Therapeutic modulation of phagocytosis in glioblastoma can activate both innate and adaptive antitumour immunity

Abstract

Tumour cell phagocytosis by antigen presenting cells (APCs) is critical to the generation of antitumour immunity. However, cancer cells can evade phagocytosis by upregulating anti-phagocytosis molecule CD47. Here, we show that CD47 blockade alone is inefficient in stimulating glioma cell phagocytosis. However, combining CD47 blockade with temozolomide results in a significant pro-phagocytosis effect due to the latter's ability to induce endoplasmic reticulum stress response. Increased tumour cell phagocytosis subsequently enhances antigen cross-presentation and activation of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in APCs, resulting in more efficient T cell priming. This bridging of innate and adaptive responses inhibits glioma growth, but also activates immune checkpoint. Sequential administration of an anti-PD1 antibody overcomes this potential adaptive resistance. Together, these findings reveal a dynamic relationship between innate and adaptive immune regulation in tumours and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Fig. 1. 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.

Fig. 2. Temozolomide (TMZ) promotes GBM cell phagocytosis by bone marrow (BM)-derived phagocytes.

a TMZ induces translocation of the ER chaperone calreticulin to the plasma membrane in both human and murine GBM cells in a concentration dependent manner. n = 6. b The membrane translocation of calreticulin induced by TMZ was transient, with peak occurring at 72-h post treatment. n = 6. c TMZ treatments did not result in significant membrane translocation of calreticulin in human or mouse normal bone marrow cells. d TMZ treatment promotes GBM cell phagocytosis by BM-derived phagocytes. n = 6. Unpaired Student’s t test. e TMZ treatment (50 μM) upregulated the mRNA expression of ER response-associated targets DDIT3, HERPUD1, and GADD45α in mouse GBM cells. n = 4. ** < 0.01, * < 0.05 (Unpaired Student’s t test). f Western blot showing TMZ treatment increased the expression levels of ER stress response-specific protein BiP, phospho-EIF2α, and CHOP in mouse GBM cells. TGN thapsigargin. g Addition of the ER stress inhibitor 4-PBT diminished the pro-phagocytosis and calreticulin translocation effect of TMZ. n = 6, * < 0.01 (Unpaired Student’s t test). Error bar = mean ± standard deviation.

Fig. 3. 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. 4. cGAS-STING pathway is essential for TMZ and anti-CD47 induced immune response.

a Combination TMZ and anti-CD47 antibody treatment against murine GBM cells (GL261) increased the production of type I interferons in APCs. IFNA interferon α, IFNB interferon β. n = 5, **p < 0.01, one-side ANOVA with Bonferroni post hoc correction. b Western blot showing combined TMZ and anti-CD47 antibody treatment resulted in the activation of cGAS-STING cytoplasmic DNA sensing pathways in BM APCs. c Combination TMZ and anti-CD47 antibody treatment promoted p65 expression and nuclear translocation in BM APCs that is dependent on STING. d STING activation is critical to the increased the production of NF-κB cytokine TNFα and IL1β in APCs in the setting of combined TMZ and anti-CD47 antibody treatment. n = 6, **p < 0.01, unpaired Student’s t test. e Immunofluorescence staining of GL261 tumors implanted in WT and STING KO animals. Nuclear p-IRF3 (Ser 396) in CD45⁺ cells is indicative of STING signaling activation. f, g Quantification of CD45⁺ cell infiltration and percentage of p-IRF3 positive CD45⁺, C57BL/6, n = 7; knockout, n = 6. Unpaired Student’s t test. h GL261 tumor volume in C57BL/6 or STING KO mice at day 20 following control or combination TMZ + aCD47 treatment, n = 5/group. i T-cell infiltration in GL261 tumors in C57BL/6 or STING KO mice at day 20 following control or combination TMZ + aCD47 treatment measured by CD3⁺ cells in total DAPI count per field of view (FOV), C57BL/6, n = 9; knockout, n = 6 unpaired Student’s t test. Representative FOV for each group on the right. All error bars = mean ± standard deviation.

Fig. 5. 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 IFNγ-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 IFNγ⁺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. 6. The anti-GBM effect of combined TMZ and anti-CD47 antibody treatment is augmented by adjuvant PD-1 blockade.

a Combination TMZ and anti-CD47 antibody treatment significantly elevated the level of IFNγ within tumors. Scale bar = 200 μm, Combo, n = 4; other groups, n = 5. **p < 0.01, one-side ANOVA with Bonferroni post hoc correction. b PD-1 (good responders, n = 7; poor responders, n = 4) and PD-L1 (good responders, n = 6; poor responders, n = 4) expression levels were notably elevated in GBM treated with combination TMZ and anti-CD47 antibody treatment. **p < 0.01, unpaired Student’s t test. c The addition of an adjuvant anti-PD1 antibody treatment prolonged animal survival in mouse with GBM. Control and aPD-1, n = 10; aCD47 + TMZ, n = 8; aCD47 + TMZ + aPD1, n = 9. **p < 0.01, log-rank test. d Proposed schematic of bridging the innate and adaptive immune responses by phagocytosis induction. Calr calreticulin. TCR T-cell receptor. All error bars = mean ± standard deviation.