Fig 2: Combined TMZ and CD47 blockade enhances GBM cell phagocytosis and cross-priming of antigen-specific T cells by BM antigen presenting cells (APCs).Combined TMZ and anti-CD47 antibody (aCD47) treatment enhances murine (a) and human (b) GBM cell phagocytosis by BM APCs. GL261, n = 8; CT-2A, n = 9; LN229, U251, U87, n = 6. **p < 0.05, one-side ANOVA with Bonferroni post hoc correction. c Blocking of calreticulin negated the enhanced phagocytosis effect of combined TMZ and anti-CD47 antibody treatment. n = 8, **p < 0.01, unpaired Student’s t test. d Immunofluorescence images of tumor infiltrating BM cells (red) and microglia (green). e Quantification of tumor-infiltrating BM cells and microglia. Control, n = 8; combo, n = 6. Unpaired Student’s t test. f Combined TMZ and anti-CD47 antibody treatment enhanced the cross-presentation of MHC-bound cOVA-derived SIINFEKL peptide on APCs. n = 8, **p < 0.01, one-side ANOVA with Bonferroni post hoc correction. g The enhanced antigen cross-presentation effect mediated by combined TMZ and anti-CD47 antibody was reduced with the addition of a calreticulin blocking peptide. GL261, n = 8, CT2A, n = 5. **p < 0.01, unpaired Student’s t test. h, i Combination TMZ and anti-CD47 antibody treatment enhanced cross-priming of cOVA antigen specific T cells. n = 6, **p < 0.01, one-side ANOVA with Bonferroni post hoc correction. Error bar = mean ± standard deviation.

Fig 3: Construction and expression of mCD47. a Schematic illustration of mCD47-GFP-Luc and GFP-Luc in a transposon vector construct. The coding sequences for murine CD47 (mCD47) or GFP-luciferase fusion gene (GFP-Luc) are shown. The locations of the promoter, the polyA signal, and the internal ribosome entry site (IRES) are indicated. b Transfection efficiency of mCD47. PC-3 cells were transfected with a Piggyback transposon with mCD47- GFP-Luc or GFP-Luc vectors, or mock transfected. Following puromycin selection, cells were stained with an isotype or a PE-conjugated anti-mCD47 antibody, and then analyzed by flow cytometry for detection of PE and GFP

Fig 4: Combination of TMZ and anti-CD47 induces anti-tumor immune response in murine GBM models.a Sequential TMZ and anti-CD47 antibody combined treatment inhibited growth of murine GBM (GL261). n = 5/group, **p < 0.01, unpaired Student’s t test vs. aCD47 or TMZ. b Sequential TMZ and anti-CD47 antibody combined treatment prolonged animal survival. Control, n = 8; other groups, n = 7. **p < 0.05, log-rank test. c Combination TMZ and anti-CD47 antibody treatment significantly increased the number of Iba1+ activated mononuclear cells within tumors, but not the total number of F4/80+ cells. Combo, n = 6; other groups, n = 5. **p < 0.01, unpaired Student’s t test. d Combined TMZ and anti-CD47 antibody treatment elevated the levels of peripheral blood cytokines. n = 5, **p < 0.05, one-side ANOVA with Bonferroni post hoc correction. e Intratumoral infiltrating CD4+ and CD8+ T cells were increased by combination TMZ and anti-CD47 antibody treatment. Scale bar = 200 µm, n = 5, **p < 0.01, one-side ANOVA with Bonferroni post hoc correction. f, g Combination TMZ and anti-CD47 antibody treatment did not result in significant changes to intratumoural regulatory T-cell content, but increased the number of IFN?-producing CD8+ T cells within tumors. Control, n = 5; combo, n = 4. **p < 0.01, unpaired Student’s t test. ns not significant. h In vivo depletion of CD8+ T cells using an anti-CD8 antibody completely eliminated the intratumoural IFN?+CD8+ T cells. Scale bar = 200 µm, Control, n = 4; combo, n = 6; combo + aCD8, n = 5. **p < 0.01, unpaired Student t test. i CD8+ T cells depletion diminished the antitumor effect of combination TMZ and anti-CD47 antibody treatment. Control, n = 5; combo, n = 10; combo + aCD8, n = 5 *p < 0.05, unpaired Student t test. All error bars = mean ± standard deviation.

Fig 5: Blockade of CD47, an anti-phagocytosis molecule over-expressed in glioblastoma (GBM), does not produce significant antitumor effect.a Immunohistochemistry analyses showing that CD47 expression is significantly elevated in GBM as compared to normal brain tissues from patient samples. Scale bar = 200 µm. n = 6/group. Unpaired student’s t test. Error bar = mean ± standard deviation. b CD47 blockade resulted in a modest increase in murine GBM cell phagocytosis by BM phagocytes. n = 5/group. **p < 0.01 compared to IgG (unpaired Student’s t test). c The tumor growth inhibitory effect of CD47 blockade was less than temozolomide. n = 8/group. d CD47 blockade did not result in improved animal survival over temozolomide treatment. n = 8/group. p = 0.1 (log-rank test). e No significant changes were noted in the intratumoral total (F4/80) or activated BM phagocytes (Iba1), CD4+ or CD8+ T cells after anti-CD47 antibody treatment. n = 8/group. Error bar = mean ± standard deviation. n.s. not significant.