Abstract A44: Disrupted transmigration of neutrophils in glioblastoma patients is augmented by steroid treatment.
Authors: Sippel Trisha R. , Russell Rae , Ung Timothy , Klaassen Marci , Waziri Allen 
Published: August 2014

Abstract
Introduction: We recently described a mechanism of peripheral immunosuppression in patients with glioblastoma (GBM), through which circulating activated neutrophils release Arginase I and suppress T cell function. The presence of activated neutrophils within the circulation of patients with GBM is of unclear etiology. To further explore this phenomenon and correlate these changes with clinical factors that could influence neutrophil biology, we performed phenotypic and functional analysis of neutrophil populations from GBM patients.
Methods: Resting neutrophils were purified from GBM patients and normal donors by dextran sedimentation of red blood cells followed by separation on a percoll gradient. In vitro activation of resting neutrophils was induced with formyl-Methionyl-Leucyl-Phenylalanine (fMLP). Circulating activated neutrophils were identified within the PBMC layer following Ficoll density centrifugation. Intratumoral neutrophils were identified within fresh human GBM samples reduced to single cell suspensions using a combination of mechanical and enzymatic disruption. Flow cytometry was used to measure expression of known markers of neutrophil activation and binding (CD11b, CD11c, CD18, CD62L, and CD66). In vitro binding studies were performed by labeling neutrophils with BCECF (2′,7′-bis-(2carboxyethyl)-5-(and-6)-carboxyfluorescein); labeled neutrophils were placed over HMEC monolayers activated with tumor necrosis factor-α (TNF-α). Binding curves were generated by serial spectrophotometric analysis after sequential washes. Clinical data, including treatment with steroids and tumor recurrence status, were retrospectively compiled for each patient.
Results: Resting neutrophils within GBM patient did not differ from normal donors in terms of baseline adhesion marker expression, adhesion marker expression after in vitro activation, or ability to adhere to endothelial cells in vitro. As expected, intratumoral neutrophils demonstrated a fully activated phenotype, expressing increased levels of CD11b, CD18 and CD66 and decreased levels of CD62L, along with a transmigrated phenotype represented by lower levels of CD11c. Interestingly, circulating activated neutrophils expressed elevated CD66 and decreased CD62L in conjunction with lower levels of CD11b, CD18 and CD11c. In review of clinical data, patients with primary GBM harbored increased numbers of circulating activated neutrophils when compared to patients with recurrent tumors. Although steroid treatment had no effect on marker expression, as no difference was detected on resting or in vitro activated neutrophils when patients were stratified according to steroid treatment, primary GBM patients treated with steroids had an increased prevalence of circulating activated neutrophils when compared to steroid-treated recurrent patients.
Conclusions: Together, these data support a tumor-specific process through which intravascular activation of neutrophils is followed by arrested transmigration and persistence of activated neutrophils within the circulation of patients with GBM. This effect appears to be augmented by treatment with steroids. Ongoing studies are focused on further exploring interactions between tumor and neutrophils at the vascular interface.
Citation Format: Trisha R. Sippel, Rae Russell, Timothy Ung, Marci Klaassen, Allen Waziri. Disrupted transmigration of neutrophils in glioblastoma patients is augmented by steroid treatment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A44.