Naomi Zakimi

Supplementary Fig. 13. UMAP markers for CyTOF gating of T cells from intracranial mouse glioblastoma allografts after radiotherapy, IL6 blockade, and immune checkpoint inhibition. a, UMAP cell density plots showing protein expression used for assigning cell types in Fig. 8c.

Supplementary Fig. 5. UMAP markers for CyTOF gating of myeloid cells from intracranial mouse glioblastoma allografts. a, UMAP cell density plots showing protein expression used for assigning cell types in Fig. 4e.

Supplementary Fig. 7. CCL2 and CXCL10 from the tumor microenvironment do not contribute to glioblastoma overall survival. a, Body weights for C57J/B6 WT mice (n=8) vs C57J/B6 Il6-/- mice (n=8) harboring intracranial SB28 glioblastoma allografts. See also Fig. 6g. b, Kaplan–Meier curves for overall survival from SB28 intracranial glioblastoma allografts in C57J/B6 WT (n=8 mice) vs C57J/B6 Ccl2-/- mice (n=8 mice) vs Cxcl10-/- mice (n=8 mice) showing no change in overall survival for mice lacking CCL2 or CXCL10 in the tumor microenvironment. Log rank test. c, Body weights for C57J/B6 WT (n=8 mice) vs C57J/B6 Ccl2-/- mice (n=8 mice) vs Cxcl10-/- mice (n=8 mice) harboring intracranial SB28 glioblastoma allografts. d, Body weights for C57J/B6 WT mice harboring SB28 intracranial glioblastoma allografts with doxycycline-induced IL6 (n=10 mice) or EV (n=10 mice) overexpression. See also Fig. 6i.

Supplementary Fig. 1. Swimmer plot. a, Clinical course and treatment history for patients included as ICI-responders and non-responders.

Supplementary Fig. 10. UMAP markers for CyTOF gating of myeloid cells from intracranial mouse glioblastoma allografts after IL6 blockade and immune checkpoint inhibition. a, UMAP cell density plots showing protein expression used for assigning cell types in Fig. 7d.

Supplementary Fig. 3. Gene circuit network changes between ICI responders and non-responders before and after ICI treatment. a, Network of gene circuits mapping differentially expressed genes between ICI responders (far left) and non-responders (middle left) post-ICI and pre-ICI specimens, and differentially expressed genes between pre-ICI (middle right) and post-ICI (far right) specimens from ICI responders and non-responders. Nodes represent pathways and edges represent shared genes between pathways (p≤0.05, FDR0.05). n = 14 match-paired glioblastoma samples from 7 patients.

Supplementary Fig. 15. Body weights for immunocompetent mice treated with radiotherapy, IL6 blockade, and immune checkpoint inhibition. a, Body weights for C57J/B6 WT mice harboring intracranial SB28 glioblastoma allografts treated with radiotherapy (RT, 18Gy/1Fx) vs RT plus αPD-1/αIL6 therapy (250 g biweekly i.p. for up to four weeks for each antibody) vs no-treatment control (n=8-10 mice/condition). See also Fig. 8g.

Supplementary Fig. 2. Human glioblastoma tissue sections pre-ICI and post-ICI. a, H&E, CD68 IHC, and CD3 IF sections for ICI responders pre-ICI (left) and post-ICI (right) showing region-of-interest (ROI) location for spatial proteomics (white circles) and spatial transcriptomics (white and red circles). b, H&E, CD68 IHC, and CD3 IF sections for ICI non-responders pre-ICI (left) and post-ICI (right) showing ROI location for spatial proteomic (white circles) and spatial transcriptomic (white and red circles) analyses. Scale bar, 1mm.

Supplementary Fig. 8. Intratumor IL6 blockade does not prolong glioblastoma overall survival and immune checkpoint inhibition in the absence of systemic IL6 blockade does not reprogram the glioblastoma immune microenvironment. a, Body weights for C57J/B6 WT mice (n=8-9 mice/condition) bearing SB28 intracranial mouse glioblastoma allografts treated with isotype control, systemic αIL6 monotherapy, systemic αPD-1 monotherapy, intratumor CED αIL6 monotherapy, or intratumor vs systemic αIL6 in combination systemic αPD-1 therapy. See also Fig. 7b. b, Heatmap of cytokine changes from multiplexed bead assays of SB28 intracranial glioblastoma allograft tumor lysates (top) or sera (bottom) after systemic monotherapy or combined antibody treatments (n=8 mice/condition) showing suppression of IL10 with all antibody treatments and suppression of IL6 with αIL6 treatments. c, Kaplan–Meier curves for overall survival from SB28 intracranial glioblastoma allografts showing no difference in overall survival after intratumor CED treatment with αIL6 (156.75 g in 15L) either alone or in combination with systemic αPD-1 therapy (250 g biweekly i.p.) compared to isotype control treatments (n=8-9 mice/condition). d, CD45+ immune cell CyTOF scaffold plot (n=104,388 cells) from SB28 intracranial glioblastoma allografts treated with isotype control at T2 vs T1 timepoints from Fig. 7b showing microglia in the tumor immune microenvironment decrease over time. e, CD45+ immune cell CyTOF scaffold plots from SB28 intracranial glioblastoma allografts treated with αPD-1 monotherapy vs isotype control at T1 (top, n=117,751 cells) or T2 (bottom, n= 194,205 cells) timepoints from Fig. 7b showing minimal change in the tumor immune microenvironment with αPD-1 monotherapy. f, CD3 IHC and Iba1 IHC sections from intracranial glioblastoma SB28 allografts in C57BL/6J mice treated with isotype control, systemic αIL6 monotherapy, or combined αPD-1/αIL6 therapy showing increases in Iba1 macrophages and CD3+ T cells following combination therapy. Scale bar, 100µm.