Cyclic hypoxia induces transcriptomic changes in mast cells leading to a hyperresponsive phenotype after FcεRI crosslinking

Mast cells (MCs) play an important role in tumor development, executing pro or antitumoral functions depending on tumor type and local conditions present in the tumor microenvironment such as cyclic hypoxia (cyH), which is a distinguishing feature of almost all solid tumors. In this study, we analyzed transcriptional changes of murine bone marrow-derived mast cells (BMMCs) exposed to an in vitro protocol of cyH, consisting of interleaved cycles of hypoxia and re-oxygenation at 1% and 21% of oxygen, respectively. Utilizing a mouse M22K microarray (Microarray Unit, Cellular Physiology Institute, UNAM, Mexico City), we identified 2512 genes in MCs subjected to cyH whose expression displayed changes compared with normoxic cells. Furthermore, functional enrichment analysis revealed that cyH-related transcriptomic alterations were for genes associated with oxidative phosphorylation and the FcεRI signaling pathway. Interestingly, after IgE/ antigen (Ag) challenge, FcεRI-dependent degranulation, calcium mobilization, and PLC-γ activity were enhanced in BMMCs exposed to cyH compared to those cells maintained in normoxic conditions. In addition, the expression of calcium signaling-related cytokines such as TNF-α, IL-4, and IL-2 was increased in BMMCs exposed to cyH after IgE/Ag challenge. Taken together, these findings indicate that cyH induces transcriptomic modifications in MCs that are translated into a phenotypic change characterized by hyperresponsiveness to IgE/Ag challenge. A two-color microarrays experiment was performed. We separately pooled equal amounts of high-quality RNA from three different cell cultures of BMMCs, which were exposed to normoxia (21%oxygen for 2.5 hours) or cyclic hypoxia (1 hour of hypoxia at 1% oxygen and 30 minutes at 21% oxygen, followed by a second hour of hypoxia at 1% oxygen.). Then, cDNA synthesis was performed and ach sample was labeled as follows: Alexa555 (Cy3) labeling was used for normoxia while Alexa647 (Cy5) labeling was used for cyclic hypoxia. Equal quantities of both samples were hybridized in the same microarray.