Role of Receptor for Advanced Glycation End product (RAGE) pathway in brain tumor macrophage function
Brain is a common site for metastatic spread of many cancers. Although immunotherapy strategies are being pursued for most cancers, such approaches may have limited efficacy for malignant brain tumors. This limitation is in part attributable to the 'immune privileged' status of the central nervous system and lack of penetration of immune cells into such tumors. Recent studies, however, indicate that several types of immune cells, including lymphocytes and macrophages, can indeed migrate into brain tumors. In contrast to brain inflammatory disease states, these cells appear to be inactive and incapable of mounting a response against tumor cells. Our own studies, for example, suggest that macrophages isolated from experimental brain tumors express low levels of stimulatory cytokines and appear to be more resistant to activation. The exact mechanism of this immune suppression is unclear, but factors secreted by tumor cells (and perhaps macrophages themselves) may be responsible for this inactivation.
Recently, STAT3 proteins have been suggested to play a role in the suppression milieu of tumor microenvironment. STAT3, which promotes tumor angiogenesis and growth, is also upregulated in tumor-infiltrating macrophages and result in their inhibition. We have shown that when STAT3 function is inhibited in macrophages, these cells become active again and can slow brain tumor growth. These findings suggest that a better understanding of STAT3 regulation in tumor macrophages may be beneficial in reversing the immunosuppressive tumor microenvironment and enhancing the effect of systemic immunotherapies.
As components of the innate immune system, macrophages are equipped with machinery to rapidly respond to acute changes in the body. For example, they express membrane receptors, such as RAGE, which are activated in response to injury, infection or oxidative stress. RAGE activation has been shown to play a role in inflammatory processes in diabetes or arthritis. Although few reports have examined the role of RAGE and RAGE ligands in cancer, to our knowledge no one has studied the function of this receptor in brain tumor macrophages.
Despite angiogenesis, or the formation of new tumor-supporting blood vessels, brain tumors often outgrow their blood supply and exhibit tissue death or necrosis. Because brain tumors express high levels of RAGE ligands (which can be released into brain tissue after tumor necrosis), we hypothesize that the RAGE pathway can play a role in macrophage function in tumors. To test this, RAGE is inhibited in macrophages after exposure to brain tumor cells. Interestingly, RAGE blockade resulted reverses macrophage suppression in response to glioma factors. Furthermore, we have noted concomitant decreases in STAT3 expression. These findings, suggest for the first time that the RAGE pathway controls STAT3 function in macrophages and potentially suppresses macrophage function.
To test this hypothesis we will perform the following experiments. First, the effect of RAGE inhibition on macrophage function will be studied in cultured cells. Macrophages will be exposed to glioma factors or RAGE ligands, and then their immune function will be evaluated by examining gene expression and their activation state. Direct comparison of macrophages under these conditions, before and after RAGE inactivation, will allow us to better characterize RAGE-dependent events that lead to macrophage activation or suppression in response to glioma factors. Results from these experiments can also identify pathways that regulate RAGE and STAT3 expression.
To confirm the results of these experiments and assure that findings are not due to in vitro artifacts (which can influence macrophage function), the role of RAGE will be studied in macrophages derived from brain tumor models. Macrophage function will be studied before and after RAGE inhibition in mice brain tumors. We will determine whether RAGE inhibition results in further activation of macrophages and modulation of the tumor immune response.
Finally, using tissue derived from the first two experiments, we will study possible mechanisms by which RAGE activation regulates STAT3 function in macrophages. Because we have shown that STAT3 inhibition can activate macrophages in our models, understanding possible interactions between RAGE and STAT3 may provide new approaches to regulate these pathways and macrophage immune function in brain tumors.
Overall, these studies will promote better understanding of two key pathways that may regulate macrophage immune function in brain tumors: RAGE and STAT3. Results from these preliminary experiments will be used to generate data that could refine our hypothesis for future grant submissions. Moreover, knowledge of macrophage immune function will optimize future immune-based strategies against tumors.
Because macrophages actively participate in other CNS pathologies such as trauma, ischemia and inflammation, our findings may be relevant to the understanding and treatment of these processes as well. For example, infiltration of brain parenchyma with migrating macrophages within 24 of ischemia can significantly contribute to cerebral edema and secondary brain injury. Release of RAGE ligands from the injured brain may also trigger macrophage migration into the brain. If so, blocking this process could potentially provide new treatments for cerebral injury.