Susceptibility of Melanoma Cells to Targeted Therapy Correlates with Protection by Blood Neutrophils.

Elevated levels of peripheral blood and tumor tissue neutrophils are associated with poorer clinical response and therapy resistance in melanoma. The underlying mechanism and the role of neutrophils in targeted therapy is still not fully understood. Serum samples of patients with advanced melanoma were collected and neutrophil-associated serum markers were measured and correlated with response to targeted therapy. Blood neutrophils from healthy donors and patients with advanced melanoma were isolated, and their phenotypes, as well as their in vitro functions, were compared. In vitro functional tests were conducted through nonadherent cocultures with melanoma cells. Protection of melanoma cell lines by neutrophils was assessed under MAPK inhibition. Blood neutrophils from advanced melanoma patients exhibited lower CD16 expression compared to healthy donors. In vitro, both healthy-donor- and patient-derived neutrophils prevented melanoma cell apoptosis upon dual MAPK inhibition. The effect depended on cell-cell contact and melanoma cell susceptibility to treatment. Interference with protease activity of neutrophils prevented melanoma cell protection during treatment in cocultures. The negative correlation between neutrophils and melanoma outcomes seems to be linked to a protumoral function of neutrophils. In vitro, neutrophils exert a direct protective effect on melanoma cells during dual MAPK inhibition. This study further hints at a crucial role of neutrophil-related protease activity in protection.

Using i-GONAD for Cell-Type-Specific and Systematic Analysis of Developmental Transcription Factors In Vivo.

Transcription factors (TFs) regulate gene expression via direct DNA binding together with cofactors and in chromatin remodeling complexes. Their function is thus regulated in a spatiotemporal and cell-type-specific manner. To analyze the functions of TFs in a cell-type-specific context, genome-wide DNA binding, as well as the identification of interacting proteins, is required. We used i-GONAD (improved genome editing via oviductal nucleic acids delivery) in mice to genetically modify TFs by adding fluorescent reporter and affinity tags that can be exploited for the imaging and enrichment of target cells as well as chromatin immunoprecipitation and pull-down assays. As proof-of-principle, we showed the functional genetic modification of the closely related developmental TFs, Bcl11a and Bcl11b, in defined cell types of newborn mice. i-GONAD is a highly efficient procedure for modifying TF-encoding genes via the integration of small insertions, such as reporter and affinity tags. The novel Bcl11a and Bcl11b mouse lines, described in this study, will be used to improve our understanding of the Bcl11 family's function in neurodevelopment and associated disease.