Phosphatidylserine: paving the way for a new era in cancer therapies

Abstract

Lipid phosphatidylserine (PS) plays a vital role in the growth and proliferation of cancer cells and has been identified as a potential target for cancer treatment. Recent studies have focused on using phosphatidylserine-targeting agents in the treatment of several classes of cancer, such as breast, lung, and prostate. The use of PS-targeting antibodies to target cancer cells while leaving healthy cells unharmed is selective. These antibodies are specifically targeted to phosphatidylserine molecules located on the exterior membrane of cancer cells, triggering a series of events that ultimately destroy the cancer cells. In addition, incorporating phosphatidylserine into the liposome membrane specifically targets cancer cells, thereby enabling more efficient drug delivery and improving cancer treatment outcomes. In general, PS has active ingredients that are currently undergoing clinical trials for potential use in treating various types of cancer. On the role of phosphatidylserine in biophysical and cancer biology, this review summarizes recent studies, as well as related prospective clinical and preclinical trials such as immunotherapy and biomarkers. A new indication of future PS implementation in cancer therapy appears to be a new era.

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1. Introduction

Cancer is a multifaceted and complex disease, which has impacted numerous individuals globally. This chronic condition is marked by the uncontrolled proliferation of cells and the infiltration and dissemination of cells from their original sites to other locations within the body.^1–3 According to Cancer Research UK, there exists a multitude of over 200 diverse forms of cancer that have the potential to occur in any anatomical region and can exert influence on individuals across various age groups, genders, and ethnic backgrounds.⁴ This phenomenon has instigated a significant shift in the conventional methodology of medical treatment, transitioning away from a generalized approach and towards a more personalized or “customized therapy” wherein the determination of treatment options is predicated upon the specific mutational configuration of a patient's tumor.⁵ Tumors can be classified as benign or malignant.⁶ Benign neoplasms are non-malignant tumors that cannot spread throughout the body. However, some neoplasms can still be dangerous based on their location, such as an intracranial neoplasm, which may present challenges in surgical removal.⁷ Whereas, malignant tumors do not remain encapsulated, show features of invasion, and metastasize,^5,8 which are often more challenging to treat and can be life-threatening.⁹

Evidence suggests that cancer is a disease of the genome at the cellular level.^1–3 Interestingly, most agents responsible for the onset of cancer, acknowledged as carcinogens, induce modifications in the sequence of DNA or mutations, commonly referred to as mutagens. Consequently, akin to all genetic disorders, the development of cancer is a consequence of modifications occurring within the DNA.¹⁰ Tobacco use, exposure to radiation or chemicals, virus infections, and specific dietary and lifestyle variables can all result in these alterations, which can either be inherited or acquired.^11,12

Despite notable advancements in cancer research and treatment, there is still a great need for new and effective therapies that can improve patient outcomes while reducing toxic side effects. In particular, the lipid composition of the plasma membrane has been associated with the advancement and progression of cancer.¹² One promising avenue of research involves targeting PS, a molecule pivotal for proliferation and survival. PS and phosphatidylcholine (PC) are two significant phospholipids that contribute to cellular membrane structural and functional properties.³ PS is a negatively charged phospholipid naturally present on the inner layer of the cell membrane, but during apoptosis or cell activation, the asymmetric arrangement of the phospholipids of the cell membrane is disrupted. With this novelty, PS is flipped to the outer leaflet of the membrane, where it serves as an “eat-me” signal for phagocytic cells.⁶ Additionally, PC is abundant in the outer leaflet of the cell membrane and contributes to membrane stability and fluidity. PS, which is available in outer plasma membranes and is subjected to a variety of stimuli, also involves the progression of various diseases,^13,14 which may be found in other membranes during cell formation.

Basically, PS is dysregulated in different cancer types, including breast, lung, and colon cancer.¹⁵ Nowadays, PS is a promising target to focus on for cancer therapy due to its involvement in cancer progression and its potential as a focal point for therapeutic intervention. Understanding the role of PS in cancer cell survival and proliferation could lead to the development of innovative treatment approaches that specifically disrupt cancer cell function while sparing normal cells.¹⁶ Recent studies suggest that the inhibition of PS synthesis or the disruption of its interaction with proteins involved in cancer cell function presents an exciting avenue for novel cancer therapy.¹⁷ In addition, PS not only contributes to cancer cell survival and proliferation but also regulates tumor progression and metastasis.^18,19 This dysregulation of PS has been associated with the ability of cancer cells to evade the immune system and promote angiogenesis, the neo-genesis of blood vessels that is crucial for tumor growth and dissemination.^20–22

Furthermore, the relationship between PS and drug resistance in cancer has garnered significant attention. Cancer cells with elevated levels of PS have been found to exhibit increased resistance to chemotherapy and targeted therapies, posing a novelty and a considerable challenge in the clinical management of cancer.²³ In addition to targeting PS directly, there is growing interest in utilizing PS as a cancer biomarker for prognosis and diagnosis.^24,25 Advancements in imaging technologies and molecular profiling techniques have enabled the detection of elevated PS levels in cancerous tissues, providing valuable insights into disease progression and the potential response to treatment.^15,26 Herein, we discuss several strategies that have been explored, including the use of antibodies, small molecules, and nanoparticles. By diminishing the levels of PS within cancer cells, these molecules can hinder their proliferation and viability. Encouraging outcomes have been observed in clinical trials by investigating PS-targeted therapies.²⁶ Furthermore, this review addresses the challenges and future perspectives of PS-targeted therapies. Despite its promising potential, several hurdles remain, including the need for precise delivery systems and a better understanding of the role of PS in different cancer types. By elucidating these aspects, we hope to pave the way for the successful integration of PS-targeted strategies into the broader framework of cancer treatment. As research continues to unravel the complexities of PS and its interactions, it holds the promise of transforming cancer treatment paradigms, making it an essential focus for future oncological research.

2. Structural organization of PS

In a cell, PS and PC are the crucial phospholipids that are essential components of cell membranes. These phospholipids are asymmetrically distributed on the cell membrane, with PS predominantly located on the inner surface of the cell membrane and PC predominantly situated on the outer side.²⁷ Cell-to-cell contact, membrane trafficking, signal transduction, and other cellular activities are all significantly impacted by the structural arrangement of PS and PC on the cell membrane.²⁸ Understanding the organization and function of PS on the cell membrane is essential for developing targeted therapies for various diseases, including cancer.^29,30

2.1. Molecular structure of PS with PC

PS is a negatively charged phospholipid composed of a serine molecule, a glycerol backbone, and two fatty acid chains. The serine molecule is attached to the glycerol backbone by a phosphate group and the fatty acid chains that form the hydrophobic tail.³¹ The hydrophilic head of PS contains a phosphate group, a serine amino acid, and a carboxyl group, which contribute to its negative charge.^32–36

The plasma membrane of mammals is composed of nearly 1000s of phospholipid molecules, out of which PS comprises approximately 2–10% of total phospholipids.³⁷ PS is a crucial phospholipid made up of two fatty acids attached by an ester linkage to the first and second carbons of the glycerol and serine linked by a phosphodiester linkage to the third carbon. It is a principal cell membrane component that is specifically present in the inner cytoplasmic leaflet of the membrane.³⁸ PC and phosphatidylethanolamine (PE) are mostly converted enzymatically by mammalian cells via a serine exchange process to produce PS by the action of 2 enzymes, i.e. S-synthase 1 (substrate PC) and PS-synthase 2 (substrate PE), generated from the endoplasmic reticulum.³⁹ PS is essential for apoptosis and cell signalling.⁴⁰ It is generally accepted that one of the primary mechanisms for eliminating apoptotic cells is PS exposure to the outside of the cell membrane, which sends a signal called “eat me signal” and marks the cell for destruction.⁴¹ The chemical structure of PS confers upon it a crucial function as an essential constituent of cell membranes. It comprises a glycerol backbone, fatty acid chains, phosphate group, and amino acid serine. The glycerol backbone is a three-carbon structure that provides a rigid framework for the careful arrangement of the other PS constituents, and 2 fatty acid chains are present in PS that provide fluidity and stability to the cell membrane.⁴² The glycerol backbone of the PS molecule is attached to a phosphate group, which is further linked to a serine amino acid, hence giving the molecule the name “phosphatidylserine.” This linkage delivers hydrophobic properties to the molecule, as shown in Fig. 1.⁴²

Fig. 1 (A) Diagram showing the structure of a typical glycerophospholipid, or PS, which has both unsaturated and saturated fatty acyl chains. The head group has one net negative charge at a physiological pH. The interaction between PS and the C2 domain of lactadherin (LactC2; blue-green-barrel structure) in a membrane bilayer is depicted in (B). The PS head groups are displayed in fuchsia, and the remaining lipids are brown. Three “fingers” of the LactC2 structure are observed, which are believed to reach the hydrophobic area of the membrane bilayer and contain hydrophobic amino acids. Reprinted from, ref. 41 Copyright CC BY 2011 MDPI.

However, PC is a neutral phospholipid composed of a choline molecule, a glycerol backbone, and two fatty acid chains.⁵ Fatty acid chains produce the hydrophobic tail of the choline molecule, which is joined to the glycerol backbone by a phosphate group. The hydrophilic head of PC contains a phosphate group, a choline molecule, and a glycerol group.^32,43 All the phospholipids are present in eukaryotic cell membranes, in which PC is the most prevalent, making up 40–50% of the total.³⁷ It is one of the main components of lung surfactants and cell membranes and is abundantly present on the outer surface of the cell membrane. It is believed that the PC transfer protein (PCTP), which belongs to the steroidogenic acute regulatory protein-related transfer (START) superfamily, moves between membranes inside the cell and plays a crucial role in cell signalling.⁴⁴ A range of fatty acids make up the glycerophosphoric acid and choline head groups that make up this phospholipid. A significant amount of dipalmitoylphosphatidylcholine can be found in animal lung PC.⁴⁵

Two methods allow mammalian species to generate PC, out of which the CDP-choline pathway is the most important. This pathway is also known as the ‘Kennedy pathway’, as described by Eugene and Kennedy.⁴⁶ Three enzymatic steps comprise the CDP pathway, which requires choline. First, choline kinase phosphorylates the choline into PC at the expense of ATP. Second, PC using CTP in the presence of cytidylyltransferase (CT) is converted into CDP-choline. Third, the conversion of CMP to diacylglycerol to create PC is catalyzed by 1,2-diacylglycerol choline phosphotransferase (Fig. 2). Through three consecutive methylations of PE by phosphatidylethanolamine N-methyltransferase (PEMT), PC can also be produced endogenously in a second pathway.⁴⁷ The relevance of the endoplasmic reticulum (ER) in PC production was highlighted by subcellular fractionation, which showed that PEMT and the last enzyme in the CDP-choline pathway are both found there.^48,49

Fig. 2 Phospholipids showing the structure of the PC. All phospholipids share the same fundamental structure but different fatty acids and head groups in place of choline. Sn 1, 2, and 3 positions are marked. Reprinted from, ref. 41 Copyright CC BY 2019 MDPI.

2.1.1. Distribution of PS and PC in the membrane. PS and PC are both found in the membrane of all cells, but their distributions are different in different cell types and the specific membrane domain. In general, PS is more abundant in the inner side of the cell membrane, where it constitutes up to 10–20% of the total phospholipids.⁵⁰ This asymmetric distribution is maintained by a class of enzymes called flippases,⁵¹ which selectively transport PS from the outer to the inner leaflet. In healthy cells, exposure to PS on the outer leaflet is minimal.⁵² In apoptotic cells, PS is translocated to the outer surface, where it serves as a signal for phagocytosis by macrophages and other immune cells.⁵³ Moreover, PC is more abundant on the outer side of the cell membrane, constituting up to 50–60% of the total phospholipids.⁵⁴ This distribution is also maintained by flippases, which selectively transport PC from the inner to the outer surface. Furthermore, PC is present in lipid rafts,⁵⁵ which are specialized membrane regions essential for cell signalling and signal transduction and rich in signalling molecules and membrane receptors.⁵⁶

In mammalian cells, PS is produced inside a particular area known as mitochondria-associated-membrane (MAM),⁵⁷ which is present in the ER and is synthesized from PC or PE by phosphatidylserine synthase-1 (PSS-1) or phosphatidylserine synthase-2 (PSS-2) through a base-exchange process with serine, as shown in Fig. 3. After blending due to the interaction of MAM and mitochondrial exterior layers, a part of the PS moves into the mitochondria.^24,58 The PS present inside the mitochondria releases carbon dioxide. PE is regulated by the PS decarboxylase (PSD) found in the prokaryotes and mitochondria of eukaryotes (Fig. 3). Further, it was found that insufficiency of PSD quality resulted in underdevelopment and severe health issues in mice.^24,58 Thus, for proper cell growth and mitochondrial functionality, the process of PE production through PS becomes crucial. The remaining PS is sent to various organelles, such as plasma film and Golgi, as shown in Fig. 3. Most transportation occurs through non-spontaneous processes, such as solvent vehicle proteins or vesicles.⁵⁸ However, the number of integrated PS molecules that enter mitochondria vs. other organelles is still a mystery.

Fig. 3 PS production inside MAM from PC either by PSS1 or PSS2. Further, a few PSs are transmitted to mitochondria and decarboxylated to form phosphatidylethanolamine by PS decarboxylase.⁵⁸

2.1.2. Interactions of PS and PC with other lipids and proteins. The presence of PS and PC in the membrane has important implications for their interactions with other lipids and proteins. PS has a high affinity for calcium ions, and its binding to calcium is known to regulate various cellular processes, such as blood clotting, synaptic transmission, and apoptosis.⁵⁹ In addition, PS can interact with other lipids, such as cholesterol and sphingolipids,⁵¹ to form specialized membrane domains known as lipid rafts. These domains are enriched in signalling molecules and membrane receptors and are essential for signal transduction and cell signalling.

PC also interacts with various lipids and proteins, including sphingomyelin,⁶⁰ which is another abundant phospholipid in the membrane. The interaction between PC and sphingomyelin is known to form a specialized type of lipid raft, known as the liquid-ordered phase or the raft phase, which has distinct biophysical properties compared to the surrounding membrane.⁶¹ These domains are essential for the organization of signalling molecules and membrane receptors, as well as for the regulation of membrane fluidity and permeability.

In addition to their interactions with lipids, PS and PC also interact with proteins in the membrane. For example, PS has been shown to interact with several proteins involved in blood clotting, including coagulation factor Va, prothrombin, and protein C.⁶² These interactions are essential for the regulation of blood clotting and thrombosis. PC, however, interacts with several membrane proteins, such as ion channels, transporters, and receptors. These interactions are essential for the regulation of cell signalling, ion homeostasis, and nutrient uptake.

2.2. Functional roles of PS and PC in cells

The distribution and interactions of PS and PC in the membrane play critical functional roles in the cell. For example, the asymmetric distribution of PS in the cell membrane is vital for the recognition and clearance of apoptotic cells by phagocytic cells.⁶³ The exposure of PS on the outer surface of the membrane serves as a signal for recognition and phagocytosis, which is an essential process for the removal of damaged or infected cells.⁵

The distribution of PC in the outer surface of the cell membrane is vital for the formation and stability of lipid rafts, which are critical for cell signalling and membrane organization. The interaction between PC and sphingomyelin in the lipid raft phase is vital for the organization of signalling molecules and membrane receptors, which is critical for signal transduction and cell signalling.⁶⁴ In addition, the fluidity and permeability of the membrane are regulated by the distribution and interactions of PS and PC with other lipids and proteins, which are essential for the regulation of cell function and adaptation to changing environmental conditions.⁵⁰

In many biological processes, PS or PC is essential for function. It is a component of the cell membrane and plays a role in endocytic internalization, intracellular vesicle movement, and the establishment of cell polarity. Furthermore, PS plays a vital role in preserving the structural and functional integrity of cell membranes.⁶⁵ Additionally, PS exposure is connected to phospholipid externalization during biological processes, such as apoptosis.^33,66 In terms of brain function, PS and DHA are essential for the structure and operation of the brain.⁶⁷ Furthermore, the synthesis of PS by PS synthase is pivotal for the growth and maintenance of neurons.⁶⁸ PS plays a function in neurotoxicity and Alzheimer's disease because it is widely distributed in neuronal membranes and is exposed on the cell surface when oxidative damage occurs⁶⁹

Moreover, PS modifies the secondary structure of protein aggregates and impacts the rates at which proteins form, especially when amyloidogenic proteins are present. Furthermore, PS plays a role in insulin production, insulin signaling transduction, and the development of problems related to diabetes. For instance, PS supplementation has demonstrated promise in preventing unfavorable ventricular remodeling and decreasing the size of myocardial infarcts,⁷⁰ indicating a possible role in cardiac health. In tumor microenvironments, PS has also been linked to the control of T cell responses, suggesting that PS may be a valuable target for cancer immunotherapy. Knowing the functioning properties of PS is critical to comprehending different disorders and creating effective treatment plans.^22,33,71,72 PS is crucial for more than just cell activities; it may also have therapeutic uses.

Regardless of the evident potential of PS-targeted therapies, notable challenges and constraints exist that necessitate attention. For instance, certain tumors may exhibit low expression levels of PS, rendering them less responsive to these treatments, as shown in Table 2.¹⁵ Furthermore, these therapies may exert off-target effects that have the potential to induce undesired adverse reactions. Nevertheless, there is a basis for optimism regarding the future of PS-targeted therapies in the field of cancer treatment. An intriguing prospect involves the combination of PS-targeted therapies with immunotherapy.¹⁵ PS and immune cell exposure in tumor microenvironments yield immunosuppression and the advancement of tumor development. Therefore, for the survival of cell proliferation, growth and symptoms linked to cancer, the PS position on the membrane is vital.^38,73

Understanding the intricate interaction of PS within the field of cancer biology and its repercussions for resistance to treatment is of utmost importance for the advancement of more efficient therapeutic approaches. Through the specific targeting of PS and the pathways associated with it, innovative treatment methods have the potential to enhance the effectiveness of current therapies and surmount drug resistance in cancer.

3. PS in cancer treatment

3.1. Exposure of PS to immune suppression

Immune suppression and inflammation provide favourable conditions for tumor development by impairing the immune system's capacity to identify and eliminate cancer cells.^74–76 Tumor cells can escape immune system detection by interfering with the stage of T-cell activation.⁷⁷ In the tumor microenvironment, PS is abnormally exposed and is essential for producing immunosuppressive signals that obstruct the development of both systemic and local antitumor immune responses.^29,76,78 PS is exposed to the outer membrane within the tumor microenvironment due to various metabolic processes linked to apoptosis. Activation of caspases, production of reactive oxygen species, and cell activation-induced calcium ion inflow are some of these processes.⁷⁹ PS exposure significantly impairs both innate and adaptive immune responses, enabling tumor cells to avoid immune detection.⁸⁰ Interestingly, PS exposure is known to be a point of control that may be addressed pharmacologically and functions as an early indicator or precursor of the several immunosuppressive signals that follow.⁸¹ The primary causes of PS exposure in the tumor microenvironment are necrotic tissue and apoptotic cells introduced by pathological states or treatment. Nevertheless, it has also been seen in living endothelial cells and extracellular vesicles derived from leukocytes, stroma, and tumors. Tyro, Axl, and Mertk (TAM receptor tyrosine kinases) are among the several PS receptors that are particularly crucial for the detection of PS and the induction of immune suppression in the tumor microenvironment.⁸² T cell/transmembrane, immunoglobulin, and mucin (TIM) are additional PS receptors.⁸³ Tim-1, Tim-3, and Tim-4 are associated with receptor-mediated immunosuppression of Th1 cells, activation of Th2 cells—a subset of T helper cells important in humoral immunity—and the uptake of apoptotic cells by macrophages and dendritic cells. The receptor tyrosine kinases (RTKs) that make up the TAM gene family are expressed on leukocytes and different kinds of tumors. Protein S or Gas6 are bridge proteins that have γ-carboxylated domains that allow TAM RTKs to connect to PS. As the TAM receptor is engaged in interaction with the receptor-binding domain, the exposed PS is directly bound by the γ-carboxylated GLA domain of Gas6 and Protein S. Therefore, when TAMRTKs on macrophages are activated, target cells exposed to PS are phagocytosed, resulting in macrophage polarization, which acts as a pro-inflammatory signal, changing the phenotype of the macrophages from “M1” to “M2,” which has pro-tumor activity, and allowing for the production of interleukin-10 and TGF-β.⁸⁴ TAM RTK activation on tumor cells is associated with resistance to chemotherapy and the ability of tumor cells to undergo epithelial plasticity. Preclinical studies have shown decreased tumor growth and metastasis when the vitamin K-dependent γ-carboxylation of Gas6 is inhibited, preventing Axl from being activated on tumor cells.⁸⁴ Additionally, PS-TAM RTK binding to tumor cells escalates the expression of programmed death-ligand 1 (PD-L1). Therefore, blocking the activation of the TIM and TAM-RTK pathways by PS can enhance immune responses against cancer, as shown in Fig. 4.⁸⁵ Furthermore, a study revealed that radiation therapy elevates PS expressions in B16 melanoma in mice bodies. The extrinsic plasma membrane surface of tumor cells, PS, hinders the immunological response to receptors found in T cells and others.^85,86 Inhibition of dendritic cell antigen representation and induction of regulatory T-cell limits T-cell activation by producing immunosuppressive cytokines, such as IL 10 or TGF-β.

Fig. 4 Immune cells are activated in the tumor microenvironment by PS-targeting antibodies interacting with exposed PS. As the article explains, oxidative stress and an immature tumor vasculature work together to cause PS to show marked dysregulation in the tumor microenvironment. This imbalance is further exacerbated by the release of PS-positive tumor exosomes and the increased apoptotic index of tumours that are actively growing. Antibodies specifically targeting PS are thought to bind to externally exposed PS molecules with selectivity and interfere with PS's ability to regulate tumor microenvironmental functions by preventing PS from connecting to PS receptors and by blocking Fcγ-mediated ADCC (antibody-dependent cell-mediated cytotoxicity). Consequently, immunogenic signals are triggered in the tumor microenvironment. Reprinted from, ref. 85 Copyright CC BY 2018 Taylor and Francis Online.

Moreover, the binding of PS to T cell receptors blocks T cells that are activated by GPR174 (G-protein coupled receptor 174 is a protein encoded by the GPR174 gene in humans). This G-protein coupling receptor shortage of T-reg cells can induce macrophage polarization and upsurge IL-10 expression.²⁴ When PS encounters macro-vesicles by PS, phagocytes clear apoptotic cells, preventing undesired inflammatory reactions and equilibrating an anti-inflammatory state in the tumor microenvironment, as shown in Fig. 5. Exposure of PS from tumor samples in patients has been demonstrated to suppress the activation of T-cell reactions²⁴ in micro-vesicles (exosomes). However, PS exposure in tumor cells also promotes anti-tumor effects through the mediation of long-term inflammation.

Fig. 5 Immunological suppression occurs in tumour cells and vesicles because of PS exposure. Immunological suppression occurs when PS is given to cancer cells because it ligates to receptors on T cells and macrophages. When PS binds to PSR on macrophages, M2-like macrophages develop and start to release TGF- and IL-10, two anti-inflammatory cytokines. TGF- and IL-10 are cytokines that inhibit the immune system and stop T cells from being activated. Reprinted from, ref. 24 Copyright 2020 Theragnostic.

As per a recent study, it has been observed that PS exposure in the body, under conditions with a large number of cells, is essential to bind IFNγ and IL-12 for the conversion of transient cytokine stimuli towards a long-lasting inflammation as a result of reducing immunosuppressive functions.⁸⁷ In conclusion, the immune response of PS and its immunosuppressive consequences is an intricate process in the tumor microenvironment. This mechanism seems to be a conserved evolutionary strategy among higher metazoans to safeguard against autoimmune complications that may arise during the regular disposal of dying host cells.⁸⁷ Although the development of agents that target PS receptors is progressing through various stages of pre-clinical and clinical investigation, advanced-stage clinical trials are currently underway to evaluate the efficacy of the PS-targeting antibody bavituximab in multiple oncology indications.

3.2. PS in biomarkers for imaging tool

The discovery and characterization of biomarkers are essential to the advancement of cancer research because they enhance the effectiveness of therapies, diagnosis, and screening. PS has demonstrated potential as a biomarker for the identification and imaging of several forms of cancer. Studies have shown that PS is present on the interface of cancer cells. This process is controlled by calcium-dependent flippases and scramblases, which are enzymes that catalyze the transfer of lipid molecules. The former moves lipids towards the cytoplasm, while the latter moves phospholipids ATP-independently between the inner and outer leaflets. For this reason, PS labelling techniques are useful for enabling tumor visualization.²⁷ Cancer cells exhibiting a lower presence of PS on their surface seem to display a heightened susceptibility to both irradiation and chemotherapeutic agents, such as gemcitabine (Gemzar)/nab-paclitaxel.⁸⁸ However, cancer cells with higher PS levels on their surfaces are more susceptible to PS-targeting anticancer therapies, such as saposin C encapsulated in a dioleoyl PS nanovesicle (SapC-DOPS).⁸⁹ Using different strategies, it has been demonstrated that cancer cells targeting PS have been imaged.^90,91 In a previous study, researchers used a compound called Annexin V-Cy to image gliosarcoma tumors in mice. When pro-apoptotic drugs were administered to the tumors, they observed that the fluorescent signal in the tumors increased three times. Zhao et al. used PGN635, a new monoclonal antibody that binds exclusively to PS, to perform in vivo optical imaging of exposed PS. The F(ab′) (2) fragment of PGN635 was linked to the near-infrared (NIR) dye IRDye800CW. After injecting 800CW-PGN635 into mice with subcutaneous or orthotopic U87 glioma xenografts, either treated with or without radiation, in vivo dynamic NIR imaging was performed. Following the injection of 800CW-PGN635 into non-irradiated subcutaneous U87 gliomas, the resulting NIR optical imaging showed a clear contrast in the tumors.⁹² Another imaging technique called positron emission tomography (PET) is employed for radioactive tracers to visualize PS. In another investigation, researchers utilized PGN635 – ⁸⁹Zr (Zirconium-89) to visualize tumors in both mice and humans. They discovered that the tumors exhibited elevated levels of radioactive tracers, thereby enabling their visualization through PET. Another method for visualizing PS is to use radio waves and magnetic fields, called magnetic resonance imaging (MRI). In a subsequent study, PGN635, along with superparamagnetic iron oxide nanoparticles (SPIO), was used to visualize tumors (breast cancer) in mice, wherein the researchers noticed that the tumors displayed a reduction in signal intensity on MRI, thereby facilitating their detection. A nano-probe known as PGN-IOL/DiR, which is made up of liposomal that binds to PS and enters cells, has a good effect on mice breast cancers of MDA-MB 231.⁸⁸ Similarly, the use of green indocyanine bonded to PS antibodies in three negative breast cancer cells enabled the tracking and mapping of apoptosis⁸⁹ and facilitated an effective therapy strategy. PS species may also be biomarkers for the diagnosis of malignancy. The 36 most frequent lipid types were evaluated in one clinical investigation of 15 patients with prostatic cancer and 13 healthy controls. The results demonstrated that a specific species, “PS (18 : 1/18 : 1)”, appeared to have significant implications for normal patients and those with prostate cancer; the two categories, % of sensitivity, differed in the combinations PS (18 : 1/18; 1), lactosylceramide (18 : 1/16 : 0) and PS (18 : 1/18 : 2).⁹⁰ A new study in patients with lung cancer evaluated lipids in aerosols between cancer and normal cells. In cancerous aerosols, an overexpose of PS was detected with declining PCs,⁹¹ which showed that PS is a viable biomarker for lung cancer in combination with other indices. PS species can also be related to the growth and progression of cancer. A method that combines PS and SapC, an endogenous sphingolipid activator protein, can be used to track several cancer cell lines. At an acidic pH, SapC has a considerable binding affinity for PS and is essential for the activation of lysosomal enzymes and the production of sphingosine and ceramide from the breakdown of sphingolipids. Because of the release of lactate from anaerobic glycolysis, tumors are known to have high levels of PS on their cell surface and to have a lower extracellular pH in comparison to normal tissues.⁹¹ Consequently, the combination of SapC and PS provides a beneficial and selective technique for targeted tumor imaging and treatment. An illustration of the various SapC-imaging modalities is presented in Fig. 6. The PS-targeting SapC-DOPS nanocarrier technology has been effectively applied to single-photon emission computed tomography (SPECT), MRI, and optical cancer cell imaging.⁹³ Chu et al.⁹⁴ used CellVue Maroon (CVM)-tagged SapC-DOPS nanovesicles, which contain the far-red fluorophore CVM, to show how to identify brain tumors and arthritic joints in mice. MRI containing iron oxide particles enclosed in SapC-DOPS nanocarriers was utilized to image neuroblastoma selectively. Winter et al. used paramagnetic gadolinium chelates, namely gadolinium-DTPA-bis(stearylamide) (Gd-DTPA-BSA)-loaded SapC-DOPS vesicles, as a targeted contrast agent for glioblastoma multiforme tumor imaging.⁹⁵ Fig. 7 presents the inferred findings from the Winter et al. experiment, which shows that the T1 of tumor cells maps before and after a 10-hour injection of Gd–DTPA–BSA/SapC-DOPS vehicles. In summary, the distinctive characteristics of polystyrene offer significant benefits in the field of imaging applications, as PS-targeting techniques allow for the specific visualization of tumors. Undoubtedly, these techniques will have a significant impact on upcoming diagnostic applications.

Fig. 6 Schematic diagram showing that many imaging modalities use saposin C-dioleoyl phosphatidylserine (SapC-DOPS)-based modalities to visualize tumors. The far-red fluorophore CVM can be attached to the SapC-DOPS nanovesicles to provide optical imaging for both in vivo and in vitro studies. The use of gadolinium chelates, such as ultrasmall superparamagnetic iron oxide (USPIO) or gadolinium-DTPA-bis (stearyl amide) (Gd-DTAPBSA), as MRI contrast agents is necessary to enable in vivo MRI. Moreover, SapC-DOPS combined with an iodine-124 contrast agent is used for in vivo PET/SPECT imaging. Reprinted from, ref. 18 Copyright CC BY 2022 MDPI.

Fig. 7 A mouse model of brain cancer is used to investigate the use of saposin C-dioleoyl phosphatidylserine (SapC-DOPS) as a carrier for contrast agents in MRI. First, a mouse with a glioma undergoes a high-resolution MRI (A). Finally, after injecting Gd-DTPA-BSA/SapC-DOPS vesicles into the tumor rim cells, and normal brain, the percentage change in T1 is evaluated (B) and (C). Reprinted from, ref. 18 Copyright CC BY 2022 MDPI.

With the finding of anxA5, a PS-binding protein⁹⁶ results in the invention of the anxA5 affinity test for evaluating apoptosis by focusing on PS that is exposed to the surface.^97,98 With the exception of the myoblast and the megakaryoblast, anxA5 interacts with dying cells rather than surviving ones throughout the body's diverse environment according to the study. In animal vitro models, some researchers have expanded the range of in vivo applications of the anxA5 affinity method.^99–101 Because AnxA5 is associated with apoptosis, macrophage infiltration, and the release of red blood cells through intraplate bleeding, it has recently been found to be a helpful marker for differentiating between stable and unstable atherosclerotic plaques in patients.¹⁰² Recent studies have suggested that a PS-binding antibody can be used for the molecular imaging of vascular endothelial cells in solid tumors.¹⁰³

3.3. PS in liposomal carriers

PS is a crucial anti-tumor mechanism in several liposomal carriers. These liposomal transporters stick with PS in the tumor microenvironment and release anti-tumor sequels through metabolism or synergistic drug effects. SapC-DOPS interacts primarily with cancer cells that contain PS and causes apoptosis by inducing ceramide accumulation and caspase activation. Research organizations have used SapC-DOPS to treat several cancers, such as brain cell tumors¹⁰⁴ and cancer cell lines from the skin,¹⁰⁵ as well as cancer cells from the pancreas.¹⁰⁶ SapC-DOPS was found to be beneficial for various metastatic tumors according to the findings. The same research discovered that cells with high top PS levels have more survival probability under radiation therapy and that this was inversely related to themselves. In response to SapC-DOPS therapies, this may have been employed in combination treatment with significant exposure to tumor cells during radiation therapies, as shown in Fig. 8.¹⁰⁷

Fig. 8 Cancer therapies involving PS. The medications aim to target PS in cancer treatment. (1) Naked antibodies bind to PS. (2) Antibody–drug conjugates bind to the PS. (3) Liposomal carriers adhere to PS. Reprinted from, ref. 24 Copyright CC BY 2020 Theragnostic.

Furthermore, in cancer cells and tumors, PC-SA is a liposomal carrier with a cationic property. PC-SA was found to be more effective when combined with typical tumor treatments in a preclinical study, and it also had anti-cancer benefits when used alone. The cancer medicines, which were camptothecin with doxorubicin encapsulated in PC-SA type liposomes, were used in this study to decrease tumor growth as PC-SA increased the half-life of anti-tumour drugs to carry a long-range in cancer treatment.¹⁰⁷

3.4. PS in targeted drug delivery

PS is expressed on the surface of dead and alive cancer cells,^108,109 as well as on the endothelial cells of blood vessels.¹¹⁰ This has led to recent speculations that PS is a viable drug-supply target. PS as a medication delivery target is further supported by clinical experience with molecular oncological imaging of PS (TDD). PS selection using 3G4 monoclonal anticorps has recently been shown to improve cytostatic treatment efficacy in mouse cancer models.^111,112 Surprisingly, 3G4 was designed to identify the plasma PS-binding protein beta2-glycoprotein 1 rather than PS. This demonstrates that when PS is delivered to the membrane in vivo, it is immediately opsonized by various endogenous PS-binding proteins, such as beta2 glycoprotein 1. In addition to AnxA5, it belongs to the same PS-binding protein family. AnxA5 competes effectively with other PS-binding proteins for PS-bound locations in vivo in humans according to the molecular imaging experience with the protein. This has been confirmed in an in vitro system using isolated proteins and PS binding assays.¹¹³ Consequently, AnxA5 is a potential therapeutic option for treating PS in vivo.

3.5. PS in molecular imaging target

With the finding of anxA5, a PS-binding protein⁹⁶ results in the invention of the anxA5 affinity test for evaluating apoptosis by focusing on PS exposed to the surface.^97,98 Apart from the myoblast and the megakaryoblast, anxA5 interacts with dying cells rather than surviving ones throughout the body's diverse environment according to the study. In animal vitro models, some researchers have expanded the range of in vivo applications of the anxA5 affinity method.^99–101 Because AnxA5 is associated with apoptosis, macrophage infiltration, and the release of red blood cells through intraplate bleeding, it has recently been found to be a helpful marker for differentiating between stable and unstable atherosclerotic plaques in patients.¹⁰² Recent research suggests that a PS-binding antibody can be used for molecular imaging of vascular endothelial cells in solid tumors, as shown in Table 1.¹⁰³

Table 1 PS-targeting imaging strategies and therapeutics, along with their findings

Strategy/approach	Description	Findings and applications
PS-targeting imaging strategies
Annexin A5 (anxA5) affinity test	Utilizes the high affinity of Annexin A5 for PS to measure apoptosis	Effective in identifying apoptotic cells in various cancer types. Widely used in molecular imaging for cancer diagnosis
Radiolabeled PS Probes	PS-binding proteins tagged with radioactive isotopes	Enables non-invasive imaging of tumor cells. Provides clear visualization of apoptotic cells in preclinical and clinical settings
PS-targeting liposomes	Liposomes designed to bind to PS-exposed cancer cells	Enhances the delivery of imaging agents to tumor sites, improving the accuracy of cancer detection
Optical imaging with PS-binding dyes	Dyes that specifically bind to PS and emit fluorescence	Allows real-time imaging of tumor progression and response to therapy. Useful in intraoperative imaging to guide surgical resection of tumors
PS-targeting therapeutics
PS-targeting antibodies	Antibodies designed to bind specifically to PS-exposed cells	Effective in delivering cytotoxic agents directly to tumor cells, reducing off-target effects. An example is bavituximab
Liposomal drug delivery systems	Liposomes encapsulating chemotherapeutic drugs that target PS	Improves the pharmacokinetics and biodistribution of drugs, enhancing their therapeutic efficacy
Antibody–drug conjugates (ADCs)	Antibodies linked to cytotoxic drugs targeting PS-exposed cells	Shows promising tumor-killing effects in preclinical models. Potential for reduced side effects compared to conventional chemotherapy
Natural plant extracts	Extracts from plants that exhibit anti-cancer properties targeting PS	Demonstrates potential in inducing apoptosis in cancer cells. Offers a natural and potentially less toxic alternative to synthetic drugs
PS-targeting nanoparticles	Nanoparticles functionalized to bind PS on tumor cells	Facilitates targeted drug delivery and reduces systemic toxicity. Shows potential in improving the efficacy of cancer treatments

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Table 2 PS is in clinical use playing an appropriate clinical significance role

Characteristics	Clinical significance of PS	Diseases	Ref.
Biomarker	Increased PS expressions	B16 melanoma	114
Biomarker	Exosomal phosphatidylserine (PS) 18 : 1/18 : 1	Prostate cancer	90
Biomarker	Presence of IgM anti-PS-prothrombin complex	Cancer-associated vasculitis	27
Biomarker	Presence of IgA anti-PS-prothrombin complex	Henoch-Schonlein purpura	115
Imaging tool	Liposomal PGN-IOL/DiR binds to PS	Breast cancer	88
Imaging tool	Indocyanine green, tagged PS antibodies	Triple-negative breast cancer	89
Immunosuppression	phosphatidylserine-micro vesicles induce TGF-β expression and suppression of macrophages	Melanoma	29
Tumoricidal target	SapC-DOPS nano vesicles- phosphatidylserine-targeting agent	Neuroblastoma	30,104
Tumoricidal target	SapC-DOPS Nano vesicles- phosphatidylserine-targeting agent	Skin cancer	105
Tumoricidal target	SapC-DOPS Nano vesicles- phosphatidylserine-targeting agent	Pancreatic cancer	93
Antitumor effect	3G4 monoclonal against PS	Breast cancer	111
Antitumor effect
	PS externalizations and DNA fragmentation by plant products (Chalepin)	Breast cancer	32,116
Antibody-based killing	Rituximab mediated killing of CD 20 positive cells	CD20-positive tumour cells	43,117

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3.6. PS in antibody–drug conjugates

Many conjugate antibody drugs are part of the development of targeting medicines that use PS. These drugs are associated with PS-targeting immunizer antibodies, which are cytotoxic medicines that produce anti-tumor effects, as shown in Fig. 9. Some immunizer antibody–drug conjugate medication forms have been proven in trials to have good tumor-suppressive activity to “bare” antibodies available on it, for example, a PS-focusing agent made using PS binding particles, which is a human Fc component that is C2A identified by IgG1. In the breast and prostate cancer models of mice, the noted usage of an Fc-Syt1 synthesized to monomethyl auristatin E, which acted as a cytotoxic chemical, had significant anticancer effects.¹¹⁸ According to some experts, the PS peptide is transferred to pH-sensitive micelles (PEGPDLLA) and then used as a chemotherapeutic drug paclitaxel (PTX) in those micelles. These pH-sensitive micelles are designed to address a corrosive set-off drug discharge framework that is appropriate for an acidic environment with a tumor. Moreover, vivo research concluded that the produced agents generated cytotoxicity and tumor cell uptake, as well as aggregation of tumors.¹¹⁹ In addition, fusion proteins containing L-methionase linked to human Annexin-V have been discovered. In comparison to L-methionine with no fusion protein present, the antibody PS has the most significant effect on tumor cell killing.¹²⁰ Furthermore, the fusion protein does not affect normal cells, indicating that technology has the potential to develop novel drugs.

Fig. 9 PS management in detail for end-to-end using manners.

3.7. PS with CD47 as a therapy for cancer

PS exposure to the phospholipid bilayer has the potential to induce phagocytic signs, which can be distinguished by immune cells, such as macrophages. In contrast, CD47 articulation on the cells can simultaneously repress the process of phagocytosis. The CD47 is a vital immune suppressive signal used by different tumor cells.¹²¹ The CD47 can act as a ligand for a category of glycoprotein known as signal regulatory protein-α or SIRPα. Upon binding of the CD47, the SIRP signaling cascade can be activated, which in turn inhibits phagocytosis⁸¹ and allows tumor cells to escape from surveillance of immune cells, such as macrophages and T cells.¹²² However, in contrast, CD47 ligation induces the articulation of PS in erythrocytes as part of a demise pathway, and CD47 has been suggested to impact PS exposure.¹²³ Again, knocking down the CDC50A, which is a subunit of ATP11C involved in the flipping of PS, can cause the tumor-associated macrophages to improve the anti-CD47 bar's activity to limit tumor growth.¹²⁴ As mentioned previously, the knockdown of CDC50A consequently increases the exposure of PS in the immortalized category T cells known as Jurkat cells. Hence, it is supposed to impact T cell function. Consequently, the CD47 barrier prevents phagocytosis; nonetheless, PS openness is used to target tumor cells associated with macrophage clearance work. When rituximab, an anti-CD20 antibody, is used with anti-CD47 medications, an anti-CD47 inhibitor, clinical trials have demonstrated some anti-cancer activities with negligible adverse effects in patients with aggressive and indolent lymphoma.¹¹⁷

4. Functions of PS in cancer

In the discipline of immunology, the goal of treatment is to pinpoint the signals that distinguish cancerous cells from non-cancerous ones: ‘eat me’ and ‘don't eat me’. The uneven distribution in eukaryotic cells results in the presence of unfavourable lipids, such as PS and PE, on the cytoplasmic side of the cell membrane and positive phospholipids, such as PC and sphingomyelin, on the outer side, which is sustainably mediated by the controlled activity of ATP-dependent and -independent enzymes referred to as scramblases, floppases, and flippases. In a tumor environment, due to various pathways, the PS is exposed to external surfaces, which results in an ‘eat me ‘signal, leading the cell towards apoptosis.^43,117 Due to PS exposure's immunosuppressive qualities, tumor cells can better elude immune surveillance by weakening both innate and adaptive immune responses. The TAM gene families, as well as T cell/transmembrane, immunoglobulin, and mucin (TIM)44, are PS receptors in the tumour microenvironment.⁸²

4.1. Apoptotic function of PS

Every day, millions of cells constantly die and regenerate in our bodies. The dead cells are then eliminated by the action of PS, which initiates a process called apoptosis that causes the dead cells to be phagocytosed.^98,125 The apoptosis pathway takes place in 2 ways: the intrinsic pathway, which takes place inside the mitochondria, and the extrinsic pathway, which takes place via numerous apoptotic factors. The intrinsic pathway is initiated by the activation of cytochrome C, which further activates caspase 3/7 liberated from mitochondria. In contrast, in the extrinsic pathway, apoptotic factors, such as TNF and Fas ligand (FasL), activate caspase 3 and promote cell apoptosis. Exposure to the cell membrane PS serves as a clear signal for the recruitment of macrophages and the engulfment of cells, indicating the initiation of apoptosis. Studies on mice and humans have shown that another protein called Xk-related protein 8 (Xkr8), also known as scramblase, becomes incompetent to expose PS during apoptosis if hypermethylated because its expression is suppressed. Moreover, other proteins, such as basigin (BSG) and neuroplastin (NPTN), belonging to the Ig superfamily, and ATPase Phospholipid Transporting 11C(ATP11C), a significant flippase protein-producing gene, are necessary for flipping of PS to the outer leaflet of the membrane. Any mutation can lead to no apoptosis and is not taken up by macrophages. Thus, both flippase and scramblase activities are necessary for PS exposure and performing programmed cell death.^23,100,126 Further, the non-apoptotic functions of PS include blood coagulation, myoblast fusion and T lymphocyte activation.

4.2. PS with TIM and TAM receptor function

Two receptor families mediate the phagocytic clearance of apoptotic cells, the TIM and TAM proteins, which are dependent on PS (PtdSer). Tim receptors are a type of membrane receptor that recognizes PS signals and exerts immune suppression.^127–129 Three receptors are encoded by the TIM gene on the human chromosome, i.e. TIM1, TIM3 and TIM4, out of which TIM 3 is highly studied in the immune-cancer biology area. Tim 3 is expressed on dendritic cells, CD8⁺ T cells, antigen-presenting cells, and other immune cells, such as monocytes and natural killer cells. It promotes the phagocytosis of apoptotic bodies, preserves immunological tolerance, and triggers inflammatory reactions. It is well-established that inhibiting TIM-3 can stop tumor development.¹³⁰ Furthermore, the mast cells, b cells and cd4⁺ t cells express the TIM-1 receptor, which also acts as a binding site for the Ebola virus. Dendritic cells and macrophages have significant levels of TIM-4 expression. Tim-4 is involved in the inflammatory reactions of the body.^131,132

According to recent research, TAM functions as an oncogene that expresses itself inappropriately in malignant cells and is essential for the migration of apoptotic bodies as well as the growth and survival of cells. The TAM encodes three tam receptors: Tyro3, AXL, and MerTK are proteins known to bind specifically to vitamin K-dependent protein S, with Tyro3 and MerTK showing an exclusive affinity for it, which triggers in T cells upon exposure to interleukin-4, while growth arrest protein 6 (Gas6) is another ligand that interacts to tam receptors. By binding to ligands, the team receptors are activated via the RTK pathway, which in turn causes the kinetic domains to become auto-phosphorylated, triggering signaling cascades and gene regulation.¹³³

4.3. Molecular role of PS in cancer therapy

Fracturing PS and its receptors is one possible avenue for cancer treatment. Clinical efforts are conducted using two methods to inactivate PS and increase antitumor activity: interrupting PS in tumor cells and aiming at PS receptors to interfere with receptor signaling. PS and its anti-cancer effects can be bound by various agents, such as liposomal vesicles (SapC-DOPS), monoclonal antibodies (bavituximab), and antibodies conjugated with medicines (monomethyl auristatin E coupled with Fc-Syt1 and Annexin-V coupled with L-methionase).^30,88 These agents work by forcing cancer cells to undergo apoptosis and controlling the body's immune system. These innovative treatments are revolutionizing the way cancer is treated and offering hope for the creation of new drugs. The PS receptors can be the target of a wide variety of compounds. Some of the compounds that can bind to and block all three of the TAM receptors are sitravatinib, LDC1267, and RXDX-106. R428 (BGB324), TP0903, KIT, FLT3, VEGFR, PDGFR, BMS777607, NPS-1034, and DP3975 are a few receptors for Axl. The MerTK inhibitors are UNC2025, UNC3133, ONO747, and MRX-2843. In addition, Pfizer-11 and 12, KRCT-7j, GL21.T, 20G7-D9, and AXL-107-MMAE are among the Tyro3 prohibitors. These are strong protein- or c small molecule inhibitors that block TAM receptors, limiting the spread and invasion of metastatic cells in the cancer microenvironment and boosting the activity of the innate and adaptive immune cells.^105,117,134 Due to their anti-tumor properties, these compounds have created new therapeutic opportunities. They can be utilized to treat malignancies of the breast, oral, cervix, lung, and pancreas in addition to lymphocyte leukemia (Tables 3 and 4).

Table 3 Small chemical inhibitors of several kinases to target TAM^{30,88,105,117}

Agents	Primary target (IC50 value)	TYRO3 IC50	MER IC50	AXL IC50	Development stage	Primary indication
Bosutinib	BCR/ABL			0.6 nM	Approved	Chronic myelogenous leukemia
Cabozantinib	MET VEGFR2			294	Approved	Medullary thyroid cancer renal cell carcinoma hepatocellular carcinoma
Crizotinib	c-MET (8 nM) ALK (20 nM)	>10 000		7 nM	Approved	Non small cell lung cancer
LDC1267	AXL, MER, TYRO3	8 nM	<5 nM	29 nM	Preclinical	Boosting NK cell activity in tumor microenvironment
RXDX-106	AXL, MER, TYRO3, MET	19 nM	29 nM	7 nM	Termination of Phase 1	Immune activation in tumor microenvironment
Sitravatinib (MGCD516)	VEGF, c-MET, AXL, MER, TYRO3		2 nM	1.5 nM	Phase 2	A potent multi-kinase inhibitor in different models of sarcoma

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Table 4 TAM-targeting selective small molecule inhibitors

Agents	Primary target (IC50 value)	TYRO3 IC50	MER IC50	AXL IC50	Development stage	Primary indication
Small molecule inhibitors for targeting MER
MRX-2843	MER, FLT3	17 nM	1.3 nM	15 nM	To overcome resistance conferring FLT3 mutations in AML	Phase 1
UNC-2025	MER, FLT3(0.80 nM)	17 nM	0.74 nM	14 nM	Leukemia	Preclinical
UNC-3133	MER	31 nM	3.0 nM	17 nM	Cancer (FLT IC50 = 6.8 nM), virus infection	Preclinical
ONO-7475	AXL, MER, FLT3		0.4 nM	2.2 nM	AML	Phase 1
Small molecule inhibitors for targeting AXL
R428 (BGB324)	AXL			14 nM	Cancer	Phase 2
TP-0903	AXL			19 nM	Chronic lymphocytic leukemia	Phase ½
BMS-777607	AXL, MET(3.9 nM)	4.3 nM	14 nM	1.1 nM	Met-dependent gastric carcinoma	Phase 2 completed
NPS1034	AXL, MET(0.80 nM)			48 nM	EGFR-resistant lung cancer cells due to MET or AXL activity	Preclinical
Small molecule inhibitors for targeting TYRO3
Pfizer compound 11	TYRO3	0.52	<10 fold than TYRO3		Thrombosis	Preclinical
Pfizer compound 12	TYRO3	0.27	<10 fold than TYRO3	10–100 fold than TYRO3	Thrombosis	Preclinical
Pfizer compound 19	TYRO3	0.010	>100 fold than TYRO3	>100 fold than TYRO3	Thrombosis	Preclinical
Pfizer compound 21	TYRO3	0.0007	10–100 fold than TYRO3	>100 fold than TYRO3	Thrombosis	Preclinical
KRCT-6j	TYRO3	0.028	12	>10	Cancer	Preclinical

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Recent research indicates that PS plays a role in the advancement of tumors. PS is externalized to the outer plasma membrane in different cancer cells; this process is controlled by calcium-dependent membrane proteins, such as flippases and scramblases. The development of tumors and resistance to chemotherapy and radiation therapy have been connected to the externalization of PS. However, PS's possible use in cancer treatment has also been investigated. According to a preclinical investigation, PS-targeting medications exhibit anticancer properties both on their own and in combination with conventional antitumor medications, where they exhibit greater potency.^132,134,135 The mechanisms of PS in cancer therapy are complex and multifaceted. PS-targeting agents have been shown to be enhanced. Furthermore, it has been discovered that administering an antibody that targets PS improves overall survival and amplifies the anti-tumor efficacy of tumor-directed radiation therapy. PS-targeting agents have also been explored as a potential cancer immunotherapy strategy, with one study showing that blocking PS externalization can restore pathogen and tumor immunity.^{33,34,136,137} Furthermore, PS synthase has been recognized as a promising therapeutic target for regulating cellular homeostasis in cancer cells.

Numerous clinical investigations have been carried out to explore the possibilities of PS-targeting medications in the management of cancer. Patients with HER2-negative breast cancer were treated with the PS-targeting combination of paclitaxel and bavituximab in a phase I clinical study.^18,108 It has been demonstrated that SapC-DOPS can efficiently target and eradicate several cancer types, including skin, lung, brain, and pancreatic cancer. Clinical trials have also shown that treatment with an antibody that targets PS improves overall survival and increases the effectiveness of tumor-directed radiation therapy. These findings imply that PS-targeting drugs may represent a fresh and exciting development in cancer treatment.

4.4. PS and PC in host immunity

PS and PC are pivotal to host immunity. A significant part of the cell membrane that separates the intracellular and extracellular environments is made up of phospholipids. In addition to their structural role, phospholipids also have signalling functions, including the modulation of immune responses.^126,138,139

PS is vital in the identification and elimination of apoptotic cells to preserve immunological tolerance and to avert autoimmunity. PS is also involved in activating immune cells, including natural killer cells and macrophages, to protect the body against bacterial and viral infections.^140,141 Exposure to PS causes pro-inflammatory cytokines to be downregulated and anti-inflammatory cytokines to be released, which resolve inflammation. In addition to its role in apoptosis, PS also plays a role in the activation of immune cells. PS can activate natural killer (NK) cells, which are a type of lymphocyte that can kill virus-infected or tumor cells without prior sensitization.^142–144 PS also enhances the phagocytic activity of macrophages and stimulates the production of reactive oxygen species (ROS), which can kill bacteria and other pathogens.

Research has shown that PS supplementation can enhance the immune response in individuals with weakened immune systems.^105,131 In a study involving elderly individuals, PS supplementation was found to improve the function of immune cells and reduce the incidence of infection. Other studies have shown that PS supplementation can boost the immune response to vaccinations and increase antibody production, which is essential for long-term protection against infectious diseases.^145–147

However, PC is the most abundant phospholipid in cell membranes and is also a significant component of lipoproteins, which transport lipids in the bloodstream. PC has been shown to modulate immune responses in several ways.⁴² For instance, PC can prevent the synthesis of pro-inflammatory cytokines, including interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α).⁴² Additionally, PC can increase the synthesis of cytokines that reduce inflammation, such as transforming growth factor beta (TGF-β) and interleukin-10 (IL-10).¹⁴⁸ PC also affects lymphocytes through immunomodulatory means. PC can inhibit the activation of T cells, which are involved in cell-mediated immunity. PC can also enhance the proliferation of B cells, which produce antibodies in the body.¹⁴⁹ PC has also been shown to improve the activity of the complement system, which is a group of proteins that plays a crucial role in the immune response. The complement system helps to identify and eliminate pathogens, and deficiencies in this system can lead to increased susceptibility to infections. Furthermore, PC can modulate the function of dendritic cells, which are specialized antigen-presenting cells that initiate adaptive immune responses.^148,150

Research has shown that PC supplementation can enhance the function of system immune cells: T cells and natural killer cells. PC can also reduce inflammation, which is a crucial aspect of the immune response.^93,104 In a study involving patients with ulcerative colitis, PC supplementation was found to reduce inflammation and improve symptoms. Further research is required to determine the optimal doses and duration of supplementation for different populations. PS and PC also play crucial roles in cancer immunity. Cancer cells can evade the immune system by expressing surface molecules that suppress immune cell activation and induce the apoptosis of immune cells.¹⁰⁵ PS and PC can modulate the immune responses to cancer cells by regulating the activation and proliferation of immune cells and by promoting the phagocytosis of cancer cells.

PS is also flipped out on the surface of cancer cells, acting as an “eat-me” signal that is recognized by immune system cells such as phagocytic cells, such as macrophages and dendritic cells.^140,141 PS activates NK cells, which recognize and kill cancer cells with low amounts of major histocompatibility complex (MHC) class I molecules, which are necessary for antigen presentation to T cells. PS also has immunomodulatory effects on dendritic cells, which are specialized antigen-presenting (sAPC) cells that are used to initiate adaptive immune responses.¹⁵¹ PS can enhance the antigen-presenting capacity of dendritic cells and promote the differentiation of r Th cells towards a Th1 phenotype. Th1 cells produce cytokines, such as IFN-γ, which activate macrophages and improve their ability to kill cancer cells.

PC can also modulate the function of T cells, which are involved in cell-mediated immunity. PC can inhibit the activation and proliferation of T cells, reducing the immune response to cancer cells. However, PC can also enhance the differentiation of regulatory T cells (T_regs), which can impair the activation and proliferation of the effector T cells. T_regs are essential for maintaining immune tolerance, but they can also nurture cancer growth by suppressing the immune response to regulate cancer cell proliferation.¹⁵² However, the role of PC in the case of cancer immunity is complex and context-dependent, and further research is needed to understand the mechanisms of the action of PC in cancer immunity.

4.5. Signaling cascade influenced by PS and PC

PS and PC play critical roles in regulating cell signalling pathways in cancer. Aberrant signalling cascades are a hallmark of cancer, and targeting these pathways is a promising approach for cancer therapy. PS and PC have been shown to influence various dysregulated signalling cascades in cancer cells, making them attractive targets for targeted cancer therapy.

PS and PC influence the PI3K/AKT/mTOR cell signaling pathway. These cell signalings, which regulate cell growth, survival, and metabolism, are frequently dysregulated in cancer cells.¹⁵³ PS can initiate the PI3K/AKT pathway by promoting the recruitment of AKT to the plasma membrane. The AKT activation in turn phosphorylates and activates the downstream targets, including mTOR, which promotes cell growth and proliferation. PC has also been shown to influence the PI3K/AKT/mTOR pathway.¹⁵³ Studies have shown that PC can activate AKT by promoting the activation of phosphoinositide-dependent kinase-1 (PDK1), which is an upstream regulator of AKT. PC has also been shown to regulate mTOR activity by modulating the expression of mTOR regulatory proteins.

Targeting the PI3K/AKT/mTOR signaling pathway has been the focus of many cancer drug development efforts.¹⁵³ Several medications targeting this system, including PI3K inhibitors, AKT inhibitors, and mTOR inhibitors, are now under clinical studies. However, the efficiency of these drug molecules has limitations, such as off-target effects and drug resistance.¹⁵⁴ Targeting PS and PC may provide a novel approach for modulating this pathway in cancer cells.

Another signaling cascade that PS and PC influence is the JAK/STAT pathway.¹⁵⁵ This pathway plays a role in controlling cell growth, differentiation, and immune function, and it is often dysregulated in cancer cells.¹⁵⁶ PS can activate the JAK/STAT signalling pathway by inducing Janus kinase (JAK), followed by the phosphorylation and activation of signal transducer and activator of transcription (STAT) proteins. PC also modulates the JAK/STAT pathway by activating STAT proteins and promoting their phosphorylation and nuclear translocation.¹⁵⁶ PC has also been shown to regulate the expression of JAK proteins, which may impact the activation of this pathway.

Targeting the signalling pathways of JAK/STAT has been the focus of many cancer drug development efforts. Several drugs targeting this pathway, including inhibitors of both JAK and STAT, have recently been used in clinical trials.¹⁵⁵ However, the efficacy of these inhibitors is often limited by off-target effects and possibly shows drug resistance. Targeting PS and PC may provide a novel approach for modulating this pathway in cancer cells.

In addition to both the JAK/STAT and PI3K/AKT/mTOR signaling pathways, PS and PC have been shown to influence other signaling cascades that are dysregulated in cancer cells.¹⁵⁵ For example, PS has been shown to turn on the MAPK/ERK signalling pathway, which is the key regulator pathway for both cell growth and cell differentiation. Again, PC has been reported to regulate developmental pathways, such as the Wnt/β-catenin pathway, which plays an important role and is also involved in the regulation of cell differentiation and cell proliferation.

Targeting PS and PC for cancer therapy has several advantages over traditional approaches that target specific proteins or pathways.^134,157,158 First, PS and PC are ubiquitous components of cell membranes, making them essential for cell survival. Therefore, targeting these phospholipids may have fewer off-target effects than drugs that target specific proteins or pathways. Second, PS and PC are highly expressed in cancer cells, making them attractive targets for cancer-specific therapies.^134,144,159 Targeting PS and PC may overcome drug resistance mechanisms that often arise with targeted therapies, as cancer cells may develop resistance to a specific protein or pathway but are unlikely to develop resistance to fundamental components of cell membranes.

Several strategies for targeting PS and PC for cancer therapy have been developed. One approach is to use agents that specifically bind to these phospholipids and initiate apoptotic pathways in cancer cells.^111,112 For example, some monoclonal antibodies, such as bavituximab, bind to PS and have been shown to prompt apoptosis in the case of cancer cells by activating the immune system. Another approach is to use lipid-based delivery of drugs to cancer cells through nanoparticles.^33,134,160 These engineered small particles are used to target PS and PC on the surface of cancer cells, allowing for the selective delivery of drugs. The current era of anti-cancer drugs with liposomal formulations, such as doxorubicin and paclitaxel, has developed target PS on the surface of cancer cells and has depicted promising results in the case of preclinical studies.^32,159,161 Finally, targeting the enzymes that synthesize PS and PC may be a promising approach for cancer therapy. For example, the enzyme PSS is responsible for the synthesis of PS, and inhibitors of PS have been reported to instigate apoptotic events in cancer cells. Similarly, the enzyme choline kinase (CK), which is responsible for the synthesis of PC, is often overexpressed in cancer cells, and inhibitors of CK have demonstrated potential results in preclinical studies. These approaches have been shown to have promising results in the case of preclinical studies, and they can offer a new avenue for developing cancer therapies.

6. Future perspective and challenges

PS has emerged as a pivotal molecule with transformative potential in the landscape of cancer therapies. As we delve deeper into understanding its intricate molecular functions and dynamic role in cancer progression, several emerging trends and future directions come to light. They are as follows:

Role of PS in cancer progression

One of the critical areas of focus in the future will be unravelling the precise role of PS in driving cancer progression. It is similarly involved in crucial cellular processes, such as proliferation, apoptosis evasion, and metastasis formation. Understanding these mechanisms provides valuable insights into designing targeted therapies that specifically disrupt PS-mediated pathways.

Dynamic interplay within the cancer microenvironment

The cancer microenvironment plays a vital role in tumor development and response to treatment. Investigating how PS actively contributes to the complex interplay within this microenvironment is a crucial area of research. Its interactions with immune cells, stromal cells, and signaling molecules offer new avenues for innovative therapeutic strategies that leverage these interactions.

Harnessing PS for targeted cancer therapies

Future research will focus on developing novel methodologies and approaches that harness the potential of PS for targeted cancer therapies. Exploring PS-targeted drug delivery systems, immunotherapies targeting PS-expressing cells, and combination therapies that synergize with PS-mediated pathways can intensify treatment efficacy and impair side effects.

Age-dependent implications of novel PS in cancer

Understanding the age-dependent implications of PS in cancer is crucial for personalized treatment strategies. Senescence-related changes in PS expression and function may impact the treatment response and toxicity profiles. Tailoring therapies based on age-specific considerations will be an essential aspect of future cancer care.

Interactions with mast cells and microglia

The intricate crosstalk between PS, mast cells, and microglia presents a promising avenue for therapeutic intervention. Investigating how PS influences immune cell behavior within the tumor microenvironment can lead to the development of immunomodulatory strategies that enhance anti-tumor immune responses.

Future horizons for next-generation cancer therapeutics

PS has tremendous potential as a cornerstone for next-generation cancer therapeutics. Continued research efforts aimed at elucidating its multifaceted roles, exploring innovative targeting approaches, and integrating age-specific and microenvironmental considerations will pave a new direction for more effective and personalized treatments for cancer.

Despite promising advancements, several challenges remain. The precise delivery of PS-targeted therapies, the heterogeneity of PS expression across different cancer types, and the need for a deeper understanding of PS's role in various cellular contexts are critical areas that require further investigation. Additionally, integrating PS-targeted therapies with existing treatment modalities to maximize therapeutic efficacy and minimize adverse effects presents a complex yet rewarding challenge.

Looking forward, the future of PS in cancer therapy is bright. With ongoing research and technological advancements, the potential for PS-targeted therapies to revolutionize cancer treatment is immense. By continuing to unravel the complexities of PS and its interactions within the tumor microenvironment, researchers can develop more effective, targeted, and personalized cancer treatments. Overall, phosphatidylserine stands at the cusp of ushering in a new paradigm in cancer therapy. Its unique properties and significant role in cancer biology offer a promising avenue for developing novel therapeutic strategies. As our understanding of PS deepens, so too does the potential for groundbreaking advancements in the fight against cancer, paving the way for a future in which cancer therapies are more effective, less invasive, and tailored to the individual needs of patients.

7. Conclusion

In tumor microenvironments, PS represents a transformative frontier in the field of cancer therapies, offering unprecedented insights and opportunities for innovative treatments. As this review has illustrated, PS is not just a structural component of cell membranes but a pivotal player in the intricate communication networks within and between cells, particularly in the context of cancer. The externalization of PS on cancer cells and apoptotic cells reveals its crucial role in immune evasion and tumor progression, making it an attractive target for therapeutic intervention. The diverse biological functions of PS, from modulating immune responses to influencing cellular signaling pathways, underscore its potential as a multifaceted therapeutic target. Current research efforts, including the development of PS-targeting antibodies and PS-binding proteins, have shown promise in disrupting these processes, thereby enhancing the ability of the immune system to recognize and eliminate cancer cells. Beyond these applications, PS is also used in antibody–drug conjugates, where it acts as a cytotoxic agent in PS-targeting antibodies to exert tumor-killing effects. These strategies, while still in the experimental stages, herald a new era of precision medicine in which treatments can be tailored to exploit the unique characteristics of cancer cells. Additional research is needed on cells and agents that detect PS exposure levels, together with other cancer medicines.

Consent for publication

All authors have read and approved the final manuscript.

Data availability

Data will be available upon request.

Conflicts of interest

The authors declare that there are no potential competing interests.

Acknowledgements

The authors acknowledge respective departments and institutions that provide facilities and support. The authors would also like to express their appreciation for the Biorender.com software.

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