Intratumoral administration of cGAMP transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site

Abstract

Stimulator of IFN genes (STING) spontaneously contributes to anti-tumor immunity by inducing type I interferons (IFNs) following sensing of tumor-derived genomic DNAs in the tumor-bearing host. Although direct injection of STING ligands such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic [G(2',5')pA(3',5')p] (cGAMP) into the tumor microenvironment exerts anti-tumor effects through strong induction of type I IFNs and activation of innate and adaptive immunity, the precise events caused by STING in the tumor microenvironment remain to be elucidated. We describe here our finding that a CD45⁺ CD11b^mid Ly6C⁺ cell subset transiently accumulated in mouse tumor microenvironment of 4T1 breast cancer, squamous cell carcinomas, CT26 colon cancer, or B16F10 melanoma tissue after intratumoral injection of cGAMP. The accumulated cells displayed a macrophage (M ) phenotype since the cells were positive for F4/80 and MHC class II and negative for Ly6G. Intratumoral cGAMP treatment did not induce Mφ accumulation in STING-deficient mice. Depletion of CD8⁺ T cell using anti-CD8 mAb impaired the anti-tumor effects of cGAMP treatment. Depletion of the Mφ using clodronate liposomes impaired the anti-tumor effects of cGAMP treatment. Functional analysis indicated that the STING-triggered tumor-migrating Mφ exhibited phagocytic activity, production of tumor necrosis factor alpha TNFα), and high expression levels of T cell-recruiting chemokines, Cxcl10 and Cxcl11, IFN-induced molecules, MX dynamin-like GTPase 1 (Mx1) and 2'-5' oligoadenylate synthetase-like 1 (Oasl1), nitric oxide synthase 2 (Nos2), and interferon beta 1 (Ifnb1). These results indicate that the STING-triggered tumor-migrating Mφ participate in the anti-tumor effects of STING-activating compounds.

Keywords: Immunotherapy; Macrophages; STING; Tumor migration; cGAMP.

Fig. 1

Increased CD11b^mid Ly6C⁺ cells in TILs by intratumoral cGAMP treatment. a 4T1, b mSCC, or c CT26-bearing mice received intratumoral injection of PBS or cGAMP on day 5. After 16–24 h of the treatment, tumor tissues were resected and TILs were collected for flow cytometric analysis using anti-CD45, anti-CD11b, and anti-Gr-1mAb. Representative flow histograms are shown. The percentages of the increased fraction (CD45⁺ CD11b^mid Gr-1^mid cells) in TILs are depicted in the rightmost panel. *P < 0.05, ***P < 0.0001, based on an unpaired t test. Data are pooled from two to three independent experiments each with two to three mice per group. d Representative flow histograms of TILs using a combination of anti-CD11b, anti-Ly6C, and anti-Ly6G mAbs. CD11b^mid or CD11b^high cells were gated in R7 or R8, respectively. e 4T1-bearing mice received intratumoral injections of PBS or cGAMP on day 5. After 16–24 h of the treatment, tumor tissues were resected and TILs were collected for flow cytometric analysis using anti-CD11b, anti-Gr1, and anti-F4/80 mAb. Representative flow histograms are shown. f Representative flow histograms of CD45-gated TILs stained with anti-CD11b and anti-Gr-1mAb (left panel). The morphological features of separately sorted fractions of CD11b^high Gr-1^high cells (upper) and CD11b^mid Gr-1^mid cells (lower) were defined by Diff-Quick staining (rightmost panel in each). Bar 10 µm

Fig. 2

Robust accumulation of CD11b^mid Ly6C⁺ F4/80⁺ Mφ in tumor tissue by intratumoral cGAMP treatment. a Percentages of the CD11b^mid Ly6C⁺ cells in TILs of 4T1-bearing mice treated with control (closed circles) or cGAMP (open circles) were evaluated at the indicated time points. *P < 0.05, based on an unpaired t test. Data are pooled from two to three independent experiments each with two to four mice per group. b Percentages of the CD11b^mid Ly6C⁺ cells in TILs of 4T1-bearing mice were assessed at 72 h after treatment of PBS or cGAMP. c Immunofluorescent staining of F4/80⁺ cells (red) in the 4T1 tumor tissues after 24 h of treatment with PBS (upper panel) or cGAMP (lower panel). Bar 100 µm. d Representative flow cytometric profiles of expressions of MHC class I and class II in the accumulated cells of the TILs collected from cGAMP-treated mice. The representative merged histograms show the fluorescent intensity of antigen-presenting cells for the isotype control (black), class I and class II (red)

Fig. 3

CD8⁺ T cell-dependent anti-tumor effect by intratumoral injections of cGAMP. a 4T1 (1.5 × 10⁵)-bearing mice received intratumoral injections of cGAMP or PBS control on days 5 and 10. The anti-tumor effects of cGAMP (N = 7, open triangles) or control (N = 8, closed triangles) treatment were assessed by measuring tumor size. b mSCC1 (2.0 × 10⁵)-bearing mice received intratumoral injections of cGAMP or PBS control on days 5 and 10. The anti-tumor effect of cGAMP (N = 11, open triangles) or control (N = 9, closed triangles) treatment of the mSCC1-bearing mice was assessed by measuring tumor size. c CT26 (2.0 × 10⁵)-bearing mice received intratumoral injections of cGAMP or PBS control on days 5 and 10. The anti-tumor effect of cGAMP (N = 11, open triangles) or control (N = 11, closed triangles) treatment of the CT26-bearing mice was assessed by measuring tumor size. d 4T1 (1.5 × 10⁵)-bearing mice that intraperitoneally received anti-CD8mAb (closed triangle) or isotype antibody (open triangle) at the day before intratumoral treatment with cGAMP on days 5 and day 10. 4T1-bearing mice in a control group (open circle) intratumorally treated with PBS without any antibodies. The anti-tumor effects of each treatment in the mice (N = 5 to 6 in each group) were assessed by measuring tumor size. *P < 0.05, based on a two-way ANOVA test. Representative data are shown from two independent experiments. e 4T1 (1.5 × 10⁵)-bearing mice intraperitoneally received the clodronate liposomes followed by intratumoral cGAMP treatment or control treatment. The anti-tumor effects of cGAMP or control treatment in the mice (N  =  6 in each group) were assessed by measuring tumor size

Fig. 4

STING signaling-dependent accumulation of Mφ in the tumor tissues and anti-tumor effects by intratumoral cGAMP treatment. B16F10 (2.0 × 10⁵)-bearing WT and STING-deficient (gt) mice received intratumoral injections of cGAMP or PBS control on days 5 and 10. a After 16–24 h of the treatment on day 5, tumor tissues were resected and TILs were collected for flow cytometric analysis using anti-CD45, anti-CD11b, and anti-Gr-1 mAb. Representative flow histograms are shown. The percentages of the increased fraction (CD45⁺ CD11b^mid Gr-1^mid cells) in TILs are depicted in the rightmost panel. *P < 0.05, based on an unpaired t test. Data are pooled from two to three independent experiments each with two to three mice per group. The anti-tumor effects of cGAMP (open triangles, WT mice; open circles, gt mice) or control treatment (closed triangles, WT mice; closed circles, gt mice) were assessed by measuring tumor size (b) and by monitoring survival of each mouse (c). *P < 0.05, based on a two-way ANOVA test for tumor growth curve. ****P < 0.0001, based on a Log-rank (Mantel–Cox) test for survival curve. Mice with tumors >300 mm² were sacrificed. Data are pooled from two independent experiments with four to six mice per group

Fig. 5

Immunological property of STING-triggered tumor-migrating Mφ (a) 4T1-bearing mice received fluorescein beads (Red) intratumorally following intratumoral cGAMP treatment. Two hours later, TILs were collected and CD45⁺ cells were isolated using a magnetic cell separation system. The isolated cells were stained with FITC-anti-Ly6C mAb and their morphological features were then microscopically analyzed following Diff-Quick staining. Bar 10 µm. b Representative flow cytometric profiles of TNFα and IL-10-producing cells gated on CD11b⁺ and Ly6C⁺ Mφ of the TILs collected from cGAMP-treated mice. The representative merged histograms show the fluorescent intensity of PE for the isotype control (black), TNFα (red), and IL-10 (blue) and each MFI level is depicted in the right most panel. *P < 0.05, based on an unpaired t test. (C, D) mRNA expression levels of Cxcl10, Cxcl11, Nos2, Ifnb1, Mx1, and Oasl1 were determined by real-time PCR in CD11b^mid Ly6C⁺ cells sorted from the tumor tissues treated with control or cGAMP. **P < 0.01. based on an unpaired t test