LIF-responsive genes in monocytes from healthy donors

LIF has an important role in immunosupression in different scenarios, such as in embryo implantation in the uterus, autoimmune disease or organ transplantation. In tumor progression it has been largely demonstrated the importance of immune system. The fact that LIF is highly expressed in certain tumor types, in addition to its immunomodulatory properties, led us to hypothesize that tumors expressing high levels of LIF might be promoting an immune-tolerant microenvironment precluding the anti-tumor immune response. To establish whether the effect of LIF was relevant in the context of human monocytes, we isolated CD14+ cells (monocytes) from peripheral blood mononuclear cells (PBMCs) from healthy donors and analyzed the effect of LIF blockade. Instead of using recombinant LIF, we studied the effect of the endogenous LIF secreted by U251 cell line. RNA was extracted from the CD14+ cells and the transcriptomic analysis was performed. We selected the GBM cell line U251 that secreted high levels of LIF. Those cells were engineered by us to express shRNAs targeting LIF, in order to knock down LIF expression and secretion. We collected the conditioned media from control and shLIF U251 cells, and incubated human monocytes (CD14+ cells isolated from peripheral blood mononuclear cells from healthy volunteers) with the conditioned media. After 5 day treatment, cells were lysed for RNA extraction and hybridized on Affymetrix microarrays to determine how the specific blockade of LIF impacted on the gene expression of monocytes.