CCL2‐ Dependent Macrophage Recruitment by Glioblastoma Stem Cells: The Role of Gα12 and YAP Signaling

Abstract

Glioblastoma multiforme (GBM), the most malignant form of brain tumor, has an average survival rate of less than two years, associated with recurrence despite conventional interventions. GBM tumors contain myeloid cells, recruited in response to inflammatory cytokines which play a role in the tumor progression and may also contribute to immune suppression. Tumor associated macrophages interact with tumor cells and conversely tumor cells contribute to generation of the inflammatory immune cell environment. Gα12 protein-coupled receptors are regulated by inflammatory mediators often generated in the tumor cell environment. Stimulation of these receptors activates the transcriptional co-activator YAP (Yes-associated protein 1) which can in turn induce inflammatory gene expression. Our previous studies showed that knockdown of YAP in GSC-23 PDX cells significantly attenuated in vitro stemness, proliferation, and migration. In vivo experiments showed that YAP knockdown PDX cells formed significantly smaller tumors and had lower lethality than wild-type cells. Our more recent in vivo studies with GSC-23 cells suggested that there is a significant inflammatory response in the tumor cell environment. In order to determine if Gα12/YAP signaling in this glioma stem cell tumor line contributed to macrophage infiltration we utilized an in vitro. Transwell migration assay with human THP-1 macrophages on the top and GSC-23 cells on the bottom. Remarkably, the presence of GSC-23 cells strongly enhanced THP-1 cell migration and this could be mimicked almost fully by replacing the cells with medium in which they were cultured for 24 hours. One of the most efficacious mediators of macrophage migration is CCL2 (MCP-1). The involvement of CCL2 and its receptor CCR2 on THP-1 cell migration was demonstrated by the ability of the CCR2 blocker RS102895 to significantly inhibit the migratory response elicited by GSC-23 cells or cell medium. To understand whether the generation of CCL2 and thus the effect of the tumor cells on macrophage recruitment was specifically regulated, we examined GSC-23 cells in which YAP or Gα12 were deleted. YAP knockout prevented GSC-23 cell mediated migration suggesting that YAP regulates production of CCL2 by the GSC-23 cells. We observed that CCL2 mRNA levels were in fact significantly lower in YAP knockdown compared to WT cells. Notably this appeared to depend on the presence of the THP-1 cells suggesting that macrophage crosstalk to the GSC-23 tumor cells controls CCL2 generation. IL-6 mRNA levels were similarly regulated by THP-1 signaling to GSC-23 cells through YAP. Using a Gliovis tool to analyze data sets of GBM tumors, we determined that there is a highly significant Pearson's correlation between YAP1 and CCL2 gene expression as well as a correlation between YAP1 and IL-6 expression. Further of studies are underway to explore mechanisms responsible for this glioma stem cell /macrophages crosstalk in co-culture experiments. We suggest that YAP mediated cytokine generation by glioma stem cells represents a critical point for therapeutic intervention to disrupt recruitment of macrophages and interfere with their potential role in creating an immunosuppressive myeloid cell tumor microenvironment.