Malignant Astrocytic Tumor Progression Potentiated by JAK-mediated Recruitment of Myeloid Cells

Abstract

Purpose: While the tumor microenvironment has been known to play an integral role in tumor progression, the function of nonresident bone marrow-derived cells (BMDC) remains to be determined in neurologic tumors. Here we identified the contribution of BMDC recruitment in mediating malignant transformation from low- to high-grade gliomas.Experimental Design: We analyzed human blood and tumor samples from patients with low- and high-grade gliomas. A spontaneous platelet-derived growth factor (PDGF) murine glioma model (RCAS) was utilized to recapitulate human disease progression. Levels of CD11b+/GR1+ BMDCs were analyzed at discrete stages of tumor progression. Using bone marrow transplantation, we determined the unique influence of BMDCs in the transition from low- to high-grade glioma. The functional role of these BMDCs was then examined using a JAK 1/2 inhibitor (AZD1480).Results: CD11b+ myeloid cells were significantly increased during tumor progression in peripheral blood and tumors of glioma patients. Increases in CD11b+/GR1+ cells were observed in murine peripheral blood, bone marrow, and tumors during low-grade to high-grade transformation. Transient blockade of CD11b+ cell expansion using a JAK 1/2 Inhibitor (AZD1480) impaired mobilization of these cells and was associated with a reduction in tumor volume, maintenance of a low-grade tumor phenotype, and prolongation in survival.Conclusions: We demonstrate that impaired recruitment of CD11b+ myeloid cells with a JAK1/2 inhibitor inhibits glioma progression in vivo and prolongs survival in a murine glioma model. Clin Cancer Res; 23(12); 3109-19. ©2016 AACR.

Conflict of interest statement

Conflict of interest: The authors declare no conflicts of interest.

©2016 American Association for Cancer Research.

Figures

Figure 1. Elevated CD11b+ expression in myeloid cells is associated with malignant human and murine gliomas

(A) Flow cytometric analysis of CD11b+ cells from the peripheral blood of patients with low (n=10) and high grade (n=10) glioma (WHO Grade II and IV) (n=20), ** p =0.01. (B) RCAS murine model depicting low to high-grade transition (3-12 weeks) and representative tumor histology at each stage. (C) Flow cytometric analysis of CD11b+/GR1+ cells in blood of RCAS mice bearing low (n=7) and high-grade tumors (n=9). Quantification of CD11b+/GR1+ cells at normal (n=4), low (n=7) and high grade tumor(n=9) stages, Student's t-test,* P<0.05. (D) Flow cytometry analysis of CD11b+/GR1+ cells in bone marrow from RCAS mice bearing low (n=7) and high-grade tumors (n=9). Quantification of percentage of CD11b+/GR1+ cells at normal (n=4), low- (n=7) and high-grade(n=9) tumor stages is shown on the right. Student's t-test,* p<0.05.

Figure 2. Bone marrow-derived cells (BMDCs) are recruited to the tumor microenvironment

(A) Representative photomicrographs of tumor cross sections from GFP+ bone marrow transplanted mice counterstained with CD31+ at low- (n=4) and high-grade stages (n=4)(4 and 8 weeks, respectively), Scale bar 50μm. (B) Representative flow cytometric analysis of brain tumor tissue from RCAS murine animals gated for GFP+ cells in low (n=4) and high grade tumors (n=4), * p<0.05. (C) Confocal microscopic analysis of high grade tumor sections (n=3 animals, >6 sections/tumor) stained for DAPI and CD11b from GFP+ bone marrow transplanted RCAS mice, Scale bar 50μm(D) Quantification of cells labeled with GFP+, CD11b+, and co-stained (GFP+/CD11b+). Percentage of CD11b+/GFP+ cells.

Figure 3. JAK 1/2 inhibition following bone marrow transplantation mitigates CD11b+/GR1+-mediated low-grade glioma transformation

(A) Schematic of RCAS low grade murine glioma bearing animals post irradiation (2wks) and GFP+ bone marrow transplantation, treated with AZD1480. (B) Representative image of T2-weighted 7T MRI of vehicle control (n=7) and AZD1480 treated (n=7) animals at 10 weeks. Animals were imaged around 7 weeks. (C) Representative photomicrographs of tumor cross sections from mice treated with vehicle control (n=5) and AZD1480 (n=5). Quantification of Average number of GFP cells per high power field in bar graph format, (n=10),Scale bar 50μm, **p=0.004. (D)Representative photomicrographs of tumor cross sections from mice treated with vehicle control (n=5) and AZD1480 (n=5) co-stained with GFP+ and CD11b+. Quantification of average number of cells per high power field in bar graph format labeled with GFP+, CD11b+ and double labeled with GFP+/CD11b+, (n=10), Scale Bar 50μm, *p<0.05.

Figure 4. JAK 1/2 inhibition prevents low to high-grade tumor progression

(A) Animal schematic depicting experimental design to examine effect of AZD1480 (treatment for 3 weeks) on tumor progression. Treatment with AZD1480 was stopped at 6 weeks (B) Representative images of T2-weighted 7T MRI of vehicle control (n=9) and AZD1480(n=9) treated animals at 6 weeks. Representative images of H&E staining of tumor sections from control (n=8) and AZD1480 (n=8) treated animals at 6 weeks, Scale Bar 100 μm. (C) Representative flow cytometry graphs of CD11b+/GR1+ from blood (n=3/group) and bone marrow (n=7/group) of vehicle control and AZD1480 treated tumor bearing mice at 6 weeks, *p=0.0386 blood, p=0.0556 BM (D) Kaplan-Meier symptom free survival curve for RCAS mice treated for 3 weeks only with vehicle control (n=9) and AZD1480,(n=9) *** P<0.0001.(E) Kaplan-Meier symptom free survival curve for RCAS mice treated indefinitely with vehicle control(n=6) and AZD1480(n=6), ***p<0.0001.