Surgical debulking promotes recruitment of macrophages and triggers glioblastoma phagocytosis in combination with CD47 blocking immunotherapy

Abstract

Surgical resection is a standard component of treatment in the clinical management of patients with glioblastoma multiforme (GBM). However, experimental therapies are rarely investigated in the context of tumor debulking in preclinical models. Here, a surgical debulking GBM xenograft model was developed in nude rats, and was used in combination with CD47 blocking immunotherapy, a novel treatment strategy that triggers phagocytosis of tumor cells by macrophages in diverse cancer types including GBM. Orthotopic patient-derived xenograft tumors expressing CD47 were resected at 4 weeks after implantation and immediately thereafter treated with anti-CD47 or control antibodies injected into the cavity. Debulking prolonged survival (median survival, 68.5 vs 42.5 days, debulking and non-debulking survival times, respectively; n = 6 animals/group; P = 0.0005). Survival was further improved in animals that underwent combination treatment with anti-CD47 mAbs (median survival, 81.5 days vs 69 days, debulking + anti-CD47 vs debulking + control IgG, respectively; P = 0.0007). Immunohistochemistical staining of tumor sections revealed an increase in recruitment of cells positive for CD68, a marker for macrophages/immune cell types, to the surgical site (50% vs 10%, debulking vs non-debulking, respectively). Finally, analysis of tumor protein lysates on antibody microarrays demonstrated an increase in pro-inflammatory cytokines, such as CXCL10, and a decrease in angiogenic proteins in debulking + anti-CD47 vs non-debulking + IgG tumors. The results indicated that surgical resection combined with anti-CD47 blocking immunotherapy promoted an inflammatory response and prolonged survival in animals, and is therefore an attractive strategy for clinical translation.

Keywords: CD47; glioblastoma; macrophage; phagocytosis; signal regulatory protein-α.

Figure 1. Survival in rats implanted with GBM is enhanced with surgical debulking

A. Representative images of implantation of spheroids with a wide bore syringe (1), burr hole drilled in the skull for implantation (2), craniectomy centered around the original burr hole in preparation for tumor debulking at week 4 (3), removal of skull bone (4), removal of tumor tissue by aspiration (5), and reinsertion of resected skull bone which was fixed with cyanoacrylate glue (6). B. Representative images of MRI and PET-CT scans of animals at one day before debulking (week 4), one day after debulking, and tumor recurrence (week 8). Circles (MRI) and arrows (PET) highlight areas of xenograft growth. C. Tumor volume (mm³) calculated from MRI scans plotted as a function of time in weeks. Tumor was resected at week 4 after implantation. D. Kaplan-Meier plots illustrating survival time (implantation = day 1) of nude rats (n = 6/group) with or without tumor debulking (P = 0.0005).

Figure 2. Increased proliferation index and vascular changes in debulking relative to non-debulking xenografts

Immunostaining performed on sections from debulking and non-debulking tumors (5 rats per group) with the antibodies indicated. A. vWF (magnification 20×) and B. quantification of the percentage of vessel area per view (P = 0.001); C. Ki67 (magnification 40×) and D. quantification of percentage of Ki-67 positive cells per view (P = 0.028). Scale bars = 100 μm.

Figure 3. P3 GBM cells uniformly express high levels of CD47

Cell suspensions were incubated with FITC-labeled anti-CD47 antibody used for treatment in rats or IgG as a control. A. Fluorescent image of P3 tumor cells incubated with FITC-labeled anti-CD47 antibody (green). DAPI (blue) was used for nuclear counterstaining. B. Analysis of CD47 expression by flow cytometry. Forward and side scatter plots of fixed cells, and percentage of cells FITC labeled (> 98%) in the gated population.

Figure 4. Nude rat bone marrow derived macrophages efficiently phagocytose P3 and P13 GBM tumor cells in an in vitro assay

A. Representative images of derived macrophages after incubation (2 h) with CFSE (green) labeled P3 and P13 tumor cells treated with anti-CD47 antibody or controls (PBS or IgG). Co-cultures were rinsed to remove free CFSE labeled P3 cells before imaging. B. Bar chart displaying the phagocytic index (number of phagocytosed tumor cells per 100 macrophages) with anti-CD47 antibody and controls (IgG and PBS) for two different GBM derived CD47+ xenografts, P3 and P13. Error bars represent SD.

Figure 5. Local injection of anti-CD47 antibody inhibits tumor growth in combination with surgical debulking

A. View of local injection of anti-CD47 antibody through the opening used to implant spheroids. B. Representative images of final MRI scans before sacrifice. C. Tumor volume derived from MRI scans and plotted as a function of time in weeks from rats treated with anti-CD47 or IgG antibodies with or without debulking. D. Survival of nude rats with GBM xenografts treated with anti-CD47 or IgG antibodies with or without debulking.

Figure 6. Macrophages coexpressing M1 (pSTAT1 and CD68) and M2 (CD163) polarization related markers are recruited to debulking xenografts

Immunostaining performed on sections from xenografts as indicated for A. CD68; B. pSTAT1; C. CD68 and pSTAT1; and D. CD163. E. Quantification of macrophages based on percentage of CD68 positive cells per view (P = 0.021). F. Quantification of macrophages based on percentage of CD163 positive cells per view (P = 0.003). Scale bar = 100μm.

Figure 7. Anti-CD47 antibody treatment alters levels of cytokines and angiogenesis-associated proteins in GBM xenografts

Protein arrays for A. cytokines and B. angiogenesis-associated proteins were incubated with protein lysates (400 μg) prepared from debulking tumor tissue treated with IgG or anti-CD47 as indicated. Data shown represent a 1000 s exposure to X-ray film.