Abstract
High-grade glioma is one of the deadliest primary tumors of the central nervous system. Despite the many novel immunotherapies currently in development, it has been difficult to achieve breakthrough results in clinical studies. The reason may be due to the suppressive tumor microenvironment of gliomas that limits the function of specific immune cells (e.g., T cells) which are currently the primary targets of immunotherapy. However, tumor-associated macrophage, which are enriched in tumors, plays an important role in the development of GBM and is becoming a research hotspot for immunotherapy. This review focuses on current research advances in the use of macrophages as therapeutic targets or therapeutic tools for gliomas, and provides some potential research directions.
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Introduction
Glioma is the most common CNS malignancy in adults with a global annual incidence of 5–6 per 100,000 people and a highly heterogeneous and aggressive nature1. And glioblastoma, the most lethal glioma, accounts for 70–75% of all diffuse glioma diagnoses. Despite the availability of conventional treatments including surgery, radiotherapy, and chemotherapy, the median survival of patients is only 14–17 months2. As a result, the development of new therapies is very crucial, for example, targeted therapy, immunotherapy and electric field therapy.
Studies over the past two decades have revealed that tumor microenvironment (TME) is a pivotal determinant of tumor behavior, and is responsible for tumor progression and metastasis3. In addition, the discovery of intracranial lymphatic vessels has led to an increased recognition of the importance of immune cells in brain tumors, challenging previous assumptions about brain tolerance and immune privilege4. Glioma is characterized by a highly suppressive and unique “cold” immune microenvironment, which includes tumor cell-derived immunosuppressive factors, exhausted cytotoxic T lymphocytes (CTLs), Treg cells, and downregulated MHC and self-presentation78. Exosomes, on the other hand, can be used as vehicles for the delivery of therapeutic agents to target cells. Understanding the complex interplay between non-coding RNA, exosomes, and tumor-associated macrophages will provide valuable insights for the development of more effective cancer treatments.
With a deeper understanding of the role of TAM in tumor development, we can develop therapies that target more specified pro-tumor functions22,79. This will help to optimize treatment efficacy while minimizing potential side effects, ultimately improving patient outcomes.
Macrophages as tools in glioma therapy
To achieve sufficient intratumoral accumulation, researchers exploit tumor-associated macrophages within the special tumor microenvironment to carry drugs or express genes80,81 (Fig. 2), for example, immune molecules and CARs (chimeric antigen receptor)82. Some details and features of these studies are presented as follows in Table 2.
Engineered macrophages as therapeutic gene vector
Macrophages have been applied for the delivery and expression of the genes of biotherapeutic substances, of which one of the most classic is IFN (interferon). IFN has been used in tumor therapy since 1986 to modulate immunity and inhibit angiogenesis. Unfortunately, its clinical application is limited due to its short half-life and high toxicity. Thus, lentiviral vector transduced IFN-α monocytes, which selectively express IFN-α under the control of GBM-specific angiopoietin receptor Tie2 promoter/enhancer elements and accumulate to tumor, were used as a vehicle for targeted delivery83. Because of the preferential activation of the Tie2 promoter in the TME, continuous, low-dose IFN-α would be released at the tumor site without inducing counterregulatory responses and systemic toxicity. IFN-α then stimulates and activates immune cells (e.g., macrophages, DCs, and T cells), inhibits angiogenesis, and suppresses tumor growth and metastasis. And a clinical study based on this technology is also underway (NCT03866109).
As for IFN-β, to exploit tumor stoma cells including TAMs in situ to secret antitumor agent IFN-β(interferon), AAV (adeno-associated virus) was injected intravenously with exosomes (exo-AAV) enhancing the ability to infect cells. The AAV encoding IFN-β was mediated by glioma stoma-specific promoter (GFAP for astrocyte and 5-NF for macrophages/microglia)84. Both types of cells can then secrete IFN-β, but the therapeutic effect of modified TAMs is weaker than that of modified astrocytes. The dilution of AAV vectors due to tumor growth may result in less effective gene expression in TAMs than in the more stable astrocytes, though AAV does effectively infect cells in CNS85. Besides, this study also mentioned the narrow therapeutic window of IFN-β. Therefore, the application of in situ genetic engineering requires the selection of a more persistent virus and a more refined TAM-specific promoter.
Macrophages have also been engineered to express BiTEs (bispecific T-cell engager) to facilitate the interactions of T cells and tumor cells via binding of a CD3ε and GBM-specific EGFRvIII (epidermal growth factor variant)86. Human monocyte-derived macrophages were transduced with lentivirus and secreted BiTEs in EGFRvIII expressing tumor site. The method resulted in the enduring expression of BiTEs, upregulated genes expression involved in T-cell activation, survival, cytokine signaling and T-cell toxicity (e.g., IL2RA, IL2RB, PRDM1, ICOS, CD40), and prevent tumor growth for 36 days87,88. Besides, immunomodulating and antigen-presenting function of engineered macrophages also help with T-cell activation in TME.
In another research, engineered microglia as the source of IL-15 was recruited to the tumor site. Researchers used rAAV2 (recombinant AAV serotype 2) carrying IL-15 to modify microglia. IL-15 (interleukin) promotes a pro-inflammatory phenotype of microglia and the cytotoxic activity of natural killer (NK) cells in TME, which also promote the production of IFN-γ, and counteracted tumor growth89. IL-12 has a similar therapeutic effect as an immunomodulator, but requires local administration to reduce systemic toxicity90. Expression of IL-12 by macrophages at subcutaneous tumor sites can improve the function of IFN cascade and activate T cells, slow tumor growth and prolong survival91. The therapeutic effect of immunomodulatory factors is confirmed. The use of macrophage carriers can improve the targeting and persistence of such therapies.
In the context of suicide gene/prodrug therapy, a novel non-viral gene vector technique based on light treatment was used to mediate the transfection of CD (cytosine deaminase) gene to macrophages in vitro. Transfected NR8383 cells could express CD with F98 glioma cells in the presence of 5-FC (5-fluorocytosine), a nontoxic precursor to 5-FU (5-fluorouracil). Because of the multi-drug resistance of NR8383 macrophages, transformed 5-FU is significantly more toxic to tumor cells than macrophages, allowing them to survive and consistently express CD92,93. This study shows high potential; however, further research is required to construct human-derived drug-resistant macrophage vectors for suicide gene therapy.
Macrophages as therapeutic drug carrier
In spite of the emergence of new chemotherapy or immunotherapy agents, passive delivered free drugs show limited efficacy because of poor diffusion into brain tumor tissue81,94. The transport of free drug is affected by blood–brain barrier, uneven tumor vasculature and the pH of the tumor microenvironment95. Tumor-targeted cell-based delivery system exploit neural stem cells, mesenchymal stem cells, and monocyte/macrophages96, among which monocytes have the widest source, and this makes monocytes/macrophages an ideal vehicle for drug delivery97. In this way, the pro-tumor microenvironment was exploited as cellular “Trojan Horses” against malignances94.
To deliver chemotherapy drugs by macrophages, the most significant aspect is to avoid the toxicity of chemotherapy drugs to the carrier. Wang et al. built ND-PG-RGD-DOX (doxorubicin) with good aqueous solubility in physiological media, and it binds to the integrin receptor avβ3 that is overexpressed on the surface of multiple cells. Nano-DOX was sequestered in the lysosomal compartment which may mechanistically contribute to monocytes’ tolerance to the drug. Monocyte took up the Nano-DOX and maintained good viability for at least 48 h. Upon recruitment to the tumor microenvironment, monocytes are induced by GBM cells to differentiate and release more Nano-DOX than in peripheral blood. The drug delivery and tumor-killing efficacy of this method has been demonstrated in orthotopic GBM xenografts95. Notably, Monocytes release Nano-DOX in the periphery no slower than in the tumor, but the exocytosis of Nano-DOX from monocytes is calcium channel dependent. Perhaps the combination with calcium channel blockers (e.g., verapamil) may limit the non-specific release of the drug in the periphery95.
Liposomes are also employed to isolate the drug and reduce toxicity to carrier cells. Using dipalmitoyl phosphatidylserine (DPPS) as a “eat me” signal, paclitaxel (PTX)-loaded liposomes were phagocyted by BV2 microglia. Microglia then cross the blood–brain barrier and deliver the drug to tumor cells via extracellular vesicles and microtubules. Owing to its high targeting performance and natural accumulation in gliomas, this cell remedy requires far less dose of PTX, and has superior antitumor effect than sole PTX-liposome or PTX therapy98. In addition, the increase in CD86/CD206, TNF-α/IL-10, and CD8/FoxP3 ratios of TAM after administration also suggested that this regime could modulate the tumor microenvironment toward a pro-inflammatory phenotype.
Conventional chemotherapy drug TMZ also faces under-delivery for GBM therapy. Mia et al. have developed a noninvasive gut-to-brain oral drug delivery system dependent on macrophages. TMZ prodrug was encapsulated in nanoparticle (NP) with β-glucans using a GSH-responsive disulfide-containing linker and were phagocytosed in situ by resident macrophages in the intestinal tract, and then delivered to brain tumor site via the lymphatic and circulatory system. Bisulfide bonds within the prodrug NPs make sure that the drugs are only released in GSH (glutathione)-overexpressing tumor microenvironment99. The TMZ that can be delivered to the intracerebral tumor tissue using prodrug NPs is five times more than that using free TMZ with some present in the liver and minimal amounts in other major organs. This treatment improved survival and reduced weight loss in mice.
Some non-chemotherapy approaches require targeted delivery of therapeutic agents. Photothermal therapy (PTT) is to induce rapid heating in tumors via gold–silica nanoshells (AuNS) mediator, which are loaded into NR8383 macrophages for delivery to the tumor site and absorbs near-infrared light to produce a therapeutic effect100,101. Photodynamic therapy (PDT) has been implemented for GBM therapy. Its effect depends on photosensitizer (PS), light and oxygen in the irradiated tumor position. Conjugated polymer nanoparticles (CPNs), as a photosensitizer, were transported by macrophages infiltrated GBM in the U87 and GL261 bearing mice. CPNs were not found to affect monocyte viability, and the using of macrophage vehicle shows superior delivery efficacy to using sole CPNs102. However, this study did not provide in vivo antitumor experimental results.
Chimeric antigen receptor-macrophage therapy
Although CAR T-cell therapy has demonstrated effectiveness and enhanced targeting for hematologic tumors, the recruitment of T cells to GBM tumor sites is limited by multiple mechanisms, including the blood–brain barrier, T-cell deletion103, and T-cell sequestration104. While several approaches have been tried to increase the infiltration of CAR-T cells in solid tumors, including GBM105,106, the suppressive tumor microenvironment could also render T-cell anergy107 and dysfunction108. As a result, creating CAR-T cells suitable for GBM treatment remains a challenge109. However, macrophages, as a crucial part of innate immune system, efficiently infiltrate into tumors, phagocyte and deplete abnormal cells, and ingest and present antigens to T cells110. These properties of monocytes/macrophages suggest expressing CAR on macrophages platform can enhance targeting and serves as a potential method of immunotherapy.
CAR-macrophage therapy is a promising area of research that has shown potential in treating various types of tumors. While very few studies have targeted GBM, one impressive method for extracranial tumors was developed by Klichinsky et al. Their CAR is based on a replication-incompetent chimeric adenoviral vector (Ad5f35), which persistently expressed CARs in macrophages and did not affect other functions. The receptor structure consists of a HER2 antigen-binding domain and an intracellular CD3ζ base domain which activate the phagocytosis of cells111. CAR-macrophages became pro-inflammatory (classically activated) phenotype after the stimulation of Ad5f35 vector, which could remodel suppressive TME. What’s more, as professional antigen-presenting cells, CAR-macrophages cross-present tumor antigens and activate T cells. Their CAR-macrophages extended the median survival time of SKOV3-burdened mice from 63 days to 88.5 days112 and CAR-M constructed using their strategy is in clinical trials (NCT04660929). CAR-macrophage therapy introduces a new means of exploiting adenovirus for tumor treatment and to some extent reshapes the immune microenvironment of tumors, suggesting that this therapy is of great significance.
Except for conventional CAR cell treatment that requires isolation, genetic modification and then reinfusion, there are new techniques to genetically engineering the intracavitary macrophages in situ to express CAR. Chen et al. constructed CD68 promoter-driven anti-CD133 CAR plasmids (pCARs) encoding the CD3ζ intracellular costimulatory domain, and used nanoporter (NP)–hydrogel superstructure for locoregional induction of CD133-specific CAR-MΦs in tumor resection cavity. Surrounding macrophages can be effectively transfected to express CARs, and then phagocytizes CD133 marked glioma stem cells and suppresses tumor growth and recurrence113. Locally engineered CAR-M cells exhibit a pro-inflammatory phenotype with only minor systemic side effects. This research provided us a new stand in CAR-macrophage therapy.
Conclusion and future perspectives
TAMs play a crucial role in the tumor immune response due to their phenotypic diversity, which can result in either tumor-promoting or tumor-suppressing effects. However, the success of therapies targeting TAMs relies on our comprehensive understanding of their properties. A deeper understanding of TAMs' role in tumor development can facilitate the identification of more precise therapeutic targets and reduce tumor resistance to treatment. Conventional treatments and immunotherapy can be evaded by tumor cells through evolution114 and immune editing115. Studies have shown that TME components, particularly TAMs, co-evolve with tumors, making it challenging to target all macrophages crudely, leading to therapeutic resistance116. Potential strategy to overcome this challenge is to design more precise treatments based on the various TAM components' functions. In addition, combining TAM clearance with engineered macrophage introduction can utilize the competitive effect between cell populations to evade treatment resistance112,117,118. These strategies highlight the need to comprehend TAMs' mechanism of action and drugs.
During TAM polarization, macrophages undergo a complex process that involves metabolic changes and changes in the expression level of human leukocyte antigen (HLA) and CCL molecules. However, our understanding of this process is not yet complete. A study has identified the temporal changes of some M2-like polarization-associated molecules (such as MEK/ERK, peroxisome proliferator-activated receptor (PPARγ)) after treatment with IL-4119, and therapies have been designed to target these signaling pathways. Gradient changes in several immune molecules (e.g., chemokine and major histocompatibility antigen) during the development of GBM have also been identified120. As a result, a deeper understanding of macrophage-related temporal changes in both intrinsic and engineered macrophages will help us gain a more specific understand of the detailed mechanisms of TAM-related therapy and develop better treatment methods.
Genetically engineered macrophage-based platforms can reduce the impact of the unique GBM tumor microenvironment on exogenous gene vectors121. However, conventional methods for modifying immune cells, such as T cells and NK cells, are not effective for monocytes/macrophages and their progenitors. Some intrinsic mechanisms of macrophages, such as restriction factors122 and the lack of corresponding receptors on the surface123, limit the function of commonly used viral vectors. Elaborate adenovirus may provide us with a way to stably express the desired gene in macrophages. Modified adenovirus recognizes a wider range of cellular markers than the commonly used coxsackie and adenovirus receptors124,125. For example, the Ad5/F35 chimeric virus has been used in preclinical and clinical studies for viral therapy of hematologic diseases as well as CAR-macrophage therapy because it recognizes the CD46 marker on macrophages and can effectively transduce them126. In addition to modified adenoviruses, modified lentiviral vectors which resist to certain restriction factors are also capable of expressing exogenous genes in monocytes and macrophages127. As for in vivo macrophage modifying, some AAV vector for gene therapy (such as AAV9) are able to cross the blood–brain barrier and can be used in combination with exosomes to enhance infection of TAM84. Several nano- or physical methods for transfection of macrophages using non-viral vectors are also under investigation93,113, although the persistence of these vectors still needs to be improved. To increase the density of expression vectors in the tumor microenvironment, we can also use oncolytic viruses that can replicate specifically in the tumor128. It is interesting to note that the vector used to modify macrophages can also affect their phenotype. Adenovirus and certain non-viral vectors can cause a shift in macrophages towards a pro-inflammatory phenotype112,113, while lentiviruses may not alter macrophage phenotype or lead to a pro-tumor phenotype91,129. The mechanism underlying these effects requires further investigation. Ultimately, the choice of vector depends on factors such as safety, efficiency, impact on macrophages, and potential toxicity of the gene being expressed.
The appropriate cell sources are crucial for macrophage therapy. However, monocytes, the primary source of macrophages, are scarce in peripheral blood, which makes it challenging to harvest enough macrophages for therapy. In such cases, induced pluripotent stem cells (iPSCs) can be used to produce macrophages with therapeutic effects, such as CAR-macrophages129,130. Moreover, self-renewing hematopoietic stem/progenitor cell (HSPC) are also potential sources of macrophages. Under certain conditions, such as bone marrow transplantation with CSF-1R blockade treatment, circulation-derived myeloid cells (CDMC) can replace microglia in brain tissue and potentially serve as a source of macrophages for therapy117. However, further validation of this approach is needed under tumor conditions. Although processing the human hematopoietic system is complex, based on the tumor tropism of macrophages, it is hypothesized that delivering HSC-derived macrophages to tumor tissue may not be as demanding as delivery to brain tissue83.
In terms of CAR therapy, several targets have been explored for GBM therapy, including interleukin-13 receptor alpha 2 (IL13Rα2), EGFRvIII, HER2, CD70, B7-H3, and others86,131,132,133, although many of them do not produce decisive outcomes in CAR-T therapy due to immunosuppressive TME, antigen drift or downregulation and heterogeneity of solid tumors. Various approaches, such as the introduction of immunosuppressants, chemokines, and increased types of CARs or CAR-T cells, have been tried to overcome these challenges105,133,134. CAR-macrophage therapy shows promise in overcoming several challenges that have hindered the application of CAR-T cells in GBM. Unlike CAR-T cells, CAR-macrophages have superior tumor infiltration capabilities and work by not only directly killing tumor cells but also stimulating the immune system, remodeling the TME, and presenting antigens113,134. As a result, CAR-macrophages may be less affected by tumor heterogeneity and downregulated CAR targets, which can hinder the effectiveness of CAR-T therapy135. However, more research is needed to fully understand the mechanisms underlying CAR-macrophage therapy. In addition, compared to CAR-T therapy, CAR-macrophage therapy has shown relatively low systemic toxicity in preclinical studies. It is hypothesized that macrophages downregulate migration-associated receptors (CCL2, CCL5) in the hypoxic TME, which may trap recruited CAR-macrophages in the tumor site and reduce systemic toxicity136. Moreover, local treatment strategies following routine surgeries for GBM may also help to reduce systemic toxicity113.
Despite the potential benefits of CAR-macrophage therapy, there are unique challenges that need to be addressed. One major challenge is maintaining the pro-inflammatory phenotype of macrophages while avoiding their pro-tumor functions. In addition, many of the current CAR-M treatments utilize the same CAR structure as in T cells, which may not be optimal for achieving both tumor cell killing and TME regulation. To overcome these challenges, it may be necessary to design CARs that can more effectively activate multiple functions of macrophages137. In conclusion, further research is needed to explore the specific mechanism of CAR-M therapy, investigate the temporal changes in the immune microenvironment after administration, and develop the optimal design of CARs suitable for macrophages.
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Acknowledgements
This study was funded by Key research and development project of science and technology department of Sichuan Province (2022YFS0321, 2022YFS0320); National Natural Science Foundation of China (82002648); General Program of the National Natural Science Foundation of China (82173175); Knowledge Innovation Program of the Chinese Academy of Sciences (JH2022007) and 1·3·5 project for disciplines of excellence–Clinical Research Incubation Project, West China Hospital, Sichuan University (2020HXFH036). Figures in this review article were created with BioRender.com.
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Tang, F., Wang, Y., Zeng, Y. et al. Tumor-associated macrophage-related strategies for glioma immunotherapy. npj Precis. Onc. 7, 78 (2023). https://doi.org/10.1038/s41698-023-00431-7
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DOI: https://doi.org/10.1038/s41698-023-00431-7
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