Background

Solid tumors are composed of both malignant cells and several nonmalignant hematopoietic and mesenchymal cells. Among the latter are tumor-associated macrophages (TAMs), which represent the most abundant subpopulation of tumor-infiltrating immune cells in the tumor microenvironment (TME) [1] and can interfere with tumor progression and neoangiogenesis. TAMs are extremely plastic immune cells, with two polarized states: classically activated M1 and alternatively activated M2 macrophages [2]. M1 macrophages play critical roles in innate host defense by producing reactive oxygen/nitrogen species (ROS/RNS) and proinflammatory cytokines such as interleukin (IL)-1β, IL‑6, and tumor necrosis factor α (TNF-α). In terms of their activity, they are generally considered as antitumor macrophages [3]. On the other hand, cytokines such as IL‑4, IL-10, and IL-13 can induce macrophage polarization to the M2 subtype, which is not only crucial for the onset of the classical Th2 immune response (i.e., humoral immunity, wound healing, tissue remodeling), but it is also key for the production of anti-inflammatory cytokines such as IL-10 and TGF‑β which foster tumor evolution. M2 macrophages are therefore considered to be protumor cells [4]. However, this “black and white” model has shown its limitations, mainly due to the existence of multiple intermediate states between M1 and M2; the polarization process is therefore dynamic, and macrophages often display characteristics of both profiles at the same time.

Besides its cytocidal effect on cancer cells, radiotherapy (RT) also plays a role in affecting the TME through multiple mechanisms, both direct and indirect, acting on different cell types. The interaction with tumor vascularization and immune cells remains crucial [5]. Endothelial damage, a central player in the induction of therapy-related inflammation, hampers CD8+ T cell infiltration into tumors and promotes the development of an immunosuppressive milieu affecting the efficacy of cancer therapies. As a consequence, suppressor cells such as M2 TAMs, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) gather together. Furthermore, hypoxic regions within the tumor are increased and hinder oxygen-dependent DNA damage, providing an even more reduced anticancer RT effect. We believe that a better understanding of the processes underpinning macrophage characterization under the influence of irradiation (IR) could be of help to establish new, effective RT schemas. Moreover, the association of RT with other available treatment options (i.e., immunotherapy) should be explored.

Methods

Search strategy

A systematic search in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA [6]) guidelines was carried out in PubMed, Google Scholar, and Scopus. It was performed from January 2000 to April 2020 in order to identify articles focused on the role of M1 and M2 macrophages in response to radiation. Medical terms referring to radiotherapy were used in combination with M1 and M2 (note that we used the “AND” Boolean logic symbol to restrict the area of investigation as follows: “macrophages and radiotherapy,” “radiation oncology,” “TAMs,” “TAM,” and “MERT”).

Screening process

All articles were screened by two independent reviewers (CB and MS). The reviewing selection process was based on title, abstract, and full text. Manuscripts exploring the role of and changes in M1 and M2 macrophages following irradiation were included in the systematic review. All study designs, with the exception of reviews, editorials, case studies, and conference abstracts/posters, were included. Languages other than English were excluded, as were studies that were not available in full-text version format. However, titles and abstracts selected by either one of the reviewers were included for additional screening. At each level of the screening process, when different opinions existed among the two reviewers as to whether to include a record or not, a mutual agreement was reached (see Fig. 1 for the flowchart). We extracted data from the included studies: for each paper, the principal author, publication year, number of patients, age, type of diagnostic imaging, treatment, and outcomes of interest were recorded. Data were summarized in evidence tables and described in the text.

Fig. 1
figure 1

Flowchart of inclusion of studies in the systematic review. RT radiotherapy (From: [6]. For more information, visit www.prisma-statement.org)

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Results

Of the 304 articles initially screened and considered potentially relevant to the topic of this study, 26 were eventually included. The selected articles were categorized according to the description of M1/M2 changes when exposed to radiotherapy. The research flow and selection process are shown in Fig. 1. We separately analyzed the manuscripts containing only radiation therapy (10 articles) and those that explore the impact of radiotherapy and concomitant drugs on macrophage status (16 articles). For convenience, based on different IR doses analyzed, we referred to high-dose irradiation (HDI) as doses higher than 10 Gy, moderate-dose irradiation (MDI) as doses ranging from 1 to 10 Gy, and low-dose irradiation (LDI) as doses lower than 1 Gy.

Role of TAMs in tumor initiation and progression

TAMs actively participate in tumor angiogenesis, matrix remodeling, invasion, immunosuppression, metastasis, and chemoresistance in various types of cancer. Several clinical studies have indicated that the presence of a tumor infiltrate characterized by high levels of TAMs represents a negative prognostic factor, as in the case of hepatocellular, ovarian, cervical, and breast cancer [7]. TAMs exhibit a wide spectrum of phenotypes, loosely categorized as the M1–M2 polarization spectrum, with M1 macrophages being generally proinflammatory and M2 macrophages presenting with anti-inflammatory and proangiogenic features. The activation of a particular macrophage profile seems to be dependent on the cytokine milieu, the production of specific growth factors, and the presence of hypoxia. While TAMs are very frequently differentiated into the M2 phenotype, the polarization process is, by definition, dynamic, and these cells very often display characteristics of both states at the same time.

Effects of RT alone on macrophages status

Macrophages are one of the most radioresistant cell types [8]. This characteristic is attributed to the production of antioxidative molecules, such as manganese superoxide dismutase (MnSOD), which are responsible for cellular resistance against damaging effects produced by radiation-induced radicals such as reactive oxygen and nitrogen species (ROS and RNS, respectively). Tsai and colleagues [8] reported that after irradiation, Arg‑1, COX‑2, and inducible nitric oxide synthase (iNOS) are overexpressed in TAMs, which stimulates tumor growth. Of note, iNOS has a dual effect on tumor expansion, depending on its levels [9, 10]: the amount produced by M1 macrophages can kill cancer cells, while at lower concentrations, enough nitric oxide (NO) is produced to ensure a vasodilative effect and an increase in blood flow within the tumor, promoting its growth [11]. However, the iNOS pathway with the substrate l‑arginine—the one responsible for the cytotoxic effect—is blocked in M2 macrophages and replaced by the synthesis of ornithine and polyamines, which favor tumor cell proliferation. Several other cytokines secreted by TAMs, such as epidermal growth factor (EGF), TGF‑β, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), also have pro-proliferative actions. As a matter of fact, the presence of TAMs within the tumor leads to faster and increased growth of the neighboring tumor cells; in the post-irradiation setting, the production of tumor necrosis factor (TNF) by activated macrophages may favor the synthesis of protective proteins against subsequent killing by oxidative stress [12]. Another consequence of IR exposure is the massive recruitment of myeloid cells to the tumor site, which is thought to be a trigger for tumor regrowth after local irradiation. In this scenario, the inhibition of CSF‑1 receptor (CSF-1R) with PLX3397, a small molecule that blocks its tyrosine kinase activity, may enhance the cytotoxic effect of concomitant IR by preventing IR-recruited myeloid cells differentiating into protumor macrophages. In the in vivo study by Stafford et al. [13], combined IR and PLX3397 therapy was compared with IR alone in two different human GBM xenograft models. Median survival was significantly improved in mice receiving the combined approach.

The main studies exploring radiotherapy’s effects on macrophages are collected in Table 1.

Table 1 Studies exploring radiotherapy’s effects on macrophages
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High-dose irradiation

Several authors have shown that HDI can expand the number of M2-like TAMs in the tumor milieu. In an in vitro experiment, 20 Gy irradiation of M1 Raw264.7 macrophages led to TAM repolarization toward a M2-like profile [24]. More specifically, IR can activate NFκB p50 and determine an increase in IL-10 levels and an inhibition of TNFα production. A subsequent in vivo experiment from the same group showed that HDI was able to recruit M2 macrophages to the tumor site [24]. Tsai et al. reported how levels of M2 TAMs can raise after delivering HDI (25 Gy single-fraction or hypofractionated RT up to a total dose of 60 Gy) to prostate cancer cells. The authors observed an upregulated mRNA expression of Arg1 and Cox‑2 in TAMs together with a low iNOS level, favoring both angiogenesis and tumor growth in a murine model [8]. Similar results in terms of M2 macrophage proliferation and subsequent release of proangiogenic molecules were obtained after irradiation of oral cancer cells with a single dose of 12 Gy [25].

HDI was also found to be responsible for an improvement in suppressor T cell activity in pancreatic cancer cells [26]. A decrease in production of iNOS and an increase in levels of Arg1, PD-L1 (which stimulated the T response), and IL-10 (causing lymphocyte anergy) were observed.

Enhanced activity of M2 TAMs following IR was also documented in a lung cancer model [14]. Notably, the radiation-induced endothelial damage caused augmented production of CCL2 in the BAL fluid, ultimately leading to M2 macrophage colonization and hyperexpression of Arg 1 and CD206 in the weeks following irradiation.

M2 TAM polarization is therefore also favored by radiation-induced tumor vascularization via the endothelial-to-mesenchymal transition (EndMT), as shown by Choi et al. [15]. Of note, authors suggest that HDI may elicit a stronger immune response than LDI, due to the rate of indirect tumor cell death resulting from vascular damage.

Overall, these results well describe how HDI polarizes TAMs in an M2-like phenotype and promotes their recruitment to the tumor site, eventually leading to induced angiogenesis and accelerated tumor growth.

Moderate-dose irradiation

MDI (i.e., 2 Gy × 5) can enhance a proinflammatory state in M1 macrophages. Classic proinflammatory markers such as human leukocyte antigen cell surface receptor (HLA-DR) and CD86 are upregulated, while anti-inflammatory molecules (which characterize the M2 phenotype) are hindered, with a reduced mRNA expression of CD163, C‑type mannose receptor 1 (MRC1), and CD206, and decreased IL-10 secretion.

Phagocytotic activity, typically associated with the M1-like phenotype, is enhanced by MDI, which, on the contrary, has no influence on the ability of cocultured macrophages to promote cancer cell invasion and angiogenesis (typically related to the M2-like profile) [27]. Ex vivo γ‑MDI of CD11b+/Gr‑1 peritoneal macrophages demonstrated augmented levels of iNOS [22] and proinflammatory activity was documented in both murine and human models after 2–4 Gy of γ‑irradiation, with increased levels of TNFα, IFNγ, IL‑6, and IL-1β mRNA expression.

Different from previous experiences which documented the activation of NFκB p65 for TAM reprogramming, Wu et al., in their research, underlined the crucial role of the kinase ATM in promoting the M1-like phenotype through the regulation of IRF5 expression [19].

Of note, in vitro studies clarified that MDI is capable of inducing an M1 phenotype in nonpolarized macrophages and enhancing this profile in those which are already polarized; on the contrary, moderate irradiation cannot reprogram M2 TAMs.

In their study, Pinto et al. [28] set up cocultures of unpolarized macrophages with both radiosensitive (RKO cells) and radioresistant colon cancer cells (SW1463 cells). In the first scenario, MDI (which consisted of a total dose of 10 Gy in five daily fractions) led to reduced mRNA expression of some proinflammatory markers (such as CCR7 and IL1β), with no changes documented for anti-inflammatory markers, while cancer cell invasion and migration were promoted. Interestingly, when macrophages were cocultured with radioresistant cells, both proinflammatory (CCR7, CD80) and anti-inflammatory markers (IL-10 and CCL18) were overexpressed, with no changes in cancer cell migration and invasion. According to these findings, unpolarized macrophages develop a different phenotype based on the type of cancer cells they interact with [28].

Low-dose irradiation

Local radiotherapy influences the activity of tumor-specific T cells through several mechanisms: it determines a higher rate of antigen release from dying tumor cells; it stimulates antigen-presenting cell subsets; and, lastly, it enhances T cell migration. In their experiments with human melanoma xenografts and human pancreatic cancer specimens exposed to LDI, Klug et al. wanted to assess whether local single irradiation of 0.5–2 Gy can be used as an adjunct strategy to improve the efficacy of multiple immunotherapeutic approaches [23]. The authors found out that, due to iNOS activity, the classic Th2 response was completely (IL‑4 and IL-13) or markedly (IL‑5, IL‑6, IL‑9, and IL-10) inhibited after tumor irradiation. Indeed, iNOS inhibition has been shown to restore the Th2 response even in irradiated tumors. The iNOS-expressing TAMs were repolarized by irradiation towards an M1-like profile, being responsible for vascular normalization, T cell proliferation, and an antitumor response. Therefore, the adoptive transfer of radiation-induced iNOS-expressing macrophages may represent a promising strategy to explore to potentiate classical immunotherapeutic approaches.

Furthermore, in specific murine models, LDI led to activation of p38 MAPK in macrophages, with an associated transitory increase in TNF‑α production [29]. After 15 min from the delivery of 0.5 Gy gamma radiation, the upregulation of MKP‑1 was responsible for inactivation of p38 MAP‑K with suppressed production of proinflammatory TNF‑α.

RT administered with concomitant agents: effects on macrophages status

Table 2 includes a list of studies, mostly preclinical, which explore the role of concomitant administration of RT and immunomodulating drugs in terms of their influence on macrophage status.

Table 2 Studies exploring the effects radiotherapy plus concomitant agents on macrophages
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As reported by Zeng and colleagues [44], T lymphocytes have a key role in mediating the effects of stereotactic radiotherapy (SRT) and immunotherapy, while both macrophages and microglia are involved in a later phase. However, it has been hypothesized that the additional benefit of the combined approach relates to M1 macrophage-mediated effects [32]. In fact, the coadministration of radiotherapy and PD‑1 checkpoint blockade can boost the immune response by increasing the number of CD8+ lymphocytes and macrophages (namely the M1/M2 ratio).

The great majority of immunomodulating molecules that have been analyzed and reported in this review act by inhibiting the signaling pathways involved in M2 polarization and hinder M2 macrophage-mediated radioresistance. For example, PM37, a phosphopeptide mimetic targeting the SH2 domain of STAT6, was shown to decrease the expression of M2 polarization markers [38]. Pretreating macrophages with PM37 reduced the radioresistance they induced in inflammatory breast cancer (IBC) cells after coculture. In another paper by Shi et al. [34], the authors noted that combining radiation with PI3Kα inhibitors resulted in a synergistic activity against esophageal squamous cancer cells and patient-derived xenografts (PDX); more specifically, this association abrogated radiation-induced survival signals in both tumor cells and the tumor microenvironment, hindering M2-like macrophage infiltration.

Discussion

The role of macrophages within the tumor milieu has gained a lot of interest in the recent literature; these immune cells show different phenotypic profiles according to the differently induced microenvironmental signals and cytokines [38]. This review captured the current knowledge about interactions between TAMs and radiation therapy.

Classically activated macrophages (M1) are mainly induced by Toll-like receptor ligands and Th1 cytokines, such as interferon IFN‑γ, while Th2 cytokines like IL‑4 and IL-13 can stimulate the adoption of an M2 profile (alternatively activated macrophages).

The acronym TAM usually refers to the M2-like phenotype, characterized by anti-inflammatory and protumoral activity. On the contrary, M1-like macrophages exhibit proinflammatory, phagocytic, and antitumor functions. M2 TAMs are responsible for augmented genetic instability, upregulated angiogenesis, and increased immunosuppressive signals, which favor metastatic spread. This cell profile is also associated with tissue remodeling and conditions characterized by augmented fibrosis, such as pulmonary fibrosis [4], due to the stimulated production of profibrotic molecules including TGF‑β, IGF‑1, and galectin‑3.

Multiple activated transcription factors and miRNAs regulate macrophages’ expression of a specific M1 or M2 phenotype. In particular, NFκB plays a key role: its active heterodimer NFκB (p50–p65) favors proinflammatory gene expression, such as TNFα, IL‑6, and IL1β, while the inactive homodimer NFκB (p50–p50) hinders the transcription process of such genes, ultimately leading to the anti-inflammatory profile which characterizes M2 macrophages [34].

In the setting of the tumor microenvironment (TME), where TAMs favor the epithelial-mesenchymal transition, these immune cells are indeed related to tumoral progression [1, 45]—both locally with enhanced tumor growth due to L‑arginine depletion [46,47,48] and systemically by increasing its metastatic potential. For all these reasons, TAM accumulation correlates with an unfavorable prognosis in many cancer types.

TAMs exert their immunosuppressive action by inhibiting T cell proliferation, thanks to the expression of specific molecules such as PD-L1 and PD-L2, which are inhibitory checkpoint regulators interacting with corresponding ligands on T cell membranes, ultimately leading to their inactivation [49, 50].

Furthermore, TAMs indirectly contribute to immunosuppression by producing chemoattractant molecules to recruit cells which further hamper the immune response, such as myeloid-derived suppressor cells (MDSCs), immature dendritic cells (DCs) and Tregs [51]. As M2 macrophages are activated by IL‑4 produced by CD4+ T cells, PMA/IL-4-treated THP‑1 cells are often used to generate TAMs [52, 53].

In this scenario, characterized by an intricate interaction between the TME immune environment and cancer cells, there is growing interest in the role of radiation.

Radiotherapy (RT) currently represents an essential component of the management of cancer patients, either alone or in combination with surgery or systemic therapies. The main goal of RT is to deliver a curative dose to the tumor while sparing the surrounding healthy tissues and organs.

Alongside the killing effect on tumor cells, different RT doses may induce modifications of the local microenvironment that can affect tumor development. IR may enhance macrophage infiltration to tumor sites, accelerating tumor progression in several ways (summarized in Fig. 2,).

Fig. 2
figure 2

Schematic representation of radiation-induced effects on macrophages. TAMs within the tumor are either present as tissue-resident macrophages or are formed after circulating monocytes are recruited and subsequently differentiated. Soluble factors such as the chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1, MCP1), complement anaphylatoxins (C3a and C5a), and colony-stimulating factor 1 (CSF 1) are well-documented signaling molecules involved in the recruitment process. Furthermore, physical changes such as upregulation of HIF‑α subunits and damaging of the extracellular matrix leads to TAM infiltration and tumor cell proliferation. Polarization of monocytes into mature macrophages phenotypically falls into a wide spectrum of either inflammatory or immunosuppressive behaviors, depending on the expression of interleukins and lipopolysaccharides. IR irradiation, miRNA micro-ribonucleic acid, CCL CC chemokine ligand, CC CC chemokine receptor, CX3CR1 C-X3‑C motif chemokine receptor 1, IL interleukin, TGF transforming growth factor, IRF4 interferon regulatory factor 4, STAT signal transducer and activator of transcription, NFkB p50/p50 nuclear factor kappa B subunit 1, miR micro-ribonucleic acid, MRC1 mannose receptor C-type 1, ECM extracellular matrix, MMP matrix metallopeptidase, CD206 mannose receptor, CD163 cell-surface glycoprotein receptor member of the scavenger receptor cysteine-rich family class B, Fizz1 resistin-like molecule alpha‑1, Ym1 rodent-specific chitinase-like protein 3, Arg1 arginase 1, VEGF vascular endothelial growth factor, TH2 type 2 helper T, DNA deoxyribonucleic acid, NF-kB nuclear factor kappa light chain enhancer of activated B cells, CD80 ligand for the proteins CD28, CD86 cluster of differentiation 86, HLA-DR human leukocyte antigen–DR isotype, IRF5 interferon regulatory factor 5, IFN‑γ interferon-gamma, VCAN versican, NOX2 nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 2, ROS oxygen-containing reactive species, ATM ataxia telangiectasia mutated, CXCL10 C-X‑C motif chemokine ligand 10, NO nitric oxide, iNOS inducible nitric oxide synthase (Created with BioRender.com)

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Wu et al. [54] demonstrated that different doses of IR can polarize macrophages to show a proinflammatory M1-like profile in xenograft tumor models and human rectal cancer specimens obtained from patients treated with neoadjuvant chemoradiation. This effect is attributed to the IR-induced activation of interferon regulatory factor 5 (IRF5): its mRNA levels and posttranslational modifications are regulated by ATM kinase, whose activation is not only decisive in radiation-elicited macrophage polarization, but which is also key for macrophage reprogramming after treatments with agents like cisplatin, γ‑interferon, and lipopolysaccharide. Furthermore, the authors demonstrated that upstream activation of NADPH oxidase 2 (NOX2)-dependent ROS, which is increased after IR exposure or IFN‑γ treatment, is also crucial for macrophages’ acquisition of a proinflammatory profile. The downregulation of this intricate pathway, at any level, can hinder macrophage activation towards a M1 phenotype, ultimately leading to poor tumor response after radiotherapy.

As demonstrated by Wang et al. [55], irradiation positively regulates IL‑6 levels; however, depletion of this cytokine was found to be associated with reduced macrophage infiltration after radiation exposure, indicating the crucial role of IL‑6 in this process.

Immunostimulating effects of IR in the tumor milieu include enhanced natural killer (NK) cell cytotoxicity and CD8+ infiltration, enhanced macrophage polarization towards an M1-like profile, reduced levels of Treg [56], and inhibition of the PD-1/PDL‑1 pathway [57].

Radioresistant tumors are characterized by a high level of macrophage infiltration, which contributes to the development of additional resistance to the cytotoxic activity of NK cells [58] through modification of tumor cell–NK cell interactions at specific ligand levels, namely PD-L1 and NKG2D [59]. It has been shown that NKG2D ligand expression on macrophages is upregulated upon coculture with NK cells [16].

Further studies will be crucial for revealing the role of THP‑1 CM in the alteration of NKG2D ligand levels (on tumor cells) and NKG2D receptor levels (on NK cells) in specific coculture systems including tumor cells, THP‑1, and NK cells. IL‑6, which is produced by THP‑1 cells, may be key in inducing tMEK/Erk activation in radioresistant cancer cells [60, 61]. Nevertheless, the role of other cytokines should also be taken into consideration for inducing tumor cells’ resistance to NK cell cytotoxicity (i.e., IL-10) [62].

In a murine model of breast cancer cells, Shiao and colleagues reported on how polarized Th2 macrophages and CD4+ T cells mediate tumor growth after radiation therapy, in part via suppression of CD8+ T cells [63]. More recently, Allen et al. [64] showed that macrophages can regulate the sensitivity of inflammatory breast cancer cells (IBCs) to radiation via increased production of IL‑6, IL‑8, IL-10, and protein kinase C zeta (PRKCZ), a previously reported modulator of radiosensitivity [65]. Irradiation promoted CT26 and 4T1 cells to secrete CCL2, which has a crucial role in recruiting TAM to the TME in a dose-dependent manner. Cheng et al. [66] observed that combining rosiglitazone treatment with irradiation significantly reduces the CCL2 level and its chemotactic effect responsible for TAM infiltration in irradiated tumors. Furthermore, the authors have highlighted that macrophage PPARc is a crucial mediator of the antitumor effect of rosiglitazone in vivo. Deletion of macrophage PPARc in mice not only facilitates tumor progression but also weakens the antitumor effects of PPARc agonists, with a concomitantly increased infiltration of CD11b+ myeloid cells and TAMs with proinflammatory and proangiogenic phenotypes [66].

TAMs, like other cells of the monocyte-derived DC system, have demonstrated phagocytic activity. A review was published recently focusing on TAMs’ phagocytic activity to improve innate anticancer immunity and promote T cell-mediated adaptive immune responses [67]. Interactions between tumor cells and TAMs that regulate phagocytosis are the result of “eat me” and “don’t eat me” signals [68]. Moreover, during radiochemotherapy treatment, there is an increased release of apoptotic tumor cells which favors activation of the efferocytosis pathway, which promotes anti-inflammatory function [69]. This process leads to rapid antigen degradation and therefore limits the cross-presentation capacity, ultimately promoting an immunosuppressive tumor microenvironment [70]. Comprehensive knowledge of these pathways will allow us to better identify targets, such as anti-CD47 and efferocytosis inhibitors, to modulate TAM phagocytic activity [67].

Macrophages can be considered rather radioresistant, as even high single doses have no significant impact on their viability, even though some hints toward increased DNA damage after exposure to ionizing radiation are found. In general, LDI seems to have a rather anti-inflammatory effect, while HDI seems to have a rather inflammatory impact on macrophage functionality. Cytokine secretion on the other hand is strongly dependent on various additional factors such as inflammatory background and radiosensitivity of the model, as well as on the applied dose.

Conclusion

TAMs contribute to tumor progression in several ways, from enhanced genetic instability to induced metastasis formation and impaired protective adaptive immunity. The plasticity of these cells makes them an attractive target for anticancer therapies, which should have the goal of polarizing TAMs to the proinflammatory and tumoricidal side of the spectrum. Radiation therapy, which exerts its main antitumor activity via cell killing, not only enhances TAM recruitment to the tumor site, but can also interfere with their characterization in multiple modalities according to different doses and schedules of administration. Assessment of the microenvironment should be included in studies combining RT with systemic therapies, as an unexpected polarization could be detrimental to their synergy. More research should be conducted in the near future to explore this potential therapeutic strategy.