Phospholipid Externalization Blockade as an Antitumor Immunotherapy

In eukaryotic cells, phospholipid asymmetry is actively maintained, with phosphatidylserine (PS) typically confined to the inner leaflet of the plasma membrane (PM), due to the active performance of the PS flippase ATP11/CDC50A complex. However, in the tumor microenvironment (TME), PS exposure on the outer leaflet occurs from multiple sources, including apoptotic tumor cells, necrotic tissue, viable endothelial cells, and tumor-derived exosomes. Especially, in apoptotic cells, the PS scamblase Xkr8 mediates PS externalization. This exposed PS plays a crucial role in immune suppression within the TME. PS binds to receptors on phagocytes, primarily macrophages and dendritic cells (DCs), triggering efferocytosis (the engulfment of PS-positive cells, usually apoptotic cells) and promoting anti-inflammatory responses.

To understand the immune suppressive role of PS exposure on tumor cells, we deleted CDC50A, the PS flippase, from the tumor. Thus, in this tumor, PS is constantly on the outer leaflet of PM, called PS^out tumor model. On the contrary, we knocked out Xkr8 from tumor cells. Thus, even tumor cells undergo apoptosis, PS can still stay in the inner leaflet of PM, called PSⁱⁿ tumor model. Taking the advantage of the PSⁱⁿ and PS^out model, we could investigate the anti-tumor immune responses of PS externalization in TME.

Using PS^out model, we found that these PS^out tumors exhibited enhanced growth, M2-polarized tumor-associated macrophages (TAMs), and reduced tumor-antigen-specific T cell infiltration. In TME, the PS receptor TIM-3 on TAMs was responsible for PS sensing. Using PSⁱⁿ model, we found that the PSⁱⁿ tumors exhibited increased anti-tumor immunity, featuring suppressed tumor progress, TAM M1 polarization, suppressed IL-10 secretion, and enhanced natural killer (NK) cell cytotoxicity. Thus, blocking PS externalization via targeting Xkr8 could serve as a promising strategy for anti-tumor immunotherapy.

However, there is no available Xkr8 inhibitor or direct anti-PS blocking antibody for therapeutic use. Thus, we developed our unique "PS all-block" strategy leveraging an engineered protein which binds only to PS, without sending signals to immune receptors, functioning as a dominant negative. The "PS all-block" can neutralize PS molecules from all sources without triggering downstream immune suppression pathways. Our data suggested that the "PS all-block" was a more effective antitumor immunotherapy compared to our successfully developed Xkr8 inhibition, as Xkr8 targeting only neutralized apoptotic PS, while the "PS all-block" approach could neutralize PS from all sources.