Myeloid-Derived Suppressor Cell Subsets Drive Glioblastoma Growth in a Sex-Specific Manner

Abstract

Myeloid-derived suppressor cells (MDSC) that block antitumor immunity are elevated in glioblastoma (GBM) patient blood and tumors. However, the distinct contributions of monocytic (mMDSC) versus granulocytic (gMDSC) subsets have yet to be determined. In mouse models of GBM, we observed that mMDSCs were enriched in the male tumors, whereas gMDSCs were elevated in the blood of females. Depletion of gMDSCs extended survival only in female mice. Using gene-expression signatures coupled with network medicine analysis, we demonstrated in preclinical models that mMDSCs could be targeted with antiproliferative agents in males, whereas gMDSC function could be inhibited by IL1β blockade in females. Analysis of patient data confirmed that proliferating mMDSCs were predominant in male tumors and that a high gMDSC/IL1β gene signature correlated with poor prognosis in female patients. These findings demonstrate that MDSC subsets differentially drive immune suppression in a sex-specific manner and can be leveraged for therapeutic intervention in GBM. SIGNIFICANCE: Sexual dimorphism at the level of MDSC subset prevalence, localization, and gene-expression profile constitutes a therapeutic opportunity. Our results indicate that chemotherapy can be used to target mMDSCs in males, whereas IL1 pathway inhibitors can provide benefit to females via inhibition of gMDSCs.See related commentary by Gabrilovich et al., p. 1100.This article is highlighted in the In This Issue feature, p. 1079.

Figure 1.

mMDSCs accumulate in the tumors of male mice, while gMDSCs accumulate systemically in female mice. A, Timeline of immune infiltration analysis. B, Frequency of CD45^high bone marrow-derived immune cells in resected tumors versus the contralateral hemisphere of sham-injected or 25,000 GL261-implanted mice on day 14 and day 21. Data shown as mean ± s.d. of n = 10/group from one of independently repeated experiments. * p<0.05, ** p<0.01 as determined by two-way ANOVA. C, Percentage of mMDSCs (CD11b⁺CD68⁻Ly6C⁺Ly6G⁻I-A/I-E⁻) and gMDSCs (CD11b⁺CD68⁻Ly6C⁻Ly6G⁺) in CD45⁺ cells of the left hemisphere from n = 17 sham-injected and n = 26 GL261-bearing animals. Data shown for individual animals, and ** p<0.01 as calculated by unpaired t-test. D, Ratio of mMDSCs to gMDSCs in the left hemisphere from n = 16 sham-injected and n = 26 GL261-bearing animals 21 days post tumor-implantation or sham injection. Data shown for individual mice combined from three independent experiments and *** p<0.001 as calculated by unpaired t-test. E, Ratio of mMDSC-to-gMDSC in the left hemisphere from n = 15 sham-injected and n = 19 SB28-bearing animals, 14 days post tumor-implantation or sham injection. Data shown for individual mice combined from two independent experiments, and *** p<0.001 as calculated by unpaired t-test. F, Percentage of mMDSCs in the CD45⁺ immune cells infiltrating the left hemisphere 21 days post-GL261 implantation or sham injection. Data shown as mean ± s.d. from three independent experiments. n = 8–9 sham-injected and n = 13 GL261-bearing mice per sex, and * p<0.05 as determined by unpaired t-test. G, Percentage of gMDSCs in the CD45⁺ immune cells infiltrating the left hemisphere 21 days post-GL261 implantation or sham injection. Data shown as mean ± s.d. from three independent experiments. n = 8–9 sham-injected and n = 13 GL261-bearing mice per sex. H, Ratio of mMDSCs to gMDSCs in the left hemisphere of sham-injected or tumor-bearing mice 21 days after the procedure. Data shown as mean ± s.d. from three independent experiments. n = 8–9 sham-injected and n = 13 GL261-bearing mice per sex, and ** p<0.01 as calculated by unpaired t-test. I, Frequency of mMDSCs and gMDSCs as a fraction of CD45+ cells in the systemic circulation of sham-injected or GL261-bearing mice. Data shown for individual mice combined from three independent experiments. n = 16 sham-injected and n = 26 GL261-bearing mice, and *** p<0.001 as determined by unpaired t-test. J, Percentage of mMDSCs in the circulation of sham-injected and GL261-bearing mice on day 21. Data shown as mean ± s.d. from three independent experiments. n = 9 sham-injected and n = 13 GL261-bearing mice per sex and * p<0.05 as determined by unpaired t-test. K, Percentage of gMDSCs in the circulation of sham-injected and GL261-bearing mice on Day 21. Data shown as mean ± s.d. from three independent experiments. n = 9 sham-injected and n = 13 GL261-bearing mice per sex and * p<0.05 as determined by unpaired t-test.

Figure 2.

gMDSCs depletion extends the survival span of female mice. A, Schematics of MDSC depletion regimen. B-G, Kaplan-Meier curves depicting survival of female and male mice treated with (B-C) anti-Gr-1, (D-E) anti-Ly6G, and (F-G) anti-Ly6C neutralizing antibodies every other day starting 7 days post-GL261 implantation with respect to the isotype-treated mice. For B, n = 9 for isotype- and n = 13 for anti-Gr-1-treated female mice; for C, n = 9 for isotype- and n = 11 for anti-Gr-1-treated male mice; for D, n = 13 for isotype- and n = 14 for anti-Ly6G-treated female mice; for E, n = 10 for isotype- and n = 10 for anti-Ly6G-treated male mice; for F, n = 9 for isotype- and n = 9 for anti-Ly6C-treated female mice; for G, n = 9 for isotype- and n = 10 for anti-Ly6C-treated male mice. Data combined from two-to-three independent experiments. Significance was determined by Gehan–Breslow–Wilcoxon test with p<0.05 being considered a significant difference. H, Representative histograms depicting ex vivo intracellular Ki-67 staining of mMDSCs and gMDSCs from blood and tumor. I, Quantification of Ki-67 staining as mean fluorescence intensity from n = 4 (2 male and 2 female) animals euthanized on day 21 post-GL261 implantation. Data corrected for background based on fluorescence-minus-one staining and shown as mean ± s.d. *** p<0.001 as determined by unpaired Student’s t-test.

Figure 3.

Proliferation of mMDSCs can be targeted by fludarabine, while IL-1 pathway inhibition is predicted to counteract gMDSC-mediated immunosuppression. A, Schematics of in vitro MDSC polarization approach. Bone marrow cells from C57BL/6 mice were co-cultured with GL261 cells in the presence of GM-CSF/IL-13 for 3 days. B, mMDSC, gMDSC and nonMDSC populations were phenotypically discriminated based on CD11b, Ly6C and Ly6G expression. C, Proliferation rates of activated T cells co-cultured with nonMDSCs, gMDSCs and mMDSCs generated by cytokine polarization, compared to unstimulated T cells. Data analyzed separately for n = 3 male and female mice and shown as mean ± s.d. *** p<0.001 as determined by two-way ANOVA. D-E, GeneOntology Enrichment Analysis using differentially expressed genes between (D) mMDSCs versus nonMDSCs and (E) gMDSCs versus nonMDSCs from n = 6 biological replicates based on log(fold change) ≤−1 and adjusted p-value <0.001. F,Potential mechanism-of-action of fludarabine and rilonacept by network inference. Differentially expressed genes of mMDSCs for fludarabine and gMDSCs for rilonacept were directly connected to the drug targets and/or through one or more common neighbors. For fludarabine, a maximum distance of 2 between drug targets and differentially expressed genes was used to visualize the network. For rilonacept, the distance was set to 4. Node size indicates log₂FC, and interaction types are color coded.

Figure 4.

MDSC subset targeting with predicted drugs confers sex-specific survival advantage. A, Treatment regimen for testing the predicted drug candidates fludarabine and anti-IL-1β neutralizing antibody. Kaplan-Meier curves depicting survival of B, male and C, female GL261-bearing mice treated with fludarabine. Data presented from two independent experiments with (B) n = 10 vehicle-treated males and n = 10 fludarabine-treated males and (C) n = 10 vehicle-treated females and n = 10 fludarabine-treated females. p<0.01 as determined by Gehan–Breslow–Wilcoxon test. Kaplan-Meier curves depicting survival of D, male and E, female GL261-bearing mice treated with anti-IL1β neutralizing or isotype control antibody. Data presented from three independent experiments with (D) n = 15 isotype control-treated males and n = 15 anti-IL-1β antibody-treated males, (E) n = 15 isotype control-treated females and n = 15 anti-IL-1β antibody-treated females. p<0.05 as determined by Gehan–Breslow–Wilcoxon test. F, Frequency of CD8+ T cells in the circulation of n = 5 vehicle- and n = 4 fludarabine-treated SB28-bearing male mice. Data shown as mean ± s.d., and *** p<0.001 as calculated by unpaired t-test. G, Frequency of CD8+ T cells in the circulation of n = 8 vehicle- and n = 7 fludarabine-treated GL261-bearing male mice. Data shown from two independent experiments as mean ± s.d., and *** p<0.001 as calculated by unpaired t-test. H, Relative percentage of tumor-infiltrating mMDSCs as a fraction of live cells in n = 5 vehicle and n = 5 fludarabine-treated SB28-bearing male mice after one cycle. Data shown as mean ± s.d. and * p<0.05 as calculated by unpaired t-test. I, Relative percentage of Ki-67+ cells in the nonimmune cells of the left hemisphere from n = 8 vehicle- and n = 7 fludarabine-treated GL261-bearing male mice after two cycles. Data shown from two independent experiments as mean ± s.d. J, Frequency of gMDSCs in the circulation of n = 8 isotype- and n = 8 anti-IL-1β-treated GL261-bearing female mice. Data shown as mean ± s.d. from two independent experiments and * p<0.05 as calculated by unpaired t-test. K, Relative percentage of Ki-67+ cells in the nonimmune cells of the left hemisphere from n = 5 isotype- and n = 5 anti-IL-1β-treated SB28-bearing female mice after one cycle. Data shown as mean ± s.d., and ** p<0.01 as calculated by unpaired t-test. L, Relative percentage of Ki-67+ cells in the nonimmune cells of the left hemisphere from n = 8 isotype- and n = 8 anti-IL-1β-treated GL261-bearing female mice after two cycles. Data shown from two independent experiments as mean ± s.d. and * p<0.05 as calculated by unpaired t-test.

Figure 5.

IDH-wild type GBM tumors from male patients have more immunosuppressive myeloid cells, which include proliferating mMDSCs. A, Fraction of IBA1+ and CD204+ area of n = 188 patients with IDH-wild type GBM from Sorensen et al., Neuropathology and Applied Neurobiology, 2018, was re-analyzed by accounting for biological sex. Data shown from n = 108 males and n = 80 females as their median. ** p<0.01 as determined by unpaired t-test. B, Percentage of CD45^high cells of the viable single cells isolated from GBM tumors. Data shown for n = 8 individual patients. C, The frequency of mMDSCs (CD11b⁺CD33⁺CD14⁺HLA-DR⁻CD68⁻) and gMDSCs (CD11b⁺CD33⁺CD66b⁺LOX-1⁺) in tumor-infiltrating leukocytes from n = 8 male patients with IDH-wild type GBM. Data shown as mean ± s.d. ** p<0.05 as determined by unpaired Student’s t-test. D, Ratio of mMDSCs to gMDSCs in tumors of n = 8 patients with IDH-wild type GBM. E, Representative histograms showing Ki-67 expression levels in matched mMDSCs and gMDSCs from the same patient, in comparison to the isotype control staining. F, Mean fluorescence intensity of Ki-67 in tumor-infiltrating mMDSCs and gMDSCs from n = 8 patients with IDH-wild type GBM. Data shown as mean ± s.d. * p<0.05 as determined by unpaired Student’s t-test.

Figure 6.

gMDSC prevalence predicts poor prognosis of female patients with GBM. A, Representative IL-1β immunohistochemistry tumor sections from male and female patients confirming IL-1β protein expression in IDH-wild type GBM. B, Quantification of IL-1β+ cells in tumors from four visual fields of n = 10 male and n = 10 female patients with IDH-wild type GBM. C, Correlation among OLR1 expression levels, patient sex and survival duration was analyzed via TCGA GBM dataset. High and low expression levels were determined based on quartiles. Data shown for n = 14 female patients with high OLR1 expression and n = 16 female patients with low OLR1 expression (left) and for n = 29 male patients with high OLR1 expression and n = 25 male patients with low OLR1 expression (right). p<0.01 as determined by Gehan–Breslow–Wilcoxon test. D, Correlation among IL-1β expression levels, patient sex and survival duration was analyzed via TCGA GBM dataset. High and low expression levels were determined based on quartiles. Data shown for n = 15 female patients with high IL-1β expression and n = 14 female patients with low IL-1β expression (left) and for n = 37 male patients with high IL-1β expression and n = 27 male patients with low IL-1β expression (right). p<0.05 as determined by Gehan–Breslow–Wilcoxon test. E, Correlation between OLR1 and IL-1β expression levels from n = 538 patients. p<0.01 based on two-sided t-test. F, Proposed model of relative mMDSC abundance and IL-1β presence in patients with GBM.