Brian Belt

Targeting Tumor-Infiltrating Macrophages Decreases Tumor-Initiating Cells, Relieves Immunosuppression, and Improves Chemotherapeutic Responses

Tumor-infiltrating immune cells can promote chemoresistance and metastatic spread in aggressive tumors. Consequently, the type and quality of immune responses present in the neoplastic stroma are highly predictive of patient outcome in several cancer types. In addition to host immune responses, intrinsic tumor cell activities that mimic stem cell properties have been linked to chemoresistance, metastatic dissemination, and the induction of immune suppression. Cancer stem cells are far from a static cell population; rather, their presence seems to be controlled by highly dynamic processes that are dependent on cues from the tumor stroma. However, the impact immune responses have on tumor stem cell differentiation or expansion is not well understood. In this study, we show that targeting tumor-infiltrating macrophages (TAM) and inflammatory monocytes by inhibiting either the myeloid cell receptors colony-stimulating factor-1 receptor (CSF1R) or chemokine (C-C motif) receptor 2 (CCR2) decreases the number of tumor-initiating cells (TIC) in pancreatic tumors. Targeting CCR2 or CSF1R improves chemotherapeutic efficacy, inhibits metastasis, and increases antitumor T-cell responses. Tumor-educated macrophages also directly enhanced the tumor-initiating capacity of pancreatic tumor cells by activating the transcription factor STAT3, thereby facilitating macrophage-mediated suppression of CD8(+) T lymphocytes. Together, our findings show how targeting TAMs can effectively overcome therapeutic resistance mediated by TICs.

Disruption of CCR5-Dependent Homing of Regulatory T Cells Inhibits Tumor Growth in a Murine Model of Pancreatic Cancer

Tumors evade immune destruction by actively inducing immune tolerance through the recruitment of CD4+CD25+Foxp3+ regulatory T cells (Treg). We have previously described increased prevalence of these cells in pancreatic adenocarcinoma, but it remains unclear what mechanisms are involved in recruiting Tregs into the tumor microenvironment. Here, we postulated that chemokines might direct Treg homing to tumor. We show, in both human pancreatic adenocarcinoma and a murine pancreatic tumor model (Pan02), that tumor cells produce increased levels of ligands for the CCR5 chemokine receptor and, reciprocally, that CD4+ Foxp3+ Tregs, compared with CD4+ Foxp3− effector T cells, preferentially express CCR5. When CCR5/CCL5 signaling is disrupted, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (N,N-dimethyl-N-{{4-{[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl}amino}}benzyl]-N,N-dimethyl-N- {{{4-{{{[2-(4-methylphenyl)-6,7-dihydro-5H-benzocycloheptan-8-yl]carbonyl}amino}}benzyl}}}tetrahydro-2H-pyran-4-aminiumchloride; TAK-779), Treg migration to tumors is reduced and tumors are smaller than in control mice. Thus, this study demonstrates the importance of Tregs in immune evasion by tumors, how blockade of Treg migration might inhibit tumor growth, and, specifically in pancreatic adenocarcinoma, the role of CCR5 in the homing of tumor-associated Tregs. Selective targeting of CCR5/CCL5 signaling may represent a novel immunomodulatory strategy for the treatment of cancer.

Inflammatory Monocyte Mobilization Decreases Patient Survival in Pancreatic Cancer: A Role for Targeting the CCL2/CCR2 Axis

Purpose: To determine the role of the CCL2/CCR2 axis and inflammatory monocytes (CCR2+/CD14+) as immunotherapeutic targets in the treatment of pancreatic cancer. Experimental Design: Survival analysis was conducted to determine if the prevalence of preoperative blood monocytes correlates with survival in patients with pancreatic cancer following tumor resection. Inflammatory monocyte prevalence in the blood and bone marrow of patients with pancreatic cancer and controls was compared. The immunosuppressive properties of inflammatory monocytes and macrophages in the blood and tumors, respectively, of patients with pancreatic cancer were assessed. CCL2 expression by human pancreatic cancer tumors was compared with normal pancreas. A novel CCR2 inhibitor (PF-04136309) was tested in an orthotopic model of murine pancreatic cancer. Results: Monocyte prevalence in the peripheral blood correlates inversely with survival, and low monocyte prevalence is an independent predictor of increased survival in patients with pancreatic cancer with resected tumors. Inflammatory monocytes are increased in the blood and decreased in the bone marrow of patients with pancreatic cancer compared with controls. An increased ratio of inflammatory monocytes in the blood versus the bone marrow is a novel predictor of decreased patient survival following tumor resection. Human pancreatic cancer produces CCL2, and immunosuppressive CCR2+ macrophages infiltrate these tumors. Patients with tumors that exhibit high CCL2 expression/low CD8 T-cell infiltrate have significantly decreased survival. In mice, CCR2 blockade depletes inflammatory monocytes and macrophages from the primary tumor and premetastatic liver resulting in enhanced antitumor immunity, decreased tumor growth, and reduced metastasis. Conclusions: Inflammatory monocyte recruitment is critical to pancreatic cancer progression, and targeting CCR2 may be an effective immunotherapeutic strategy in this disease. Clin Cancer Res; 19(13); 3404–15. ©2013 AACR.

Pancreatic adenocarcinoma induces bone marrow mobilization of myeloid-derived suppressor cells which promote primary tumor growth

PurposeMyeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells that are upregulated in cancer. Little is known about the prevalence and importance of MDSC in pancreas adenocarcinoma (PA).Experimental designPeripheral blood, bone marrow, and tumor samples were collected from pancreatic cancer patients, analyzed for MDSC (CD15+CD11b+) by flow cytometry and compared to cancer-free controls. The suppressive capacity of MDSC (CD11b+Gr-1+) and the effectiveness of MDSC depletion were assessed in C57BL/6 mice inoculated with Pan02, a murine PA, and treated with placebo or zoledronic acid, a potent aminobisphosphonate previously shown to target MDSC. The tumor microenvironment was analyzed for MDSC (Gr1+CD11b+), effector T cells, and tumor cytokine levels.ResultsPatients with PA demonstrated increased frequency of MDSC in the bone marrow and peripheral circulation which correlated with disease stage. Normal pancreas tissue showed no MDSC infiltrate, while human tumors avidly recruited MDSC. Murine tumors similarly recruited MDSC that suppressed CD8+ T cells in vitro and accelerated tumor growth in vivo. Treatment with zoledronic acid impaired intratumoral MDSC accumulation resulting in delayed tumor growth rate, prolonged median survival, and increased recruitment of T cells to the tumor. This was associated with a more robust type 1 response with increased levels of IFN-γ and decreased levels of IL-10.ConclusionsMDSC are important mediators of tumor-induced immunosuppression in pancreatic cancer. Inhibiting MDSC accumulation with zoledronic acid improves the host anti-tumor response in animal studies suggesting that efforts to block MDSC may represent a novel treatment strategy for pancreatic cancer.

Targeting tumour-associated macrophages with CCR2 inhibition in combination with FOLFIRINOX in patients with borderline resectable and locally advanced pancreatic cancer: a single-centre, open-label, dose-finding, non-randomised, phase 1b trial

BACKGROUND In pancreatic ductal adenocarcinoma, the CCL2-CCR2 chemokine axis is used to recruit tumour-associated macrophages for construction of an immunosuppressive tumour microenvironment. This pathway has prognostic implications in pancreatic cancer, and blockade of CCR2 restores anti-tumour immunity in preclinical models. We aimed to establish the safety, tolerability, and recommended phase 2 oral dose of the CCR2 inhibitor PF-04136309 in combination with FOLFIRINOX chemotherapy (oxaliplatin and irinotecan plus leucovorin and fluorouracil). METHODS We did this open-label, dose-finding, non-randomised, phase 1b study at one centre in the USA. We enrolled treatment-naive patients aged 18 years or older with borderline resectable or locally advanced biopsy-proven pancreatic ductal adenocarcinoma, an Eastern Cooperative Oncology Group performance status of 1 or less, measurable disease as defined by Response Evaluation Criteria in Solid Tumors version 1.1, and normal end-organ function. Patients were allocated to receive either FOLFIRINOX alone (oxaliplatin 85 mg/m(2), irinotecan 180 mg/m(2), leucovorin 400 mg/m(2), and bolus fluorouracil 400 mg/m(2), followed by 2400 mg/m(2) 46-h continuous infusion), administered every 2 weeks for a total of six treatment cycles, or in combination with oral PF-04136309, administered at a starting dose of 500 mg twice daily in a standard 3 + 3 dose de-escalation design. Both FOLFIRINOX and PF-04136309 were simultaneously initiated with a total treatment duration of 12 weeks. The primary endpoints were the safety, tolerability, and recommended phase 2 dose of PF-04136309 plus FOLFIRINOX, with an expansion phase planned at the recommended dose. We analysed the primary outcome by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01413022. RESULTS Between April 19, 2012, and Nov 12, 2014, we treated 47 patients with FOLFIRINOX alone (n=8) or with FOLFIRINOX plus PF-04136309 (n=39). One patient had a dose-limiting toxic effect in the dose de-escalation group receiving FOLFIRINOX plus PF-04136309 at 500 mg twice daily (n=6); this dose was established as the recommended phase 2 dose. We pooled patients in the expansion-phase group (n=33) with those in the dose de-escalation group that received PF-04136309 at the recommended phase 2 dose for assessment of treatment-related toxicity. Six (75%) of the eight patients receiving FOLFIRINOX alone were assessed for treatment toxicity, after exclusion of two (25%) patients due to insurance coverage issues. The median duration of follow-up for treatment toxicity was 72·0 days (IQR 49·5-89·0) in the FOLFIRINOX alone group and 77·0 days (70·0-90·5) in the FOLFIRINOX plus PF-04136309 group. No treatment-related deaths occurred. Two (5%) patients in the FOLFIRINOX plus PF-04136309 group stopped treatment earlier than planned due to treatment-related toxic effects. Grade 3 or higher adverse events reported in at least 10% of the patients receiving PF-04136309 included neutropenia (n=27), febrile neutropenia (n=7), lymphopenia (n=4), diarrhoea (n=6), and hypokalaemia (n=7). Grade 3 or higher adverse events reported in at least 10% of patients receiving FOLFIRINOX alone were neutropenia (n=6), febrile neutropenia (n=1), anaemia (n=2), lymphopenia (n=1), diarrhoea (n=2), hypoalbuminaemia (n=1), and hypokalaemia (n=3). Therapy was terminated because of treatment-related toxicity in one (17%) of the six patients receiving FOLFIRINOX alone. 16 (49%) of 33 patients receiving FOLFIRINOX plus PF-04136309 who had undergone repeat imaging achieved an objective tumour response, with local tumour control achieved in 32 (97%) patients. In the FOLFIRINOX alone group, none of the five patients with repeat imaging achieved an objective response, although four (80%) of those patients achieved stable disease. INTERPRETATION CCR2-targeted therapy with PF-04136309 in combination with FOLFIRINOX is safe and tolerable. FUNDING Washington University-Pfizer Biomedical Collaborative.

Induction of Th17 Cells in the Tumor Microenvironment Improves Survival in a Murine Model of Pancreatic Cancer

An important mechanism by which pancreatic cancer avoids antitumor immunity is by recruiting regulatory T cells (Tregs) to the tumor microenvironment. Recent studies suggest that suppressor Tregs and effector Th17 cells share a common lineage and differentiate based on the presence of certain cytokines in the microenvironment. Because IL-6 in the presence of TGF-β has been shown to inhibit Treg development and induce Th17 cells, we hypothesized that altering the tumor cytokine environment could induce Th17 and reverse tumor-associated immune suppression. Pan02 murine pancreatic tumor cells that secrete TGF-β were transduced with the gene encoding IL-6. C57BL/6 mice were injected s.c. with wild-type (WT), empty vector (EV), or IL-6–transduced Pan02 cells (IL-6 Pan02) to investigate the impact of IL-6 secretion in the tumor microenvironment. Mice bearing IL-6 Pan02 tumors demonstrated significant delay in tumor growth and better overall median survival compared with mice bearing WT or EV Pan02 tumors. Immunohistochemical analysis demonstrated an increase in Th17 cells (CD4+IL-23R+ cells and CD4+IL-17+ cells) in tumors of the IL-6 Pan02 group compared with WT or EV Pan02 tumors. The upregulation of IL-17–secreting CD4+ tumor-infiltrating lymphocytes was substantiated at the cellular level by flow cytometry and ELISPOT assay and mRNA level for retinoic acid-related orphan receptor γt and IL-23R by RT-PCR. Thus, the addition of IL-6 to the tumor microenvironment skews the balance toward Th17 cells in a murine model of pancreatic cancer. The delayed tumor growth and improved survival suggests that induction of Th17 in the tumor microenvironment produces an antitumor effect.

Tumor-derived TGF-β Mediates Conversion of CD4+Foxp3+ Regulatory T Cells in a Murine Model of Pancreas Cancer

CD4+25+Foxp3+ regulatory T cells (Treg) play a critical role in the induction of tolerance to tumor-associated antigens and suppression of antitumor immunity. How Treg are induced in cancer is poorly understood. We reported previously that Treg are significantly elevated in the peripheral blood of patients with pancreas cancer and that in a murine pancreas cancer model induction of Treg seems to be transforming growth factor (TGF)-β dependent. Here we provide additional evidence that Treg are increased locally within the tumor microenvironment by a mechanism that seems dependent on TGF-β receptor expression and the presence of tumor derived TGF-β. The murine pancreas cancer cell line Pan02 produces high levels of TGF-β both in vitro and in vivo. In contrast, the esophageal murine cancer cell line, Eso2, does not. Immunohistochemical staining of Foxp3 in explanted tumors shows an identifiable population of Treg in the Pan02 (TGF-β positive) tumors but not Eso2 (TGF-β negative). Naive CD4+25−Foxp3− T cells, when adoptively transferred into Rag−/− mice, are converted into Foxp3+ Treg in the presence of Pan02 but not Eso2 tumors. Induction of Treg in Pan02 mice is blocked by systemic injection of an anti–TGF-β antibody. If Rag−/− mice are instead reconstituted with naive CD4+25− T cells expressing a mutated TGF-β receptor, induction of Foxp3+ Treg in Pan02 bearing mice is blocked. Collectively, these observations further support the role of TGF-β in the induction of Treg in pancreas adenocarcinoma.

Tumor-induced STAT3 activation in monocytic myeloid-derived suppressor cells enhances stemness and mesenchymal properties in human pancreatic cancer

Pancreatic cancer (PC) mobilizes myeloid cells from the bone marrow to the tumor where they promote tumor growth and proliferation. Cancer stem cells (CSCs) are a population of tumor cells that are responsible for tumor initiation. Aldehyde dehydrogenase-1 activity in PC identifies CSCs, and its activity has been correlated with poor overall prognosis in human PC. Myeloid cells have been shown to impact tumor stemness, but the impact of immunosuppressive tumor-infiltrating granulocytic and monocytic myeloid-derived suppressor cells (Mo-MDSC) on ALDH1Bright CSCs and epithelial to mesenchymal transition is not well understood. In this study, we demonstrate that Mo-MDSC (CD11b+/Gr1+/Ly6G−/Ly6Chi) significantly increase the frequency of ALDH1Bright CSCs in a mouse model of PC. Additionally, there was significant upregulation of genes associated with epithelial to mesenchymal transition. We also found that human PC converts CD14+ peripheral blood monocytes into Mo-MDSC (CD14+/HLA-DRlow/−) in vitro, and this transformation is dependent on the activation of the STAT3 pathway. In turn, these Mo-MDSC increase the frequency of ALDH1Bright CSCs and promote mesenchymal features of tumor cells. Finally, blockade of STAT3 activation reversed the increase in ALDH1Bright CSCs. These data suggest that the PC tumor microenvironment transforms monocytes to Mo-MDSC by STAT3 activation, and these cells increase the frequency of ALDH1Bright CSCs. Therefore, targeting STAT3 activation may be an effective therapeutic strategy in targeting CSCs in PC.

Myeloid-Derived Suppressor Cells: General Characteristics and Relevance to Clinical Management of Pancreatic Cancer

Recent studies describe a heterogeneous population of cells of the myeloid lineage, termed myeloid derived suppressor cells (MDSC), which are observed with increased prevalence in the peripheral blood and tumor microenvironment of cancer patients, including pancreatic cancer. Accumulation of MDSC in the peripheral circulation has been related to extent of disease, and correlates with stage. MDSC have primarily been implicated in promoting tumor growth by suppressing antitumor immunity. There is also compelling evidence MDSC are also involved in angiogenesis and metastatic spread. Two main subsets of MDSC have been identified in cancer patients: a monocytic subset, characterized by expression of CD14, and a granulocytic subset characterized by expression of CD15. Both subsets of MDSC actively suppress host immunity through a variety of mechanisms including production of reactive oxygen species and arginase. Just as in humans, accumulation of monocytic and granulocytic MDSC has been noted in the bone marrow, spleen, peripheral circulation, and tumors of tumor bearing mice. Successful targeting of MDSC in mice is associated with improved immune responses, delayed tumor growth, improved survival, and increased efficacy of vaccine therapy. By further elucidating mechanisms of MDSC recruitment and maintenance in the tumor environment, strategies could be developed to reverse immune tolerance to tumor. We discuss here what is currently known about MDSC as well as some potential strategies targeting MDSC in the context of our work on pancreatic cancer and recent literature. Due to the number of new reports on MDSC, the most pertinent ones have been selected.

Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system.