Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

Abstract

Background: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C-type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.

The Posttraumatic Increase of the Adhesion GPCR EMR2/ADGRE2 on Circulating Neutrophils Is Not Related to Injury Severity

“…To enrich neutrophils, density gradient centrifugation at 550 g/30 min using Polymorphprep was used (Serumwerk Bernburg, Germany), but yielded insufficient purity. A multistep protocol including the removal of platelets and a two-time density gradient of histopaque 1119 and 1077 (Sigma-Aldrich GmbH, Taufkirchen, Germany) at 700 g/45 min centrifugation [27] avoided the release of cell debris via red blood cells lysis. However, damaged/dying cells and cell debris were still present, as seen through unbiased scatter analysis in flow cytometry; thus, neutrophils were truly enriched only up to ~70%.…”