Macrophage PI3Kγ Drives Pancreatic Ductal Adenocarcinoma Progression

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a low 5-year survival rate, yet new immunotherapeutic modalities may offer hope for this and other intractable cancers. Here, we report that inhibitory targeting of PI3Kγ, a key macrophage lipid kinase, stimulates antitumor immune responses, leading to improved survival and responsiveness to standard-of-care chemotherapy in animal models of PDAC. PI3Kγ selectively drives immunosuppressive transcriptional programming in macrophages that inhibits adaptive immune responses and promotes tumor cell invasion and desmoplasia in PDAC. Blockade of PI3Kγ in PDAC-bearing mice reprograms tumor-associated macrophages to stimulate CD8(+) T-cell-mediated tumor suppression and to inhibit tumor cell invasion, metastasis, and desmoplasia. These data indicate the central role that macrophage PI3Kγ plays in PDAC progression and demonstrate that pharmacologic inhibition of PI3Kγ represents a new therapeutic modality for this devastating tumor type.

Significance: We report here that PI3Kγ regulates macrophage transcriptional programming, leading to T-cell suppression, desmoplasia, and metastasis in pancreas adenocarcinoma. Genetic or pharmacologic inhibition of PI3Kγ restores antitumor immune responses and improves responsiveness to standard-of-care chemotherapy. PI3Kγ represents a new therapeutic immune target for pancreas cancer. Cancer Discov; 6(8); 870-85. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Conflict of interest statement

Conflicts: None

2016 American Association for Cancer Research.

Figures

Figure 1. PI3Kγ is a marker of pancreatic ductal adenocarcinoma associated macrophages

A. Immunofluorescent staining of F4/80+ macrophages (red) counterstained with DAPI (blue) in pancreata from normal mice, LSL-KRasG12D; Pdx-1Cre (KC) mice, LSL-KRasG12D/+; LSL-Trp53R172H/+; Pdx-1Cre (KPC) mice and mice that were implanted orthotopically with LSL-KRasG12D/+; LSL-Trp53R172H/+Pdx-1Cre (Orthotopic) tumors. Bar indicates 50μm. B. Quantification of the increase in F4/80+ macrophages over time in pancreata that were orthotopically implanted with LMP tumor cells (n=10), *p<0.01. C. Representative histopathologic characterization of human invasive pancreatic ductal adenocarcinoma tissues (n=7) and normal pancreas (n=8) showing immune reactivity for the macrophage marker CD68, with hematoxylin counterstaining. Scale bar indicates 100μm. D. Quantification of CD68+ macrophages/100X microscopic field in tissue sections from normal human pancreata (n=7) and from invasive pancreatic ductal adenocarcinomas (n=8). Statistical significance was determined via Wilcoxon rank-sum test. E. Immunostaining of human invasive pancreatic ductal adenocarcinomas for expression of PI3Kγ (green) and CD68+ macrophages (red). Tissues were counterstained with DAPI (blue) to detect nuclei. Arrows indicate examples of overlap (yellow) of PI3Kγ and CD68 immunostaining. Scale bar indicates 50μm. F. Western blot of p110γ, p110γ, p110α and actin in murine bone marrow derived macrophages (MΦ), tumor associated macrophages (TAM), CD19+ B cells, CD90+ T cells and LMP and p53 2.1.1 murine PDAC cells.

Figure 2. PDAC tumor growth and metastasis depend on macrophage PI3Kγ

A. Panc02, K8484 or p53 2.1.1 PDAC tumor cells were orthotopically implanted into syngeneic WT or p110γ−/− mice according to the depicted schema. B. Western blot comparing p110γ and actin expression in WT and p110γ−/− macrophages and in vitro cultured p53 2.1.1 PDAC cells. C–E. Weights of normal pancreata as well as pancreata containing tumors from WT and p110γ−/− mice 28 days after orthotopic implantation with (C) Panc02, (D) K8484 or (E) p53 2.1.1 PDAC cells. Significance testing was performed by nonparametric t test. F. Incidence of kidney, diaphragm and liver metastases in WT and p110γ−/− animals implanted with Panc02 cells. Significance testing was performed by Fisher’s exact test. G. Images of kidney, diaphragm and liver metastases from F. Arrowheads indicate metastatic foci. Scale bar indicates 50 μm. H. Quantification of CD11b+Gr1− macrophages and CD11b+Gr1+ myeloid cells by flow cytometry in representative tumors from WT (n=3) and p110γ−/− (n=5) mice. Significance testing was performed by unpaired t test. I. Representative Facs plots from WT and p110γ−/− orthotopic tumors. J. Quantification of CD4 and CD8 T cells in tumors from WT (n=3) and p110γ−/− (n=5) mice. Significance testing was performed by unpaired t test. K. Immunofluorescence images of CD8+ T cells in WT and p110γ−/− tumors. Scale bar indicates 50μm.

Figure 3. PI3Kγ inhibition promotes survival of mice with PDAC

A. Kaplan-Meier plot of survival of KC mice (n=29; median survival=36 weeks) compared to KC;p110γ−/− mice (n=15; median survival=43.9 weeks). B. Kaplan-Meier plot of survival of KPC animals (n=26; median survival=26 weeks) compared to KPC; p110γ−/− mice (n=21; median survival=35.5 weeks). Significance testing was performed by parametric Student’s t test. C. Images of hematoxylin and eosin staining and fluorescence immunostaining to detect amylase, claudin and cytokeratin 19 in tissue sections of pancreata tissue harvested at sacrifice in B. Images representative of the group are shown. Scale bar indicates 50 μm. D. Incidence of invasive carcinoma in animals from B. Significance testing was performed by Fisher’s exact test. E. Percent normal acinar tissue in animals from B. F. Incidence of metastasis in animals from B. Significance testing was performed by Fisher’s exact test.

Figure 4. Pharmacological inhibitors of PI3Kγ suppress PDAC growth and metastasis

A. Merged brightfield and red fluorescent images of mCherry-labeled LMP orthotopic pancreatic tumors from mice that were treated with PI3Kγ inhibitor TG100-115 or chemically similar control according to the schema depicted. B. Mean area of fluorescence from A. C. Mean weight of pancreata from A. D. Incidence of metastases in animals from A. E. Weight of normal pancreas and TG100-115 and control treated p53 2.1.1 orthotopic tumors. F. Images of tumors from TG100-115 and control treated animals from E. Arrows indicate necrotic tissue. G. Weight of PDAC tumors from animals treated with saline, TG100-115, gemcitabine and gemcitabine+TG100-115 according to the depicted schema. Significance testing was performed by one-sided Anova with Tukey’s posthoc multiple pairwise testing. H. Incidence of metastases in animals from G. Significance testing was performed by Fisher’s exact test. I. Effect of TG100-115 (n=9; median survival = 33 days), gemcitabine (n=9; median survival = 32 days) and combination gemcitabine + TG100-115 treatment (n=9; median survival = 38 days) on survival of implanted PDAC tumors compared to control treated animals (n=9; median survival = 28 days). J. Kaplan-Meier survival plot of KPC animals that were treated from week 10 to week 25 with TG100-115 (n=5; median survival = 28.5 weeks) compared to control treated KPC (n=5; median survival = 21 weeks) and untreated KPC mice (n=11; median survival= 24.3 weeks). K. Incidence of metastasis in TG100-115 (n=15) treated and control treated KPC animals (n=14). Significance testing was performed by Fisher’s exact test. L. Images of metastases in liver cross sections from control and TG100-115 treated tumors from K. Significance testing was performed by Anova or unpaired t-test.

Figure 5. PI3Kγ drives tumor associated macrophage polarization and immune suppression in vitro and in vivo

A–B. Relative mRNA expression of immune response genes in (A) orthotopic WT and p110γ−/− Panc02 tumors and (B) purified TAM from orthotopic WT and p110γ−/− Panc02 tumors. C. Relative mRNA expression of immune response genes in p110γ−/− or TG100-115 and control treated in vitro macrophages that were polarized with IL-4 for 48h. *p<0.05. D. Mean weight of tumors from WT or p110γ−/− mice implanted with p53 2.1.1 tumor cells admixed 1:1 with macrophages isolated from p53 2.1.1 tumors grown p110γ−/− and WT mice (n=8). Significance testing was performed by one-way Anova with Tukey’s posthoc multiple pairwise testing. E. mRNA expression of Ifng, Tgfb and Il10 in T cells isolated from orthotopic WT and p110γ−/− Panc02 tumors. F–G. In vitro proliferation of anti-CD3, anti-CD3+CD28 or IL2 + anti-CD3+ CD28 treated T cells isolated from spleens of (F) naive mice or (G) p53 2.1.1 PDAC tumor bearing mice. H. p53 2.1.1 tumor growth in WT, p110γ−/−, CD8−/−, and p110γ−/−;CD8−/− animals (n=10). Significance testing was performed by one-way Anova with Tukey’s posthoc multiple pairwise testing.

Figure 6. PI3Kγ controls macrophage PDGF expression to promote PDAC tumor cell invasion

A. Chemotaxis of LMP PDAC cells towards conditioned medium from IL-4 stimulated WT or p110γ−/− macrophages (IL-4 CM). B. Relative mRNA expression of select chemotactic factors in IL-4 stimulated p110γ−/− vs WT macrophages as determined by RNA sequencing and expressed as Log2-fold change from WT. C. Chemotaxis of LMP PDAC cells towards conditioned medium from IL-4 stimulated WT macrophages (WT IL4 CM) or RPMI medium in the presence or absence of dilutions of the PDGFR inhibitor Imatinib. D. Chemotaxis of LMP PDAC cells towards RPMI or conditioned medium from IL-4 stimulated WT macrophages (WT IL4 CM) in the presence or absence of the PDGF inhibitor Fovista. E. Chemotaxis of LMP PDAC cells towards medium (RPMI) or conditioned medium from IL-4 stimulated WT or p110γ −/− macrophages in the presence or absence of 100 ng/ml CCL2, SCF, PDGF-AA or PDGF-BB. F. Concentration of PDGF-BB in conditioned medium from WT and p110γ−/− basal (mCSF-stimulated) and IL-4 stimulated macrophages. G. Concentration of PDGF-BB in lysates from p53 2.1.1 orthotopic pancreatic tumors grown in WT and p110γ−/− mice. H. Relative mRNA expression of Pdgfb in tumor-derived CD11b+Gr1− macrophages, CD11b+Gr1lo monocytes, CD11b+Gr1hi neutrophils and CD11b−Gr1− cells (tumor cells). Significance testing was performed by Anova or parametric Student’s t test.

Figure 7. PI3Kγ promotes macrophage PDGF-BB expression to control PDAC fibrosis

A. Masson’s Trichrome staining of tissue sections of pancreata from WT and p110γ−/− KC and KPC animals. Scale bar, 100μm. B. Masson’s Trichrome staining of sections of pancreata from control and TG100-115 treated KPC animals. C–D. Images (C) and quantification (D) of picrosirius red staining of sections of pancreata from KPC tumors grown in WT and p110γ−/− animals. Scale bar 100μm. E. Collagen I immunostaining of LMP tumors from animals treated with the PI3Kγ inhibitor TG100-115 or chemically similar inert control. F. Western blot and quantification of collagen I protein expression in LMP tumors from animals treated with the PI3Kγ inhibitor TG100-115 or chemically similar inert control. G. Relative collagen I mRNA expression in normal pancreata and LMP tumors from animals treated with the PI3Kγ inhibitor TG100-115 or chemically similar inert control. H. Relative collagen I mRNA expression in primary murine fibroblasts incubated in the presence or absence of stimulus free conditioned medium from IL-4 stimulated WT and p110γ−/− macrophages. I. Relative collagen I mRNA expression in fibroblasts incubated in the presence or absence of stimulus free conditioned medium from IL-4 stimulated WT macrophages in the absence or presence of anti-TGFβ, anti-PDGF-BB or Imatinib. Significance testing was performed by parametric Student’s t test.