lobe, and the occipital bone, far from tumor site, showed the highest erosion. b, Established criteria to measure skull density and thickness at defined anatomical regions indicated by arrows (bregma, lambda, mid-occipital, sub-occipital). c, SB28 tumor had calcification within the tumor as visualized by microCT. d, Statistical analysis of bone density showed significant reduction in bregma and lambda at early-stage/half-time tumor as compared with sham. SB28 had higher erosion extent than GL261. e, Statistical analysis of bone thickness showed significant reduction of bone thickness in lambda, bregma and sub-occipital in SB28 tumors and to lesser extent in GL261 at early-stage/half-time tumor. f, Statistical analysis of bone density showing significant reduction of bone density in bregma, lambda and sub-occipital in last-stage tumor. g, Statistical analysis of bone thickness showing significant reduction of bone density in lambda and sub-occipital in both SB28 and GL261, in addition to SB28 mid-occipital. Number of animals in statistics: in d,e, early-stage/half-time (4 mice sham; 9 mice GL261; 7 mice SB28); in f,g, late-stage tumor (6 mice sham; 6 mice GL261; 8 mice SB28). Data are presented means ± s.e.m. In d–f, significance was assessed by a one-way ANOVA with Tukey’s multiple comparison test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). See Supplementary Fig. 2. Scale bars: 1,000 µm. HU, Hounsfield unit. Source data

Fig. 2. GBM induces specific bone morphometry changes in the calvarium and increases skull channel openings.

a, High-resolution microCT (9 µm) of dissected skull from two GBM models (GL261 and SB28), during early-stage (day 8, day 15) and late-stage tumor progression. Aged mice (78–104 weeks) were used for late-stage only. Occipital bone distant from tumor site was visualized with skull channels highlighted and used for bone morphometry analysis. b–e, Statistical analysis of bone morphometry showing significant reduction in BV and Tb.Th in GL261 at days 8 and 15, and in late-stage SB28. Number of animals in statistics: b, day 8 (5 mice sham; 4 mice GL261; 6 mice SB28); c, day 15 (5 mice sham; 4 mice GL261; 4 mice SB28); d, late-stage tumor (5 mice sham; 4 mice GL261; 5 mice SB28); e, aged late-stage tumor (3 mice sham; 3 mice GL261; 3 mice SB28). Data are presented means ± s.e.m. In b–e, significance was assessed by a one-way ANOVA with Tukey’s multiple comparison test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). f, Statistical analysis quantification of the skull channel diameters at early-stage (day 8 and 15) and late-stage tumor. Three animals per cohort (except GL261 day 15) were used for each of the experimental groups (day 8, day 15 and late-stage). Data are presented means ± s.e.m. One-way ANOVA with Tukey’s multiple comparison test (*P < 0.05; **P < 0.01). g, Statistical analysis of skull channel counts showing significant increase for early-stage and late-stage tumors, GL261 and SB28. Three animals per experimental group (except GL261 day 15 were 4 animals). Data are presented means ± s.e.m. One-way ANOVA with Tukey’s multiple comparison test (*P < 0.05; **P < 0.01). Scale bars: 500 µm. NS, not significant. Source data

Fig. 3. Patients with GBM showed a reduced skull thickness compared with age- and gender-matched controls.

CT of patients with GBM and control patients without any tumor history (majority were stroke, subdural and subarachnoid hemorrhage, hematoma and epilepsy) were used for the analysis. a, Representative 3D visualization of the five anatomic areas that were analyzed for bone thickness and density. b, Sagittal CT section pointing to anatomic areas used in the analysis. c, Reduction in skull thickness was highly significant in lambda and mid-occipital, followed by crossing point of midorbital axes and bregma. d, The change in bone density was not significant. In c and d, sample size was 26 patients with GBM and 22 age- and gender-matched controls. Data are presented as means ± s.e.m. Significance was assessed by Mann–Whitney U test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). e, Linear fit model for tumor volume (cm³) and bone thickness on average measurements of bregma thickness, bregma to midorbital left side, bregma to midorbital right side, lambda and mid-occipital (3 measurements in mm). See also Supplementary Fig. 3. Student’s t-test was used for analyzing volume against bone thickness. A P value < 0.05 was set as statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001). Scale bars: 10 mm. Source data

Fig. 4. GBM induces dynamic changes in skull OC number during tumor progression and disturbs OC cluster formation and distribution in the skull.

a, A representative light sheet imaging of SB28 showing segmented OC volume during tumor progression (day 8, day 15 and late-stage tumor). Red signal, OCs expressing TRAP-tdTomato; green, tumor cells. Scale bar: 1,000 µm. b, A representative maximum intensity projection of intravital multiphoton microscopic tiling of skull in TRAP-tdTomato transgenic mice. The skull of the same contralateral side was imaged in sham animal, GL261 and SB28 tumor. Red signal, OCs expressing TRAP-tdTomato; blue, bone tissues; green, tumor cells (crossed to contralateral hemisphere in SB28). Scale bar: 400 µm. Experiment was repeated four times. c, Distance between neighboring OCs was increased in both GL261 and SB28, and to a higher extent in SB28 than GL261 suggesting major loss of OC number in SB28. The distance between every pair of segmented OCs was calculated and the distribution was plotted. The OCs were divided into three groups according to their size: 900–3,000 µm³, small cells; 3,000–2,0000 µm³, big cells; >20,000 µm³, cell cluster. d, Representative images of segmented OCs in the skull classified according to their size. The OCs were divided into three groups according to their size: 900–3,000 µm³, small cells; 3,000–20,000 µm³, big cells; >20,000 µm³, cell cluster. e, GL261 tumors induce a decrease in skull OC numbers at days 8 and 15, and a resurgence in late-stage tumor. A longitudinal quantification in GL261 skull showed that the OC numbers declined significantly at early-stage (day 8) and half-time (day 15) of tumor progression compared with sham (P < 0.0001), then rebounded and increased in numbers significantly in late-stage tumor compared with days 8 and 15 (P < 0.0001), but still less than sham control (P < 0.05). OCs were calculated as areas of OCs per imaging area (mean = 3.72 sham; 2.737 GL261 day 8; 2.503 GL261 day 15; 3.102 GL261 late-stage). f, Quantification of segmented cells in GL261 skull according to their size showed that all three types were significantly decreased at days 8 and 15, compared with sham skull, then rebounded and increased in number during the late-stage of tumor progression to be higher than day 15 in all three subsets (P < 0.0001 for small and big size cells, P < 0.05 for cell cluster), but it was significant only for small size cells compared with sham skull. g, SB28 tumors induce a decrease in skull OC numbers during tumor progression, with a small resurgence at day 15. A longitudinal quantification in SB28 skull showed that the OC numbers declined significantly at early-stage (day 8) and half-time (day 15) of tumor progression compared with sham (P < 0.0001). All three types of tdTomato⁺ cells were significantly decreased in number compared with sham skull (mean = 3.720 sham; 0.6348 SB28 day 8; 0.5491 SB28 day 15; 0.5068 SB28 late-stage). h, Quantification of segmented cells in SB28 skull according to their size showed that all three types were significantly decreased at days 8 and 15 and late-stage compared with sham skull. A resurgence was recorded in the big size and cell cluster by day 15, but all three types of tdTomato⁺ cells were significantly decreased in number compared with sham controls (P < 0.0001). Multiphoton images used in c–h were collected from four times repeated experiments, 12 tiling images from: day 8, 3 mice GL261; 3 mice SB28; day 15, 3 mice GL261; 3 mice SB28; late-stage, 6 mice GL261; 4 mice SB28; and 3 mice sham. Data are presented as means ± s.e.m. Significance was assessed using a one-way ANOVA with Tukey’s multiple comparison test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). See also Supplementary Fig. 6. SHG, second harmonic generation. Source data

Fig. 5. Global disturbance in myeloid lineage of the SM in response to GBM shown by scRNA-seq and confirmed by FACS.

a,b, 2D UMAPs and stacked bar charts visualizing the main myeloid cell populations within CD45⁺ cells in the SM and BM of sham control compared with GL261 and SB28 derived from scRNA-seq. a, In homeostatic SM, neutrophil subsets made up the largest myeloid populations, with Mature Neutro. and Transit. Neutro. 1 being the majority, and they almost doubled in response to GBM, whereas Pro. Neutro. increased twice to reach 8% in SB28 and four times in GL261 (16%). Prol. Monocytes, macrophages, Acp5⁺ macrophage, MEPs and GMPs made up a small subset of 0.2–4% which also increased in response to GBM, except for macrophages in SB28 SM, and DCs stayed almost the same (~2%) in SM. b, In sham BM, similar to SM, neutrophil subsets made the largest cell population, but with some differences including: Pre. Neutro. was the largest population in sham BM, slightly increased in GL261 BM and decreased in SB28 BM, whereas Mature Neutro increased three times, and Transit. Neutro. 1 doubled in GL261, but was only slightly elevated in SB28 BM. MEPs and GMPs as well ST-HSCs and Hemato. Prog. in BM were reduced in response to GBM, opposite to their population in SM, whereas Erythroid. Prog. increased in BM and decreased in SM. c, A dot plot depicting normalized expression of different marker genes used to annotate individual cell clusters within the myeloid lineage. Gene markers are shown on the x axis and cell types on the y axis. d, Flow cytometry analysis confirming that GBM alters the SM and BM homeostasis, pushing it towards myelopoiesis. Spectral flow cytometric analysis of CD45⁺ population in sham SM, GL261 SM and SB28 SM (top) as well as in sham BM, GL261 BM and SB28 BM (bottom). FACS analysis further recapitulated and validated the increase in percentage of neutrophils in the SM and the concurrent decrease in percentage of the lymphoid compartment. Lin., lineage. Source data

Fig. 6. GBM induces distinct DEGs and pathways across myeloid lineages in SM and BM.

a, Venn diagrams showing DEGs and differential pathways induced by two GBM models, GL261 and SB28, in SM as well as BM compared with sham SM and sham BM, respectively. GL261 and SB28 induced common as well as tumor-specific DEGs and pathways in both SM and BM. pyDESEQ2 was used to identify DEGs in all cell clusters. Venn diagram depicts DEGs with more than 1.2-fold change in upregulation/downregulation. For pathway enrichment, GSEA was used to identify enriched GO pathways and only pathways having FDR Q value less than 0.25 were retained for analysis. b, Stacked bar charts showing significantly upregulated and downregulated pathways induced in each cell type by both GL261 and SB28 in SM (left) and BM (right). There was overall upregulation of pathways in SM while BM had downregulation across most cell types. Prol. Monocytes in SM observed most downregulated pathways while Transit. Neutro. 2 in BM observed most upregulated pathways. c, A heatmap depicting NES of select pathways that were downregulated or upregulated across different cell types in SM in response to both GL261 and SB28. Average NES combined from GL261 and SB28 is plotted. d, A heatmap depicting NES of select pathways that were downregulated or upregulated across different cell types in BM in response to both GL261 and SB28. Average NES combined from GL261 and SB28 is plotted. e, 3D PCA plot showing pseudo bulk transcriptomic profile of different samples. Each sample was randomly split into three pseudo replicates and raw counts were summed for each gene to generate bulk RNA profiles with three pseudo replicates for each of the samples. pyDESEQ2 was used to analyze these bulk RNA profiles and normalized counts were then used to perform PCA. Subsequently, the first three principal components were plotted in 3D space. f, Venn diagrams showing DEGs and differential pathways between SM and BM in sham, GL261 and SB28. Sham SM had a significant number of DEGs as well as differential pathways compared with sham BM which remained differential even in the presence of tumor. SB28 induced more DEGs as well as differential pathways compared with GL261. pyDESEQ2 was used to identify DEGs in all cell clusters. Venn diagram depicts DEGs with more than 1.2-fold change in upregulation or downregulation. For pathway enrichment, GSEA was used to identify enriched GO pathways and only pathways having FDR Q value less than 0.25 were retained for analysis. g, A heatmap depicting NES of select pathways that were downregulated or upregulated across different cell types in SM versus BM. NESs for both GL261 and SB28 are plotted unless a pathway is specific only to GL261 or SB28. Gray boxes depict nonenrichment of a pathway in a given cell type and condition. NES, normalized enrichment score.

Fig. 7. scRNA-seq and flow cytometry analysis shows a dramatic change in the lymphoid lineage within the SM and BM in response to GBM.

a, 2D UMAPs and stacked bar charts depicting different cell populations and their percentages within the lymphoid subset present in sham SM compared with GL261 and SB28 SM. b, 2D UMAPs and stacked bar charts of corresponding BM derived from scRNA-seq. a,b, In homeostatic SM, B cells were the dominant population within the lymphoid subset, with some differences between SM and BM including higher Pre-B. cells in SM than BM, while this last one had higher Late Pro. B cells which almost disappeared in response to tumor. Early developmental B cell subsets were the most decreased cell types in both SM and BM. While immature B cells were much higher in SM (biggest B cell population) versus BM and reduced almost by 50% in SM and not affect in BM or increased in case of GL261 tumor. Mature B cells also seemed to expand in BM of both tumors while decreasing in SM, and NK cells, NKT cells and T cells were also present in small frequencies. In response to both tumors, T cells, regulatory T cells (T_reg cells) and NKT cells increased in the SM while the percentages of NK cells, plasma B cells and NKT cells were increased only in SB28. c, A dot plot summarizing the expression of marker genes utilized to annotate the lymphoid clusters. d, Flow cytometry analysis confirming that GBM decreases SM and BM lymphopoiesis tremendously. Spectral flow cytometric analysis of lymphoid populations in sham SM, compared with GL261 and SB28 SM (top), as well as in sham BM, GL261 BM and SB28 BM (bottom). Flow cytometry analysis of the lymphoid subset validated the decrease in percentage of B cells and the increase in percentage of CD4 and CD8 T cells, in addition to NK/NKT cells in the SM in SB28 and GL261. To simplify the analysis, all six B cell subsets were merged as one cluster (B cells). e, Venn diagrams showing DEGs and differential pathways induced by two GBM models, GL261 and SB28, in SM as well as BM compared with sham SM and sham BM, respectively, across the lymphoid lineage. GL261 and SB28 induced common as well as tumor-specific DEGs and pathways in both SM and BM. pyDESEQ2 was used to identify DEGs in all cell clusters. Venn diagram depicts DEGs with more than 1.2-fold change in upregulation/downregulation. For pathway enrichment, GSEA was used to identify enriched GO pathways and only pathways having FDR Q value less than 0.25 were retained for analysis. f, Stacked bar charts showing significantly upregulated and downregulated pathways induced in different lymphoid lineage cell types by both GL261 and SB28 in SM (left) and BM (right). There was overall upregulation of pathways in SM while BM had downregulation across most cell types. All the B cells subsets were affected in both SM and BM in response to the tumor. g, A heatmap depicting NES of select pathways that were downregulated or upregulated across different cell types in SM (left) or BM (right) in response to both GL261 and SB28. Average NES combined from GL261 and SB28 is plotted. DN T cells, double negative T cells; ILCs, innate lymphoid cells. Source data

Fig. 8. OC inhibition enhances tumor progression and reduces the efficacy of checkpoint inhibitor in mesenchymal GBM mouse model.

a, Schematic drawing of in vivo inhibition of OC-like macrophages. Treatment started at day 9 (SB28) or day 10 (GL261) after stereotactic tumor injection, three times a week, and microCT was done for the late-stage tumor. b, Kaplan-Meier survival in SB28 tumor-bearing mice. Treatment started at day 9 with 3 mg kg⁻¹ s.c. injection of Zol three times a week, and treatment lasted 2 weeks. Zol treatment enhanced tumor progression significantly (10 mice Zol group, 5 control, experiment repeated twice). c, Anti-RANKL antibody s.c. injection (5 mg kg⁻¹) abolished the survival benefit of anti-PD.L1 treatment (200 µg per mouse) in SB28 tumor (5 control, 7 aPD-L1, 6 aRANKL, 8 aPD-L1 + aRANKL). P value 0.0113 calculated using log-rank (Mantel–Cox) test. d, Coronal and transaxial views of microCT imaging showing the efficacy of Zol in inhibiting skull bone resorption in last-stage SB28 tumor-bearing mice. aRANKL did not stop bone resorption, and neither combination did, opposite to aPD-L1-treated mice. microCT was performed on the same day. e, Flow cytometry analysis of SB28 tumor showing the effect of combining aPD-L1 with Zol or aRANKL. PD-L1 single treatment increased the number of proliferative T cells (KI67⁺) and functional T cells expressing IFNγ, GzmB and TNF significantly in the tumor, but upon combining it with OC inhibitors (Zol or aRANKL), this abolished the significant difference. f, Flow cytometry analysis of SB28 tumor showing that Zol or aRANKL treatment increased inflammatory neutrophils in the tumor. These neutrophils increased the expression of PD-L1, Tim3 and TNF significantly, while aPD-L1 treatment abolished them from the tumor, and they started to show again upon combining Zol or aRANKL with aPD-L1. For e and f, 5–10 animals per group (10 mice control, 5 Zol, 5 PD-L1, 7 PD-L1 + Zol, 8 PD-L1 + aRANKL, 5 aRANKL) were used for analysis. Data are presented as means ± s.e.m. Significance was assessed using a one-way ANOVA with Tukey’s multiple comparison test (*P < 0.05; **P < 0.01; ***P < 0.001).

Extended Data Fig. 1. SB28 exhibits Mes subtype signature while GL261 expressed hybrid signature of Mes and PN.

(a and b) RNA Expression profiles of GL261 and SB28, respectively. Molecular subtype of GL261 and SB28 were discerned based on a panel of 12 genes according to Behnan et al. (39).

Extended Data Fig. 2. Additional representative microCT images of GBM induced bone resorption in early-and late-stage glioma at the osteogenic edges adjacent to the skull sutures.

The bone erosion existed in other non-glioma origin with different pattern and was absent in subcutaneous (sc) glioma models. (a) microCT images of early-stage/half-time brain tumors, GL261 and SB28, bearing mice compared to age-matched sham control showing bone erosion in GBM models. (b) microCT image of additional animals bearing late-stage brain tumors, GL261 and SB28, compared to age-matched sham control showing expanded bone erosion in last stage tumor. (c) microCT images of sc-glioma models, GL261-sc and SB28-sc, compared to intracranial (ic) glioma of the same cell line, GL261-ic and SB28-ic. In addition, i.c injected breast cancer cell line, 4T1-ic and (mouse embryonic fibroblasts) MEF-ic as representative of non-glioma origin tumor.

Extended Data Fig. 3. GBM does not induce bone erosion in the fumers.

(a) MicroCT image of fumer from mice bearing brain tumors, GL261 and SB28, compared to age-matched sham control. (b–d) Statistical analysis of bone morphometry showed no significant reduction in BV and Tb.Th in femer. Number of animals in statistics: in (b), Day 8 (6 mice/sham; 5 mice/GL261; 5 mice/SB28); in (c), Day 15 (6 mice/sham; 4 mice/GL261; 6 mice/SB28); in (d), Late-stage tumor (6 mice/sham; 3 mice/GL261; 7 mice/SB28). Data are presented means ± SEM. In (b–d), significance was assessed by a one-way ANOVA with Tukey’s multiple comparison test (ns, no-significant; *, p < 0.05; **, p < 0.01; *** p < 0.001; ****, p < 0.0001). Source data

Extended Data Fig. 4. Bone Thickness across gender and ages in control and GBM patients.

(a) Bone thickness at defined anatomical locations classified according to gender in control dataset (Top) or GBM dataset (Bottom). Female subjects in control dataset had higher bone thickness at Mid-occipital compared to Males, while it was not significant at other locations. In the GBM dataset, female subjects had higher bone thickness only at intersection of Right-eye midorbital axis with coronal suture. No significant difference was observed at other anatomical locations, including Mid-Occipital. (b) Scatter plot of bone thickness and age in control dataset (Top) or GBM dataset (Bottom). No significant correlation was observed between bone thickness and age in both control and GBM datasets. Sample size was 11 female- and 11 male- GBM patients, 10 female- and 12 male- control, 3 measurements for each anatomic point. Mann-Whitney U Test was used to calculate p-values for bone thickness differences between Females and Males. Spearman correlation was calculated for bone thickness vs age correlations. (*, p < 0.05; **, p < 0.01; *** p < 0.001). Four GBM patients were missing age and gender information and were excluded from this analysis. Source data

Extended Data Fig. 5. Human GBM CT bone thickness according to tumor location showing no impact of tumor location on skull thickness except Lmbda point upon having tumor in left frontal region.

Patients with tumor in Left Frontal region had more bone thickness compared to those in Right Frontal region. 12 patients had MRI and CT. Data are presented as means ± SEM. Significance was assessed a one-way ANOVA with Tukey’s multiple comparison test (*, p < 0.05). Source data

Extended Data Fig. 6. GBM reduce the number of OCs in the skull during tumor progression in GL261 and SB28 glioma models.

(a) A representative Maximum Intensity Projection of Light Sheet microscopic of skull in TRAP-tdTomato transgenic mice. Skulls from SB28 and GL261 at Day-8, -15, late-stage were imaged, and sham animals without tumor were used as control. Red signal, OCs expressing TRAP-tdTomato; green, Autofluorescent. Scale bar, 1000 µm. (b) Representative images of segmented OCs in the skull (Surrounded by dotted line). OCs expressing TRAP-tdTomato; white, Segmentaed OC surfaces. Scale bar, 1000 µm. GL261 and SB281 tumors induce a decrease in total volume of skull OCs at Day-8 and -15, and a resurgence in late-stage tumor.

Extended Data Fig. 7. Flow cytometry analysis showing functional characteristics of Macrophages and DCs in SM and BM of SB28 tumor bearing mice.

(a, b) Percentage of total BM macrophages (c, d) Percentage of total SM macrophages out of total CD45+ cells in SB28 and their expression of MHC-II and IL10 showing higher expression of MHC-II and IL10 in SB28 compared to sham control. (e, f) Percentage of total BM DCs (g, h) Percentage of total SM DCs out of CD45+ cells showing no difference in MHC II expression in both BM and SM DCs, while IL10 was significant higher in SM derived DCs, and not significant in BM. Number of animals in statistics: 4 mice/sham; 5 mice/SB28. Data are presented as means ± SEM. Significance was assessed as Mann-Whitney U test (*, p < 0.05; **, p < 0.01; *** p < 0.001; ****, p < 0.0001). Source data

Extended Data Fig. 8. Flow cytometry analysis showing functional characteristics of macrophages and DCs in SM and BM of GL261 tumor bearing mice.

(a) Percentage of total BM macrophages (b) Percentage of total SM macrophages in GL261 and their expression of MHC-II, IL10, IL17, PDL1 and proliferation marker KI67 showing only MHCII was higher in both GL261 BM and SM macrophages compared to sham control. (c) Percentage of total BM DCs (d) Percentage of total SM DCs showing significant increase in MHCII and PDL1 expression in DCs from GL261 BM, while SM DCs increased IL10 expression. Number of animals in statistics: in (a), 5 mice/sham; 6 mice/GL261; in (b), 8 mice/sham; 4 mice/GL261; in (c), 7 mice/sham; 6 mice/GL261; in (d), 8 mice/sham; 4 mice/GL261. Data are presented means ± SEM. Significance was assessed as Mann-Whitney U test (*, p < 0.05; **, p < 0.01; *** p < 0.001). Source data

Extended Data Fig. 9. Flow cytometry analysis showing functional characteristics of Neutrophils and Monocytes in GL261 and SB28.

(a–c) Percentage of total BM and SM Neutrophils out of CD45 showing only SM Neutrophils have higher proliferation marker in response to GL261 (b–d) Percentage of total BM and SM Monocytes out of total CD45+ cells showing higher expression of KI67 in both BM and SM in response to GL261. and their expression of MHC-II and IL10 showing higher expression of MHC-II and IL10 in SB28 compared to sham control. (e) Percentage of total BM Neutrophils (f) Percentage of total SM Neutrophils in response to SB28 tumor showing significant increase in SM neutrophils frequency, not BM neutrophils, but its increased expression of KI67 was not significant no significant difference in Ki67. PDL1 expression was equal in BM of SB28 vs Sham and SM of SB28 vs sham, but SM seems to express higher PDL1 level. Also, Monocytes from SM, not BM and DCs showed higher KI67 expression. Number of animals in statistics: in (a), 5 mice/sham; 6 mice/GL261; in (b), 7 mice/sham; 6 mice/GL261; in (c), 7 mice/sham; 4 mice/GL261; in (d), 7 mice/sham; 4 mice/GL261; in (e), 5 mice/sham; 8 mice/GL261; in (f), 7 mice/sham; 7 mice/GL261. Data are presented as means ± SEM. Significance was assessed as Mann-Whitney U test (*, p < 0.05; **, p < 0.01). Source data