IFNγ and CCL2 Cooperate to Redirect Tumor-Infiltrating Monocytes to Degrade Fibrosis and Enhance Chemotherapy Efficacy in Pancreatic Carcinoma

Abstract

Dense fibrosis and a robust macrophage infiltrate are key therapeutic barriers in pancreatic ductal adenocarcinoma (PDAC). CD40 activation can circumvent these barriers by inducing macrophages, originating from peripheral blood monocytes, to deplete fibrosis. The precise mechanism and therapeutic implications of this antifibrotic activity, though, remain unclear. Here, we report that IFNγ and CCL2 released systemically in response to a CD40 agonist cooperate to redirect a subset of Ly6C(+)CCR2(+)monocytes/macrophages to infiltrate tumors and deplete fibrosis. Whereas CCL2 is required for Ly6C(+)monocyte/macrophage infiltration, IFNγ is necessary for tumor-infiltrating monocytes/macrophages to shift the profile of matrix metalloproteinases (MMP) in tumors, leading to MMP-dependent fibrosis degradation. In addition, MMP13-dependent loss of extracellular matrix components induced by a CD40 agonist increased PDAC sensitivity to chemotherapy. Our findings demonstrate that fibrosis in PDAC is a bidirectional process that can be rapidly altered by manipulating a subset of tumor-infiltrating monocytes, leading to enhanced chemotherapy efficacy.

Significance: We report that CD40 agonists improve chemotherapy efficacy in pancreatic carcinoma by redirecting tumor-infiltrating monocytes/macrophages to induce fibrosis degradation that is dependent on MMPs. These findings provide novel insight into the plasticity of monocytes/macrophages in cancer and their capacity to regulate fibrosis and modulate chemotherapy efficacy in pancreatic carcinoma.

Figure 1. Monocyte subsets show distinct trafficking patterns in response to agonist CD40 therapy in KPC mice

(A) Peripheral blood counts for F4/80⁺Gr-1^neg resident monocytes (RM) and F4/80⁺Gr-1⁺ inflammatory monocytes (IM) in control (Ctrl) littermates and tumor-bearing KPC mice. Bar shows mean; n=17-25 per group, *, P<0.05; ****, P<0.0001. (B) Percent change in monocyte subsets within blood of KPC mice one day after treatment. n=4-7 per group, *, P<0.05. (C) KPC mice were treated with unlabeled liposomes (Ctrl) versus DiI-labeled liposomes (DiI⁺ Lipo) and blood was analyzed 24 hours later. Shown is a representative histogram of CD45⁺CD19^negF4/80⁺ monocytes labeled with DiI-liposomes in vivo. (D) Representative FACS plot showing relationship between labeling with DiI-liposomes in vivo and Ly6C expression to identify subsets of CD45⁺CD19^negF4/80⁺ monocytes. (E) KPC mice were injected i.p. with DiI-labeled liposomes one hour before treatment with istoype control or anti-CD40 antibodies. Tumor tissue was analyzed 48 hours later. Shown is immunofluorescence imaging of peritumoral lymph nodes and tumor to detect monocyte/macrophages labeled with DiI-labeled liposomes (red) and EpCAM⁺ tumor cells (green). n=3 mice. One representative image of three is shown per group. Scale bar, 100 μm. (F) Representative images of anti-Gr-1 stained tumor tissue from KPC mice one day after treatment. Scale bar, 100 μm. Quantification of (G) Ly6C⁺ myeloid cells (n=4-5 mice per group) and (H) Ly6G⁺ cells (n≥3 mice group) within tumor tissue of KPC mice one day after treatment. *, P<0.05, unpaired 2-tailed t-test.

Figure 2. Anti-fibrotic activity of a CD40 agonist requires CCL2-dependent inflammatory monocyte recruitment to tumor tissue

KPC mice were treated with isotype control antibody, anti-CD40, or anti-CD40 in combination with clodronate encapsulated liposomes (CEL), anti-Gr-1, anti-Ly6C or anti-CCL2. Shown are (A) representative images of PDAC sections stained with Masson's trichrome to detect extracellular matrix deposition (blue) one day after treatment and (B) quantification of extracellular matrix. n = 3-9 mice per group. Scale bar, 100 μm. (C) Quantification of Ly6C⁺ myeloid cells in PDAC tumors of KPC mice one day after treatment. n=3-4 mice per group. (D) Plasma CCL2 levels in PDAC patients at baseline (Pre) and 24 hours post treatment with the fully human CD40 agonist CP-870,893 n=5-13 subjects per group. Significance determined using Mann-Whitney test. (E) Serum CCL2 levels in KPC mice one day after treatment with isotype control (Ctrl) or anti-CD40 antibodies administered with or without CEL. n=7-14 mice per group. (F) Relative Ccl2 mRNA levels in PDAC implanted tumors from mice at one day after treatment. n=9-10 mice per group. (G) Ratio of peripheral blood:bone marrow F4/80⁺ Ly6C⁺ inflammatory monocytes (IM) one day after treatment. n = 4-6 mice per group. Significance testing was performed using unpaired 2-tailed Student's t test, unless otherwise specified. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure 3. IFN-γ/STAT-1 signaling is necessary to redirect inflammatory monocytes with anti-fibrotic activity

(A) Wild-type non-tumor bearing mice and KPC tumor-bearing mice were treated with isotype control (Ctrl) or anti-CD40 antibody. Serum was collected 24 hours after treatment for analysis. Shown are serum levels (pg/mL) of IFN-γ. n=17-20 mice per group for wild-type and n=10 mice per group for KPC. **, P<0.01; ***, P< 0.001. (B) Plasma cytokine levels were determined in PDAC patients at baseline (Pre) and 24 hours (Day +1) post treatment with the fully human CD40 agonist CP-870,893. Shown are levels (pg/mL) for IFN-γ. n=13 patients at baseline; n=5 patients after CP-870,893; **, P < 0.01; Mann-Whitney test. (C) Intracellular flow cytometry detection of pSTAT1 in murine peripheral blood leukocyte subsets one day after treatment. MFI, mean fluorescence intensity. n=5 per group, from two independent experiments; *, P<0.05, unpaired t-test. (D) Shown is pSTAT1 expression in human monocytes treated with plasma collected from PDAC patients at baseline (Pre) and multiple time points after CP-870,893 treatment. n=3; ****, P<0.0001, one-way Anova with Bonferroni correction for multiple pairwise comparison testing. (E) Representative immunofluorescence images showing co-localization of pSTAT1 in Ly6C⁺ inflammatory monocytes in PDAC tumors of KPC mice one day after treatment. Scale bar, 50 μm. (F) Quantification of pSTAT1 expression detected one day after treatment by immunofluorescence staining of tumor sections. n=4-5 mice per group; ****, P < 0.0001; unpaired 2-tailed t-test. (G) Quantification of extracellular matrix protein within tumors. Tumor sections were analyzed one day after treatment with control (Ctrl) or anti-CD40 antibodies with or without anti-IFN-γ antibody neutralization. n=5-9 mice per group; *, P < 0.05; **, P < 0.01; unpaired 2-tailed t-test determined from 4-6 images per mouse.

Figure 4. CD40 agonist induces a shifts in the MMP gene expression profile in tumors that is dependent on inflammatory monocytes and IFN-γ

(A) Heat map showing global gene expression in tumor homogenates from mice with implanted PDAC tumors one day after treatment with control (n = 9) or anti-CD40 (n = 10). Statistical significance was determined using one-way Anova with Bonferroni correction for multiple comparisons testing. P values are shown to right of heat map. (B) Tumor-bearing mice were treated with control or anti-CD40 with or without depletion of Ly6C⁺ inflammatory monocytes or Ly6G⁺ granulocytes. Shown is relative transcript expression for Mmp3, Mmp9, Mmp10, Mmp12, and Mmp13. n=5-10 mice per group. (C) qRT-pCR analysis from tumor homogenate cDNA isolated one day after treatment. Displayed are fold changes in Mmp genes relative to control treatment. For panels B-C, significance testing was performed using unpaired 2-tailed Student's t test. *, P < 0.05; **, P<0.01; ****, P<0.0001.

Figure 5. Anti-fibrotic activity of a CD40 agonist is dependent on MMPs

Mice with implanted PDAC tumors were analyzed one day after treatment with isotype control or anti-CD40 antibody with or without anti-Ly6C or anti-IFN-γ antibodies. (A) Shown is quantification by immunofluorescence staining of MMP13 expressing cells in tumors. n=5-12 mice per group. (B) Representative immunofluorescence images showing MMP13 (green) expression by F4/80⁺ (red, top panel) and FAP⁺ (red, bottom panel) cells in tumors one day after treatment with anti-CD40. Scale bar, 10 μm. (C) Representative immunofluorescence images showing MMP14 (green) expression by F4/80⁺ (red) cells in tumors after treatment with control (top panel) and anti-CD40 (bottom panel). Scale bar, 20 μm. (D) Mice with implanted tumors were analyzed one day after treatment with isotype control or anti-CD40 with or without a broad spectrum MMP inhibitor (iMMP; Actinonin). Representative immunofluorescence images show type I collagen (TIC, red) among Ly6C⁺ inflammatory monocytes (green) one day after treatment. Nuclei are stained with DAPI (blue). One representative image is shown out of five images per tumor section per group. Scale bar, 100 μm. (E) Quantification of type I collagen and (F) fibronectin within tumors after isotype control (Ctrl) or anti-CD40 treatment with or without MMP inhibitor (iMMP; Actinonin). n=3-4 mice per group. (G) Quantification of Ly6C⁺ cells in tumor tissue after treatment. n=3-4 mice per group. (H) Quantification of extracellular matrix detected in tumors by Masson's Trichrome one day after treatment with isotype control or anti-CD40 antibodies with or without selective inhibitors of MMP13 (iMMP13 #1, WAY-170523; iMMP13 #2, 544678-85-5) or a broad spectrum MMP inhibitor (iMMP, Actinonin). n=4-8 per group. For panels A and E-H, significance testing was performed using unpaired 2-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 6. CD40 agonist improves gemcitabine efficacy in PDAC

(A) Representative H&E, Masson's Trichrome, and gross images of implanted PDAC tumors obtained from mice at defined time points after treatment with anti-CD40 antibodies with comparison to isotype control (Ctrl). Scale bars, 500 μm (H&E) and 100 μm (Masson's Trichrome). (B) Quantification of collagen content in PDAC tumors detected by Masson's trichrome relative to control treatment. n=7-8 tumors per group. (C) Quantification of tumor necrosis seen on H&E imaging. n=7-8 tumors per group. (D) Mice with implanted PDAC tumors were treated with anti-CD40 on day 0 followed by gemcitabine on day 2 or 5 as indicated with comparison to gemcitabine alone. Shown is quantification of tumor necrosis and cellular proliferation (Ki67) detected by H&E and immunohistochemical staining, respectively, at one day after gemcitabine treatment. n=5 mice per group. (E) Mice with implanted PDAC tumors were treated with or without anti-CD40 antibody on day 0 followed by gemcitabine on day 5. Shown is fold change in tumor volume from time of gemcitabine treatment to 6 days later. n = 8-9 mice per group. (F) Effect of anti-Ly6C, anti-IFN-γ, broad spectrum MMP inhibitor (iMMP, Actinonin) and MMP13 specific inhibitors (iMMP13 #1, WAY-170523; iMMP13 #2, 544678-85-5) on tumor necrosis induced with gemcitabine administered 5 days after anti-CD40 therapy with comparison to anti-CD40 and gemcitabine alone. Tumor necrosis is quantified one day after treatment with gemcitabine. For panels B-F, significance testing was performed using one-way Anova with Bonferroni correction for multiple pairwise comparisons or unpaired 2-tailed Student's t test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure 7. Proposed step-wise model for anti-fibrotic activity induced with a CD40 agonist in PDAC

Treatment with a CD40 agonist induces the systemic release of IFN-γ which activates CCR2⁺ inflammatory monocytes (IM) and polarizes their phenotype toward anti-fibrotic (Step 1: Polarization). Inflammatory monocytes are then recruited to the tumor microenvironment in a CCL2-dependent manner that is regulated by resident monocytes/macrophages (RM) residing outside of the tumor microenvironment (Step 2: Trafficking). Within the tumor microenvironment, tumor infiltrating inflammatory monocytes alter the MMP profile leading to rapid degradation of extracellular matrix proteins including type I collagen and fibronectin (Step 3: Effector Activity). Together, depletion of extracellular matrix components induced by inflammatory monocytes enhances the sensitivity of tumors to gemcitabine chemotherapy.