TMIC-115. Reversing glioblastoma-induced changes in bone marrow hematopoiesis slows tumor growth

Abstract While the contribution and polarization of immune cell populations in glioblastoma has been well studied, the reciprocal effect of this malignancy on immune reservoirs remains less understood. To that end, we orthotopically implanted C57BL/6J mice with syngeneic GL261 tumors and analyzed their femurs by flow cytometry at 0, 1, and 2 weeks post-implantation (n=5/ea). Neutrophil production was acutely expanded at week 1 compared to week 0, reflected as 1.26-fold greater neutrophil abundance (72.1% vs 56.9% of marrow cells, p=0.001) and accelerated maturation (70.0% vs 64.7% CXCR2+ mature, p=0.011); effects waned by week 2 (p>0.05). In vitro culture of FACS-isolated immature murine neutrophils (Lin-CD11b+Gr1+CXCR2-) confirmed that tumor-secreted proteins (GL261-conditioned media) accelerated maturation compared to RPMI at 12h (56.2% vs 40.3%, p<0.001) through 48h (9.8% vs 6.9%, p=0.025). Given recent work from our lab demonstrating that neutrophil immaturity is critical for their polarization into antitumoral neutrophil-dendritic ‘hybrids’, we hypothesized that reversing tumor-accelerated neutropoiesis would pose clinical benefit. In our testing of neutrophil maturation in vitro, we found that CXCR3 signaling potently inhibited murine neutrophil plasticity; conversely, antibody-mediated blockade of this receptor quadrupled neutrophil immaturity (30.3% vs 7.0%, p<0.001) and hybrid polarization (12.8 vs 3.6 induced hybrids per 1000 cells, p=0.002). Similarly, in vivo, CXCR3 blockade (200 µg αCXCR3, i.p., every 48h) curbed glioma-induced neutrophil expansion by 51.3%vs isotype (p=0.027) and entirely abrogated glioma-accelerated neutrophil maturation (p=0.005), resulting in greater accumulation of fully-polarized CD11c+MHCII+ hybrid neutrophils intratumorally (8.7% vs 3.2% of MHCII+ neutrophils, p=0.022). Longitudinally, this slowed tumor growth (as measured by bioluminescent imaging; 5.7E5 vs 1.2E6 at 16 days, p=0.020) and prolonged survival (22 vs 19 days, p<0.001; n=10/ea). Critically, our work highlights that glioblastoma-immune interactions extend beyond the microenvironment, even affecting developing marrow cells; in turn, treatments that reverse these changes – such as αCXCR3 – hold therapeutic promise and merit exploration.