Research ArticleCancer

Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies

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Science Translational Medicine  03 Jul 2019:
Vol. 11, Issue 499, eaau9240
DOI: 10.1126/scitranslmed.aau9240
  • Fig. 1 PDAC has a dense CD11b+ myeloid infiltrate.

    (A) Representative images of human PDAC and adjacent normal tissues assessed for CD11b+ myeloid cells at 2.5×, 10×, and 20× (inset). Graphs show the frequency of the subsets in human PDAC and adjacent normal tissues from the same surgical sample (n = 13 paired samples). (B to D) CD11b expression determined by CyTOF analysis of human PDAC tissue samples. (B) Representative CyTOF tSNE (t-distributed stochastic neighbor embedding) plot showing monocytes, granulocytes, macrophages, T cells, and B cells. (C) Expression analysis of CD11b in leukocyte populations. (D) Relative frequencies of the composition of CD11b+ cells (n = 7). (E) Representative images of CD15+ and CD14+ monocytes and macrophages by IHC.Graphs show the frequency of positive cells in human PDAC and adjacent normal tissues from the same surgical sample (n = 12 to 15 per paired samples). (F and G) CD11b expression analysis of murine PDAC tissues. (F) Representative flow cytometry plots showing expression of CD11b on pregated tumor-infiltrating immune cell populations in an orthotopic KP2 PDAC model. (G) Graphs depicting the cell composition of total leukocytes (left) and CD11b+ cells, as well as the number of CD11b+ cells per total number of leukocytes (right) (n = 8 mice per graph). G-MDSCs, granulocytic-myeloid derived suppressor cells; Mo-MDSCs, monocytic-myeloid derived suppressor cells. (H and I) Leukocyte proximity analysis. (H) Representative images of CD8a and CD11b (brown) costaining with CK19 (pink). Histogram of relative CD8a+ or CD11b+ cell numbers binned by cellular distances from CK19+ cells. (I) Mean number of positive cells per area within 60 μm of the CK19+ tumor cells (n = 23 PDAC samples). Graphs show the mean ± SE; *P < 0.05 by two-tailed t test.

  • Fig. 2 Changes in myeloid infiltrates after ADH-503 treatment.

    (A) Chemical structure of ADH-503. (B) A computational model of ADH-503 bound integrin CD11b/CD18 based on the published structure of αAβ2 (54). The integrin chains CD11b (yellow), CD18 (green and red), and the αA-domain of CD11b (blue) are labeled. The model also displays ADH-503 (space-filling model) docked in the activation-sensitive allosteric pocket of the CD11b αA-domain. (C) Plasma concentration-time data after oral gavage administration of ADH-503 at 30 mg/kg in male rats on days 1 and 5. n = 4 per group. (D) Representative flow cytometry plots of PE+ beads taken up by CD11b+ cells in PDAC tissue from the KP2 orthotopic PDAC model. (E) Quantification of PE+ beads taken up by tumor-infiltrating CD11b+ cells, monocytes, granulocytes, or macrophages with and without ADH-503 treatment (n = 3 per group). (F to H) RNA-seq expression analysis of bone marrow–derived macrophages treated with PDAC conditioned media ± ADH-503 for 7 hours. (F) Heat map of differentially expressed genes, (G) gene ontology (GO) table, and (H) select gene changes are depicted. (I) Q-PCR mRNA expression analysis of bone marrow–derived macrophages treated with PDAC conditioned media ± ADH-503 or vehicle for 7 hours. Changes in gene expression are depicted as the fold change from the vehicle baseline. Graphs show the mean ± SE; *P < 0.05 by two-tailed t test.

  • Fig. 3 ADH-503 alters innate responses in PDAC tissues.

    (A to D) Relative frequencies of tumor-infiltrating granulocytes, monocytes, eosinophils, B cells, NK cells, and macrophages in orthotopic KP2 or KI PDAC models 10 days after treatment with ADH-503 or vehicle (n = 6 per group). (E) Representative immunofluorescent images of GR1 and CD68 in PDAC tissues from KPC mice treated with vehicle or ADH-503 for 14 days. Quantification is shown as the cell number per area in PDAC tissues from KPC mice treated for 14 days or until end-stage tumors developed (n = 5 to 7 mice per group). Rx indicates treatment. (F and G) Flow cytometry analysis of antigen presentation markers on TAMs. Data are shown as histograms of geometric mean fluorescent intensity (Geo-MFI) data on TAMs in orthotopic KP2 and KI PDAC models treated with ADH-503 or vehicle for 10 or 12 days (n = 5 to 7 per group). (H) Q-PCR analysis on TAMs isolated by fluorescence-activated cell sorting (FACS) from orthotopic KP PDAC tumors 10 days after treatment with ADH-503. Data are shown as the fold change from the vehicle baseline (n = 4 samples per group). Graphs are shown as the mean ± SE; *P < 0.05 by two-tailed t test. All flow cytometry data are representative of two to three independent in vivo experiments using both tumor models. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.

  • Fig. 4 CD11b agonism stimulates T cell infiltration and function through augmentation of cDC1s.

    (A and B) Frequencies of tumor-infiltrating CD8a+ CTLs, FOXP3+ Tregs, and CD4+ effectors in orthotopic KP2 PDAC tissues from mice treated 10 to 12 days with ADH-503 or vehicle. Graphs show the mean CTL number and subsets of CD8+ CTLs marked by CD44HiCD62LLow, Ki-67, PD-1Hi, Tim3+PD-1+, or Eomes+PD-1+ (n = 5 to 6 per group). (C) Measurement of PD-L1 expression by flow cytometry of the CD45 population in orthotopic KP2 PDAC tissues 10 days after treatment with ADH-503 or vehicle. Representative histograms and Geo-MFI are shown. (D) Frequencies and phenotypes of tumor-infiltrating CD8a+ CTLs in orthotopic KI PDAC tissues from mice treated 12 days with ADH-503 or vehicle. Graphs show the mean CTL number and subsets of CD8a+ CTLs marked by CD44HiCD62LLow, Ki-67, or PD-1Hi (n = 5 to 6 per group). (E) Representative IHC images of CD8a (brown) and CK19 (pink) in PDAC tissues derived from KPC mice treated with vehicle or ADH-503 for 14 days. The histogram shows the relative CD8a+ cell number or frequency binned by cellular distances from CK19+ cells. Mean number of positive cells per area within 60 μm of CK19+ tumor cells (n = 6 per group). (F and G) Frequencies and quantification of ovalbumin (OVA)–specific dextramer+ cells in PDAC tissues (F) and dLNs (G) using the orthotopic KP2-OVA PDAC model in mice treated with vehicle or ADH-503 for 10 days. (H) Frequencies of CD11b+ and CD103+ DCs and MHC-I and MHC-II expression in CD103+ cDCs in KP2 PDAC tissues from mice treated with vehicle or ADH-503 for 12 days. Mean cell percentages and Geo-MFI data are shown. (I) Quantification of CD8+ T cells in KP2-OVA PDAC tissues from wild-type and BATF3-deficient mice treated with vehicle or ADH-503 for 10 days. Mean cell number per area and representative IHC images are shown. Graphs show the mean ± SE; *P < 0.05 by two-tailed t test (A to H) or Mann-Whitney test (G), depending on the data distribution or Kolmogorov-Smirnov test for immune cell proximity (E). All flow cytometry data are representative of two to three independent in vivo experiments using at least two PDAC models.

  • Fig. 5 CD11b agonism delays tumor progression.

    (A) Tumor growth in syngeneic orthotopic models of PDAC KP2, KI, and KP2-OVA shown by tumor weights 10 days after treatment with vehicle or ADH-503 (n = 7 to 10 per group). (B) Changes in tumor volume as measured by ultrasound imaging. Representative ultrasound images of KI PDAC tumors and mean percent changes in tumor volume are depicted 14 days after treatment. Yellow line depicts tumor area. (C) Kaplan-Meier survival analysis of orthotopic tumors KI tumors treated with vehicle or ADH-503 (n = 7 to 8 per group). (D) Genetic KPC mice were treated with vehicle or ADH-503. Tumor weight of mice treated for 14 days and Kaplan-Meier survival analysis are shown (n = 6 to 7 or 10 to 12 per group, respectively). (E) Subcutaneous (SubQ) KP2 tumor growth in wild-type or CD11b-deficient animals treated with vehicle or ADH-503 once the tumor reached 75 to 100 cm3. Tumor volume was measured by calipers (n = 7 to 8 per group). (F) Orthotopic KP-OVA tumor burden measured 10 days after treatment with vehicle or ADH-503 in wild-type or BATF3-deficient mice or in wild-type mice treated with CD4- and/or CD8-depleting IgGs or neutralizing IgGs against CXCR3 (n = 5 to 10 per group). (G) Analysis of PDAC pathology. Shown are representative hematoxylin and eosin (H&E) images with histological grading, IHC results of tumor and stromal Ki-67 staining, tumor cleaved caspase 3 staining, Sirius red–stained collagen density, and smooth muscle actin+ (SMA+) or FAP+ fibroblasts in PDAC tissues from KPC tumors treated with vehicle or ADH-503 for 14 days or at end stage (n = 5 to 10 mice per group). Bar graphs show the mean ± SE; *P < 0.05 by two-tailed t test, log-rank test or analysis of variance (ANOVA) as appropriate. Tumor burden data are representative of two to three independent in vivo experiments.

  • Fig. 6 CD11b agonism improves the efficacy of chemotherapy.

    (A) Changes in tumor volume as measured by ultrasound imaging. Animals were enrolled when the orthotopic KI tumor was greater than 0.4 cm in diameter and subsequently treated with vehicle or ADH-503 ± GEM/PTX. Representative ultrasound images and mean percent change in tumor volume are shown 12 days after treatment (n = 8 to 10 per group). (B) Kaplan-Meier survival analysis of mice from (A) (n = 8 to 10 per group). (C) Quantification of the percentage of mice bearing overt liver metastases on gross examination (n = 7 to 10 per group). (D) Changes in tumor volume as measured by ultrasound imaging. Animals were enrolled when the orthotopic KI tumor was greater than 0.4 cm in diameter and subsequently treated with vehicle or ADH-503 ± RT (4 Gy × 5). Representative ultrasound images and mean percent change in tumor volume are shown 12 days after treatment (n = 8 to 10 per group). Bar graphs show the mean ± SE; *P < 0.05 by two-tailed t test or log-rank test.

  • Fig. 7 CD11b agonism renders PDAC tumors responsive to checkpoint immunotherapy.

    (A) Tumor burden 12 to 14 days after treatment with vehicle or ADH-503 ± anti–PD-1 in orthotopic KP2-OVA or KI models (n = 8 to 10 per group). Dashed line depicts tumor burden from five parallel mice taken at start of treatment. (B) Analyses of survival and rechallenge. KI tumor-bearing mice from (A) were assessed for Kaplan-Meier survival analysis (left), pathological analysis of the pancreas was performed in mice surviving more than 120 days (middle), and mice treated with this regimen were rechallenged with KI tumor cells subcutaneously (right). “Control” mice were animals who had never been exposed to KI tumor cells before subcutaneous injection (n = 8 to 10 per group). (C) Tumor burden 12 days after treatment with vehicle or ADH-503 ± anti-41BB in mice bearing established (>0.4 cm) orthotopic KI PDAC tumors (n = 7 to 9 per group). (D) Analyses of survival and rechallenge. KI tumor-bearing mice from (C) were assessed for Kaplan-Meier survival analysis (left), pathological analysis of the pancreas was performed in mice surviving more than 120 days (middle), and mice were rechallenged with KI tumor cells subcutaneously (right). Control mice were animals who had never been exposed to KI tumor cells before subcutaneous injection (n = 4 to 10 per group). (E) Kaplan-Meier survival analysis of genetic KPC mice treated with vehicle or ADH-503 ± “immunotherapy” [GEM (50 mg/kg) + anti–PD-1 + anti-CTLA4]. Mice were enrolled in the study when tumors were greater than 0.4 cm in diameter (n = 10 to 15 per group). (F) Analysis of CD8a+ cells that infiltrated into PDAC tissues in end-stage KPC tumors from mice in (E). Representative images and quantitation results are shown. (G) Comparison of ADH-503 and CCR2 inhibition (CCR2i, PF-04136309). Tumor burden 14 days after treatment with vehicle, ADH-503, or CCR2i ± anti–PD-1 in mice bearing established (>0.4 cm) orthotopic KI PDAC tumors (n = 7 to 9 per group). Data are depicted as the change in tumor burden compared to five untreated animals euthanized at the beginning of treatment. (H) Comparison of ADH-503 and CSF1/CSF1R or granulocyte inhibition. Left: The observed changes in tumor volume 14 days after treatment with vehicle, ADH-503, anti-CSF1 IgG (5A1), or Ly6G-depleting IgG (1A8) ± anti–PD-1 in mice bearing established (>0.4 cm) orthotopic KI PDAC tumors. Right: The Kaplan-Meier survival analysis in the same animals. Bar graphs show the mean ± SE; *P < 0.05 by two-tailed t test, log-rank test, or ANOVA as appropriate. ** indicates comparison between ADH-503 and ADH-503 + immunotherapy in (B), (D), and (E); ** indicates comparison between ADH-503 and ADH-503 + αPD-1 in (B); ** indicates comparison between ADH-503 and ADH-503 + α41BB in (D); ** indicates comparison between ADH-503 and ADH-503 + immunotherapy in (E).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/499/eaau9240/DC1

    Materials and Methods

    Fig. S1. PDAC has a dense CD11b+ myeloid infiltrate.

    Fig. S2. ADH-503 in vivo properties.

    Fig. S3. ADH-503 alters innate responses in PDAC tissues.

    Fig. S4. CD11b agonism stimulates T cell and cDC1 infiltration and function.

    Fig. S5. ADH-503 direct effects on PDAC cells.

    Fig. S6. CD11b agonism in combination with chemotherapy.

    Fig. S7. Tumor wet weights.

    Table S1. Mouse antibodies for flow cytometry and FACS.

    Table S2. Tissue IHC and immunofluorescence antibodies.

    Table S3. Antibodies for CyTOF staining.

    Data file S1. Primary data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. PDAC has a dense CD11b+ myeloid infiltrate.
    • Fig. S2. ADH-503 in vivo properties.
    • Fig. S3. ADH-503 alters innate responses in PDAC tissues.
    • Fig. S4. CD11b agonism stimulates T cell and cDC1 infiltration and function.
    • Fig. S5. ADH-503 direct effects on PDAC cells.
    • Fig. S6. CD11b agonism in combination with chemotherapy.
    • Fig. S7. Tumor wet weights.
    • Table S1. Mouse antibodies for flow cytometry and FACS.
    • Table S2. Tissue IHC and immunofluorescence antibodies.
    • Table S3. Antibodies for CyTOF staining.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

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