Melanoma
.jpg "Steven H. Sun, MD, MS photo")
Steven H. Sun, MD, MS
Resident Physician
Ohio State University
Columbus, Ohio, United States
Disclosure: Disclosure information not submitted.
Myeloid-derived suppressor cells (MDSC) expand in cancer patients and MDSC exert their immunosuppressive effects in large part through the production of reactive nitrogen and oxygen species, which combine to form peroxynitrite (PNT). PNT inactivates signaling proteins through tyrosine nitration. Targeting MDSC via PNT inhibitors is an avenue to improve the response to immunotherapy.
Methods:
Melanoma patients and healthy donors were consented to participate in an IRB-approved clinical registry. Peripheral blood mononuclear cells (PBMC) and MDSC were isolated from blood samples via flow cytometry. PBMC and MDSC were cultured with immunoglobulin-G (IgG) labeled 10 µm TentaGel beads. Upon immune cell recognition of/reaction with IgG, PNT fluorescence was detected via PS3, a novel sensor compound invented by our group. Cells cultured with PS3 and beads were treated for 4 hr with ibrutinib (10 nM, 1 µM), an FDA-approved Bruton’s tyrosine kinase inhibitor, or with dasatinib (50 nM, 500 nM), an FDA-approved Src tyrosine kinase inhibitor. Fluorescence produced by PNT generation was measured using a Clariostar plate reader.
Results:
MDSC were significantly elevated in Stage 3, 4 melanoma patients compared to normals (12.0% vs. 3.8%; p< 0.01). Purified MDSC had significantly higher PNT production compared to unmanipulated PBMC (9.8 vs 2.7-fold increase over control). PBMC from cancer patients produced significantly more PNT than those from healthy donors (10.9 vs 2.8-fold increase over control). Ibrutinib inhibited PNT output by 30% and 50% at 10 nM and 1 µM. Dasatinib inhibited PNT by 50% and 90% at 50 and 500 nM. PNT was accurately detected by this sensor system and correlated strongly with circulating MDSC levels (R2=0.9518).
Conclusions: PBMC PNT output could be reproducibly quantified and correlated with MDSC levels. Ibrutinib and dasatinib significantly inhibited PNT production. This sensor system allows for translational studies of novel compounds that inhibit PNT and could be paired with new immunotherapies.