Macrophage-Mediated Tumor Cell Phagocytosis: Opportunity for Nanomedicine Intervention

Abstract

Macrophages are one of the most abundant non-malignant cells in the tumor microenvironment, playing critical roles in mediating tumor immunity. As important innate immune cells, macrophages possess the potential to engulf tumor cells and present tumor-specific antigens for adaptive antitumor immunity induction, leading to growing interest in targeting macrophage phagocytosis for cancer immunotherapy. Nevertheless, live tumor cells have evolved to evade phagocytosis by macrophages via the extensive expression of anti-phagocytic molecules, such as CD47. In addition, macrophages also rapidly recognize and engulf apoptotic cells (efferocytosis) in the tumor microenvironment, which inhibits inflammatory responses and facilitates immune escape of tumor cells. Thus, intervention of macrophage phagocytosis by blocking anti-phagocytic signals on live tumor cells or inhibiting tumor efferocytosis presents a promising strategy for the development of cancer immunotherapies. Here, the regulation of macrophage-mediated tumor cell phagocytosis is first summarized, followed by an overview of strategies targeting macrophage phagocytosis for the development of antitumor therapies. Given the potential off-target effects associated with the administration of traditional therapeutics (for example, monoclonal antibodies, small molecule inhibitors), we highlight the opportunity for nanomedicine in macrophage phagocytosis intervention.

Table of Contents

This review introduces the regulation of macrophage-mediated tumor cell phagocytosis, including anti-phagocytic checkpoints for the immune evasion of live tumor cells and the process of efferocytosis, followed by summarizing the emerging therapeutic strategies by promoting phagocytosis of live tumor cells or inhibiting efferocytosis. Given the limitations of current therapeutics, this review further highlight the opportunity of nanomedicine for phagocytosis intervention.

1. Introduction

Cancer progression is determined by both tumor cells and the microenvironment in which malignant cells reside^[1]. The tumor microenvironment is comprised of cellular and acellular components beyond tumor cells, including blood vessels, immune cells, fibroblasts and extracellular matrix^[2]. Infiltrating and tissue resident macrophages constitute a major cell population of the tumor microenvironment that can account for up to 50% of some solid tumors^[3], suggesting macrophages can be a critical mediator of tumor immunity. Indeed, previous studies demonstrate that the presence of tumor-associated macrophages is associated with tumorigenesis, limited therapy response and poor prognosis in most solid tumor types^[4]. Notably, tumor-associated macrophages are not a single cell population but highly dynamic and heterogeneous, regulated by specific local stimuli^{[4c, 5]}. Generally, the activation states of macrophages consist of a spectrum of phenotypes between M1 and M2^[4b]. M1 macrophages are stimulated with lipopolysaccharides (LPS), expressing high levels of proinflammatory cytokines, such as INFγ and TNFα, and further presenting the capability of tumor cell killing as well as adaptive immune system activation. In contrast, M2 macrophages, activated by IL-4 or IL-13, are characterized with the expression of anti-inflammatory cytokines, including IL-10, and are viewed to promote tumor initiation and progression. As the majority of tumor-associated macrophages are polarized to M2 state, creating an immunosuppressive microenvironment^[3a], numerous therapeutic investigations focus on the depletion or phenotype repolarization of macrophages to acquire microenvironment remodeling^[6]. However, macrophages are also important immune cells with the role in phagocytizing^[7], leading to growing interest in targeting macrophage-mediated tumor cell phagocytosis to achieve substantial therapeutic activity.

Immunotherapy targeting T cell immune checkpoints has been extensively investigated since the FDA approval of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody ipilimumab for the treatment of metastatic melanoma in 2011^[8]. Although adaptive immune checkpoint inhibitors, including anti-CTLA-4, anti-programmed cell death 1 (PD-1), and anti-programmed cell death ligand 1 (PD-L1), have shown significant therapeutic efficacy in multiple cancer types by interrupting T cell inhibitory pathways, the response rates to these inhibitors in cancer patients remain suboptimal^[9], emphasizing the importance of innate immune cell activity and improved cancer antigen processing. As a result, a new type of therapeutic strategy based on tumor cell phagocytosis regulation has emerged since the identification of the “don’t eat me” signal CD47^[10]. Macrophages, as one of the antigen presenting cell (APC) types in the innate immune system, play important roles in the induction of antitumor immunity via phagocytosis of tumor cells, which further serves as a bridge between the innate and adaptive immunity through the presentation of tumor-specific antigens^[11]. However, tumor cells have evolved to evade engulfment by phagocytes via the expression of anti-phagocytic molecules^[12], leading to immune escape. In addition, macrophages are endowed with the ability to clear dead cells^[13]. As a result, macrophages in the tumor rapidly recognize and engulf dying/apoptotic tumor cells (efferocytosis), which supports the resolution of inflammation and generates a tumor tolerant environment^[14]. Thus, targeting anti-phagocytic signals in live tumor cells or inhibiting efferocytosis in the tumor microenvironment could be promising approaches for the development of tumor immunotherapies.

Given the limitations of traditional drugs (e.g., small molecules), such as low targeting ability and potential systemic toxicity, nanomedicine has attracted increasing interests in terms of the generation of therapeutic agents for cancer treatment^[15]. In the past several decades, therapies based on nanomaterials have been widely investigated due to its capability to overcome multiple systemic delivery barriers and complete the delivery of therapeutic drugs by designing nanoparticles with specific size, shape, surface charge, hydrophilic properties, and other modifications^[16]. For instance, nanoparticles can achieve relatively long term circulation in the blood via polyethylene glycol (PEG) modification^[17] and further accumulate in the rodent tumor tissue through enhanced permeability and retention effect^[18]. In addition, nanoparticles can be modified with active targeting ligands to specifically target a distinct type of cells and achieve the controlled release of encapsulated drug via stimuli responsiveness, which reduces the off-target effect of traditional drugs and further enhances the therapeutic efficacy. Moreover, nanoparticles can easily integrate multiple drugs to achieve improved therapeutic potential^{[15a, 19]}. Therefore, the development of nanomedicine is a promising approach to amplify the effectiveness of cancer therapies. To date, numerous drug delivery systems have been established, including lipid-based nanoparticles, polymeric nanoparticles, protein nanoparticles, inorganic nanoparticles, and extracellular vesicles^[20]. In preclinical and clinical studies, nanomaterial-based delivery systems have been successfully used to deliver multiple therapeutic drugs ranging from small molecules to macromolecular compounds, such as proteins and nucleic acids, for the treatment of different cancers^{[15a, 15b, 16b]}.

In this Review, we focus on the phagocytosis activity of tumor-associated macrophages and discuss how to use nanotechnology to govern the engulfment of tumor cells for developing therapeutics.

2. Regulation of Macrophage Phagocytosis against Tumor Cells

2.1. Anti-Phagocytic Checkpoints in the Tumor Microenvironment

Macrophages are the main phagocytic population in tumors^{[4b, 21]}, whereas tumor cells have the ability to avoid phagocytic elimination depending on the expression of anti-phagocytic ligands, which are also known as “don’t eat me” signals. To date, four regulatory molecules on tumor cells have been identified with the capability in inhibiting phagocytic clearance, including CD47, PD-L1, major histocompatibility class I complex (MHC-I), and CD24 (Figure 1). The associated binding receptors that can be found on macrophages are signal-regulatory protein α (SIRPα), PD-1, leukocyte immunoglobulin-like receptor 1 (LILRB1), and sialic-acid-binding Ig-like lectin 10 (Siglec-10), respectively.

Figure 1.

Anti-phagocytic checkpoints in the tumor microenvironment. The expression of “don’t eat me” signals on tumor cells, including CD47, PD-L1, MHC-I, and CD24, protect tumor cells from phagocytic clearance by interacting with their cognate receptors on macrophages.

CD47 is a surface glycoprotein, originally known as integrin-associated protein (IAP)^[22]. Notably, CD47 is expressed on the membrane of all normal cells throughout the body. This molecule was described for the first time on ovarian cancer cells in 1992^[23], but the interaction of CD47 with SIRPα was not identified until 1999^[10b]. SIRPα is widely expressed on the surface of myeloid cells, including macrophages, granulocytes, monocytes and dendritic cells^{[10b, 24]}. It has been shown that the expression of SIRPα is to counteract signals that myeloid cells receive via activating surface receptors, such as Fc receptors, complement receptors and the lipoprotein-related protein^[25]. Structure analysis reveals that SIRPα has an extracellular immunoglobulin domain, enabling ligand binding. In addition, the cytosolic domain of SIRPα includes an immunoreceptor tyrosine-based inhibitory motif (ITIM), which confers its properties as an inhibitory receptor^[26]. The CD47-SIRPα interaction promotes the phosphorylation of ITIM, which further recruits and activates the inhibitory tyrosine phosphatases SH2-containing protein tyrosine phosphatase 1 (SHP1) and SHP2^[27]. Within macrophages, the SHP phosphatases leads to the dephosphorylation and disruption of myosin elements, thus inhibiting cytoskeleton rearrangement and preventing macrophage engulfment^[28]. CD47 is important for tumor cells to evade immune clearance. It is well established that the expression of CD47 is elevated in a wide range of tumor cell types, and is negatively associated with prognosis in many cancers^[29]. Therefore, the CD47-SIRPα axis has been regarded as a promising target for cancer therapy. Of note, considering that the CD47-SIRPα axis is able to regulate the activity of all types of myeloid cells, the anticancer effect of CD47 blockade could result from several cell types, including macrophages and neutrophils^[30].

PD-1 and its ligands, including PD-L1 and PD-L2, have attracted much interest as T cell immune checkpoints^[31]. Initially, PD-1 is identified as a coinhibitory molecule expressed on the membrane of T lymphocytes and PD-L1 is widely expressed by tumor cells. Engagement of PD-1 and its cognate ligand PD-L1 activates the downstream signal of PD-1, thereby causing immunosuppression by inhibiting T cell activation and proliferation^[32]. However, PD-1 expression was also found on tumor-associated macrophages and the PD-1-PD-L1 axis was further identified as an anti-phagocytic checkpoint in 2017^[33]. In this study, the authors found that the expression of PD-1 on tumor-associated macrophages increased with the tumor progression, and negatively regulated the phagocytosis of tumor cells. As a result, PD-1⁻ macrophages exhibited a higher level of phagocytosis activity against tumor cells compared with PD-1⁺ macrophages. Besides, blockade of PD-1 or PD-L1 with antibodies resulted in enhanced tumor cell phagocytosis and inhibited tumor progression in a macrophage-dependent manner^[33]. These findings indicate that the PD-1-PD-L1 axis not only serves as a checkpoint of adaptive immunity, but also promotes the evasion of tumor cells from macrophage-mediated phagocytosis. While the results provide new insights into the function of traditional immune checkpoints and the anti-phagocytic activity of PD-1/PD-L1 has attracted much attention as novel therapeutic targets^[34], the precise signaling pathway that regulates the anti-phagocytosis function of PD-1 on macrophages remains to be elucidated. A recent study further indicates that PD-1 is also expressed on myeloid cells and the targeted ablation of PD-1 in myeloid cells induces antitumor efficacy with improved T cell functionality^[35], demonstrating the multiple mechanisms of PD-1 in dampening antitumor immune responses.

MHC-I, as one of the two primary classes of MHC expressed by many nucleated cells, has the ability to present antigens to T cells^[36]. Nevertheless, the expression of the beta-2 microglobulin subunit (B2M) of MHC-Ⅰ on tumor cells was found to correlate with the resistance to macrophage-mediated phagocytosis in 2018^[37]. Subsequent investigation indicated that the anti-phagocytic capability of MHC-I depends on its interaction with LILRB1 on macrophages^[37]. Depletion of MHC-I on tumor cells sensitized tumor cells to macrophage engulfment in vitro and in vivo, suggesting that cancers with compromised MHC expression could be ideal candidates for macrophage-based immunotherapy. Moreover, the receptor LILRB1 is also expressed on NK cells^[38]. The engagement of NK cell-expressed LILRB1 with MHC-I inhibits the tumor cell killing activity of NK cells^[38a], which suggests that the deletion of MHC-I may also sensitize tumor cells to killing by NK cells.

Recently, Barkal et al.^[39] demonstrated that tumor cell-expressed CD24 is a dominant innate immune checkpoint in ovarian cancer and breast cancer, which promotes immune evasion of cancer cells through its interaction with tumor-associated macrophage-expressed Siglec-10. CD24 is a highly glycosylated glycosylphosphatidylinositol-anchored surface protein^[40], which has been shown to interact with Siglec-10 on innate immune cells to inhibit tissue damage-induced inflammatory responses^[41]. Although the expression of CD24 in solid tumors has been discovered^[42], its role in mediating tumor immune responses is not well explored. In this study, the authors demonstrated that cancer cells can harness CD24 to avoid phagocytic clearance by macrophages that express Siglec-10. Importantly, macrophages stimulated with TGFβ and IL-10 (M2-like) express higher levels of Siglec-10, and are less phagocytic than unstimulated macrophages. However, coculture of breast cancer cells deficient in CD24 with M2-like macrophages with Siglec-10 can induce sufficient phagocytosis of cancer cells, suggesting M2 polarized macrophages also maintain their phagocytosis activity against cancer cells as long as the anti-phagocytic signaling is blocked. As a result, therapeutic blockade of CD24 with antibodies enhanced phagocytosis against cancer cells and exerted significant anticancer effect. Collectively, CD24 is a newly identified anti-phagocytic signal that protects cancer cells from Siglec-10-expressing macrophages, indicating an alternative target in cancer immunotherapy.

While cancer cells generally express higher levels of anti-phagocytic signals than normal cells, cellular changes associated with malignant transformation may lead to the exposure of pro-phagocytic ligands on cancer cells to activate phagocytosis. The pro-phagocytic signals include calreticulin, signaling lymphocytic activation molecule family member 7 (SLAMF7), and tumor-associated neoantigens, which have been well reviewed elsewhere^[7b].

2.2. Macrophage-Mediated Clearance of Apoptotic Cells

Apoptotic or dying cells, including apoptotic tumor cells in the tumor microenvironment, are recognized and engulfed by phagocytic cells, which is a process termed efferocytosis. Swift engulfment of apoptotic cells by efferocytosis maintains tissue homeostasis and subsequently supports the resolution of inflammation. Although efferocytosis can be accomplished by many types of phagocytes (e.g., macrophages, immature dendritic cells, neutrophils, and epithelial cells), macrophages are the principal phagocytes that deal with cell death^[13]. Basically, the process of efferocytosis requires a series of coordinated molecular events, finally resulting in the engulfment of apoptotic cells^[43].

2.2.1. Find Me Signals and Correlated Receptors on Phagocytes

In the early process of apoptosis, dying cells emit “find me” signals to recruit macrophages into the site of cell death, enabling rapid recognition of apoptotic cells by phagocytes. To date, four “find me” signals have been identified, including lysophosphatidylcholine (LPC)^[44], sphingosine-1-phosphate (S1P)^[45], nucleotides (e.g., ATP and UTP)^[46], and chemokine C-X₃-C motif ligand 1 (CX3CL1)^[47]. Corresponding to the release of “find me” signals, macrophages will detect and respond to these signals with an array of receptor systems^[48]. For lysophospholipids, the G protein-coupled receptor (GPCR) G2A on macrophages mediates the response to LPC^[49], as wild-type but not G2A-deficient mouse macrophages migrated toward LPC. However, the molecular pathway that transduces LPC signaling is not yet determined. Intriguingly, five S1P receptors (S1PR1-S1PR5) have been discovered in both mice and humans but the in vivo data that how these receptors coordinate to regulate the macrophage chemotaxis are much limited^{[13, 50]}. Besides, the expression of P2Y purinoreceptors on macrophages can bind with nucleotides, which drives the phagocytes to migrate towards dead cells^[51]. As an example, Ellliott et al. found that triphosphate nucleotides attract phagocytes in vivo through P2Y₂ receptor binding, and ablation of triphosphate nucleotides caused accumulated dead cells^[46], indicating the importance of “find me” signals in the clearance of apoptotic cells. CX3CL1, also known as fractalkine, was found to be released by apoptotic lymphocytes, which further stimulated the recruitment of macrophages by binding with its cognate receptor CX3CR1^[47]. Of note, inhibition of the CX3CL1/CX3CR1 axis induced partial inhibition of macrophage attraction, suggesting this signaling is one of the mechanisms in regulating phagocyte recruitment.

2.2.2. Eat Me Signals and Phagocytic Receptors

Once macrophages migrate to the site and are close to dying cells, they rely on specific cell surface molecules to identify apoptotic cells, which are also called “eat me” signals. While several candidates of the “eat me” signal have been proposed, the most widely investigated signal is phosphatidylserine (PtdSer)^{[13, 52]}, which is a component of the cellular membrane. For healthy cells, PtdSer is confined to the inner leaflet of the plasma membrane. However, it is rapidly translocated from the inner to the outer leaflet of the plasma membrane with the involvement of a set of phospholipid translocases (scramblases)^[53], which serves as an indicator to show that the cell has died by apoptosis.

Two types of phagocyte receptors that mediate apoptotic cell recognition have been identified, based on whether these receptors bind to PtdSer directly or not (Figure 2). For instance, macrophage receptors, including T cell immunoglobulin and mucin domain-containing molecule 4 (TIM4)^[54], brain angiogenesis inhibitor 1 (BAI1)^[55], C300b^[56], and stabilin 2^[57], bind directly to externalized PtdSer, leading to apoptotic cell recognition and uptake. Alternatively, some phagocytic receptors do not bind to PtdSer directly, but rely on a “bridging ligand” to achieve the recognition of PtdSer. For example, the TAM receptors (Tyro3, Axl, and MerTK) are a family of receptor tyrosine kinases that do not bind to the phospholipid directly, but instead depend on their extracellular activating ligands, including growth arrest-specific Gas6 and protein S (ProS1), for this activity^{[13–14, 58]}. Notably, Gas6 binds to and activate all three TAM receptors, but ProS1 only shows the ability to activate Tyro3 and MerTK^[59]. Importantly, the TAM receptors and their activating ligands are the most broadly expressed PtdSer recognition system in macrophages and MerTK has been shown to be a crucial regulator of macrophage-mediated efferocytosis throughout the body^[60]. For instance, mice with dysfunctional TAM receptors, especially with deficient MerTK, showed significant accumulation of apoptotic cells in multiple tissues^{[13, 61]}. Of note, TIM-4 also requires cooperation with MerTK to facilitate engulfment of apoptotic cells since it lacks an intracellular domain^[62]. Without the expression of MerTK, TIM-4 can only tether apoptotic cells but does not show phagocytosis capability. However, the mechanism regulating this interaction has not been clearly demonstrated. PtdSer can be also recognized by the glycoprotein milk fat globule-EGF factor 8 (MFGE8), which further functions to bridge PtdSer of dying cells to receptors expressed by phagocytic macrophages, such as αvβ3 and αvβ5 integrins^[63]. Consistent with its activity, mice with a loss of MFGE8 showed compromised activity in apoptotic cell elimination, resulting in autoimmune diseases^[64].

Figure 2.

Macrophage-mediated phagocytosis of apoptotic cells. Externalized PtdSer on apoptotic cells, as the most widely studied “eat me” signal, can be recognized by macrophages with the assistance of various receptors. Phagocytic receptors, including TIM4, BAI1, CD300b, and stabilin 2, can directly bind to PtdSer for apoptotic cell recognition. TAM receptors (Tyro3, Axl, and MerTK) and αvβ3/αvβ5 integrins, however, rely on bridging ligands (Gas6/Pros1 and MFGE8) for the recognition of externalized PtdSer.

Following apoptotic cell recognition and tethering, cytoskeletal rearrangement within macrophages occurs, leading to cell motility and phagosome formation to eventually engulf the apoptotic cell. Although it has been shown the ELMO/Dock180/Rac1 pathway is involved to complete the engulfment process^{[14b, 65]}, the precise molecular signaling pathways responsible for the initiation of cytoskeletal rearrangement and phagosome formation remain to be determined.

While live cells avoid phagocytic clearance via the expression of “don’t eat me” signals, dying cells tend to reduce the presentation of surface-associated anti-phagocytic determinants and increase the “eat me” signals^[66], promoting the recognition and clearance of dying cells. Collectively, rapid clearance of dying/apoptotic cells in the tumor microenvironment through efferocytosis hinders the inflammatory response, which facilitates immune escape and promotes tumorigenesis and progression^[67]. In contrast, uncleared apoptotic tumor cells undergo secondary necrosis and release damage-associated molecular patterns (DAMPs) to activate the innate immune system and further induce adaptive antitumor activity^[68]. Therefore, blockade of efferocytosis in the tumor tissue could switch the immunogenically silent cell apoptosis to immunogenic cell death, leading to robust innate and adaptive antitumor immunity.

3. Therapeutic Strategies Targeting Macrophage Phagocytosis

3.1. Targeting Anti-Phagocytic Checkpoints

3.1.1. Blockade of Anti-Phagocytic Signaling to Promote Phagocytosis of Live Tumor Cells

Malignant cells express higher levels of anti-phagocytic signals^{[29a, 29b]}, which plays a key role in mediating immune evasion of tumor cells. For instance, CD47, as the most heavily studied anti-phagocytic signal, is overexpressed in various types of tumors^[69], and forms a formidable barrier for the clearance of tumor cells. Instead, CD47-deficient cells are critically sensitive to phagocytic clearance^{[10a, 66]}, suggesting that blockade of CD47 is a promising therapeutic strategy for the development of antitumor therapies. Indeed, therapeutic reagents intended for blocking the interaction of CD47-SIRPα resulted in phagocytosis of live tumor cells and inhibited tumor growth in many tumor models^{[29b, 70]}. In fact, several strategies have been explored in terms of disturbing the CD47-SIRPα axis. A well-established strategy is the use of anti-CD47 or anti-SIRPα antibodies. Anti-CD47 antibody shows the capability to significantly enhance the phagocytosis of tumor cells by macrophages^{[69b, 71]}. Clodronate is a bisphosphonate that, once internalized, specifically kills macrophages^[72]. Mice treated with clodronate liposomes abrogated the antitumor efficacy of anti-CD47 antibody^[29b], suggesting a macrophage-dependent antitumor response in the application of anti-CD47. Alternatively, treatment with anti-SIRPα antibody leads to facilitated macrophage phagocytic efficacy and suppressed the tumor progression in a colon cancer model^[73]. In addition, recombinant CD47 or SIRPα proteins and their variants can compete with the endogenous proteins, thus attenuating the interaction of CD47 and macrophage-expressed SIRPα. For example, Lin et al. showed that the extracellular domains of the human SIRPα (hSIRP(ext)) expressed by E. coli competed with endogenous SIRPα to bind with CD47 on the membrane of leukemia cells, leading to the blockade of CD47-SIRPα signaling in vitro^[74]. Further studies demonstrated that an engineered high-affinity SIRPα variant potently antagonized CD47 on tumor cells, and remarkably enhanced the efficacy of therapeutic anticancer antibodies in vivo^[75]. Similarly, exosomes with SIRPα variants dramatically increased macrophage-mediated tumor cell engulfment, and further induced robust T cell-based antitumor activity^[76]. Another therapeutic approach is to regulate the expression of anti-phagocytic checkpoints through direct or indirect inhibition of the mRNA translation. Application of siRNA delivery directing CD47 attenuation has shown improved tumor cell phagocytosis and inhibited tumor metastasis^[77]. Moreover, a hypoxia-inducible factor 1α (HIF-1α) target protein BNIP3 is correlated with the expression of CD47 as the silencing of BNIP3 leads to decreased protein levels of CD47^[78], suggesting an alternative target for blocking the CD47-SIRPα signaling.

3.1.2. Simultaneous Blockade of Innate and Adaptive Checkpoints

Notably, CD47 blockade with antibodies not only directly eliminate live tumor cells through macrophage-mediated phagocytosis, but also primes antigen-specific CD8+ T cells by facilitating the cross-presentation of tumor-associated antigens through macrophage-expressed MHC-I^[79]. Other investigation suggests that anti-CD47 antibody can decrease the activity of Treg cells, thereby enhancing the activation of CD8+ T cells to achieve efficient antitumor potential^[65]. These results indicate that the adaptive immune system, especially CD8+ cytotoxic T cell response, is also involved to generate antitumor efficacy in anti-CD47 therapy. Considering that the expression of adaptive immune checkpoints in the tumor microenvironment attenuates the antitumor capability of T cells, the combination of adaptive checkpoint blockade with interference of CD47-SIRPα signaling can be a promising therapeutic strategy to elicit synergistic anticancer effect. Indeed, the combination of anti-CD47 antibody with PD-L1 blockade achieved improved antitumor efficacy in a B16F10 melanoma model compared with CD47 or PD-L1 blockade alone^[80]. Similarly, a fusion protein targeting both CD47-SIRPα signaling and PD-1-PD-L1 axis was also reported to generate enhanced therapeutic response^[81].

Although the synergistic therapeutic efficacy of this combination strategy has been confirmed in many tumor models, the in vivo mechanism of action remains elusive due to the identification of PD-1-PD-L1 axis as both innate and adaptive immune checkpoints^[33]. PD-1/PD-L1 blockade could also improve the phagocytosis of tumor cells in addition to facilitating T cell-based antitumor response. Therefore, further studies might be required to clearly demonstrate the contribution of innate and adaptive antitumor immunity when PD-1/PD-L1 blockade is employed as a treatment.

3.1.3. Combination of Anti-Phagocytic Checkpoint Blockade with Therapeutic Antibodies to Enhance Antibody-Dependent Cellular Phagocytosis

Tumor-specific monoclonal antibodies can target tumor cells and interact with the Fcγ receptors on macrophages to induce antibody-dependent cellular phagocytosis (ADCP)^[82], thus bypassing the anti-phagocytic checkpoints. For example, both trastuzumab (a humanized anti-HER2 monoclonal antibody for breast and gastric cancers) and rituximab (a chimeric anti-CD20 monoclonal antibody for lymphoma and leukemia) can induce ADCP to achieve therapeutic efficacy, and have been widely investigated in the clinic^[83]. However, a recent study demonstrated that ADCP upregulated PD-L1 and indoleamine 2,3-dioxygenase (IDO) in macrophages^[84], which causes immunosuppression and suggests immune checkpoint blockade should be considered to obtain the optimal antitumor immunity of therapeutic monoclonal antibodies. Alternatively, blockade of the CD47-SIRPα signaling can also synergize with tumor-specific antibodies to promote phagocytic clearance of tumor cells. For instance, The combination of CD47-blocking antibody with rituximab resulted in improved tumor regression in a xenograft model of human lymphoma^[30a]. Similarly, a bispecific antibody targeting both CD47 and CD20 demonstrated synergistic phagocytosis activity and antitumor effect in B cell lymphoma compared with either single agent^[85]. Additional studies further confirmed that the combination of anti-CD47 antibody with therapeutic antibodies, including trastuzumab and cetuximab (an anti-EGF receptor monoclonal antibody), enhanced tumor cell engulfment and increased the overall therapeutic efficacy in many solid tumor types^[75]. Therapeutics targeting anti-phagocytic checkpoints that are under clinical investigation are presented in Table 1.

Table 1.

Summary of clinical trials targeting anti-phagocytic checkpoints

Drug	Target	Clinical Trials	Phase	Cancer types	Strategy	Status
Hu5F9-G4 (Humanized anti-CD47 antibody)	CD47	NCT02216409	Phase 1	Solid tumors	Single agent	Completed
		NCT02678338	Phase 1	Acute myeloid leukemia	Single agent	Completed
		NCT03248479	Phase 1	Acute myeloid leukemia	Single agent or combined with Azacitidine	Ongoing
		NCT2953509	Phase 1/2	Relapsed and refractory lymphoma	Combined with Rituximab	Ongoing
		NCT02953782	Phase 1/2	Colorectal neoplasms, solid tumors	Combined with Cetuximab	Ongoing
CC-90002 (Humanized CD47-blocking antibody)	CD47	NCT02367196	Phase 1	Hematologic neoplasms	Single agent or combined with Rituximab	Ongoing
		NCT02641002	Phase 1	Myeloid leukemia	Single agent	Ongoing
TTI-621 (SIRPα-Fc fusion protein)	CD47	NCT02663518	Phase 1	Hematologic malignancies, solid tumors	Single agent or combined with Rituximab/Nivolumab	Ongoing
		NCT02890368	Phase 1	Solid tumors	Combined with PD-1/PD-L1 inhibitors	Ongoing
ALX148 (SIRPα variant)	CD47	NCT03013218	Phase 1	Solid tumors, Non-Hodgkin lymphoma	Combined with Pembrolizumab/Trastuzumab/Tuximab	Ongoing
HX009 (anti-PD-1/CD47 infusion protein)	CD47 PD-1	NCT04097769	Phase 1	Advanced solid tumors	Single agent	Ongoing
SRF231 (Fully human anti-CD47 antibody)	CD47	NCT03512340	Phase 1	Advanced solid tumors, hematologic cancers	Single agent	Ongoing
IBI188 (Anti-CD47 antibody)	CD47	NCT03717103	Phase 1	Advanced malignancies	Single agent or combined with Rituximab	Ongoing
AO-176 (Humanized anti-CD47 antibody)	CD47	NCT03834948	Phase 1	Solid tumors	Single agent	Ongoing

3.2. Targeting Efferocytosis to Inhibit the Clearance of Apoptotic Tumor Cells

3.2.1. PtdSer Targeting

The anti-inflammatory environment generated by efferocytosis in the tumor is under-appreciated for cancer therapy. Actually, increasing evidence has demonstrated that efferocytosis is correlated with tumor progression^[86], suggesting the therapeutic potential by inhibiting tumor efferocytosis. Therapies targeting the “eat me” signaling pathway, such as PtdSer targeting, has been widely studied. For instance, Annexin V, which blocks externalized PtdSer from interacting with phagocytic receptors on phagocytes, reduces efferocytosis and efficiently inhibits tumor progression^[87]. Blockade of PtdSer with Annexin V or targeting antibodies increases the immunogenicity of dying tumor cells, which further creates an immune-responsive microenvironment by promoting antigen presentation and M1 macrophage polarization^[88]. To date, therapeutic effect with PtdSer targeting has been observed in many preclinical tumor models, including lung, pancreatic, and breast cancers^{[88b, 89]}. Besides, the antitumor efficacy of bavituximab, an antibody specifically binding to PtdSer, has been evaluated in multiple clinical trials^{[88a, 90]}. The phase 1 and 2 clinical trials demonstrated the safety and tolerability of bavituximab in combination with paclitaxel in breast cancer patients and advanced non-small cell lung cancer (NSCLC) patients. However, the phase 3 study of bavituximab plus docetaxel for the treatment of advanced non-squamous NSCLC patients demonstrated that the addition of bavituximab did not exert superior therapeutic effect compared with docetaxel treatment alone, suggesting the therapeutic potential of bavituximab requires further investigation.

3.2.2. Phagocytic Receptor Targeting

Given the pivotal role of phagocytic receptors on macrophages in apoptotic cell phagocytosis, blockade of the receptors is a promising approach to obtain antitumor effect. Previous studies have demonstrated that TAM receptors are overexpressed in many cancer types, which facilitate the clearance of apoptotic tumor cells through efferocytosis and are associated with poor survival^[14b]. Efferocytosis further promotes the expression of Tyro3, Axl, and MerTK on macrophages and skews macrophage polarization towards an immunosuppressive phenotype^[14a]. Therefore, targeting the TAM receptors to block efferocytosis may suppress the induction of anti-inflammatory responses in the tumor microenvironment, presenting a strong candidate for the development of anticancer therapeutics. In fact, several studies have reported that MerTK expressed by tumor-associated macrophages is important for the generation of a more aggressive tumor microenvironment, and its inhibition is effective for cancer therapy^{[86a, 91]}. For example, reduced tumor growth and metastasis have been observed in some solid tumor models after MerTK blockade in tumor leukocytes, including breast cancer, melanoma and colon cancer^[92]. Notably, transplantation of MerTK^−/− bone marrow into the mice with metastatic breast cancer decreased the tumor progression, but the transplantation of wild-type bone marrow did not show any therapeutic effect ^[92]. Moreover, tumor-associated macrophage-expressed MerTK correlates with increased tumor resistance to radiotherapy^[93], suggesting the inhibition of MerTK can improve its antitumor efficacy. Importantly, inhibition or genetic ablation of MerTK not only decreases efferocytosis, but also promotes the repolarization of tumor-associated macrophages to an antitumor phenotype with the production of proinflammatory cytokines^[14a], indicating the multiple antitumor activities of MerTK blockade. Targeting other TAM receptors achieved similar anticancer effect. For instance, Axl inhibitors have been shown to decrease the migration of tumor cells^[94]. Further, Axl inhibition can synergize with other therapies (e.g., targeted therapies) to improve the therapeutic efficacy^[95].

The design and development of inhibitors that specifically binds to one of the TAM receptors is difficult given the similar structure of all three receptors. As such, Axl inhibitors may also have the activity to inhibit MerTK and Tyro3^[96]. However, the relatively low specificity of inhibitors allows multiple receptor targeting, which may enhance the therapeutic effect since all TAM receptors are involved in efferocytosis and the production of anti-inflammatory responses. A great number of inhibitors that target TAM receptors have been developed. Currently, five types of Axl inhibitors have been reported, including small molecule inhibitors, monoclonal antibodies, soluble receptors, nucleotide aptamers, and natural compounds^{[65, 97]}. A small molecule inhibitor R428, also known as BGB324, has been widely used in preclinical investigations and is currently in clinical trials^{[96, 98]}. The therapeutic efficacy of nucleotide aptamers and antibodies are also under evaluation in preclinical studies^{[65, 98b]}. The development of MerTK inhibitors is ongoing as well. Small molecule inhibitors, including UNC2025, UNC1062, UNC1666, and UNC2250, have been reported to target MerTK and block tumor progression in preclinical tumor models^[99]. UNC2025 has shown antitumor effect in multiple tumor models, such as NSCLC and glioblastoma^{[99a, 100]}. However, these small molecule inhibitors mainly focused on targeting tumor cell-expressed MerTK, rather than macrophages or other phagocytes in the tumor microenvironment. As a result, whether these inhibitors targeted macrophages to contribute to the antitumor efficacy is not determined. Blockade of MerTK with functional antibodies also presents therapeutic potential. A monoclonal antibody targeting MerTK (Mer590) has been reported in 2014, and was shown to rapidly reduce the surface levels of MerTK^[101]. Recently, Zhou et al. also generated an anti-MerTK antibody to block this phagocytic receptor on tumor-associated macrophages, which increased the tumor immunogenicity by inhibiting MerTK-mediated efferocytosis and further enhanced antitumor T cell response by inducing a type I interferon response^[102]. This study strongly supports the concept that phagocytic clearance of dying tumor cells through efferocytosis favors immune tolerance and tumor progression. It also highlights the potential of targeting phagocytic receptors on macrophages for cancer immunotherapy. Representative therapeutic agents developed for efferocytosis inhibition are summarized in Table 2.

Table 2.

Representative therapeutic agents targeting efferocytosis

Inhibitor	Drug type	Primary target	Stage	Malignancies targeted	Ref
Annexin V	Natural occurring ligand	PtdSer	Preclinical	Lymphoma	[87]
Bavituximab	Antibody	PtdSer	Phase 3 (Completed)	Lung cancer, breast cancer	[90a, 90b]
R428	Small molecule	Axl	Phase 1/2	Breast cancer, head and neck cancer, melanoma, pancreatic cancer	[96, 98]
DP-3975	Small molecule	Axl	Preclinical	Mesothelioma	[97a]
GL21.T	RNA aptamer	Axl	Preclinical	Lung cancer	[97b]
UNC2025	Small molecule	MerTK (Tumor cell)	Preclinical	NSCLC, glioblastoma melanoma	[99a, 100]
UNC1062	Small molecule	MerTK (Tumor cell)	Preclinical	NSCLC, melanoma	[99b, 99d]
UNC1666	Small molecule	MerTK (Tumor cell)	Preclinical	AML	[99c]
UNC2250	Small molecule	MerTK (Tumor cell)	Preclinical	NSCLC	[99e]
Mer590	Antibody	MerTK (Tumor cell)	Preclinical	NSCLC	[101]
Anti-MerTK	Antibody	MerTK (Tumor-associated macrophage)	Preclinical	Colon cancer, breast cancer	[102]

NSCLC: non-small cell lung cancer, AML: acute lymphoblastic leukemia

4. Opportunity for Nanomedicine Intervention

Although various therapeutic strategies have been proposed in modulating macrophage phagocytosis against live or dying tumor cells, side effects have limited their further clinical translation. For example, although CD47 blockade with monoclonal antibodies increases phagocytosis of live tumor cells, systemic CD47 blockade can result in anemia and thrombocytopenia because red blood cells and platelets also express CD47. Besides, the expression of CD47 by these normal cells creates an antigen “sink” which limits the accumulation of anti-CD47 antibodies at the tumor site^[103]. These drawbacks provide an opportunity for nanomedicine to intervene in macrophage-mediated phagocytosis for achieving the optimal antitumor immunity. In the following parts we will discuss the potential application of nanomedicine for phagocytosis manipulation and highlight specific examples where nanoparticle-based therapies were used to enable improved cancer therapy.

4.1. Nanomedicine for Improving Phagocytosis against Live Tumor Cells

4.1.1. Recombinant Protein- or Antibody-Integrated Nanoparticles for Blocking the Anti-Phagocytic Checkpoints

The extensive application of antibodies for anti-phagocytic checkpoint blockade, especially for the CD47-SIRPα signaling inhibition, promotes the development of nanomedicine for phagocytosis manipulation, thereby leading to increased bioavailability of the antibodies at the tumor site and improved macrophage phagocytosis of tumor cells. Lipid-based nanoparticles are one of the most frequently used delivery systems in nanomedicine due to their biocompatibility and the ability to deliver multiple compounds, including proteins and therapeutic genes^[16b]. Kulkarni et al. have developed a lipid-based nanosystem with the integration of anti-SIRPα antibodies (Figure 3) for cancer therapy^[104]. Of note, this study designed an amphiphile with the capability to inhibit CSF-1R, which further forms bilayers with co-lipids and results in a supramolecular structure, termed AK750. Subsequently, SIRPα-blocking antibodies were successfully integrated to the surface of the supramolecule via conjugation to the terminal ends of the PEG chains with a carbodiimide cross-linker. While AK750 itself exerted antitumor efficacy through inhibiting the CSF-1R signaling and skewing macrophages towards an M1 phenotype, the integration of anti-SIRPα antibodies to the supramolecule showed dramatically enhanced antitumor activity compared with AK750 treatment alone^[104]. With mechanistic investigation, the authors demonstrated that the treatment with anti-SIRPα-AK750 significantly increased macrophage phagocytosis against tumor cells compared to IgG-AK750 treatment, suggesting the blockade of anti-phagocytic signaling can synergize with macrophage repolarization to exert improved efficacy in tumor inhibition. Recently, a lipid-based phagocytosis nanoenhancer (LPN) that simultaneously engages macrophages and tumor cells was also reported^[105]. The LPN was tethered with antibodies for both CD47 and SIRPα, which results in the simultaneous blockade of CD47 and SIRPα in the tumor. The interruption of the CD47-SIRPα signaling increased the phagocytic activity of macrophages due to increased contact between macrophages and tumor cells. Additionally, treatment with LPN promoted the infiltration of effector T cell, NK cells and monocytes into the tumor, facilitating the tumor inhibition. Minimal changes of the body weight in mice treated with LPN were observe, indicating its relatively low systemic toxicity^[105].

Figure 3.

The design of the anti-SIRPα-AK750 nanoparticles for cancer therapy. A) Cancer cells use the CD47-SIRPα interaction to inhibit macrophage phagocytosis and the CSF-1R signaling further promotes the polarization of macrophages to a protumor M2 phenotype. B) Schematic demonstration of the anticancer effect of the dual-function nanoparticle anti-SIRPα-AK750. Anti-SIRPα-AK750 repolarizes macrophages to an antitumor M1 phenotype following sustained inhibition of CSF-1R signaling and improves macrophage-mediated phagocytosis of tumor cells with the blockade of CD47-SIRPα interaction. All panels adapted with permission.^[104] Copyright 2018, Springer Nature.

Anti-phagocytic checkpoint blockade with extracellular vesicles has gained considerable interests. Exosomes are nano-sized membrane-bound extracellular vesicles, which can be secreted by various types of cells and have specific enriched content such as small molecules, proteins and nucleic acids^[20a]. Owing to their feature as endogenous nanoparticles, exosomes show improved biocompatibility, higher stability, and lower immunogenicity, compared with other synthetic nanocarriers^[20a], resulting in an alternative platform for the development of cancer therapies. An exosome with surface-modified SIRPα variants that can bind to both human and mouse CD47 has been designed^[76]. The engineered exosomes interfered the interaction of CD47 and SIRPα between tumor cells and bone marrow derived macrophages, leading to enhanced phagocytosis of tumor cells in vitro. Moreover, intravenous injection of the exosomes significantly suppressed the tumor progression in immune-competent mice engrafted with CT26.CL25 cells, but only showed a slight effect on tumor growth in immuno-deficient nude mice, suggesting the important involvement of T cell immunity for CD47 blockade therapy. To completely abolish the “don’t eat me” signaling in the tumor microenvironment, Nie et al. synthesized a novel responsive exosome nano-bioconjugates with antibodies targeting both CD47 and SIRPα for synergistic cancer therapy^[106]. To obtain these exosome nano-bioconjugates, azide-modified M1 exosomes were first collected from the macrophages which were polarized to an antitumor M1 phenotype by using Mn²⁺ as an inducer following the cell membrane modification with azide groups through the intrinsic biosynthesis and metabolic incorporation of phospholipids. Subsequently, the azide-modified exosomes were simultaneously conjugated with the dibenzocyclooctynes (DBCO)-modified anti-CD47 antibody and anti-SIRPα antibody linked with pH-sensitive benzoic-imine bonds. Followed by systemic administration, the exosomes can accumulate in the tumor tissue and release conjugated antibodies through the cleavage of benzoic-imine bond in the acidic microenvironment, thus leading to the blockade of the anti-phagocytic signaling and improving the engulfment of tumor cells. Besides, the native M1 exosomes re-educated M2 macrophages towards the antitumor M1 phenotype, which synergized with the antibodies to inhibit cancer progression (Figure 4).

Figure 4.

Schematic illustration of the preparation and antitumor effect of the engineered M1 exosomes with antibodies targeting both CD47 and SIRPα. Adapted with permission.^[106] Copyright 2020, Wiley-VCH.

In addition to lipid-based nanoparticles and exosomes, inorganic nanoparticles also hold great potential for the application of antibodies to improve tumor cell phagocytosis by macrophages due to the features that their particle size, shape and surface modifications can be precisely controlled. As an example, Chen et al.^[107] reported an in situ sprayed fibrin gel containing anti-CD47 antibody-loaded CaCO₃ nanoparticles (aCD47@CaCO₃), which can gradually dissolve and release the encapsulated anti-CD47 antibodies in the acidic tumor microenvironment within the tumor resection cavity after surgery, thus promoting the activation of M1 macrophages possibly by modulating the acidity of the tumor microenvironment and inducing macrophage phagocytosis of cancer cells by blocking the CD47-SIRPα interaction, while reducing the systemic toxicity of anti-CD47 antibody (Figure 5). Nevertheless, dendritic cells also express SIRPα, whether aCD47@CaCO₃ promoted dendritic cell-based phagocytosis of tumor cells to facilitate dendritic cell maturation in vivo remains to be determined. Moreover, a recent study demonstrated that CD47-targeted bismuth selenide nanoparticles (Ab-PEG-Bi₂Se₃)^[108] can increase phagocytosis of tumor cells by macrophages with inhibited crosstalk between CD47 and SIRPα. Meanwhile, Ab-PEG-Bi₂Se₃ served as a nanoagent for photothermal therapy due to its excellent photothermal performance. This study presents an example to combine phagocytosis manipulation with other therapies via designing nanoparticles for improved cancer immunotherapy.

Figure 5.

Schematic demonstration of the in situ sprayed bioresponsive therapeutic fibrin gel containing aCD47@CaCO3 nanoparticles for the inhibition of local tumor recurrence after surgery. The aCD47@CaCO3 nanoparticles are embedded in the fibrin gel to serve as a release reservoir and a proton scavenger for regulating the acidity of the tumor microenvironment. The fibrinogen solution containing aCD47@CaCO3 nanoparticles and thrombin solution can be quickly mixed within the tumor resection cavity and gradually release the encapsulated anti-CD47 to promote macrophage phagocytosis of cancer cells. Adapted with permission.^[107] Copyright 2019, Springer Nature.

Engineering of bacteria to deliver therapeutic payloads in vivo has emerged for cancer immunotherapy, which may drive a new era for the development of drug delivery systems. To block the anti-phagocytic signals in the tumor microenvironment, Chowdhury and colleagues reported a non-pathogenic E. coli strain for the local delivery of an potent nanobody antagonist of CD47^[103], which has an approximately 200-fold higher binding affinity than commercially used monoclonal antibodies for mouse CD47. Notably, the engineered bacteria contained a synchronized lysis circuit (eSLC) that undergoes quorum lysis in the tumor microenvironment, thus releasing the encoded nanobody for CD47 blockade. This system allows for the safe and local delivery of immunotherapeutic agents and further induces their controlled release, leading to durable and systemic antitumor immunity.

4.1.2. Nanoparticle-Based Combination Therapies

Cancer cells use both innate and adaptive checkpoints to evade immune surveillance. Thus, the generation of nanoparticles that simultaneously targets innate and adaptive checkpoints has great potential to exert synergistic antitumor response. Given the low toxicity and biocompatibility of protein-based nanoparticles, various proteins have been used for developing delivery systems. Chen et al.^[109] reported an reactive oxygen species (ROS) responsive protein complex of anti-PD-1 (aPD-1) and anti-CD47 (aCD47) antibodies for cancer combination therapy (Figure 6). This albumin-based complex was formulated with anti-PD-1 in the core and anti-CD47 in the shell (aPD1@aCD47 complex), which leverages the abundant ROS in the tumor microenvironment to sequentially release anti-CD47 and anti-PD-1 antibodies. The combination strategy induced the activation of both innate and adaptive immune systems, leading to significant antitumor response in melanoma cancer models. In addition to focusing on blocking the checkpoint on T cells, delivering drugs to induce immunogenic cancer cell death was also proposed to combine with CD47 blockade for cancer treatment. For instance, Lee et al.^[110] designed a nanocage (FHSirpα-dox) that not only presents SIRPα variants for enhancing cancer cell phagocytosis, but also delivers doxorubicin to induce immunogenic cell death of cancer cells, therefore enabling the release of neoantigens and danger signals in dying cancer cells to cross-prime tumor-specific T cells. Treatment with the nanocages generated durable and robust immune response, which significantly inhibited tumor progression, resulting in complete tumor eradication in most tumor-bearing mice.

Figure 6.

The design of ROS-responsive aPD1@aCD47 protein complex and its synergistic antitumor effect by sequentially releasing anti-CD47 and anti-PD1 in the tumor microenvironment. Adapted with permission.^[109] Copyright 2019, American Chemical Society.

4.1.3. Gene Delivery for Anti-Phagocytic Checkpoint Intervention

In addition to the antibody-based therapy, delivery of therapeutic nucleic acids, such as short hairpin RNA (shRNA) or small interfering RNA (siRNA), is an alternative approach to block the anti-phagocytic signals, enabling tumor inhibition induced by macrophage phagocytosis of tumor cells. Li et al. ^[111] found that CD47 was highly expressed in glioma cells, especially glioma stem cells, which is related to poor therapeutic outcome. To validate the therapeutic efficacy of CD47 blockade, the authors used a lentiviral vector loaded with short hairpin RNA shCD47 to knockdown CD47 in glioma cells, which reduced their proliferation activity and tumor formation capability. However, the in vivo therapeutic efficacy of this delivery system was not evaluated. In addition to the application of viral vectors, multiple nonviral vector-based gene delivery systems for CD47 blockade have been also exploited. Systemic delivery of siRNA for CD47 with a liposome-protamine-hyaluronic acid (LPH) nanoparticle formulation efficiently silenced CD47 in the tumor tissue and significantly inhibited the growth and metastasis of melanoma tumors^[77], indicating CD47 plays a key role for tumor cells to evade the immune surveillance in melanoma and targeting this molecule with RNA interference is an efficacious approach for cancer therapy. Compared to reduced levels of red blood cells and platelets after anti-CD47 antibody administration, repeated injection of siRNA loaded LPH nanoparticles maintained the blood parameters, suggesting the safety of this formulation and the potential to co-deliver siCD47 and other therapeutic agents for synergistic antitumor activity.

Dual blockade of CD47 and PD-L1 with antibodies has demonstrated enhanced therapeutic efficacy in melanoma and colon carcinoma, opening up the possibility that simultaneous blockade of innate and adaptive checkpoints with gene delivery could enable improved antitumor immunity. To avoid the undesired side effects associated with the administration of antibodies, Lian et al.^[112] designed EpCAM (epithelial cell adhesion molecule)-targeted cationic liposome loaded with both siCD47 and siPD-L1 (LPP-P4-Ep), which can target cancer cells with highly expressed EpCAM and efficiently inhibit the expression of CD47 and PD-L1. This combination strategy significantly enhanced the antitumor effect compared with CD47 or PD-L1 blockade alone and did not cause severe toxicity, which provides an approach for the treatment of tumors with highly expressed CD47 and PD-L1. Polymeric nanoparticles are also widely used for the codelivery of therapeutic agents due to their high drug-loading efficiency and flexibility for surface modification. For instance, Chen et al. established a tumor acidity-responsive nanoparticle delivery system to carry both CD47 siRNA and CCL25 (NP-siCD47/CCL25)^[113]. In the slightly acidic tumor microenvironment, this system can sequentially release CCL25 in tumor stroma and CD47 siRNA into tumor cells. The intratumoral delivery of CCL25 promoted CCR9+ T cell infiltration, which further enhanced siCD47-based immunotherapy in a murine TNBC model and highlighted the great potential of CCR9+ T cells in mediating antitumor immunity.

Of note, delivery of plasmid DNA (pDNA) or messenger RNA (mRNA) encoding a trap protein has emerged as a novel approach for the blockade of immunosuppressive mediators. A small trapping protein for C-X-C motif chemokine 12 (CXCL12), a key chemokine that inhibits T cell infiltration, has been designed based on known anti-CXCL12 antibody sequences in 2016^[114]. To avoid the potential systemic off-target toxicity, the authors further developed a lipid calcium phosphate (LCP) nanoparticle to deliver plasmid DNA encoding the CXCL12 protein trap into the liver for the treatment of colorectal liver metastasis. As a result, delivery of the plasmid yielded liver-specific expression of CXCL12 trap, which reversed the immunosuppressive microenvironment and significantly reduced the occurrence of liver metastasis. Subsequently, Miao et al.^[115] developed a trimeric PD-L1 trap with the extracellular PD-L1-binding domain of PD-1 (Figure 7), which shows more than 1,000 times higher affinity to mouse PD-L1 compared with endogenous PD-1. Local and transient expression of the PD-L1 trap by liposome-protamine-DNA (LPD) nanoparticle-based pDNA delivery synergized with tumor-expressed CXCL12 trap for pancreatic cancer therapy in an orthotopic pancreatic tumor model, leading to a promising platform for the immune microenvironment modification. Since then, nanoparticle-mediated tumor-selective expression of traps for interleukin 10^[116], Wnt family member 5A^[117] and lipopolysaccharide^[118] has been successfully exploited for cancer immunotherapy in diverse mouse models, suggesting the feasibility to interfere the anti-phagocytic signaling with locally expressed trap protein. A summary of nanoparticles that have been successfully applied for anti-phagocytic signaling intervention is also presented in Table 3.

Figure 7.

Delivery of the plasmid DNA encoding PD-L1 trap for immune microenvironment modulation. A) Schematic illustration of the trimeric PD-L1 trap derived from the extracellular PD-L1-binding domain of PD-1. B) Fabrication of lipid-protamine-DNA nanoparticles for the delivery of PD-L1 trap plasmid. A) Adapted with permission.^[115] Copyright 2017, American Chemical Society.

Table 3.

Summary of delivery systems developed for anti-phagocytic signaling intervention

Strategy	Nanoparticle		Drug	Target	Ref
Blockade of anti-phagocytic checkpoints with recombinant proteins or antibodies	Lipid-based nanoparticle	Anti-SIRPα-AK750	Anti-SIRPα	SIRPα	[104]
		Lipid-based phagocytosis nanoenhancer	Anti-SIRPα Anti-CD47	SIRPα CD47	[105]
	Extracellular vesicle	Surface-modified exosome	SIRPα variant	CD47	[76]
		Exosome nano-bioconjugate	Anti-CD47 Anti- SIRPα	CD47 SIRPα	[106]
	Inorganic nanoparticle	Anti-CD47@CaCO3	Anti-CD47	CD47	[107]
		Ab-PEG-Bi2Se3	Anti-CD47	CD47	[108]
	Programmable bacterium	eSLC–CD47nb	Nanobody against CD47	CD47	[103]
	Protein nanoparticles	Albumin-based complex aPD-1@aCD47	Anti-CD47 Anti-PD-1	CD47 PD-1	[109]
		FHSirpα-dox nanocage	SIRPα variant Doxorubicin	CD47 -	[110]
Gene delivery for anti-phagocytic checkpoint intervention	Lentiviral vector	-	shCD47	CD47	[111]
	Liposome	LPH(CD47)	siCD47	CD47	[77]
		LPP-P4-Ep	siCD47 siPD-L1	CD47 PD-L1	[112]
	Polymeric nanoparticle	NP-siCD47/CCL25	siCD47 CCL25	CD47 CCR9	[113]

4.2. Development of Nanoparticles for Efferocytosis Intervention

Blockade of the “eat me” signal PtdSer on apoptotic tumor cells is one of the strategies to inhibit efferocytosis in the tumor microenvironment. However, administration of antibodies targeting PtdSer would not only inhibit phagocytic clearance of dying tumor cells, but also indiscriminately block all PtdSer-dependent phagocytic processes, which could induce interfered antigen presentation on the host^[119]. Therefore, nanotechnologies can be applied for the targeted delivery of therapeutic antibodies for PtdSer blockade, leading to selective inhibition of tumor cell efferocytosis. In addition, anti-PtdSer antibodies to block the recognition of apoptotic cells have shown improved therapeutic efficacy in combination with chemotherapy and anti-PD-1 therapy in preclinical studies^{[89b, 120]}, indicating an opportunity for developing nanoparticles to achieve the co-delivery of anti-PtdSer antibodies and other therapies for synergistic cancer therapy. Unfortunately, this combination strategy has not been assessed in vivo, whether the nanomaterial-based therapy can enhance benefits derived from free drug-based therapy remains to be determined.

Of note, most MerTK inhibitors are small molecules without specificity, and their systemic administration could induce undesired effects as MerTK inhibition in the retina causes blindness^[65], which suggests a potential approach to reduce the risk of side effects by developing nanomedicine for targeted drug delivery. Besides, inhibitors targeting TAM receptors have been successfully used to combine with traditional cytotoxic agents or adaptive checkpoint inhibitors to improve therapeutic efficacy. One such example is that the MerTK inhibitor UNC2025 increased sensitivity to methotrexate in a murine xenograft model^[121]. Combined treatment of the pan-TAM tyrosine kinase inhibitor BMS-777607 with anti-PD-1 antibody achieved enhanced antitumor response compared with either monotherapy in a triple-negative breast cancer model^[122]. Similarly, anti-MerTK antibody synergized with PD-1 blockade therapy to inhibit tumor progression in models of colon cancer and breast cancer^[102]. Further, the combination of sitravatinib (a broad spectrum inhibitor for several tyrosine kinases including TAM receptors) with nivolumab (an anti-PD-1 monoclonal antibody) is under a phase III trial in patients with advanced NSCLC who have previously experienced disease progression on or after platinum-based chemotherapy and checkpoint inhibitor therapy (NCT03906071). These preclinical and clinical studies provide the rationale to design nanoparticles for loading TAM receptor-targeting agents with other therapies or employing TAM receptor inhibitors in conjugation with cytotoxic chemotherapy or targeted therapies to enhance the antitumor efficacy.

An alternative for blocking TAM receptors is to silence their expression by delivering non-coding RNAs. miR-34a is naturally occurring tumor suppressor that is downregulated in a wide range of tumor types and one of its direct targets is Axl^[123]. Overexpression of miR-34a results in decreased Axl mRNA and protein levels^[123b], which presents an additional method to block the activity of Axl in vivo. Correspondingly, A nanoparticle-based miR-34a mimic (MRX34) has been developed, which is a liposomal formulation of miR-34a and a potential first-in-class miRNA mimic cancer therapy. The phase I study to assess the maximum tolerated dose, safety, pharmacokinetics, and clinical activity of this microRNA-based cancer therapy has been performed in patients with advanced solid tumors^[124]. The results showed that MRX34 treatment was tolerable under adequate dexamethasone premedication in most patients. However, the trial was terminated early due to serious immune-mediated adverse events, which is believed to be caused by the delivery vehicles. As a result, the application of this miRNA-based nanomedicine requires an improved delivery method, creating an opportunity for the development and clinical translation of delivery systems that can avoid the systemic immune activation. Delivery of siRNA is another way to target Axl and silence its expression. Considering that Axl expression is associated with the progression and metastasis of ovarian and uterine cancer, Mills et al.^[125] used a nanoparticle-based delivery platform p5RHH to deliver siAxl for the treatment of these tumors. Of interest, the siRNA delivery system p5RHH was developed by selectively modifying a natural cytolytic peptide melittin^[126], which is the pore forming component of honey bee venom. In this study, the authors demonstrated p5RHH-siAxl treatment significantly reduced the tumor metastasis and showed marginal toxicity, thereby providing a promising therapy for the treatment of uterine and ovarian cancer patients. While these studies were aimed at targeting the tumor cells, they demonstrate that opportunities exist for nanomedicine to target TAM receptors on tumor-associated macrophages for efferocytosis intervention.

5. Conclusion and Future Outlook

Targeting the phagocytosis activity of macrophages against tumor cells is one of the strategies to achieve efficient anticancer immunity. Generally, live tumor cells rely on the expression of “don’t eat me” signals to evade phagocytic clearance by macrophages, leading to a feasible anticancer approach by blocking the anti-phagocytic signals or their cognate receptors to enable phagocytosis and immune recognition of tumor cells. The CD47-SIRPα axis is the first anti-phagocytic checkpoint identified in the tumor microenvironment and its inhibition presents a promising therapeutic strategy for cancer treatment. Since then, more anti-phagocytic checkpoints have been identified, including PD-L1-PD1, MHC-I-LILRB1 and CD24-Siglec-10 axes, providing new targets for enhancing macrophage-mediated phagocytosis of tumor cells and subsequent antitumor response. Although therapeutics that target additional phagocytosis determinants represent an intriguing research avenue for cancer immunotherapy, diverse questions remain to be elucidated. First, the potency of each anti-phagocytic checkpoint and their mechanical interactions are yet unexplored. Although tumor cells rely on “don’t eat me” signals to evade phagocytic elimination by macrophages, different tumors or different tumor cells in the same tumor may show distinct expression profile of the anti-phagocytic determinants. It has been shown that the expression of CD24 and CD47 is inversely related in patients with diffuse large B cell lymphoma^[39], indicating the antitumor efficacy of treatment targeting CD24 or CD47 may vary between different tumors or cancer patients. Therefore, extensive investigation of the collective expression of “don’t eat me” signals in the tumor microenvironment is required when determining the optimal treatment for particular cancer patients. Besides, innate immune checkpoints can also function as adaptive checkpoints, such as the PD-L1-PD-1 axis. The engagement of PD-L1 and PD-1 on T cells induces the exhaustion and apoptosis of cytotoxic T cell, whereas for macrophages, tumor cell-expressed PD-L1 uses PD-1 to evade phagocytosis. How the same regulator can respond diversely in different cell types is not clear and elucidating this issue would provide new insights in the development of immune checkpoint inhibitors.

In contrast to live tumor cells, apoptotic tumor cells attract macrophages with the presentation of specific “find me” and “eat me” signals to complete the clearance of apoptotic cells through efferocytosis. Rapid clearance of apoptotic cells in the tumor microenvironment resolves inflammation and generates an immune tolerant environment, promoting the tumor progression. Blockade of efferocytosis, conversely, induces accumulated apoptotic tumor cells, which further undergo secondary necrosis and activate the innate immune system with the release of danger signals, thus producing antitumor immunity.

To facilitate the engulfment of live tumor cells or blockade of efferocytosis in the tumor microenvironment, numerous therapeutic agents have been developed and some have entered different clinical trial phases. Nevertheless, the inefficiency or side effects induced by low specificity limits their further application. The development of nanomedicine, however, allows for the effective targeting of specific tissues or cells, thereby inducing stronger therapeutic efficacy compared with free drugs. Actually, multiple forms of nanomedicine have been used to address clinical issues, such as the exploitation of Doxil^® (PEGylated liposome doxorubicin) and Abraxane^® (albumin-bound paclitaxel nanoparticles), which have been approved by FDA for the treatment of various cancers. Therefore, nanomedicine is an attractive field for the clinical translation of traditional therapies and is anticipated to drive the evolution of therapeutic research. Exploration of various combination strategies based on nanoparticles could further enable improved therapeutic efficacy compared with the monotherapy. However, there are also challenges for translating nanoparticle-based therapies into real clinical use. Preclinical studies by using mouse models usually cannot mimic the complexity of human tumors, which suggests that nanoparticle-based therapies work in mice may induce little therapeutic response in human patients. In particular, the relatively insufficient targeting and accumulation of nanoparticles in the tumor present a major limitation for nanomedicine translation, which may be overcome by analyzing the nanoparticle-biological interactions and further optimizing the delivery systems^[127]. Besides, more studies about the systemic toxicity and optimal dosing of nanoparticles are required.

Biography

Xuefei Zhou received his Ph.D. degree in Chemical Engineering at Zhejiang University in 2020. Currently, He works as a Distinguished Associate Research Fellow in the Fourth Affiliated Hospital, Zhejiang University School of Medicine. His research interests are in developing nanomaterials for cancer gene therapy and immunotherapy.

Xiangrui Liu received his Ph.D. degree in Pharmaceutical Science at University of Nottingham in 2011 and is currently an Associate Professor in School of Basic Medical Sciences at Zhejiang University. His research interests include anticancer pharmacology, therapeutic gene delivery, nanoparticle-based cancer chemo-immunotherapy and the development of therapies targeting non-malignant cells in the tumor.

Leaf Huang is a Fred Eshelman Distinguished Professor in the Eshelman School of Pharmacy, University of North Carolina at Chapel Hill. His research focuses on the development of nanoparticle platforms for targeted drug delivery and cancer immunotherapy with immunogenic cell death (ICD) inducers or tumor microenvironment modulators.