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. 2024 Jun 22;4(4):401–414. doi: 10.1007/s43657-023-00154-6

Modulating and Imaging Macrophage Reprogramming for Cancer Immunotherapy

Jialu Wang 1, Yafang Lu 1, Ren Zhang 1, Zhenzhen Cai 2, Zhan Fan 1, Yilun Xu 2, Zheng Liu 2,✉, Zhihong Zhang 1,2,✉
PMCID: PMC11584841  PMID: 39583310

Abstract

Cancer immunotherapy has made great progress in effectively attacking or eliminating cancer. However, the challenges posed by the low reactivity of some solid tumors still remain. Macrophages, as a key component of the tumor microenvironment (TME), play an important role in determining the progression of solid tumors due to their plasticity and heterogeneity. Targeting and reprogramming macrophages in TME to desired phenotypes offers an innovative and promising approach for cancer immunotherapy. Meanwhile, the rapid development of in vivo molecular imaging techniques provides us with powerful tools to study macrophages. In this review, we summarize the current progress in macrophage reprogramming from conceptual roadmaps to therapeutic approaches, including monoclonal antibody drugs, small molecule drugs, gene therapy, and chimeric antigen receptor-engineered macrophages (CAR-M). More importantly, we highlight the significance of molecular imaging in observing and understanding the process of macrophage reprogramming during cancer immunotherapy. Finally, we introduce the therapeutic applications of imaging and reprogramming macrophages in three solid tumors. In the future, the integration of molecular imaging into the development of novel macrophage reprogramming strategies holds great promise for precise clinical cancer immunotherapy.

Keywords: Macrophage reprogramming, Imaging techniques, Immunotherapy, Polarized phenotypes, Solid tumors

Introduction

As a large group of diseases, cancers caused 10 million deaths globally in 2020 (Siegel et al. 2021). Up to date, cancer immunotherapy has become a highly debated topic in the field of cancer treatment (Wang et al. 2021). The main focus of immunotherapy is to enhance the immune responses of various immune cells within the tumor microenvironment (TME) to effectively eliminate cancer cells (Li et al. 2021a). Among the immune cells present in TME, macrophages account for over one-third of the total population (Duan and Luo 2021). Therefore, targeting macrophages has shown promising potential in improving the efficacy of cancer immunotherapy.

In general, macrophages are often divided into two types: classically activated M1-type and alternatively activated M2-type. This classic dichotomy is based on various stimuli, surface molecules, cytokine secretion patterns, metabolic signals, and cell–cell interactions (Fig. 1). Macrophages exhibit notable characteristics, primarily their remarkable plasticity and heterogeneity, enabling them to transition between distinct phenotypes and execute a range of functions (Loke and Lin 2022). Although the classical dichotomy of macrophage phenotypes has faced challenges from advanced technologies such as single-cell RNA-seq (scRNA-seq) (Xue et al. 2022), cellular indexing of transcriptomes and epitopes by sequencing (Ma et al. 2022b), mass cytometry (Ding et al. 2023), and hyperspectral image (Strack 2023). The M1/M2 classification remains crucial in many inflammation-related diseases, especially in cancer, which always reduces the M1/M2 ratio to achieve immune evasion (Gharavi et al. 2022; Noonepalle et al. 2023). During early-stage cancer, a strong immune system with powerful adaptive and innate immunity exhibits a higher proportion of M1 type macrophages to surveil and eliminate abnormal cells. This corresponds to a Th1 immune response by secreting higher levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-2, IL-6, IL-12, and IL-23 (Chen et al. 2023).

Fig. 1.

Fig. 1

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The typical markers and morphology of polarized macrophages. The polarization states of macrophages exhibit various markers and morphologies, represented by the M1 and M2 phenotypes. The representative markers of M1/M2 macrophages consist of cluster of differentiation (CD), interleukin (IL), CC chemokine ligand (CCL), CXC chemokine ligand (CXCL), toll-like receptor (TLR), transcription factors (TFs), and other effectors. In terms of morphology, M1 macrophages typically have a round and flattened appearance. This distinct shape contributes to their slower movement speeds and denser cell-to-cell contacts, which allow them to exert pro-inflammatory effects, such as killing tumors and other infectious pathogens. In contrast, M2 macrophages exhibit more elongated and distinct synapses, facilitating faster movement for tissue repair, angiogenesis, and other anti-inflammatory effects

However, with the development of immune selection, the proliferation of abnormal cells gradually loses its major histocompatibility complex class I (MHC-I) and major histocompatibility complex class II (MHC-II) antigens and decreases the amount of tumor antigens in the equilibrium phase (Lerner et al. 2023). By this time, tumor-derived soluble factors switch M1 type macrophages to M2 type macrophages and shape a tumor-promoting TME, in which M2 type macrophages protect cancer cells from immune cell attack, allowing for their progression and metastasis (Fig. 2) (Qian et al. 2023). Therefore, although the majority of cancer immunotherapies harness T lymphocytes to destroy tumors, it is believed that targeting macrophages to switch their dysfunctional phenotypes also holds promising therapeutic potential in cancer immunotherapy.

Fig. 2.

Fig. 2

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The intervention of polarized macrophages with cancer cells. During early-stage cancer, a strong immune system with powerful adaptive and innate immunity exhibits a higher percentage of M1 type macrophages to surveil and clear abnormal cells, which correspond to Th1 immune responses by secreting higher levels of pro-inflammatory cytokines, such as TNF-α and IL-1β, IL-2, IL-6, IL-12, and IL-23. However, with the development of immune selection, the proliferation of abnormal cells gradually loses their major histocompatibility complex class I (MHC-I) and major histocompatibility complex class II (MHC-II) antigens and decreases the amount of tumor antigens in the equilibrium phase. By this time, tumor-derived soluble factors switch M1 type macrophages to M2 type macrophages and shape a tumor-promoting TME, in which M2 type macrophages assist cancer cells in evading immune attack, thereby promoting tumor progression and metastasis

At present, various strategies have been adopted to develop macrophage-targeted therapies, including macrophage depletion (Cassetta and Pollard 2023), macrophage phagocytosis enhancement (Hu et al. 2023), and modifying macrophage recruitment (Bied et al. 2023). Though these strategies have been moved forward in several preclinical trial phases and partly showed promising preclinical results, the off-tumor toxicity and other side effects limit their applications in cancer immunotherapy. In recent years, a novel targeting macrophage strategy called “macrophage reprogramming” has represented a more viable option to address these issues. Macrophage reprogramming, also known as macrophage repolarization, is defined as a continuous dynamic phenotype switch between the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. Macrophage reprogramming aims to modulate the activation dysregulation of macrophages and get controllable phenotypic alterations in diseases. The destination of macrophage reprogramming is the reconstruction of a strong and well-balanced immune environment to eradicate diseases. Importantly, this strategy modulates the macrophages themselves to maintain the healthy homeostasis of tissues in diseases.

The heterogeneity and plasticity of macrophages increase the difficulty of targeting macrophage therapies. One difficulty lies in accurately tracking and evaluating the effects of targeted therapies. Several conventional biochemical techniques mainly focus on a certain moment in macrophage-targeted therapies, often overlooking the global dynamic structural and functional changes of polarized macrophages. Molecular imaging supplements this missing spatiotemporal dynamic information by providing long-term tracking, high sensitivity and specificity imaging (Hurt et al. 2023). Moreover, molecular imaging provides a variety of powerful non-invasive tools for dynamically monitoring biological processes, including optical imaging (Pahlevaninezhad et al. 2022), photothermal imaging (Fu et al. 2023), photoacoustic imaging (Lin and Wang 2022), raman imaging (Shi et al. 2022), and so on. Based on a series of advanced and highly sensitive fluorescent dyes or proteins, imaging techniques have been applied in cancer imaging, neuroscience detection, metabolism measurement, response monitoring of clinical treatment, and other preclinical and clinical applications (Rowe and Pomper 2022). Nowadays, molecular imaging has been widely used in real-time monitoring of polarized macrophages during various biological events.

In this review, we comprehensively summarize recent advances in the field of macrophage reprogramming, including distinct roadmaps, approaches, and applications from preclinical experiments to clinical trials. Moreover, we discuss various molecular imaging approaches that can be used to monitor these biological processes in real time. Overall, this review provides an extensive overview of modulating and imaging macrophage reprogramming for cancer immunotherapy.

The Main Roadmaps of Macrophage Reprogramming

Generally, the imbalanced phenotypes of macrophages frequently occur in various diseases and can further worsen the progression of these diseases through molecular cascades and the immune network (Mass et al. 2023). To address these excessively polarized macrophages, macrophage reprogramming has emerged as a superior strategy for correcting inappropriate deviations. At present, this developing reprogramming strategy aims to obtain controllable promotion of the desired pro-inflammatory M1 or anti-inflammatory M2 phenotypes in specific diseases (Ma et al. 2023). The core roadmaps of macrophage reprogramming include: (1) targeting and modulating genomic DNA and epigenetics; (2) targeting cell surface and intracellular molecules to activate or block them; and (3) regenerating desired cellular microenvironments with specific physicochemical properties.

Targeting Genomic DNA and Epigenetics

The activation and polarization of macrophages involve a complex interplay of signaling and effector molecules. The gene regulatory network of polarized macrophages plays a central role in these biological processes, which includes transcriptional, post-transcriptional regulation, translation, post-translational modifications, and correlative signal transduction of activation and polarization-associated genes (Revel et al. 2022). Epigenetic modifications are increasingly recognized as an important mechanism for regulating gene expression. In the TME, cancer cells extend their survival advantages through epigenetic modifications (Sun et al. 2022). Correspondingly, immune cells also take advantage of this mechanism to boost antitumor immune responses. Considering the strong heterogeneity of macrophages, more and more research has been directed toward exploring epigenetic pathways for macrophage reprogramming, such as DNA methylation, histone modifications, and noncoding RNAs.

Targeting Cell Surface and Intracellular Molecules

The heterogeneity and plasticity of macrophages largely depend on cell surface molecules and cell signaling, which include cell surface receptors, ligand-receptor pairs, and signaling pathway modulators (Zhang et al. 2023). Considering the complexity, dynamics, and universality of macrophages throughout the body, targeting specific cell populations with appropriate cell surface molecules, especially clinically relevant surface receptors, will enable the development of more precise reprogramming strategies to treat diseases without off-target-related adverse effects. The major cell surface receptors involved in the pro-inflammatory M1 phenotype include CD16, CD32, CD64, CD68, CD80, CD86, CD369, MHC-II, and so on (Cheng et al. 2022). M2 macrophages are characterized by up-regulation of several surface molecules, such as the mannose receptor (Fig. 1). Targeting these receptors and their ligands have advanced to preclinical trials and shown promising effects.

Regenerating Cellular Microenvironment

More and more evidence proves that the discriminative activation and polarization of macrophages largely depend on the different physiological and pathological microenvironments, especially in the TME (Christofides et al. 2022). The microenvironment of macrophages consists of extracellular matrix, distinct cellular populations in terms of phenotype and spatial distribution, soluble proteins such as growth factors, and physicochemical signal molecules. These factors influence cellular phenotypes and functions through various physical, mechanical, and biochemical mechanisms like pH, oxygen, and shear stress (Zhao et al. 2022). Additionally, macrophages secrete a series of cytokines to influence the microenvironment, and ultimately stimulate cell proliferation. Therefore, manipulating the cellular microenvironment will be more conducive to macrophage activation and polarization.

The Therapeutic Approaches of Macrophage Reprogramming

As stated in the previous paragraph, it summarizes the main directions of macrophage reprogramming. Based on these promising roadmaps, some therapeutic approaches have been developed to reprogram macrophages, which include monoclonal antibody drugs, small molecule drugs, gene therapy, and chimeric antigen receptor engineered macrophages (CAR-M). Some of these methods have been adopted in preclinical trials and achieved certain promising outcomes.

Monoclonal Antibody Drugs

Since the approval of the first monoclonal antibody drug Orthoclone OKT3 (muromonab-CD3) by the Food and Drug Administration (FDA) in 1986, over 100 antibody drugs have been approved for disease treatment (Attwood et al. 2020). These advancements have greatly accelerated the development of monoclonal antibody drugs for modifying macrophage reprogramming. In most common cancers, the presence of anti-inflammatory M2 phenotype tumor-associated macrophages (TAMs) always leads to the failure of conventional cancer therapies (Zhang et al. 2021b). Therefore, a series of monoclonal antibody drugs with high specificity have been developed to target and reprogram TAMs from the anti-inflammatory M2 phenotype to the pro-inflammatory M1 phenotype in cancer immunotherapies.

The general approach to developing monoclonal antibody drugs involves targeting the cell surface signaling molecules on macrophages. Liu et al. (2023) reported that CD40 monoclonal antibodies promoted M1 polarization through the modulation of glutamine metabolism and fatty acid oxidation. Additionally, the key mechanism of therapeutic antibodies is antibody-dependent cellular phagocytosis (ADCP) (Kamber et al. 2021). Cao et al. (2022) found that paclitaxel efficiently reprogrammed macrophages into the M1 phenotype, and this reprogramming enhanced ADCP when combined with cetuximab and rituximab. Interestingly, this phenomenon differed from the efferocytosis of apoptotic cancer cells induced by M2 macrophages. These findings underscore the significance of macrophage reprogramming in determining the long-term efficacy of anticancer monoclonal antibodies.

Small Molecule Drugs

Small molecule drugs, characterized by low molecular weights and simple structures, possess the capability to effectively permeate cells, which exerting a wide range of biological effects (Childs-Disney et al. 2022). The common applications of small molecule drugs are developed to target signaling molecules. Among these, colony-stimulating factor 1 (CSF1) has been recognized as one of the most important molecule targets in macrophage reprogramming. The CSF1/CSF1R signaling inhibitor pexidartinib (PLX3397) has been approved by the FDA to reprogram TAMs in tenosynovial giant cell tumors. Fujiwara et al. (2021) found PLX3397 significantly exhibited versatile immune modulating effects to reprogram TAMs by suppressing the phosphorylation of extracellular signal-regulated protein kinases 1/2 （pERK1/2） activation and the polarization of M2 TAMs. Moreover, other CSF1/CSF1R signaling inhibitors, such as BLZ945, PLX7486, ARRY382, DCC3014, and JNJ40346527 (Edicotinib), have also achieved significant progress in various cancer treatments (Kosti et al. 2022).

In addition, internal signaling molecules in macrophages are also potential targets for reprogramming. For example, vorinostat, a small molecule drug, was developed to inhibit histone deacetylases to switch macrophages from an M2 phenotype to an M1 phenotype (Li et al. 2021b). Similarly, TG100-115, a PI3Kγ/δ pathway inhibitor, has been identified to suppress tumor growth and metastasis by reconstructing TME and reprogramming macrophages (Sullivan et al. 2018). Based on similar design principles, a series of internal signaling pathways, such as the nuclear factor kappa B (NF-κB) pathway, the mitogen-activated protein kinase (MAPK) signalling pathway, the janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway, the adenosine monophosphate-activated kinase (AMPK)-peroxisome proliferatoractivated receptor (PPAR)-dependent pathway, and the nuclear factor erythroid-2 related factor 2 (Nrf2)/heme oxygenase (HO-1) signaling pathway, have been targeted for the design of small molecule drugs.

Gene Therapy

With the development of genetics and molecular biology, gene therapy has become an attractive therapeutic approach from bench to bedside. Nowadays, there are several developing gene therapy methods to reprogram macrophages, including delivery of therapeutic nucleic acids (TNAs), direct gene editing, and epigenetic regulation (Gupta et al. 2021).

The TNAs have been explored for their role in regulating ILs that are associated with polarized macrophages. Li et al. (2022) designed an engineered exosome that encapsulated IL-10 pDNA to reprogram macrophages. This approach aimed to reduce the levels of pro-inflammatory cytokines (such as IL-1β and TNF-α) and increase the expression of IL-10 cytokines. In addition to pDNA, miRNA and mRNA are also widely adopted for inducing reprogramming in macrophages (Song et al. 2022; Zhang et al. 2019). Furthermore, the advent of gene-editing technology has accelerated the development of human gene therapy (Zou et al. 2022). For example, Lin et al. (2022) developed a gene delivery system to reprogram macrophages through clustered regularly interspaced short palindromic repeats (CRISPR)-mediated CD47 blockade and IL-12 overexpression in situ. This combination gene therapy successfully remolded the TME, promoting the production of IL-12 and synergistically reprogramming TAMs to the M1 phenotype, thereby enhancing the macrophage-mediated anti-tumor immunotherapy effects.

Some researchers also pay attention to regulating the epigenetics of macrophages. Lauterbach et al. (2019) found that targeting the ATP-citrate lyase, which was associated with histone acetylation, could treat certain cancers by reprogramming macrophages. Moreover, Zhang et al. (2021a) discovered that glycoprotein A repetitions predominant and DNA methylation-mediated mechanisms metabolically reprogrammed TAMs in pancreatic cancer cells. Furthermore, Qian et al. (2017) developed the M2-like TAM dual-targeting nanoparticles (M2NPs) loaded with CSF-1R small interfering RNA and scavenger receptor B type 1 peptides to eliminate M2-like TAMs. As expected, this strategy specifically targeted M2-like TAMs and elicited more powerful anti-cancer immune responses compared to other tissue-resident macrophages in the liver, spleen, and lung.

CAR-M

Chimeric antigen receptor-engineered T cells (CAR-T) therapy has been developed to treat hematological malignancy and has achieved revolutionary clinical responses (Lu and Jiang 2022). The success of CAR-T has accelerated the development of similar immune cell therapies, such as chimeric antigen receptor-engineered natural killer cells (CAR-NK), chimeric antigen receptor-engineered natural killer T cells (CAR-NKT), CAR-M, CAR-Treg, and CAR-γδT (Lin et al. 2021). Considering the extensive distribution of macrophages in most solid tumors, CAR-M, as a unique therapeutic approach, has achieved promising preclinical and clinical effects.

Klichinsky et al. (2020) developed anti-human epidermal growth factor receptor 2 (HER2) CAR-M (CT-0508), which genetically manipulated human macrophages by CAR technology to acquire constant pro-inflammatory (M1) phenotype macrophages against tumors. This work has achieved encouraging progress in first-in-human phase 1 clinical trials (NCT04660929) and acquired the fast-track designation of the FDA in 2021 (Mukhopadhyay 2020). Based on nanobiotechnology, Kang et al. (2021) designed polymer nanocarriers delivering plasmids encoding CAR and interferon-γ to manipulate macrophages in vivo. This delicate design effectively reprogrammed the TAMs from the M2 phenotype to the M1 phenotype and successfully accomplished the CAR-M in situ. Meanwhile, the CAR-M remodeled the TME and suppressed tumor growth by augmenting the immune functions of M1 TAMs and tumor-specific cytotoxic T lymphocytes. Moreover, Pierini et al. (2022) developed a combination therapy that mixed CAR-M and PD-1/PD-L1 T cell checkpoint axis blockade. This combination therapy obtained much better results in reprogramming macrophages to enhance the anti-tumor effects.

The Molecular Imaging of Macrophage Reprogramming

It is difficult for traditional biochemical methods to obtain accurate and long-term research data that contain spatiotemporal dynamics information in health and disease states, especially in the occurrence and development of tumors. With the continuous development of molecular imaging techniques, represented by intravital imaging (IVI) (Entenberg et al. 2023), imaging macrophage reprogramming provides a more intuitive and visual method to explore the regulation of macrophage activation and polarization in situ.

It is crucial for imaging macrophage reprogramming to characterize different activation states of macrophages in real-time. Surface markers related to macrophages and some metabolic molecules are commonly adopted as markers for distinguishing polarized macrophages (Jiang et al. 2022; Lee and Bensinger 2022). However, few studies have reported the application of visualizing gene expression to differentiate macrophage polarization in vivo. The weak expression of these proteins makes it difficult to distinguish the real molecule signals from the background. Some signal amplification strategies have been employed to improve the signal intensity, such as antibody labeling. Ramos et al. (2022) identified the folate receptor beta positive (FOLR2+) tissue-resident macrophages in healthy tissue and cancers. Furthermore, they used confocal living imaging to image their cytokinetics connections with CD8+ T cells by labeling fluorescently coupled antibodies. Zhang et al. (2017) developed Dylight755-N-hydroxysuccinimide-conjugated anti-CD206 monoclonal antibodies and 85 MBq Na125I-conjugated anti-CD206 monoclonal antibodies, which were used for imaging M2 macrophages in vivo through near-infrared fluorescence imaging or single photon emission computed tomography.

Molecular Imaging of Macrophage Metabolism

An alternative strategy for monitoring macrophage reprogramming is metabolic imaging, which involves detecting various metabolic products, such as the activity of cellular enzymes (Lung et al. 2022). During macrophage reprogramming, nitric oxide (NO) produced by inducible nitric oxide synthase has been widely used as a signature molecule for M1 polarization (Fig. 3). A series of exogenous synthetic or endogenous gene-coding probes have been developed to characterize polarized macrophages by detecting NO. For exogenous probes, Zhao et al. (2021) developed a FRET-NO probe and Hoe-Rh-NO for endogenous NO imaging, and Liang et al. (2022) developed a two-photon ratiometric fluorescent probe for NO imaging. Li et al. (2020a) developed a highly sensitive NO probe for imaging inflammation reactions in the stroke process, and Zhou et al. (2020) developed a smart NO probe, PYSNO, for NO imaging in myocardial fibrosis tissue. Currently, fluorescence probes with endogenous NO gene encoding have lower detective sensitivity compared to exogenous NO probes. However, their non-invasive nature enables them to more accurately reflect the real-time status of macrophage reprogramming.

Fig. 3.

Fig. 3

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The representative methods of targeted imaging polarized macrophages. a Representative structure of exogenous and endogenous probes for the detection of nitric oxide (NO) and reactive oxygen species (ROS) as well as matrix metalloproteinase (MMP) activity associated with macrophage activity. The different fluorescent probes are classified according to their targets: NO, ROS, and MMP. Reproduced with permission from reference (Dickinson et al. 2010). b Fluorescent probes for imaging the phagocytosis of macrophages. The probes are named according to their targets: phagosome pH: PhagoGreen, CD11b: CDnir7, Slc18b1: CDg16, GLUT1: CDr17. c Imaging of the mitochondrial organization is correlated with the activation status of macrophages. There are some representative structured illumination microscopy images of mitochondria (red color, MitoTracker Red CMXRos) in bone marrow-derived macrophages (BMDMs) (scale bar in upper image, 10 µm; scale bar in lower image, 2 µm). Reproduced with permission from reference (Li et al. 2020b)

Despite releasing NO, activated macrophages also release reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) (Fig. 3). Deguchi et al. (2006) utilized an activated near-infrared fluorescent probe with high sensitivity and high-resolution to detect gelatinase enzyme activity (MMP-2 and MMP-9) in polarized macrophages in vivo. In addition to detecting protein expression, the expression of intracellular RNA also exhibits significant differences between M1 and M2 activation. Yang et al. (2019) have developed a new method that is suitable for real-time detection of RNA in living cells. In the future, RNA-based molecular probes might also be gradually applied to distinguish macrophages with different activation states.

Molecular Imaging of Macrophage Morphology

It has been reported that macrophages, due to their strong heterogeneity and plasticity, always exhibit irregular morphological changes associated with various polarization states (Ackermann et al. 2022). In recent years, the development of novel technologies, represented by machine learning and deep learning techniques, has greatly improved the ability to distinguish macrophage polarization states through imaging their morphology. For example, Kröger et al. (2022) developed a two-photon excited lifetime imaging method that efficiently distinguished polarized macrophages from dermal cells in vivo using specific machine learning algorithms. Li et al. (2020b) analyzed the morphology of mitochondrial organization in bone marrow-derived macrophages (BMDMs) and polarized macrophages by extracting characteristic parameters, such as mean length of branches, mean network size (the mean number of branches per network), and mitochondrial footprints (mitochondrial coverage area). These parameters were successfully used to recognize and distinguish macrophages with different activation states in vivo. Pavillon et al. (2018) obtained morphological parameters of macrophages through non-invasive and labeling-free methods such as quantitative phase microscopy, spontaneous fluorescence imaging, and molecular indicators from Raman spectroscopy. They then constructed a multivariate statistical model based on logistic regression to visualize macrophage reprogramming.

Molecular Imaging of Macrophage Phagocytosis

In addition to the irregular cell morphology of polarized macrophages, recent research has reported that various polarized macrophages have significant differences in the phagocytosis of fluorescent compounds. Lin et al. (2019) developed a novel nanoparticle named “Nanopomegranate” with fast self-assembly, dye aggregates, and dual-modality “off/on” capability. In particular, this nanopomegranate showed better photostability, extinction coefficient, and labeling efficiency (98.8%) for visualizing kupffer cells (KCs) in the liver compared to commercial FluoSpheres. Further, Deng et al. (2021) designed an innovative drawer-type abdominal window and combined it with nanopomegranate to achieve more than 10 days of IVI in KCs of the liver using intravital fluorescence and photoacoustic imaging. Kang et al. (2014) developed a near-infrared probe named CDnir7 for visualizing polarized macrophages in vivo using multiple imaging methods, such as in vivo imaging system spectrum, fluorescence molecular tomography, and multi-spectral optoacoustic tomography. Vazquez-Romero et al. (2013) synthesized a series of BODIPY derivatives with high fluorescence quantum yield and excellent cell permeability using a multi-component reaction method. Among them, a fluorescent probe called PhagoGreen, characterized by high sensitivity to low pH, selectively imaged the phagocytosis of reprogramming macrophages through responding to the acidification of macrophage phagosomes. To image activated macrophages, Park et al. (2019) screened CDg16 from more than 8000 compounds and further identified Slc18b1/SLC18B1 molecules that interacted with CDg16, regulating the phagocytosis of activated macrophages. Although the CDg16 probe provided a valuable tool for IVI of macrophage activation, it could not distinguish the specific polarized phenotype of macrophages. Based on the CDg16 probe, Cho et al. (2022) constructed a luminescent-carbohydrate library and screened the CDr17 probe, which distinguished M1 polarized macrophages through glucose transporter 1 translocation. These fluorescent probes, which are stable in cells and independent of molecular binding, mainly rely on the Gating Oriented-Live Cell Distinction of ion channels of cell-surface membranes and provide a novel strategy for IVI of reprogramming macrophages (Choi et al. 2019).

The Therapeutic Applications of Macrophage Reprogramming

Hepatic Carcinoma (HCC)

The microenvironment of HCC is abundantly infiltrated by various macrophage populations, such as resident macrophages, KCs, and monocyte-derived macrophages (Ohshiro et al. 2023). The activation and polarization states of these various macrophage populations directly affect the development of HCC, especially the anti-inflammatory M2 phenotype TAMs. Targeting and reprogramming liver macrophages, converting them from the M2 to M1 phenotype has been regarded as a valuable approach in liver cancer immunotherapy.

Li et al. (2017) tested a novel CC chemokine receptor 2 (CCR2) antagonist in murine hepatocellular carcinoma cells and found that the blockade or knockout of CCR2 significantly reconstructed TME by reprogramming M2 TAMs and activating the CD8+ cytotoxic T cells. Moreover, Wang et al. (2022) designed a nanodrug to reprogram TAMs with an NF-κB inhibitor and PD-L1 antibody in liver cancer. Interestingly, some traditional drugs are now being used to reprogram macrophages in HCC. De Oliveira et al. (2019) used IVI to visualize the disease progression of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis-HCC in zebrafish models. Their data showed that metformin specifically reprogrammed macrophages and increased T-cell density to restore tumor surveillance. Nowadays, a variety of novel preclinical and clinical methods have been developed to reprogram macrophages in the treatment of HCC, which pay more attention to interdisciplinary approaches and new technologies. These studies further highlight the important role of reprogramming macrophages in HCC.

Lung Carcinoma

Lung carcinoma accounts for almost 25% of all cancer deaths in 2022 (Giaquinto et al. 2022). Thorsson et al. (2019) performed immunogenomic analysis from The Cancer Genome Atlas, which included 33 diverse cancer types and found that macrophage phenotypes, especially M2 TAMs, were closely correlated with poor prognosis in lung cancer patients. Therefore, targeting and reprogramming macrophages may provide a new powerful therapy for treating lung cancer.

Zhou et al. (2022) performed a high-throughput screening and found that carfilzomib, a selective proteasome inhibitor, reprogrammed the M2 macrophages into the M1 phenotype by inducing endoplasmic reticulum stress and activating NF-κB. In addition to conventional anticancer drugs, some novel nanodrugs have been developed for lung cancer treatment in preclinical trials. Su et al. (2022) designed Au@PG nanoparticles to reprogram macrophages by transforming anti-inflammatory M2 macrophages into pro-inflammatory M1 macrophages in lung cancer models. As mentioned above, monoclonal antibodies serve as potential therapeutic agents. Yu et al. (2022) developed an anti-Chi3L1-humanized antibody and evaluated its antitumor and antimetastatic effects. They found that this monoclonal antibody inhibited M2 TAMs through signal transducer and activator of transcription 6, while having no effect on M1-polarized macrophages in lung cancer. La Fleur et al. (2021) identified tumor-derived IL-37 and macrophage receptors with collagenous structure (MARCO) as potential therapeutic targets in lung cancer. The blocking antibodies to IL-37 and MARCO remodeled the TME and reprogrammed the TAMs to kill lung cancer cells. In conclusion, these various studies prove that reprogramming macrophages is beneficial for treating lung cancer patients.

Breast Carcinoma

Breast carcinoma almost accounts for 12.5% of all new annual cancer cases globally (Dolatkhah et al. 2020). Nearly 30% of patients develop recurrent disease due to therapy resistance, which eventually metastasizes in distant organs and becomes the main cause of death (Pan et al. 2017). Moreover, a large number of studies, especially the widely applied scRNA-seq, have identified the versatile role of macrophages connected with the progression to malignancy of breast cancer. These macrophages mediate tumor cell migration, invasion, and intravasation by suppressing anti-tumor immunity (Zhang et al. 2021b). Therefore, developing novel immunotherapy to target and reprogram macrophages may effectively inhibit resistance, recurrence, and metastasis of breast cancer, thus improving treatment outcomes and the results of combination therapies.

At present, the prerequisite for targeting and reprogramming macrophages is to accurately identify the function of macrophages in breast cancer. Wang et al. (2023) developed a visible mesoporous silica nanoparticle with apolipoprotein A-1 (ApoA-1) mimetic peptide R4F and indocyanine green. This multifunctional nanoparticle was not only efficiently taken up to label 4T1 cells and macrophages but also decreased M2-like TAMs and killed cancer cells by photoacoustic imaging and photothermal therapy. Nishida-Aoki and Gujral (2022) developed an in vitro TAM polarization system and screened phenotypic kinase inhibitors that significantly decreased the populations of M2 TAMs in breast cancer. Additionally, using epigenetic regulation is also common in reprogramming TAMs for treating breast cancer. Ma et al. (2022a) proved that knockout of miR-182 suppressed the growth of breast cancer by reducing M2 TAM populations, which involved suppressing toll-like receptor (TLR)-4, and inactivated NF-κB pathways. Furthermore, antagomiR-182 effectively elicited macrophage reprogramming and tumor suppression in breast cancer. Mehta et al. (2021) demonstrated that macrophage populations were the most abundant immune cell type in breast cancer through scRNA-seq. Then, combined with multi-omics profiling, they found that poly (ADP-ribose) polymerase inhibitors indistinguishably reprogrammed both macrophage populations by metabolic pathways. Additionally, they evaluated the therapeutic effects of combination therapy with poly (ADP-ribose) polymerase inhibitors and CSF1R-blocking antibodies. The results showed that the combination therapy induced a durable reprogramming of TME, providing a promising therapeutic strategy for breast cancer. Overall, targeting and reprogramming macrophages in breast cancer is a novel therapeutic method with great potential for clinical treatment.

Conclusions and Perspectives

The development of cancer involves multistep biological processes, including proliferation, survival, invasion, and metastasis. Immunotherapy is committed to developing specific cancer therapies for each of these processes. As the most abundant immune cell population in TME, macrophages are the most potential treatment targets for cancers. Among various targeting macrophage immunotherapies, macrophage reprogramming has been considered the most promising next-generation immunotherapy due to its precision and low side effects. In this review, we summarize the current progress in macrophage reprogramming from conceptual roadmaps to therapeutic approaches and applications. Specifically, we focus on molecular imaging of macrophage reprogramming. Molecular imaging allows us to directly observe various molecular events during the macrophage reprogramming process in cancer immunotherapy (Table 1). In the future, integrating molecular imaging to develop new macrophage reprogramming methods will become a promising direction for precise clinical cancer immunotherapy (Fig. 4).

Table 1.

A schematic summary of modulating and imaging macrophage reprogramming

Polaration Targets Cancers Events Methods Imaging Models References
M1 CSF-1R, SRB1 Melanoma tumors Dual-targeting reprogramming TAMs Gene therapy Confocal intravital microscopy Mice Qian et al. (2017)
Mesothelin, HER2 Ovarian cancer Phagocytic activity CAR-M Bioluminescence, imaging flow cytometry Mice, Human Klichinsky et al. (2020)
CD20, EGFR, HER2 Lymphoma, epidermoid carcinoma, breast adenocarcinoma ADCP Monoclonal antibody drugs Confocal microscopy, IVIS Mice, BMDM Li et al. (2021a)
PD-1,TLR7/8 Colorectal cancer High-throughput and low-cost phenotypic screening Small molecule drugs Fluorescence reflectance imaging, intravital imaging Mice Rodell et al. (2018)
ASF1A, PD-1 Lung cancer In vivo epigenetic CRISPR screen Gene therapy MRI Mice Li et al. (2020b)
M2 CD206, NO Breast cancer Early metastasis Small molecule drugs The NIR-II FL and bioluminescence Mice, RAW264.7 Yuan et al. (2023)
SIRPα, CD47 Breast cancer, melanoma TAM repolarization Gene therapy Confocal imaging Mice Rao et al. (2020b)
m6A modification

Melanoma,

lung cancer

 Epigenetic regulation Gene therapy Bioluminescence Mice Yin et al. (2021)
CD24, Siglec-10 Ovarian cancer, breast cancer Anti-phagocytic Monoclonal antibody drugs Bioluminescence Mice, Human Barkal et al. (2019)
MUC1, CD47 Pancreatic cancer Phagocytic activity CAR-M Bioluminescence, TEM, IVIS Human, Mice, BMDMs, RAW 264.7 cells Liu et al. (2023)
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CSF1R colony-stimulating factor 1 receptor, HER2 human epidermal growth factor receptor 2, CD20 cluster of differentiate 20, EGFR epidermal growth factor receptor, PD-1 programmed cell death protein 1, TLR7/8 toll-like receptor 7/8, ASF1A anti-silencing function 1A histone chaperone, CD206 the mannose receptor, NO nitric oxide, SIRPα signal regulatory protein α, CD47 cluster of differentiation 47, M6A N6-methyladenosine, CD24 heat stable antigen (HSA) receptor, Siglec-10 sialic acid binding Ig like lectin 10, MUC1 mucin 1, ADCP antibody-dependent cellular phagocytosis, IVIS in vivo imaging system, MRI magnetic resonance imaging, TEM transmission electron microscopy, BMDM bone marrow-derived macrophage

Fig. 4.

Fig. 4

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Targeted modulation and imaging of macrophage reprogramming

Modulating and imaging macrophage reprogramming provides an innovative solution to visually understand this specific process. However, there remains a long way to go for good clinical practice. Firstly, due to the great plasticity and heterogeneity of macrophages, existing macrophage-based cancer immunotherapies exhibit a lack of specificity, potentially disrupting the body's immune homeostasis and increasing the risk of treatment failure in cancer patients. Besides, the individual differences between tumors and patients limit the therapeutic effects of macrophage reprogramming as a universal method. To overcome this obstacle, identifying new single-specific target molecules in macrophages via advanced technologies such as scRNA-seq is a potential direction for precise macrophage targeting. Secondly, the safety of reprogramming macrophages by various methods should be thoroughly tracked and evaluated. This requires joint efforts by regulatory agencies and scientists to ensure that patients around the world have early access to safe and high-quality cancer immunotherapies. Finally, the in vivo efficiency of inducing macrophage reprogramming (such as gene-editing efficacy, expansion efficiency, and immune efficacy) remains a challenge, limiting their applications in preclinical and clinical trials. Emerging gene-editing techniques, such as optimized CRISPR-Cas9 technology, base editing methods, and prime editing, might offer new strategies for solving these problems.

On the other hand, there are still some challenges in molecular imaging technologies to image macrophage reprogramming, such as non-invasion, real-time, precise labeling, long-term tracking, and penetration depth. To address these questions, some strategies have been adopted, including: (1) designing novel multimodality probes that combine diverse imaging techniques, such as fluorescence imaging integrated with magnetic resonance imaging and positron emission tomography. (2) Developing advanced imaging instruments and contrast agents. (3) Cooperating with new computer image processing technologies, such as deep learning and machine learning. In the future, these enhanced macrophage reprogramming imaging methods will achieve more precise and individualized treatment of therapeutic drugs by dynamically visualizing and guiding in real-time.

To sum up, with the development of molecular imaging and molecular biology, imaging macrophage reprogramming will significantly enhance our understanding to this specific biological event. Based on these findings, more and more innovative probe sensors and cancer immunotherapies will be developed to detect and eliminate solid tumors in the future.

Acknowledgements

This work was supported by the State Key Program of National Natural Science Foundation of China (32330048), the National Natural Science Foundation of China (82371857), and the Hainan University Scientific Research Foundation (KJRC2023B09).

Abbreviations

TME

Tumor microenvironment

TAMs

Tumor-associated macrophages

CAR-M

Chimeric antigen receptor-engineered macrophages

CD

Cluster of differentiation

IL

Interleukin

TNF

Tumor necrosis factor

TLR

Toll-like receptor

MHC-I

Major histocompatibility complex class I

MHC-II

Major histocompatibility complex class II

ADCP

Antibody-dependent cellular phagocytosis

CSF1

Colony-stimulating factor 1

CAR-T

Chimeric antigen receptor-engineered T cells

IVI

Intravital imaging

NO

Nitric oxide

ROS

Reactive oxygen species

MMP

Matrix metalloproteinase

KCs

Kupffer cells

BMDMs

Bone marrow-derived macrophages

NF-κB

Nuclear factor kappa B

HCC

Hepatic carcinoma

Authors' Contributions

ZHZ and ZL provided the concept, designed the outline, and directed the writing of the paper. JLW performed the literature survey, drafted the manuscript, and finished the figures and tables. YFL, RZ, and ZZC participated in the literature collection. ZF and YLX offered crucial content revisions and language polishing. All authors contributed to the article and approved the submitted version and final version.

Data Availability

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Declarations

Conflict of Interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical Approval

Not applicable.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Contributor Information

Zheng Liu, Email: liu-zheng@hainanu.edu.cn.

Zhihong Zhang, Email: czyzzh@mail.hust.edu.cn.

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Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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