Context-dependent activation of STING-interferon signaling by CD11b agonists enhances anti-tumor immunity

Abstract

Chronic activation of inflammatory pathways and suppressed interferon are hallmarks of immunosuppressive tumors. Previous studies have shown that CD11b integrin agonists could enhance anti-tumor immunity through myeloid reprograming, but the underlying mechanisms remain unclear. Herein we find that CD11b agonists alter tumor-associated macrophage (TAM) phenotypes by repressing NF-κB signaling and activating interferon gene expression simultaneously. Repression of NF-κB signaling involves degradation of p65 protein and is context independent. In contrast, CD11b agonism induces STING/STAT1 pathway-mediated interferon gene expression through FAK-mediated mitochondrial dysfunction, with the magnitude of induction dependent on the tumor microenvironment and amplified by cytotoxic therapies. Using tissues from phase I clinical studies, we demonstrate that GB1275 treatment activates STING and STAT1 signaling in TAMs in human tumors. These findings suggest potential mechanism-based therapeutic strategies for CD11b agonists and identify patient populations more likely to benefit.

Keywords: CD11b; NF-κB; STING; immunotherapy; pancreatic cancer; tumor-associated macrophages.

Figure 1.. GB1275 anti-tumor activity is dependent on TAM reprograming.

(A) UMAP visualization of 54,000 TAMs from different cancer types, including PDAC, early gastric cancer (EGC), basal cell carcinoma (BCC), colorectal cancer (CRC), uveal melanoma (UVM), renal cancer (RCA), breast cancer (BRAC), ovarian serous cystadenocarcinoma (OVC), skin cutaneous melanoma (SKCM), and lung adenocarcinoma (LUAD). Clusters are annotated for cancer types. (B) GSEA identified pathway enrichment in TAMs from each cancer type. (C) Schematic of in vitro macrophage system. (D) QPCR mRNA expression analysis of BMDMs treated with TCM for 6 hours. Changes in gene expression are depicted as the fold change from the vehicle. (E-F) QPCR mRNA expression analysis of BMDMs isolated from wild-type(E) or CD11b-null mice (F) treated with TCM ± GB1275 for 7 hours. Changes in gene expressions are depicted as the fold change from the vehicle. (G) WT or CD11b-null mice bearing syngeneic orthotopic KP2 tumors were treated with vehicle or GB1275 for 14 days. Tumor volume and frequencies of tumor-infiltrating TAMs, CD8⁺ T cells, and Ki67⁺ CD8⁺ T cells are depicted (n = 6–8/group). (H) Mice bearing orthotopic KI tumors treated with lgG+ PBS or αCSF-1 IgG+ Clodronate as depicted. Tumor volume and frequencies of PDAC-infiltrating TAMs, CD8⁺ T cells, and Ki67⁺ CD8⁺ T cells at day 19 are depicted (n = 7–9/group). All graphs depict the mean ± SEM and *denotes a value of p < 0.05 using the two-sided t-test. In vitro data are representative of three independent experiments. Also see Figure S1.

Figure 2.. TAMs CD11b activation results in more proliferative effector T cells.

(A) Syngeneic KI orthotopic model and treatment (left). Tumor burden in each group (right) (n = 8/group). (B) Frequencies of TAMs and CD8⁺ T cells from A (n = 7/group). (C) CyTOF UMAP plot of tumor-infiltrating T cells. (D) Subpopulations of CD8⁺ T cells and CD4⁺ T cells. (E) Median expressions of Ki67 and GZMB in CD8⁺ T cells. (F) Percentage of individual subclusters in CD8⁺ T cells. (G) Syngeneic KP2-OVA orthotopic model treated with vehicle or GB1275 for 12 days, and frequencies of GZMB⁺ CD8⁺ T cells (n = 7/group). (H) Gate of functional assay of CD8⁺ T cells from G (left). Percentage of functional CD8⁺ T cells (right) (n = 7/group). Graphs depict the mean ± SEM and *denotes a value of p < 0.05 using a two-sided t-test for comparisons between two groups. Also see Figure S2.

Figure 3.. GB1275 downregulates NFκB/IL-1.

(A) UMAP scRNAseq plots of the whole TAM/Monocytes population (left), Itgam expression in this population (right). (B) UMAP scRNAseq plots of subclusters from TAM/monocyte population in 14 days-vehicle and GB1275 treated KP2 syngeneic model (left), Right, percentages of individual clusters. (C) GSEA identified pathway enrichment in the whole TAM/monocyte population (p<0.05). (D) GSEA identified pathway enrichment in four subclusters (p<0.05). (E) Volcano plot depicting GB1275- changed differentially expressed genes within the NFκB/IL-1 signaling pathway from the whole TAM/monocyte population. (F) QPCR mRNA expression analysis of BMDMs treated with GB1275 for 7 hours. Changes in gene expression are depicted as the fold change from the vehicle baseline. (G) Representative immunoblot for pp65, total p65, pIκB, total IκB, and β-ACTIN (loading control) in BMDMs treated with GB1275 ± TCM for 7 hours. (H) Representative immunoblot for total p65 and β-ACTIN (loading control) in BMDMs treated with GB1275 for 7 hours after 1 hour-MG132 pretreatment. (I) BMDMs were treated with GB1275 for 1 or 7 hours after 1 hour-MG132 pretreatment. Immunoblot for p65 from total lysates and polymer-ubiquitin from purified p65 protein. (J) Representative mpIHC staining for p65, CD11b, F4/80 and CK19 in tumors from 14 days-vehicle and GB1275 treated KPC mice, KP2 (K), and KI (L) orthotopic models. Scale bar, 100μm. Right, percentage of np65⁺ TAMs (n = 7–8 mice per group). (M) Kaplan–Meier survival curves for the top 80 downregulated NFκB/ IL-1 signaling-related genes from scRNAseq (E) in TCGA patient dataset for pancreatic adenocarcinoma (PAAD). Graphs show the mean ± SEM; *denotes p < 0.05 using the two-sided t-test for comparisons between two groups or log-rank test for Kaplan–Meier survival curves. In vitro data are representative of three independent experiments. Also see Figure S3, Table S1,2,3.

Figure 4.. GB1275 increases IFN/CXCL transcription via STING.

(A) QPCR mRNA expression analysis of BMDMs treated with different TCM ± GB1275 for 7 hours. Changes in gene expression are depicted as the fold change from the vehicle baseline. (B) Heat map of protein expression in BMDMs treated with vehicle or GB1275 ± TCM for 4 and 7 hours (left). Quantification of STING expression (right). (C) Heat map of integrin signaling-related protein expression. (D) QPCR mRNA expression analysis of BMDMs treated with TCM ± GB1275 for 7 hours after 1 hour-FAKi pretreatment. Changes in gene expression are depicted as the fold change from the vehicle baseline. (E) GSEA identified pathway enrichment in cluster TAM1 in 3B (p<0.05). (F) Intracellular total ROS in BMDMs stimulated by TCM + GB1275 for different time points (left). Quantification of ROS production (right). (G) BMDMs were treated by TCM ± GB1275 for 4 hours after 1 hour-FAKi pretreatment. Representative immunoblots for pFAK, SIRT3, and β-ACTIN (loading control). (H) Intracellular total ROS in BMDMs transfected with Sirt3 siRNA or ctrl siRNA for 24 hours prior to 7-hour TCM ± GB1275 (left). Quantification of ROS production (right). (I) QPCR analysis of mitochondrial genes released in cytoplasm from BMDMs treated with vehicle or GB1275 ± TCM for indicated time periods. Changes in gene expression are depicted as the fold change from the vehicle baseline. (J) QPCR mRNA expression analysis of BMDMs treated with TCM ± GB1275 for 7 hours after 1 hour-EtBr pretreatment. Changes in gene expression are depicted as the fold change from the vehicle baseline. (K) Representative immunoblots for STING, total IRF3, pIRF3, pSTAT1, total STAT1, and β-ACTIN (loading control) in BMDMs treated with TCM ± GB1275 for the indicated time periods (left). Quantification of STING and pIRF3 relative expression (right). (L) QPCR mRNA expression analysis of BMDMs isolated from wild-type or STING-null mice treated with TCM ± GB1275 for 7 hours. Changes in gene expression are depicted as the fold change from the vehicle baseline. (M) C57/BL-6 mice were lethally irradiated and adoptively transferred with BM from either wild-type mice or STING-null mice. KP2-syngeneic tumors were established on above mice and treated with vehicle or GB1275 for 14 days. Right, tumor growth curve expressed as percentages of tumor volume changes (n = 7–8/group). (N) Tumor-infiltrative CD8⁺ T cell frequencies from the above mice (n = 5–6/group). (O) The proposed model: GB1275 regulated two separate signaling pathways in macrophages, including p65/IL-1 inhibition and STING/IFN/CXCL axis activation. Graphs show the mean ± SEM; *denotes p < 0.05 using the two-sided t-test between two groups. In vitro data are representative of three independent experiments. Also see Figure S4, Table S4.

Figure 5.. STING activation by GB1275 in mouse models and tumor biopsies.

(A) Representative mpIHC staining for pSTAT1, STING, F4/80, CD11b, and CK19 in tumors from 14-day vehicle and GB1275 treated KPC mice. Scale bar, 100μm. Right, percentages of STING⁺ CD11b⁺ cells, STING⁺ TAMs, pSTAT1⁺ TAMs, and pSTAT1⁻ p65⁺ TAMs (n = 7–9 mice per group). (B) Representative mpIHC staining for STING, CD163, CK19, and CD8α in human PDAC TMAs. Scale bar, 50μm. (C) Average percentage of STING⁺ TAMs from the TMAs. (D) Scatter plot showing Spearman’s correlation between the percentage of STING⁺ macrophages and CD8⁺ T cells from B. (E) Kaplan–Meier survival curves for patients with high STING⁺ TAM infiltration and low STING⁺ TAM infiltration from B. (F) Representative mpIHC staining for STING, pSTAT1, p65, CD163, and pan-keratin (PanK) in 11 paired tumor biopsies from patients. Scale bar, 50μm. (G) Relative fold changes of STING⁺, STING^hi macrophages, pSTAT1⁺, pSTAT1^hi macrophages, and nuclear p65⁺ macrophages in paired pre- and post-groups. (H) Scatter plots showing Spearman’s correlations between the percentage of STING^hi macrophages and pSTAT1^hi macrophages in all 22 tumor biopsies. Scatter plots showing Spearman’s correlation between the percentages of STING^hi macrophages (out of total cells) and CD8⁺ T cells in all 22 tumor biopsies (right). Graphs show the mean ± SEM; *denotes p < 0.05 using the two-sided t-test between two groups, one sample t-tests, and Wilcoxon tests, or the log-rank test for Kaplan–Meier survival curves. Also see Figure S5, Table S5.

Figure 6.. Chemotherapy or radiation therapy combined with GB1275, amplifies STING/IFN signaling.

(A) Concentrated protein (> 3 kDa) and metabolites (< 3 kDa) in TCM were separated by a protein concentrator (left). QPCR mRNA expression analysis of BMDMs treated with different fractions of TCM ± GB1275 for 7 hours. Changes in gene expression are depicted as the fold change from the vehicle baseline (right). (B) KP2 cells were treated with either gemcitabine (left) or radiation (right). TCM was made from above cells. QPCR mRNA expression analysis of BMDMs treated with the above mentioned TCM ± GB1275 for 7 hours. Changes in gene expression are depicted as the fold changes from the vehicle baseline. (C) Tumor growth of KP2 syngeneic model treated with vehicle or GB1275 ± chemotherapy (left). Mean percent change in tumor volume on day 12 (n = 9–10/group) (middle). Right, Kaplan–Meier survival analysis (n = 9–10/group). (D) QPCR mRNA expression analysis of tissue from C (n = 6–8/group). Changes in gene expression are depicted as fold changes from the vehicle baseline. (E) IFNβ, CXCL10, 11, and IL-1β production in tissues from a syngeneic KP2-OVA orthotopic model treated with vehicle or GB1275 + chemotherapy for 12 days. (n = 7–8 group). (F) Syngeneic KP2-OVA orthotopic model treated with vehicle, GB1275, chemotherapy, or combination for 12 days. Frequencies of tumor-infiltrating CD8⁺ T cells, Dex⁺ CD8⁺ T cells, and proliferative Dex⁺ CD8⁺ T cells (n = 6/group). (G) Frequencies of Dex⁺ CD8⁺ T cells, proliferative CD8⁺ T cells, and proliferative Dex⁺ CD8⁺ T cells in dLN from F. (H) C57/BL-6 mice were lethally irradiated and adoptively transferred with BM from either wild-type mice or STING-null mice. KP2-OVA orthotopic model was established on above mice and treated with chemotherapy ± GB1275 for 12 days. Frequencies of tumor-infiltrating CD8⁺ T cells, Dex⁺ CD8⁺ T cells, and proliferative Dex⁺ CD8⁺ T cells (n = 6/group). (I) Frequencies of Dex⁺ CD8⁺ T cells and proliferative Dex⁺ CD8⁺ T cells in dLN from H. The graphs show mean ± SEM; *denotes p < 0.05 using the two-sided t-test between two groups or log-rank test for Kaplan–Meier survival curves. In vitro data are representative of three independent experiments. Also see Figure S6.

Figure 7.. STING agonist synergizes with GB1275 and remodels TME.

(A) Schematic of several combinations of GB1275. (B) Heat map of relative mRNA expression from BMDMs after treatment (A). Percentages of changes in gene expression are depicted as the fold change from the vehicle baseline. (C) Tumor burden from the KP2-OVA syngeneic mouse model treated with vehicle or GB1275 ± TLR7 agonist or TLR9 agonist for 14 days (n = 9–10/group). (D) Immunohistochemical staining for CD8⁺ T cells in tumors from 14-day vehicle, GB1275, TLR9 agonist or combination-treated KPC mice. Right, average percentage of CD8⁺ T. (E) Tumor burden from the KP2 syngeneic mouse model treated with vehicle or GB1275 ± ADUS100 for 19 days (n = 10/group). Frequencies of tumor-infiltrating CD8⁺ T cells and macrophages (n = 6/group). (F) Immunohistochemical staining for CD8⁺ T cells in tumors from 14-day vehicle, GB1275, ADUS100, or combination-treated KPC mice. Scale bar, 100μm. Right, average percentage of CD8⁺ T cells (n = 6–7/group). (G) Tumor burden from KP2 syngeneic mouse model treated with ADU-S100 or combination of GB1275 ± CD4/CD8⁺ T cell depletion for 12 days (left). (H) KP2 syngeneic model treated with GB1275, ADU-S100, or combination for 14 days (left). CyTOF UMAP plot of tumor-infiltrating T cells, including CD8⁺ T cells, Th, and Tregs (n = 6/group). (I) Percentages of individual subclusters in CD8⁺ T cells. (J) Median expressions of GZMB and CTLA-4 in CD8⁺ T cells. (K) CyTOF UMAP plot of tumor-infiltrating myeloid cells, including TAMs, granulocytes, monocytes, and dendritic cells from H (n = 6/group) (left). Percentages of individual subclusters in TAMs (right). (L) Tumor growth of KP2 syngeneic model treated with vehicle or GB1275 ± radiation therapy (6 Gy × 5) ± ADU-S100 (left). Mean percent change in tumor volume on day 12 (n = 10/group) (middle). (right) Kaplan–Meier survival analyses (n =10/group). Graphs show the mean ± SEM; *denotes p < 0.05 using the two-sided t-test between two groups, analysis of variance or log-rank test for Kaplan–Meier survival curves. In vitro data are representative of three independent experiments. Also see Figure S7.