Macrophages as a “weapon” in anticancer cellular immunotherapy

Abstract

Anticancer immunotherapy is a treatment that activates the immune system to fight the tumor. Immunotherapy has several advantages over other cancer treatments in that anticancer immunotherapy displays high specificity, low side effects, and can combine with various conventional therapies. In recent years, oncologists have shown increasing interest in using macrophages for adoptive cell therapy and predict a bright future of macrophage‐directed therapy for eliminating cancer. The focus of increased research interest is the classically activated M1 macrophages exhibiting pronounced tumoricidal activity, and the alternatively activated M2 tumor‐associated macrophages, which otherwise help malignant cells evading attack by the immune system. M1 macrophages may represent an effective weapon in anticancer cellular immunotherapy, and the use of autoimmune macrophages properly prepared for antitumor administration is one of the promising ways for personalized therapy of cancer patients. The present report mainly discusses some modern aspects of the problem in application of activated M1 macrophage in anticancer therapy and reviews relevant publications up to 2021.

1. INTRODUCTION

The immune system of vertebrates has a rather complex organization—specific organs, tissues, a large number of different types of cells, and a vast number of chemical factors are involved in the implementation of protection against. The main task performed by the immune system is the detection, neutralization, and suppression of nonself objects such as microorganisms, tumor cells, or toxic chemical compounds. The function of the immune system is very flexible and depending on the conditions and the organisms encountered, it can be activated or suppressed. The development of any immune response is carried out with the obligatory interaction of almost all types of cells that make up its composition, and consists of a cascade of strictly organized and successively replacing stages.

Since Ilya Mechnikov had discovered the phenomenon of phagocytosis and subsequently created the cellular (or phagocytic) theory of immunity ¹ in 1882, many important findings have taken place in the field of immunology research, many of which have won Nobel prizes. The results of these discoveries are of great practical importance and many of the discoveries have been introduced into everyday medical practice. Furthermore, several new directions pertaining to a more complete understanding of the role and function of immune cells in living organisms have emerged.

One of such rapidly developing areas is the cancer immunotherapy, which has received great attention in recent years. However, the mechanisms of the immune system are far from being fully understood, despite the significant success achieved in this area. That is why the studies of cellular interaction and the development of an immune response are substantial areas of modern immunology, cell biology, and biomedicine.

Anticancer immunotherapy is a treatment that activates the patient's immune system to fight the tumor in the patient. It is also called biological therapy or biotherapy. The basic idea of immunotherapy is simple: to help the body defend itself against harmful “invaders.” However, tumor cells can be insidious; they often find a way to hide from the immune system. They also use multiple ways to turn off the body's defenses when the immune system tries to attack the tumor. In general, immunotherapy is aimed at counteracting these evasions from immune attacks.

There are different types of immunotherapies; each of them has specific mechanism to enhance the immune response. For modern cancer immunotherapy, the following approaches are used ² : (1) tumor‐targeting monoclonal antibodies, (2) adoptive cells transfer (cell therapy), (3) dendritic cell (DC)‐based interventions, (4) oncolytic viruses, (5) immunostimulatory cytokines, (6) inhibitors of immunosuppressive metabolism, (7) pattern recognition receptor (PRR) agonists, (8) immunogenic cell death (ICD) inducers, (9) cancer vaccines, and others.

Immunotherapy has several advantages over other cancer treatments. This type of anticancer therapy displays high specificity, low side effects, and can combine with various conventional therapies. In addition, immunotherapy has gained significant success in the postoperative treatments of cancer, prevention of metastases, and cancer recurrences. In general, adoptive cell therapy consists of several steps including collection and separation of circulating or tumor‐infiltrating immune cells, modification, activation, and the reintroduction of these cells to the patient. ³ In anticancer cellular therapy, it is possible to use populations of cells that directly kill tumor cells, such as different T‐lymphocytes including engineered chimeric antigen receptor (CAR) T cells, and NK cells. Also, other immune cells may participate cellular antitumor immunity. These are B cells and dendritic cells responsible for immune recognition and presentation of tumor‐associated antigens (TAA), expressing costimulatory molecules, and producing TAA‐specific antibodies. These experimental approaches are being actively developed and are well described in many reviews. ⁴ , ⁵ , ⁶ , ⁷

In recent years, oncologists have shown increasing interest in using macrophages for adoptive cell therapy and have predicted a bright future of macrophage‐directed therapy for eliminating cancer. The focus of increased research interest is the classically activated M1 macrophages exhibiting pronounced tumoricidal activity, and the alternatively activated M2 tumor‐associated macrophages (TAMs), which otherwise help malignant cells evading attack by the immune system. ⁸ The present report mainly covers some current aspects of the problem in activated M1 phenotype macrophage application in anticancer therapy and reviews relevant publications up to 2021.

2. MACROPHAGE ACTIVATION AND POLARIZATION

Macrophages (or the “big eaters”) are specialized cells in the body of animals and humans capable of actively capturing and digesting bacteria, the remains of dead cells, and other particles that are foreign or toxic to the body. They also regulate lymphocyte activation and proliferation and they are essential in the activation process of T‐ and B‐lymphocytes by antigens and allogenic cells. Macrophages are present in virtually every organ/tissue where they act as the first line of immune defense against pathogens and play an important role in maintaining tissue homeostasis. Activation is the most important step in the functional maturation of macrophages. Certain cytokines such as interferons, interleukins, growth factors, chemokines, and tumor necrosis factors have an activating effect. IFN‐γ, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), macrophage colony‐stimulating factor, macrophage migration inhibitory factor, and TNF‐α, as well as growth hormones and bacterial endotoxins or cell wall proteins are known to activate macrophages. After activation, macrophages become larger, number of pseudopodia increases, and the plasma membrane becomes more folded. The term “activated macrophage” in its broadest sense means that it has an increased ability to kill microorganisms or tumor cells. ¹⁰

Macrophages are activated by a variety of signaling molecules that cause their differentiation into various functional types (Figure 1). In the light of modern concepts, macrophages are divided into several phenotypes, depending on the properties acquired during activation. Classically activated macrophages (M1 phenotype) are stimulated by lipopolysaccharides (LPS), as well as IFN‐γ in conjunction with LPS. Their main functions are to destroy pathogenic microorganisms and induce an inflammatory response. Polarization toward the M1 direction is accompanied by the secretion of proinflammatory mediators. They express receptors for IL‐1 (IL‐1R1), TLR, and costimulatory molecules, the activation of which ensures the amplification of the inflammatory response. ¹¹ , ¹² , ¹³ , ¹⁴

FIGURE 1.

Characterization of M1 and M2 phenotypes of activated macrophages. Adapted from Genard et al. ⁹ The figure was created with BioRender.com

The bactericidal properties of M1 macrophages are determined by the production of free nitrogen and oxygen radicals generated by iNOS and the NADPH‐oxidase complex. ¹⁵ , ¹⁶ IL‐12 secreted by M1 macrophages also plays a key role in Th1 polarization, whereas IL‐1β and IL‐23 direct the immune response along the Th17 pathway. ¹¹ , ¹⁴ Recent studies have shown that M1 macrophages in addition to proinflammatory functions exhibit reparative properties as they secrete vascular endothelial growth factor (VEGF) to stimulate angiogenesis and the formation of granulation tissues. ¹⁷

Alternative activation of macrophages (M2 macrophages) is observed when the cells are stimulated by interleukins, glucocorticoids, immune complexes, TLR agonists, etc. They are found in zones of helminth invasion, fibrosis loci, healing skin wounds, and neoplastic formations. M2 macrophages are capable of active proliferation in situ. They express higher levels of receptors including: CD36 scavenger receptor for apoptotic cells, CD206 mannose receptor, CD301 receptor for galactose, and N‐acetylglucosamine, CD163 receptor for the hemoglobin–haptoglobin complex. Macrophages of this type are characterized by a low IL‐12/IL‐10 secretion ratio. Furthermore, M2 phenotype macrophages are subdivided into M2a, M2b, M2c, and M2d subtypes depending on their markers and functions. ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²²

The nature of macrophage activation is not rigidly determined and stable. The possibility of transformation of the M1 phenotype into M2 was shown with a range of cytokines and as a result of efferocytosis. After uptake of apoptotic cells, macrophages sharply reduce the syntheses and secretions of inflammatory mediators such as CCL2, CCL3, CXCL1, CXCL 2, TNF‐α, GM‐CSF, IL‐1β, IL‐8 and multiply increase the production of TGF‐β. The reverse transformation of the M2 phenotype into M1 is assumed with the development of obesity. ¹⁶ , ²¹ , ²³

3. HOW DO MACROPHAGES RECOGNIZE AND KILL CANCER CELLS?

Macrophages are the type of phagocytes that, due to their motility, can enter the site of injury, although some macrophages are found in tissues. These are large, granular cells with high destructive potential. In addition to destroying pathogens and alien cells, macrophages are capable of cleaving foreign agents into peptides and presenting those peptides by the main histocompatibility complex II class (MHC II). T cells that recognize peptides presented on MHC and costimulatory signals by antigen presenting cells (such as macrophages) are activated. Thus, macrophages are responsible for the destruction of foreign targets and activation of the T cell‐mediated immune responses. ²⁴ Macrophages and other myeloid cells can move to the tumor, extricate themselves from the bloodstream, and infiltrate into the tumor. ²⁵

It is believed that activated macrophages recognize and distinguish tumor cells from normal cells due to the difference in the composition of cell membranes. Such tumor markers include an increased content of phosphatidylserine. Additional mechanisms may include the recognition of altered carbohydrate structures (or glycosylation) on the cell surfaces of tumor cells. Certain tumor antigens, such as carcinoembryonic antigen and Tn antigen are carbohydrate structures recognized by lectin‐like receptors on the cell membranes of macrophages. ²⁶

Currently, the molecular mechanisms of the antitumor activity of macrophages are not fully understood. It is known that macrophages can kill cancer cells by several mechanisms. These mechanisms include (1) indirect killing by recruitment of other immune cells that can lyse the cancer cells, (2) cytolysis of cancer cells through antibody (Ab) dependent cellular cytotoxicity, and (3) direct killing through the release of harmful products (such as oxygen radicals) (Figure 2).

FIGURE 2.

Mechanisms of macrophage‐mediated cancer cell killing: 1. Direct killing; 2. Cytolysis through antibody dependent cellular cytotoxicity; 3. Indirect killing. The figure was created with BioRender.com

At this moment, it is known that the innate immune receptor Dectin‐1 expressed on dendritic cells and macrophages is critical to NK‐mediated indirect killing of tumor cells that express N‐glycan structures at high levels. Receptor recognition of these tumor cells causes the activation of the IRF5 transcription factor and downstream gene induction for the full‐blown tumoricidal activity of NK cells. The affected genes include several membrane‐bound proteins among which Inam (termed as Fam26f) is known to activate NK cells via its homophilic interaction. It is confirmed that NK cells are required to orchestrate with macrophages for tumor cell killing, wherein activation of the IRF5 transcription factor by Dectin‐1 signaling instigated by receptor recognition of N‐glycan structures on tumor cells is critical. ²⁷

Macrophage can phagocytose antibody‐coated cancer cells after macrophages bind to the Fc portion of the antibodies. Based on in vitro studies using mononuclear cells, it has been appreciated that myeloid cells such as macrophages are crucial for antibody‐regulated killing of tumor cells. ²⁸ , ²⁹ For example, mouse IgG2a is the most potent antibody isotype for the activation of effector cells through its intermediate‐affinity interaction with the Fc receptor, FcRIV. ³⁰ , ³¹ FcRIV utilizes FcRγ, the FcR common γ‐subunit, to transmit signal into the cell. As such, antibodies may activate macrophages via the FcRIV‐FcRγ pathway to mediate both antibody‐dependent, macrophage‐mediated cytotoxicity and further contribute to NK cell activation.

It is known that M1 macrophages exhibit phagocytic and antigen‐presenting activity, produce proinflammatory cytokines, and exert cytotoxic functions. M1 macrophages can kill target cells directly via mechanisms dependent on reactive oxygen species (ROS), reactive nitrogen species, and IL‐1β and TNF‐α production. M1 macrophages are known to promote also indirect cytotoxicity by activating other immune cells, such as NK cells and T cells. ³² , ³³ , ³⁴

TNF‐α playing a key role in inflammation and apoptosis of cancer cells can induce cancer cell apoptosis through the tumor necrosis factor receptor 1 (TFNR1) signaling pathway. ³⁵ TNF‐α binds to the TFNR1 receptor triggering the conversion of Complex I to Complex II ³⁶ in which Complex I is composed of TNFR1, tumor necrosis factor receptor type 1‐associated DEATH domain protein (TRADD), receptor‐interacting protein 1 (RIP1), and TNF receptor‐associated factor 2 (TRAF2). Activated RIP1 binds to Fas‐associated protein with death domain and procaspase‐8 and ‐10 to form Complex II. Complex II induces ROS production and activation of caspase‐3 and caspase‐7. ³⁷ , ³⁸ TNF‐α also induces cancer cell apoptosis through the MAPK‐JNK pathway. ³⁷ Thus, TNF‐α is considered to be an effective anticancer cytokine. ³⁹ , ⁴⁰

ROS and NO are signal molecules that play an important role in many physiological and pathological processes. Several studies have shown that ROS and NO generated by activated macrophages can kill different tumor cells by producing nitrosative/oxidative stress, inducing DNA damage, cytotoxicity, and apoptosis. ⁴¹ , ⁴² , ⁴³ , ⁴⁴ So, ROS and nitric oxide generated in activated macrophages and released into the environment could also contribute to the anticancer effect. The image of macrophages attack the cancer cell is represented in Figure 3.

FIGURE 3.

Human Hs578T triple‐negative breast cancer cell (indicated by arrow) surrounded and attacked by RAW264.7 macrophages stained with DiO fluorescent probe. (courtesy of Dr. Yun‐Ming Wang, National Yang Ming Chiao Tung University, Hsinchu 30010, Taiwan)

4. HOW DO TUMOR CELLS ESCAPE THE ANTITUMOR EFFECT OF MACROPHAGES?

Although macrophages exhibit significant tumoricidal and phagocytic activity of tumor cells in vitro, in reality, tumor cells can deceive the immune system and lull the action of macrophages. To date, four regulatory molecules expressed on tumor cells have been identified with the capability of inhibiting phagocytic clearance, including CD47, PD‐L1, major histocompatibility complex (MHC) class I, and CD24. Signal‐regulatory protein α (SIRPα), PD‐1, leukocyte immunoglobulin‐like receptor 1 (LILRB1), and sialic‐acid‐binding Ig‐like lectin 10 (Siglec‐10), the respective binding receptors ⁴⁵ are expressed on macrophage surface (Figure 4). The cancer cells often overexpress the membrane‐bound protein CD47, which is often called the “don't‐eat‐me” signal. This signal suppresses the phagocytic activity of macrophages upon binding to SIRPα (signal regulatory protein α)‐receptor present on phagocytes. ⁴⁶ , ⁴⁷ , ⁴⁸ Blocking CD47‐SIRPα binding promotes phagocytosis of tumor cells by macrophages and induces antitumor responses in different xenograft models. ⁴⁶ , ⁴⁹

FIGURE 4.

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. Adapted from Zhou et al. ⁴⁵ The figure was created with BioRender.com

In addition, a second mechanism for evading from macrophages has recently been discovered. The central role in the control of the phagocytic function of macrophages was found to play by the MHC class I. The common MHC class I component β2‐microglobulin (β2m) expressed by cancer cells can protect cancer cells from phagocytosis. This effect is mediated by the inhibitory receptor LILRB1 overexpressed on the TAM. Disruption of either MHC class I or LILRB1 potentiates phagocytosis of tumor cells both in vitro and in vivo, which indicates MHC I and LILRB1 are important regulators of the macrophages, and are potential targets of anticancer immunotherapy. ⁵⁰

PD‐1 is a membrane protein of the immunoglobulin superfamily that is involved in the cellular differentiation of immune cells. PD‐1 and its ligands, PD‐L1 and PD‐L2, play an important role in the negative regulation of the immune system by preventing the activation of T lymphocytes. ⁵¹ , ⁵² More recently, PD‐1 has been found in TAMs, and its expression correlates with tumor growth. ⁵³ It was found that PD‐1 negatively regulates phagocytosis of tumor cells. PD‐1 ⁻ macrophages exhibit a higher level of phagocytic activity compared to PD‐1 ⁺ macrophages. Blockade of PD‐1 or PD‐L1 with Abs leads to increased phagocytosis of tumor cells and suppresses tumor growth. Thus, the PD‐1 not only serves as a checkpoint for T cells but also contributes to the evasion of tumor cells from the killing by macrophages.

It was found that some tumor cells overexpress a glycosylated surface protein, CD24, which interacts with Siglec‐10 (Sialic acid‐binding Ig‐like lectin 10) on immune cells to suppress inflammatory responses caused by tissue damage. ⁵⁴ , ⁵⁵ , ⁵⁶ Some cancer cells use CD24 to avoid phagocytosis by macrophages expressing Siglec‐10. ⁵⁷ Tumor‐associated M2 macrophages express higher levels of Siglec‐10 and are less phagocytic than M1 macrophages.

However, macrophages can bypass the inhibitory activity mediated by CD47 against cancer cells. For this to take effect, macrophages must be preactivated. It has recently been found that stimulation of macrophages with oligodeoxynucleotide CpG, a Toll‐like receptor agonist, alters the central carbon metabolism in macrophages to confer antitumor activity, even when cancer cells express CD47. This activation of macrophages requires oxidation of fatty acids and shunting of intermediate products of the tricarboxylic acid cycle for de novo lipid biosynthesis. This integration of metabolic inputs is supported by carnitine palmitoyltransferase 1A and adenosine triphosphate citrate lyase, which together confer antitumor potential on macrophages to overcome inhibitory CD47‐signal on cancer cells. ⁵⁸

5. USE OF BIOLOGICALLY ACTIVE COMPOUNDS FOR MACROPHAGE ACTIVATION, POLARIZATION, AND REPROGRAMMING

It is well known that bacterial LPS potently activate macrophages via Toll‐like receptors. ⁵⁹ However, the use of LPS for the activation and polarization of macrophages for anticancer immunotherapy in patients is practically unwarranted due to the unpredictable and detrimental effect of LPS on the immune system. In this regard, the search for new methods to obtain stimulated macrophages continues. Researchers have been trying to stimulate macrophages to enhance their antimicrobial and antitumor properties for several past decades. Since the 80s–90s of the last century, the scientific literature has been full of various publications describing the immunomodulatory activity of various extracts and individual compounds. The attention of scientists has been attracted by natural substances isolated from microbial, animal, and plant sources. For instance, human fibronectin and C‐reactive protein have been studied as stimulants of macrophages in vitro, ⁶⁰ , ⁶¹ as well as beta‐1,3‐D‐glucan from yeast cell walls, ⁶² lipophilic muramyldipeptide analogs, ⁶³ lipoprotein containing lipophilic muramyl tripeptide, ⁶⁴ lipopeptide analog of a fragment from the cell wall of gram‐negative bacteria. ⁶⁵

The extracts and individual compounds isolated from plants included in the list of traditional medicine were of particular interest. Among them are plants growing in Japan and China. It was found that an extract from Crassocephalum crepidioides, a plant distributed in Okinawa Islands, stimulated macrophages to enhance the expression of iNOs and increase the synthesis of NO, and activated antitumor activity against murine Sarcoma 180 (S‐180) cancer cells. ⁶⁶ It was found that some compounds derived from plants in traditional medicine are able not only to effectively activate and polarize M1 macrophages but also to transdifferentiate tumor‐associated M2 macrophages into a M1 phenotype. For example, baicalein (5,6,7‐trihydroxyflavone), isolated from the Chinese herb Scutellaria baicalensis root, can block TGF‐β1 via inhibiting PI3K/Akt pathway in M2 macrophages and repolarize them to a M1 phenotype in breast cancer tissues. ⁶⁷ A extracts from the root of Panax notoginseng can reeducate M2‐like macrophages toward M1 differentiation. ⁶⁸ Individual Ginsenoside Rb3 from Panax ginseng has protective functions against acute lung injury via M1/M2 phenotypic switch. ⁶⁹ Emodin (1,3,8‐trihydroxy‐6‐methylanthraquinone) is a natural anthraquinone derivative from many Chinese herbs. This compound bidirectionally regulates both M1 and M2 phenotype programs via different mechanisms including suppression of STAT6 and C/EBPβ signaling to increase H3K27m3 on the M2‐related genes. Emodin also restrains excessive M1 or M2 macrophages. ⁷⁰ Osthole [7‐methoxy‐8‐(3‐methyl‐2‐butenyl)‐2H‐1‐benzopyran‐2‐one] is a coumarin member isolated from Cnidiummonnieri (Fructus Cnidii), found to decrease M2‐like macrophages in pancreatic tumors by inhibition of STAT6 and p‐ERK1/2‐C/EBP β. ⁷¹

Special attention is paid to compounds of marine origin, isolated from algae, microorganisms, echinoderms, and other organisms. As a rule, these compounds possess a wide spectrum of biological activity, including immunomodulatory and antitumor activities. It was found that several “marine” compounds can regulate macrophages and lead to the polarization of macrophages in both the M1 and M2 phenotypes. ⁷²

Recent studies on the membrane‐type diterpenoids isolated from soft coral species Briareum violaceum in Taiwan demonstrated suppressive effects on iNOS release from the cells, suggesting a potential to shift the M1 phenotype toward the M2 type. ⁷³ Polysaccharides isolated from gorgonian Pseudopterogorgia americana induces the expression of TNF‐α, IL‐6, and COX‐2 in mouse macrophages, but had no effect on the expression of iNOS and NO production. These compounds decrease expression of proinflammatory cytokines in LPS‐activated macrophages, indicating a potential in reprogramming of macrophage polarization toward the M1‐type. ⁷⁴ The purine alkaloid homarine, a major metabolite found in water extracts of Portugal sea anemones, Actinia equine and Anemonia sulcata, has a great potential in modulating macrophage polarization. ⁷⁵ Crustaceans of the order Decapoda (crab, shrimp, prawn, and lobster) are a valuable source of plain polysaccharide and chitosan, which may promote the drug delivery targeting M1 or M2 macrophages. ⁷⁶ It is known that triterpene glycosides of sea cucumbers have a pronounced immunomodulatory effect. Triterpene glycosides are capable of activating macrophages both in vivo and in vitro and polarizing them into the M1 phenotype by affecting the pathway mediated by purinergic P2X4 receptors. ⁷⁷ , ⁷⁸ , ⁷⁹ Increased cell adhesion, spreading and motility, lysosomal and bactericidal activity, proinflammatory cytokine release, expression of iNOs, and increase in ROS and NO levels are found in cells treated with triterpene glycosides.

The above examples give a clear understanding that sea organisms are attractive source of biologically active substances in general, and compounds‐modulators of macrophage activity and their phenotype, in particular.

6. PROSPECTS FOR THE USE OF MACROPHAGES AS A “WEAPON” IN THE FIGHT AGAINST CANCER

6.1. Nano‐immunotherapy

In recent years, strategies have been actively developed that allow the delivery of immunotherapeutic and anticancer drugs to tumor targets. Nanoparticles (NPs) are promising carriers in this way. ⁸⁰

Due to the advent of new biomaterials with unique properties, complex systems of multifunctional NPs are now being developed, some carrying specific antibodies and receptors, and/or a variety of cytotoxins and cytostatics, which are expected to expand the theranostic applications for cancers. ⁸¹ With the growing understanding of the crucial role of immune cells in cancer development, the focus of nanotherapeutic agents is now shifting toward modulating the activity of immune cells for anticancer treatments.

Macrophages are major phagocytic cells with a powerful ability to detect and absorb NPs. This ability means that macrophages could interfere with the intended delivery of drugs in the NPs. But on the other hand, they are a convenient “container” for NPs that can be strategically used in cancer therapy using cell‐mediated delivery of therapeutic cargo. Such macrophage‐mediated delivery can be achieved through intracellular uptake and subsequent release of the cargo at the tumor sites. It has been shown that monocytes/macrophages loaded with gold nanoshells (Au‐NS) can be recruited into a breast tumor spheroid using an in vivo mouse model of cancer. The subsequent death of cancer cells can be subsequently initiated by photo‐induced ablation of macrophages loaded with such NPs. ⁸² , ⁸³

Alternative approach can be the attachment of therapeutic NPs to the surface of macrophages in the form of a nanoscale “cell backpack.” It was convincingly demonstrated that in mouse inflammation models such backpacks on macrophage cells can enter and accumulate in the target organ, but not the reticuloendothelial system which removes NPs from the bloodstream. This makes it possible to carry out long‐term cell therapy procedures without harming the carrier cells. ⁸⁴ In addition, it was demonstrated that macrophage‐associated cellular backpacks are capable of a prolonged controlled release of a model protein in the targeted tissues. ⁸⁵

In parallel, methods to use NPs for direct interaction with macrophages and their activation or reprogramming are actively sought. For example, NPs were used to reprogram TAMs from the protumor M2 to the antitumor M1 phenotype. In particular, chitosan/poly(γ‐glutamic acid) NPs were shown to effectively reprogram M2 macrophage induced by interleukin‐10 to M1 phenotype. After contact with NPs, these macrophages decreased CD163 expression and increased TNF‐α secretion. ⁸⁶ Another interesting approach use low doses of photodynamic sensitization and Temoporfin NPs to repolarize M2‐d THP‐1 cells into M1 phenotype. ⁸⁷ NPs of various designs are now being created that carry anti‐SIRPα and anti‐PD‐L1 or anti‐CD47 antibodies on their surfaces for double suppression of the “don't‐eat‐me” signals subsequent phagocytosis of tumor cells by macrophages. More detailed information on the creation and application of such NPs can be obtained in a recent review. ⁴⁵

6.2. Engineered macrophages for cancer therapy

One of the promising approaches in cell therapy for cancer is the use of CAR initially applied to modify T cells. However, CAR T‐cell therapy has serious limitations for solid tumors since these cells show limited capacity to enter the tumors. ⁸⁸ , ⁸⁹ On the contrary, macrophages can infiltrate tumors, making them promising killers for solid cancers. Given these circumstances, a first‐generation anti‐CD19 CAR encoding the CD3ζ intracellular domain, which is required for antibody dependent cellular phagocytosis in macrophages, have recently been genetically engineered in the human THP‐1 macrophages. ⁹⁰ The anti‐CD19 CAR confers a persistent proinflammatory M1 phenotype on the macrophages. The CAR‐engineered macrophages express proinflammatory cytokines and chemokines, convert M2 macrophages to M1, activate the antigen presentation mechanism, recruit and present antigen to T cells, and resist immunosuppressive cytokines. In addition, they exhibit pronounced antigen‐specific phagocytosis and in vitro tumor suppression. In two mouse models of solid tumor xenograft, a single infusion of CAR‐macrophages reduced the growth of human tumors and increased the overall survival of tumor‐bearing animals. In humanized mouse models, it was also shown that genetically modified macrophages induce a proinflammatory tumor microenvironment and enhance the antitumor activity of T cells. ⁹⁰ Based on these results, Carisma Therapeutics plans to begin the first human trials of autologous‐CAR macrophage therapy directed against tumors expressing human epidermal growth factor receptor 2. ⁹¹

In a series of recent studies, it was found that the suppression of SIRPα on macrophages from the bone marrow of mice and humans leads to the blocking of recognition of its own “marker of self,” CD47, on all other cells. The authors used human lung A549 solid tumor models established in NSG mice. Systemic iv injections cause highly phagocytic marrow‐derived macrophages that have both SIRPα inhibition and tumor‐targeting Abs preloaded into Fc receptors to safely engorge and accumulate in solid tumors and significant regression of tumor growth within 1–2 weeks before differentiation to ineffective TAMs. This study provides the first in vivo demonstration that injections of human macrophages can induce tumor regression by around 80% or more. ⁹² , ⁹³

An integrated nanotechnology/biology strategy for cancer immunotherapy that uses arginine nanoparticles (Arg‐NPs) to deliver CRISPR‐Cas9 gene editing machinery into cells to generate SIRP‐α knockout macrophages and block its binding to CD47 was recently reported. ⁹⁴ The NP system efficiently codelivers single guide RNA and Cas9 protein required to knockout the “don't‐eat‐me” signal in RAW 264.7 macrophages. Turning off this signal increased the phagocytosis of human osteosarcoma U2OS cancer cells by fourfold. Authors argued that CRISPR/Cas9 knockout of SIRP‐1α a strategy promising for the creation of “weaponized” macrophages for cancer immunotherapy.

7. CONCLUSION

Macrophages are a unique type of cells that make up a cellular army and are called upon to fight against an alien invasion in the body. These cells, like well‐armed border guards, are on alert at the most advanced lines, tracking the enemy's presence. As professional soldiers, at the first signs of aggression, they can cuddle to the ground (adhesion and spreading on the vessels walls and tissues) and crawl toward the enemy (motility), receiving various commands on the move (cytokine signaling) and putting effective weapons (synthesis of ROS, NO, tumor necrosis factor, and activation of phagocytosis), becoming active and combat‐ready macrophages of the M1 phenotype. Using a well‐organized molecular warning system “friend or foe,” they recognize tumor cells and destroy them by various mechanisms. In direct contact with tumor cells, macrophages “shoot” and “bombard” them with cytotoxic molecules, and after a successful military operation they clear the battlefield by phagocytosing the remains of tumor cells. In addition, they present the distinguishing marks of the enemy in the form of tumor antigens to other combat arms (T and B cells) so that these cells also take an active part in the defense of the body, using additional mechanisms of fighting the enemy in the form of cytotoxic molecules, produced by cytotoxic T lymphocytes and NK cells, and specific antibodies produced by B lymphocytes.

However, the enemy is cunning and insidious. On the one hand, tumor cells can use “stealth technology” and change the “friend or foe” recognition system on their surface and become invisible to macrophages and other participants in cellular defense. On the other hand, tumors can “fool” macrophages, lure them over to their side, and recruit them into their service in the form of M2 macrophages or TAMs. Therefore, the effectiveness of the fight against tumors by macrophages is significantly reduced, and an urgent task to researchers is to help them in this antitumor fight. In this regard, scientists are trying to find new ways of additional activation of a larger number of macrophages and their programming into a combat‐ready M1 phenotype. For this purpose, a large amount of synthetic and natural chemical compounds from various sources are being investigated, and a series of biologically active molecules have already been found that can additionally stimulate the antitumor activity of macrophages. Scientists are actively looking for ways to influence the “cheated” M2 macrophages to reprogram them back to the antitumor M1 phenotype. In addition, researchers are learning to use macrophages to deliver additional anticancer agents in the form of cytotoxins, cytostatics, and blocking antibodies. Finally, scientists are learning to genetically modify macrophages so that they ignore the “don't‐eat‐me” signal on tumor cells and overcome the barrier created by tumors.

Now, there is a clear understanding that the future in the successful fight against human neoplastic neoplasms is associated with the development of cellular technologies and the creation of effective methods of cellular antitumor immunotherapy. Several advantages such as the ability to obtain the required number of cells from patients for subsequent autologous transplantation and the ability to infiltrate solid tumors make macrophages a rather attractive immune cell for oncologists. Therefore, macrophages are effective “weapon” in anticancer cellular immunotherapy, and the use of autoimmune macrophages properly prepared for antitumor administration is a promising way for personalized therapy of cancer patients.

CONFLICT OF INTEREST

The authors declared no potential conflicts of interest.

Aminin D, Wang Y‐M. Macrophages as a “weapon” in anticancer cellular immunotherapy. Kaohsiung J Med Sci. 2021;37:749–758. 10.1002/kjm2.12405