GM-CSF has been employed as an adjuvant to cancer immunotherapy with mixed results based on dosage. We previously showed that GM-CSF regulated tumor angiogenesis by stimulating soluble vascular endothelial growth factor (VEGF) receptor-1 from monocytes/macrophages in a dose-dependent manner that neutralized free VEGF, and intratumoral injections of high-dose GM-CSF ablated blood vessels and worsened hypoxia in orthotopic polyoma middle T Ag (PyMT) triple-negative breast cancer (TNBC). In this study, we assessed both immunoregulatory and oxygen-regulatory components of low-dose versus high-dose GM-CSF to compare effects on tumor oxygen, vasculature, and antitumor immunity. We performed intratumoral injections of low-dose GM-CSF or saline controls for 3 wk in FVB/N PyMT TNBC. Low-dose GM-CSF uniquely reduced tumor hypoxia and normalized tumor vasculature by increasing NG2+ pericyte coverage on CD31+ endothelial cells. Priming of “cold,” anti-PD1–resistant PyMT tumors with low-dose GM-CSF (hypoxia reduced) sensitized tumors to anti-PD1, whereas high-dose GM-CSF (hypoxia exacerbated) did not. Low-dose GM-CSF reduced hypoxic and inflammatory tumor-associated macrophage (TAM) transcriptional profiles; however, no phenotypic modulation of TAMs or tumor-infiltrating lymphocytes were observed by flow cytometry. In contrast, high-dose GM-CSF priming increased infiltration of TAMs lacking the MHC class IIhi phenotype or immunostimulatory marker expression, indicating an immunosuppressive phenotype under hypoxia. However, in anti-PD1 (programmed cell death 1)–susceptible BALB/c 4T1 tumors (considered hot versus PyMT), high-dose GM-CSF increased MHC class IIhi TAMs and immunostimulatory molecules, suggesting disparate effects of high-dose GM-CSF across PyMT versus 4T1 TNBC models. Our data demonstrate a (to our knowledge) novel role for low-dose GM-CSF in reducing tumor hypoxia for synergy with anti-PD1 and highlight why dosage and setting of GM-CSF in cancer immunotherapy regimens require careful consideration.

Pancreatic adenocarcinoma (PDAC) has a dismal survival rate due to limited effective therapies. Although studies have focused on the influence of innate immune cells on adaptive immune cell functions, few have explored interactions between innate immune cells, which modulate the PDAC tumor microenvironment (TME). Macrophages are responsible for the clearance of neutrophil-mediated inflammation in physiologic, resolving immune responses; however, both of these cell types coexist in the TME, suggesting a failure of macrophages to clear neutrophils in PDAC. We sought to determine how neutrophil extracellular traps (NET), neutrophil release of decondensed chromatin, and intracellular contents affect monocyte/macrophage populations in the PDAC TME. Utilizing samples from patients with PDAC, we demonstrated elevated levels of the monocyte chemokine CCL2 in plasma, as well as elevated NET citrullinated histone H3 and the pan-macrophage marker CD68 in the PDAC TME via fluorescent IHC. To determine how NETs affected macrophage populations in the PDAC TME, we targeted NETs with DNase I treatment to digest extracellular DNA released from NETs or with genetic knockout of PAD4, an enzyme required for NET formation. NET depletion resulted in an elevation in the pan-macrophage marker F4/80. The depletion led to an increased T-cell stimulatory signal, CD80, whereas the protumor macrophage marker CD206 was decreased. We further demonstrated that macrophages in the NET-deficient PDAC TME may be recruited through the CCL2/CCR2 axis, and CCL2 was released from tumor cells and macrophages in the presence of IFNγ. Taken together, our findings reveal that inhibition of NETs can prime the innate immune response toward an antitumor phenotype.

Background/Objectives: An anastomotic leak (AL) following colorectal surgery is one of the most feared complications due to its associated morbidity and mortality. Early detection of ALs remains difficult, as the development of clinical signs of deterioration can be a late finding. This is particularly problematic in patients with poor access to care after discharge. C-reactive protein (CRP) is a systemic marker of inflammation that has been proposed as an early AL screening. However, absolute cut-off values have been shown to have limited sensitivity and specificity. We propose the use of CRP trends for early AL detection. Methods: A retrospective chart review of patients undergoing surgery requiring at least one anastomosis at a single tertiary care center was performed. Patients with two or fewer postoperative CRP values were excluded. Postoperative CRP trends were compared between control and AL patients using a mixed model with a Geisser–Greenhouse correction. Results: CRP trends differed significantly between AL and control patients, with a 10% CRP increase after postoperative day two showing 100% sensitivity and 84% specificity for an AL as well as a 100% negative predictive value. Accepted CRP cut-off values on postoperative days three and four had sensitivities of only 71.4% and 80% and specificities of 70.0% and 76.5%, respectively. CRP trends differed in AL versus control patients despite the surgical approach or presence of additional procedures. Conclusions: Daily monitoring of CRP trends (versus absolute cut-offs) may enhance early anastomotic leak detection and aid in discharge decision-making, particularly important in rural settings with limited healthcare access.

Pancreatic adenocarcinoma (PDAC) has a dismal survival rate due to limited effective therapies. Although studies have focused on the influence of innate immune cells on adaptive immune cell functions, few have explored interactions between innate immune cells, which modulate the PDAC tumor microenvironment (TME). Macrophages are responsible for the clearance of neutrophil-mediated inflammation in physiologic, resolving immune responses; however, both of these cell types coexist in the TME, suggesting a failure of macrophages to clear neutrophils in PDAC. We sought to determine how neutrophil extracellular traps (NET), neutrophil release of decondensed chromatin, and intracellular contents affect monocyte/macrophage populations in the PDAC TME. Utilizing samples from patients with PDAC, we demonstrated elevated levels of the monocyte chemokine CCL2 in plasma, as well as elevated NET citrullinated histone H3 and the pan-macrophage marker CD68 in the PDAC TME via fluorescent IHC. To determine how NETs affected macrophage populations in the PDAC TME, we targeted NETs with DNase I treatment to digest extracellular DNA released from NETs or with genetic knockout of PAD4, an enzyme required for NET formation. NET depletion resulted in an elevation in the pan-macrophage marker F4/80. The depletion led to an increased T-cell stimulatory signal, CD80, whereas the protumor macrophage marker CD206 was decreased. We further demonstrated that macrophages in the NET-deficient PDAC TME may be recruited through the CCL2/CCR2 axis, and CCL2 was released from tumor cells and macrophages in the presence of IFNγ. Taken together, our findings reveal that inhibition of NETs can prime the innate immune response toward an antitumor phenotype.

Supplemental Figure 7 from Neutrophil Extracellular Traps Modulate Recruitment and Immunosuppression of Macrophages in Pancreatic Adenocarcinoma

Supplemental Figure 5 from Neutrophil Extracellular Traps Modulate Recruitment and Immunosuppression of Macrophages in Pancreatic Adenocarcinoma