Our current knowledge of how a phagocyte manages engulfment and digestion of apoptotic corpses remains sparse. Understanding the mechanisms by which a phagocyte engulfs and digests corpses is particularly important, because immune evasion by cancer relies on phagocytosis of cancer cells to establish a tolerogenic microenvironment. We identified a network of solute carrier (SLC) proteins in phagocytes engulfing apoptotic Jurkat lymphoma cells associated with distinct physiological processes and demonstrated functional relevance for two SLCs: SLC2A1 (facilitates corpse uptake via glucose transport) and SLC16A1 (required for export of the anti-inflammatory molecule lactate). In this application, we show that phagocytes regulate several distinct functional programs in response to corpse engulfment and digestion. One of these programs includes SLC12A2 which, together with the upstream kinases WNK1/OSR1/SPAK, act as a physiological ?brake? on apoptotic Jurkat cell engulfment via chloride sensing/flux and cell volume regulation. This application proposes to test the hypothesis that deletion/ inhibition of the SLC12 pathway will lead to increased lung adenocarcinoma clearance in vitro/ in vivo and induce a clinically beneficial switch from ?tolerogenic? to ?immunogenic? cancer cell clearance. Preliminarily, SLC12A2-deficient phagocytes actively engulfing apoptotic Jurkat cells exhibited suppression of the canonical anti-inflammatory transcriptional signature and induction of a pro-inflammatory signature highlighted by a robust type I interferon response. Furthermore, using bioactive small molecules targeting SLC12A2 or WNK1, we provide proof-of-principle that targeting this pathway leads to boosted Jurkat cell clearance in vivo. These studies will provide further insight into a previously unknown cell clearance regulatory pathway, the role of this pathway in lung adenocarcinoma development/ progression, and explore the hypothesis that genetic/ pharmaceutical perturbation of the SLC12 pathway will promote increased cancer cell clearance and a stronger anti-cancer immune response. During the mentored phase of this application, I will master experimental techniques for the lungs such as intratracheal injection and bronchioalveolar lavage fluid collection as well as establish crucial lung adenocarcinoma models proposed herein. I will establish new conditional deletion mice to evaluate genetic perturbation of the SLC12 pathway on the development/ progression of lung adenocarcinoma. Together with colleagues, we will develop new tools to assess the importance of volume regulation during tumor cell clearance in vivo, allowing us to understand how phagocytes establish immune tolerance during lung adenocarcinoma development/ progression. With the guidance of my mentoring committee, I will strengthen my scientific and professional skillsets in preparation for the independent phase. During the independent phase, I will explore targeting the SLC12 pathway during lung adenocarcinoma development/ progression using small molecules. Further, I will combine my areas of expertise in functional genomics, T cell biology/antigen presentation, and apoptotic cell clearance together with the tools developed during the mentored phase to understand how lung cancer exploits phagocyte volume regulation to evade the immune response. The proposed project will promote my scientific and professional training and allow me to successfully establish an independent academic research program.
The question of how a phagocyte regulates its own physiology during apoptotic cancer cell clearance is unknown and of great importance because immune tolerance of developing/ progressing cancer depends on rapid removal of apoptotic cancer cells. Phagocytes utilize chloride flux through opposing solute carrier channels to either initiate apoptotic cell uptake or inhibit subsequent corpse uptake, respectively. This proposal aims to evaluate the role of this pathway in phagocytes during lung adenocarcinoma development/ progression and assess the therapeutic potential of targeting this pathway to promote both increased cancer cell clearance and a stronger anti-tumor immune response.
