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The ability of disseminated cancer cells to evade the immune response is a critical step for efficient metastatic progression. Protection against an immune attack is often provided by the tumor microenvironment that suppresses and excludes cytotoxic CD8+ T cells. Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive metastatic disease with unmet needs, yet the immunoprotective role of the metastatic tumor microenvironment in pancreatic cancer is not completely understood. In this study, we find that macrophage-derived granulin contributes to cytotoxic CD8+ T-cell exclusion in metastatic livers. Granulin expression by macrophages was induced in response to colony-stimulating factor 1. Genetic depletion of granulin reduced the formation of a fibrotic stroma, thereby allowing T-cell entry at the metastatic site. Although metastatic PDAC tumors are largely resistant to anti–PD-1 therapy, blockade of PD-1 in granulin-depleted tumors restored the antitumor immune defense and dramatically decreased metastatic tumor burden. These findings suggest that targeting granulin may serve as a potential therapeutic strategy to restore CD8+ T-cell infiltration in metastatic PDAC, thereby converting PDAC metastatic tumors, which are refractory to immune checkpoint inhibitors, into tumors that respond to immune checkpoint inhibition therapies.Significance: These findings uncover a mechanism by which metastatic PDAC tumors evade the immune response and provide the rationale for targeting granulin in combination with immune checkpoint inhibitors for the treatment of metastatic PDAC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4253/F1.large.jpg. Cancer Res; 78(15); 4253–69. ©2018 AACR.

Fig. S1. Primary M2-like macrophages express IGF-1 and IGF-2. Fig. S2. In the absence of chemotherapy, addition of MCM, IGF blockade or recombinant IGF does not affect proliferation or survival of pancreatic cancer cells. Fig.S3. MCM enhances resistance of pancreatic cancer cells to gemcitabine, 5-FU and paclitaxel in an IGF-dependent manner. Fig. S4. Biopsies from pancreatic cancer patients express activated Insulin and IGF1 receptors and are infiltrated by macrophages. Fig. S5. Mouse orthotopic pancreatic tumors are rich in macrophages and myofibroblasts. Fig. S6. Tumors from KPC mouse model express activated Insulin/IGF1 receptors. Fig. S7. Gating strategy used to FACS-sort tumor cells, immune cells and stromal cells. Fig. S8. Primary pancreatic myofibroblasts secrete IGF-1 and IGF-2. Fig. S9. IGF-1 and IGF-2 are expressed in the tumor microenvironment of PDA patients. Fig. S10. Gating strategies used to analyse tumor infiltrated immune cell populations. Fig. S11. IGF blockade decreases activation of Insulin and IGF1 receptors in pancreatic tumors. Fig. S12. Combination of 5-FU and paclitaxel with BI 836845 in a syngeneic orthotopic pancreatic cancer model.

Supplementary Figure legends. Table S1. Clinical information from 40 consented PDA patient samples. Table S2. Clinical information from 16 consented PDA patient samples. Supplementary Materials and Methods.

Files in this Data Supplement: - Supplementary Figures. Figure S1: Loss of CD8+ T cell numbers during metastatic progression in PDAC patients and genetically engineered mouse model of pancreatic cancer. Figure S2: Colony stimulating factor 1 (CSF-1) is expressed by disseminated PDAC cells, and its cognate receptor CSF-1R is expressed by metastasis associated macrophages. Figure S3: Pharmacological blockade of CSF-1R using BLZ945 reprograms MAMs, restores T cell infiltration, and reduces metastasis. Figure S4: CSF-1 inhibition reduces desmoplasia, enhances CD8+ T cell infiltration and sensitizes metastatic PDAC to αPD-1 treatment. Figure S5: Administration of Digoxin, a transcriptional inhibitor of HIF-1α, promotes M1-like MAM reprogramming and reduces Granulin level in vivo. Figure S6: αPD-1 therapy dramatically reduces metastatic tumor burden in the absence of granulin, an effect which depends on a high abundance of immune stimulatory MAMs. Figure S7: Reduction in liver fibrosis improves CD8+ T cell infiltration and enhances αPD-1 therapy in metastatic pancreatic cancer. Figure S8: Macrophage-derived granulin drives resistance to immune checkpoint inhibition in metastatic pancreatic cancer.

Tumor-associated macrophages (TAM) and myofibroblasts are key drivers in cancer that are associated with drug resistance in many cancers, including pancreatic ductal adenocarcinoma (PDAC). However, our understanding of the molecular mechanisms by which TAM and fibroblasts contribute to chemoresistance is unclear. In this study, we found that TAM and myofibroblasts directly support chemoresistance of pancreatic cancer cells by secreting insulin-like growth factors (IGF) 1 and 2, which activate insulin/IGF receptors on pancreatic cancer cells. Immunohistochemical analysis of biopsies from patients with pancreatic cancer revealed that 72% of the patients expressed activated insulin/IGF receptors on tumor cells, and this positively correlates with increased CD163+ TAM infiltration. In vivo, we found that TAM and myofibroblasts were the main sources of IGF production, and pharmacologic blockade of IGF sensitized pancreatic tumors to gemcitabine. These findings suggest that inhibition of IGF in combination with chemotherapy could benefit patients with PDAC, and that insulin/IGF1R activation may be used as a biomarker to identify patients for such therapeutic intervention. Cancer Res; 76(23); 6851–63. ©2016 AACR.

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease and cytotoxic chemotherapy is the standard of care treatment for patients with advanced disease. Here, we investigate how the microenvironment in PDAC liver metastases reacts to chemotherapy and its role in metastatic disease progression post-treatment, an area which is poorly understood.DesignThe impact of chemotherapy on metastatic disease progression and immune cell infiltrates was characterised using flow and mass cytometry combined with transcriptional and histopathological analysis in experimental PDAC liver metastases mouse models. Findings were validated in patient derived liver metastases and in an autochthonous PDAC mouse model. Human and murine primary cell cocultures and ex vivo patient-derived liver explants were deployed to gain mechanistical insights on whether and how chemotherapy affects the metastatic tumour microenvironment.ResultsWe show that in vivo, chemotherapy induces an initial infiltration of proinflammatory macrophages into the liver and activates cytotoxic T cells, leading only to a temporary restraining of metastatic disease progression. However, after stopping treatment, neutrophils are recruited to the metastatic liver via CXCL1 and 2 secretion by metastatic tumour cells. These neutrophils express growth arrest specific 6 (Gas6) which leads to AXL receptor activation on tumour cells enabling their regrowth. Disruption of neutrophil infiltration or inhibition of the Gas6/AXL signalling axis in combination with chemotherapy inhibits metastatic growth. Chemotherapy increases Gas6 expression in circulating neutrophils from patients with metastatic pancreatic cancer and recombinant Gas6 is sufficient to promote tumour cell proliferation ex vivo, in patient-derived metastatic liver explants.ConclusionCombining chemotherapy with Gas6/AXL or neutrophil targeted therapy could provide a therapeutic benefit for patients with metastatic pancreatic cancer.

Data from in vitro and animal studies support a neuroprotective role of glatiramer acetate (GA) in multiple sclerosis (MS). We investigated prospectively whether treatment with GA leads to clinical and paraclinical changes associated with neuroprotection in patients with relapsing-remitting (RR) MS. Primary aim of this clinical study was to determine serum BDNF levels in RR-MS patients who were started on GA as compared to patients who remained therapy-naive throughout 24 months. Secondary outcomes included relapses and EDSS, cognition, quality of life, fatigue and depression, BDNF expression levels on peripheral immune cells (FACS, RT-PCR), serum anti-myelin basic peptide (MBP) antibody status, evoked potential and cerebral MRI studies. While GA treatment did not alter serum levels or expression levels on peripheral immune cells of BDNF over time it resulted in a transient increase of serum IgG antibody response to MBP, mainly due to subtype IgG1 (p

Corrigendum: Macrophage-secreted granulin supports pancreatic cancer metastasis by inducing liver fibrosis