Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC), which not only mediate immune suppression but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive anti-tumor T cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics.

Regulatory T cells (Treg) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. In mice, depletion of Treg by Ab therapy leads to more efficient tumor rejection. Treg-mediated suppression of antitumor immune responses may partly explain the poor clinical response to vaccine-based immunotherapy for human cancer. In this study, we measured the prevalence of Treg that coexpress CD4 and CD25 in the PBLs, tumor-infiltrating lymphocytes, and regional lymph node lymphocytes from 65 patients with either pancreas or breast cancer. In breast cancer patients (n = 35), pancreas cancer patients (n = 30), and normal donors (n = 35), the prevalence of Treg were 16.6% (SE 1.22), 13.2% (SE 1.13), and 8.6% (SE 0.71) of the total CD4+ cells, respectively. The prevalence of Treg were significantly higher in breast cancer patients (p < 0.01) and pancreas cancer patients (p < 0.01) when compared with normal donors. In tumor-infiltrating lymphocytes and lymph node lymphocytes, the Treg prevalence were 20.2% (SE 3.93) and 20.1% (SE 4.3), respectively. Treg constitutively coexpressed CTLA-4 and CD45RO markers, and secreted TGF-β and IL-10 but did not secrete IFN-γ. When cocultured with activated CD8+ cells or CD4+25− cells, Treg potently suppressed their proliferation and secretion of IFN-γ. We conclude that the prevalence of Treg is increased in the peripheral blood as well as in the tumor microenvironment of patients with invasive breast or pancreas cancers. These Treg may mitigate the immune response against cancer, and may partly explain the poor immune response against tumor Ags.

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 appears 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 demonstrate that targeting tumor-infiltrating macrophages and inflammatory monocytes by inhibiting either the myeloid cell receptors CSF1R or CCR2 decreases the number of tumor-initiating cells in pancreatic tumors. Targeting CCR2 or CSF1R improves chemotherapeutic efficacy, inhibits metastasis and increases anti-tumor 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 tumor-infiltrating macrophages can effectively overcome therapeutic resistance mediated by tumor-initiating cells.

Purpose To determine the role of the CCL2/CCR2 axis and inflammatory monocytes (IM; CCR2+/CD14+) as immunotherapeutic targets in the treatment of pancreatic cancer (PC). Experimental Design Survival analysis was performed to determine if the prevalence of pre-operative blood monocytes correlates with survival in PC patients following tumor resection. IM prevalence in the blood and bone marrow of PC patients and controls was compared. The immunosuppressive properties of IM and macrophages in the blood and tumors, respectively, of PC patients were assessed. CCL2 expression by human PC tumors was compared to normal pancreas. A novel CCR2 inhibitor (PF-04136309) was tested in an orthotopic model of murine PC. Results Monocyte prevalence in the peripheral blood correlates inversely with survival, and low monocyte prevalence is an independent predictor of increased survival in PC patients with resected tumors. IM are increased in the blood and decreased in the bone marrow of PC patients compared to controls. An increased ratio of IM in the blood versus the bone marrow is a novel predictor of decreased patient survival following tumor resection. Human PC 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 IM and macrophages from the primary tumor and premetastatic liver resulting in enhanced anti-tumor immunity, decreased tumor growth, and reduced metastasis. Conclusions IM recruitment is critical to PC progression, and targeting CCR2 may be an effective immunotherapeutic strategy in this disease.

Background Pancreatic ductal adenocarcinoma utilizes the CCL2/CCR2 chemokine axis to facilitate recruitment of tumor associated macrophages to sculpt an immunosuppressive tumor microenvironment. This pathway has prognostic implications in pancreas cancer, and blockade of CCR2 restores anti-tumor immunity in pre-clinical models. This provided the rationale for a clinical study in pancreatic adenocarcinoma to determine the safety and recommended phase 2 oral dosage of the CCR2 inhibitor PF-04136309 in combination with chemotherapy (FOLFIRINOX). Methods In this single-center, open label, phase Ib clinical trial patients age ≥ 18 years with treatment naïve borderline resectable or locally advanced, biopsy-proven pancreatic ductal adenocarcinoma, Eastern Cooperative Oncology Group performance status <2, measurable disease by Response Evaluation Criteria in Solid Tumors Version 1.1, and normal end organ function were eligible for enrollment. FOLFIRINOX (oxaliplatin, 85 mg/m2; irinotecan, 180 mg/m2; leucovorin, 400 mg/m2, and bolus fluorouracil 400 mg/m2 followed by 2,400 mg/m2 46 hour continuous infusion) was administered every 2 weeks for a total of six treatment cycles. To determine the recommended phase 2 dose, PF-04136309 was orally administered at a starting dose of 500 mg twice daily in a standard 3+3 dose de-escalation design with an expansion phase planned at the recommended phase 2 dose. Both FOLFIRINOX and PF-04136309 were simultaneously initiated with a total treatment duration of 3 months. The primary endpoints were to determine the recommended phase 2 dose and toxicity of PF-04136309 in combination with FOLFIRINOX. All patients in the dose de-escalation and expansion phase received the recommended phase 2 dose of PF-04136309 were combined for assessment of treatment toxicity by an intention to treat analysis. For tissue specimen comparison in corollary studies, a group of patients receiving FOLFIRINOX alone were enrolled and evaluated for treatment related toxicity. This study has been completed and is registered at ClinicalTrials.gov; number NCT01413022. Results From April 19th, 2012 through November 12th, 2014 a total of 47 patients were enrolled. The dose de-escalation group (n=6) received PF-04136309 at 500 mg administered orally twice daily. No dose-limiting toxicities were observed and this was established as the recommended phase 2 dose. The expansion phase cohort (n=33) and patients in the dose de-escalation arm receiving PF-04136309 at the recommended phase 2 dose (n=6) were combined for assessment of treatment related toxicity. No therapy related deaths occurring during the study interval. Early termination as the result of treatment related toxicity occurred in 2 of the 39 patients (5%) in the FOLFIRINOX plus PF-04136309 arm. Grade ≥3 adverse events reported in ≥10% of the patients receiving PF-04136309 included neutropenia in 27 patients (69%), febrile neutropenia in 7 patients (18%), lymphopenia in 4 patients (10%), diarrhea in 6 patients (15%), and hypokalemia in 7 patients (18%). Among...