Work

Fractalkine inhibition improves survival and reduces tumor immune cell infiltration in orthotopic murine glioma models

Year: 2025

Type: article

Abstract: We sought to examine the effect of AZD8797-induced CX3CR1 inhibition on tumor macrophage infiltration and survival in a murine glioma model. We treated mice with AZD8797 and monitored them for the development of neurologic symptoms and survival. Survival was longer in the AZD8797-treated mice compared to that of control mice (41 vs 22 days, P = .005). We observed significant downregulation of CX3CR1 in AZD8797-treated tumor specimens (P < .0001), accompanied by a significant decrease in macrophage infiltration and CCL2 levels. Thus, pharmacologic inhibition of CX3CR1 signaling with AZD8797 improved survival and attenuated immune infiltration in our murine glioma model. The CX3CR1/CX3CL1 signaling axis has been shown to be an important mediator of tumor behavior in glioma. A common CX3CR1 variant, V249I, is associated with decreased signaling activity and a lower number of CX3CL1 binding sites. This polymorphism has been shown to improve survival in patients with low-grade glioma (LGG)1 and high-grade2 glioma (HGG). Our group has shown that LGG patients with the V249I variant have reduced M2 macrophage infiltration, CCL2 presence, and microvessel density,1 while others have shown that HGG patients with this variant also have reduced peritumoral microglial infiltration.2 Given these findings and their association with decreased CX3CR1 signaling, inhibition of the CX3CR1 axis represents a potential opportunity for novel anti-glioma therapy. We sought to investigate the effects of AZD8797, an allosteric, noncompetitive inhibitor of CX3CR1, on tumor immune cell infiltration and survival in orthotopic mouse models of glioblastoma.3 We obtained 4- to 8-week-old C57BL/6 mice in accordance with our institutional Animal Care and Use Committee. The right frontal lobes of mice were implanted with 5 × 104 mCherry-Luciferase labeled GL-261 cells. Mice were allocated in a sex-balanced fashion to the treatment arm injected intraperitoneally daily with 80 µg/kg AZD 8797 dissolved in dimethyl sulfoxide (DMSO), as per the experimental protocol of Chen et al.4 Mice in the control arm were injected intraperitoneally daily with phosphate-buffered solution dissolved in DMSO. Mice were treated daily beginning the day after tumor implantation for 30 days or until the development of symptomatic tumor burden, at which point mice were humanely sacrificed and their brains collected for further study. Blocked specimens were then stained for Iba1 (1:500 Millipore), CX3CR1 (1:200 Invitrogen), and CCL2 (1:250 Millipore) as published earlier5 and processed for imaging using a Zeiss Confocal microscope. First, to verify the bioavailability of the treatment, we injected 4 mice with 80 µg/kg of AZD8797 intraperitoneally and humanely sacrificed them at prespecified time points (see Figure 1A and B). We detected measurable levels of the drug in mouse plasma and brain tissue 1 h after injection. Median survival was 22 days in the control group (n = 8) and 41 days in the AZD8797 treatment group (n = 9) (P = .005, log-rank test, see Figure 1C). The brains of 6 mice (three AZD-treated and 3 control) were available for histologic analysis (see Figure 1D and E). To measure the effects of AZD8797 on CX3CXR1/CX3CL1 signaling, we performed immunofluorescence assays on histologic sections of tumors from affected mice. Immunofluorescence staining revealed a significantly decreased number of CX3CR1-positive cells in AZD8797-treated mouse tumors compared with tumors from DMSO-treated mice (32.3% vs 4.0%, P < .0001) (see Figure 1F). Immunofluorescence revealed significantly decreased numbers of Iba1-positive cells in the brains of mice treated with AZD8797 compared to those of controls (Figure 1F, 32.5% vs. 14.3%, P < .0001). Therefore, the inhibition of the CX3CR1 axis is associated with decreased microglial infiltration into tumors in our murine orthotopic glioma model. Presence of AZD8797 in mouse (A) brain tissue and (B) plasma 1 h following intraperitoneal administration. (C) Improved survival of immuno-deficient mice implanted with orthotopic GL-261 cells (41 days vs 22 days, P = .0046). (D) Immunoflourescence staining of CX3CR1, Iba1, and DAPI in AZD8797-treated and DMSO-treated mouse tumor specimens. (E) Immunofluorescence staining of CCL2 and DAPI in AZD8797-treated and DMSO-treated mouse tumor specimens. (F) A significant reduction in CX3CR1 positivity, Iba1 macrophage infiltration, and CCL2 expression was observed in AZD8797-treated mouse tumors. To help determine the mechanism by which CX3CR1 inhibition leads to improved survival, we performed immunofluorescence assays of CCL2 on histologic sections of tumors from affected mice. We detected significantly lower expression of CCL2 in the tumors of mice treated with AZD8797 compared to those of controls (Figure 1F, 69.3% vs 29.0%, P < .0001). This decrease is likely due to the decrease in infiltrating macrophages observed in treated tumor specimens. Given the role of CCL2 in recruiting immunosuppressive regulatory T cells and myeloid-derived suppressor cells, it is likely that reduced levels of this chemokine contributed to improved survival in our AZD8797-treated mice. Infiltrating immune cells can account for as much as a third of the tumor bulk in glioblastoma (GBM), where they contribute to tumor cell survival, proliferation, and migration.6 Of these, tumor-associated macrophages and microglia (TAMs) are the most frequent.7 The number of TAMs has been associated with higher tumor grade and the proportion of glioma stem cells.7 As GBM is able to harness host immunity to protect itself, therapies aimed at rescuing immune cells from tumor influence may represent another treatment option. One possible treatment option is “re-education” of the microenvironment via CSF1R inhibition. Pyonteck et al.8 demonstrated decreased tumor growth and improved survival in murine GBM models treated with the CSF1R inhibitor BLZ945. Treatment did not affect the number of TAMs, which were found to be sustained by multiple tumor-supplied chemokines, but rather reduced their M2 polarization and abrogated their pro-tumoral signaling.8 Conversely, Lee et al.1 demonstrate a significant role of the CX3CR1/CX3CL1 axis in GBM. Histologic samples from patients heterozygous or homozygous for the V249I variant had significantly lower infiltration by M2-polarized macrophages, associated with peritumoral immunosuppression, and lower CD31-positive microvessel density.1 Indeed, levels of the family of CX3CR cytokines are implicated in TAM recruitment.7 V249I variant tumors were also shown to have reduced expression of CCL2, implicated in TAM recruitment (and subsequent immunosuppression), and matrix metalloproteinase 9 (MMP-9), associated with tumor invasion.1 Furthermore, co-expression of CX3CR1 and CX3CL1 in a murine glioma model led to worse survival and higher tumor grade compared to controls.1 It is increasingly understood that chemokines play an important role in glioma. In particular, CCL2 expression has been shown to drive regulatory T cell and myeloid-derived suppressor cell recruitment and is associated with worse overall survival.9 Antibody-mediated blocking of the CCL2 signaling axis has been shown to improve survival in murine glioma models.10 Thus, it is likely that the survival improvement seen in AZD8797-treated mice is at least partly mediated by decreased macrophage infiltration, as evidenced by decreased Iba1 staining and thus decreased CCL2 expression. E.G.: Data review, manuscript and figure preparation. M.M.: Manuscript review, experiment conduction. A.P., K.M., Y.Y.: Experiment conduction, manuscript review. K.L.: manuscript review. G.R.: manuscript review, project oversight. None declared. None declared. Our project was carried out according to standards set forth by the Baylor College of Medicine Institutional Animal Care and Use Committee. Data generated as part of this project will be made available upon request.

Source: Neuro-Oncology Advances

DOI: 10.1093/noajnl/vdaf165