# The Presence of Neutrophil Extracellular Traps (NETs) in Brain Tumor Vessels Is Linked to Platelet Aggregates and Podoplanin in the Tumor Microenvironment

## TL;DR

This study explores how neutrophil extracellular traps in brain tumor vessels are connected to platelet clusters and podoplanin, which may contribute to blood clot formation.

## Contribution

The study identifies a novel link between podoplanin-induced platelet activation and neutrophil extracellular traps in the tumor microenvironment.

## Key findings

- H3Cit+ tumor vessels were significantly associated with increased intravascular platelet clusters.
- H3Cit+ tumor vessels were significantly associated with podoplanin expression in tumor tissue.
- H3Cit+ tumor vessels correlated with higher D-dimer levels and tumor-infiltrating neutrophils.

## Abstract

Several mechanisms can induce cancer-associated thrombosis. In brain tumors, podoplanin upregulation on cancer cells appears to play a pivotal role in the development of venous thromboembolism (VTE). Of particular note, podoplanin is able to activate platelets via the C-type lectin-like receptor 2 (CLEC-2). Generally, inflammation is also involved in clot formation. For example, so-called neutrophil extracellular traps (NETs) can be released by neutrophils upon multiple triggers (e.g., activated platelets) and then lead to venous thrombosis. In this study, prothrombotic NET components in brain tumor vessels were linked to local procoagulant characteristics such as intravascular platelet clusters and podoplanin expression. These results highlight a possible relationship between podoplanin-induced platelet activation and NET formation, which might enhance hypercoagulability and thrombus development.

Background: Multiple mechanisms might lead to cancer-related hypercoagulability. In brain tumors, podoplanin, via its ability to activate platelets, seems to play a crucial role in developing venous thromboembolism (VTE). Different stimuli (including activated platelets) can trigger the release of prothrombotic neutrophil extracellular traps (NETs) by neutrophils. It remains to be elucidated whether podoplanin-induced platelet aggregates might also impact NET formation and subsequent hypercoagulability and thrombosis. Methods: Patients with glioma were enrolled in this prospective observational cohort study. The primary endpoint was VTE. Immunohistochemical staining of NETs (via citrullinated histone H3 [H3Cit]) and neutrophils (via myeloperoxidase [MPO]) was conducted in glioma specimens and correlated with intravascular platelet clusters (via CD61) and podoplanin. Results: In total, 154 patients were included. H3Cit+ tumor vessels were found in 45/154 cases. H3Cit were significantly associated with increased intravascular platelet clusters (CD61− vs. CD61+ vs. CD61++ vs. CD61+++: 3.7% (1/27) vs. 18.6% (11/59) vs. 39.4% (13/33) vs. 57.1% (20/35), p < 0.001) and podoplanin expression (PDPN− vs. PDPN+: 14.3% (7/49) vs. 36.2% (38/105), p = 0.007) in the tumor tissue. Furthermore, H3Cit+ tumor vessels were significantly associated with tumor-infiltrating MPO+ neutrophils (H3Cit− vs. H3Cit+, median [Q1-Q3]: 6.0 [3.3–12.3] vs. 12.5 [5.9–22.0] cells/mm2, p < 0.001) and with D-dimer levels (H3Cit− vs. H3Cit+: 0.53 [0.32–1.10] vs. 0.84 [0.46–2.75] µg/mL, p = 0.034). The VTE risk was not linked to H3Cit+ tumor vessels (p = 0.613, log-rank). Conclusions: H3Cit in tumor vessels was not associated with VTE. However, H3Cit was linked to a local procoagulant phenotype in glioma, thereby potentially contributing to a systemic hypercoagulable state and thrombus formation.