Tumor-associated macrophages: Shifting bad prognosis to improved efficacy in cancer therapies?

Macrophages are innate immune cells that play an important role in the response to damaged tissue and pathogenic infection. During activation, signals from the local environment induce macrophage polarization towards either the classical pro-infl ammatory phenotype (M1) or towards the alternative anti-infl ammatory phenotype (M2). In cancer, M2 tumor-associated macrophages (TAMs) are associated with a poor prognosis. Notably, the Tumor Microenvironment (TME) is known to promote the M2 phenotype by dampening anti-tumor immune responses and thus promoting tumoral growth. Recent studies have demonstrated that TAMs play a major role in cancer cells resistance to chemoand radiotherapies leading to ineffective treatment strategies. This raises the importance of including macrophage targeting strategies, either to dampen their activities or to re-educate them toward pro-infl ammatory phenotype, to improve the effi ciency of current and future treatments. Therefore, this minireview aims to highlight recent discoveries demonstrating how macrophages induce cancer resistance to therapies and how re-educated TAMs could be used to improve treatment outcomes. Review Article

Moreover, these cells exhibit an increased expression of major histocompatibility complex type II (MHC-II) and the costimulatory molecules CD80/86 which are associated with an enhanced ability for presenting antigens [11]. Conversely, M2 macrophage polarization (often referred to as alternatively activated macrophages) occurs under Th2 cytokines via IL-4, IL-10, IL-13 or tumor growth factor-beta (TGF-) cytokines [12]. M2 macrophages exhibit an anti-infl ammatory phenotype and are associated with a lower expression of MHC-II and CD80/86 but a higher expression of CD206 (a mannose receptor). This subset produces anti-infl ammatory cytokines such as IL-10 and TGF- known to dampen the immune cell response [12]. The M2 macrophage subset is mainly implicated in tissue repair processes (e.g., by boosting angiogenesis via the production of Vascular Epithelial Growth Factors (VEGFs)) and immune tolerance thus promoting a decline in local infl ammation [13]. Regarding their phagocytic capacity, both M1 and M2 are more effi cient than M0 macrophages [8], but M1 exhibits higher phagocytosis activity compared to M2 [8,14].
This duality and complex balance of M1 and M2 macrophage activation is crucial in several pathological conditions, including cancer.
In this mini-review, we provide an update of recent advances regarding the role of macrophages in cancer progression and the acquisition of resistance to therapeutic strategies focusing mainly on chemo-, hormone-and radiotherapies.

Macrophages and their role in cancer development
The role of macrophages during solid tumor development has been largely described [15,16]. Thus, in this section, we will briefl y discuss the role of tumor-associated macrophages (TAMs), the main mechanisms for their polarization within tumors, and how this polarization impacts tumoral growth or regression.
Macrophages represent the most important leukocyte population to infi ltrate the tumor tissue. Once in the tumor, macrophages have a dual role depending on their polarization.
In general, it is accepted that the pro-infl ammatory M1polarized phenotype promote an anti-tumor immune response whilst the anti-infl ammatory properties of M2-polarized macrophages are associated with pro-tumor functions by dampening immune system responses and promoting metastasis in solid tumors [17][18][19]. M2 macrophages induce tumor cell proliferation and angiogenesis by producing growth factors that drive metastatic dissemination and tumoral growth [17]. Moreover, M2 macrophages produce TGF- and IL-10 cytokines known to dampen immune cells activation, consequently inhibiting anti-tumoral immune responses [20]. When recruited to the Tumor Microenvironment (TME), the mature macrophages are converted into tumor associated macrophages referred to as TAM. The TME is known to predominantly polarize TAMs towards the M2 phenotype with a small fraction of M1 [21,22]. In addition to enhancing the polarization towards pro-tumoral M2 macrophages, cancer cells also develop mechanisms to escape the immune system. TAM s with an M1 pro-infl ammatory phenotype tend to correlate with a favorable prognosis and longer survival for patients, whilst an increased accumulation of TAMs with an M2 anti-infl ammatory phenotype in tumor tissue is now commonly associated with worse patient outcomes for several tumor types, including; glioma, head and neck, lung, pancreatic, breast, ovarian, colorectal, liver, melanoma, and bladder cancer [32][33][34][35][36][37][38][39][40][41][42]. It h as thus become increasingly apparent that the role of TAMs in current treatment modalities, such as chemo-and radiotherapy, must be considered to have therapeutic implications and could be deemed a potential target for treatment strategies.

Involvement of TAMs in cancer therapy resistance
The M2 phenotype TAM association with bad prognoses is not only restricted to their ability to dampen the antitumor immune response and promote cancer cell proliferation and metastasis, but also to induce resistance to therapies by decreasing the effi cacy of current treatment strategies. Here, we describe several recent studies describing how TAMs have hindered therapeutic strategies in different types of cancers by providing resistance to chemo-,hormone and radio-therapies. that macrophages produce and release miRNAs that modulate the TME and, consequently, cancer cell sensitivity towards treatment. For example, a study by Zhu et al. showcased that TAMs produced exosomes enriched with miR-223 which could be transferred to ovarian epithelial cancer cells [43]. By transferring miR-223, macrophages are able to downregulate the phosphatase and tensin homolog (PTEN) protein expression in ovarian cancer cells, thus promoting PI3K/Akt signaling pathway known to play a role in cancer cell survival and provide cisplatin resistance [43].

Role of TAMs in chemo-and hormonotherapy resistance
TAMs in colorectal tumors have been demonstrated to exhibit low levels of cellular miR-155, which is known to decrease Janus kinase (JAK)2/STAT3 phosphorylation, leading to increased IL-6 production by macrophages [44].
TAMs, polarized to the M2 phenotype, are known to produce TGF- and IL-10 [12]. Recent studies have demonstrated that the production of IL-10 by TAMs provides resistance to paclitaxel and carboplatin in breast cancer [51]. A study by Wei, activation thus dampening apoptosis induction [55]. Although the exact mechanism is not well understood, the resistance is hypothesized to be cell-contact independent and instead seems to be related to the secretome of TAMs which affects multiple signaling pathways. Another recent study demonstrated TAMs secreting nucleosides as the induction of pancreatic cancer cell resistance to gemcitabine [56]. Gemcitabine is a deoxycytidine analog which, upon incorporated into the DNA during replication, leads to cell death [57]. In this work, the authors observed that deoxycytidine release from TAMs can lead to gemcitabine resistance of pancreatic cancer cells [56].

Role of TAMs in radiotherapy resistance
Radiation therapy has been shown to induce Immunogenic Cell Death (ICD) wherein the release of tumor antigens, at the radiation site, induces immune responses. This leads to the accumulation of myeloid cells, the release of infl ammatory cytokines (e.g. IL-1), monocyte/macrophage recruitment factors (e.g. IL-34 and colony-stimulating factor 1, CSF1), and pro-fi brotic mediators (e.g. TGF-) [58]. ICD induction has been shown to contribute to anti-tumor immunity and has been seen as a promising exploitable process for cancer treatments. Unfortunately, downstream components of the immune system, such as TAMs, have been shown to either promote or suppress ICD.
Fractionated radiation therapy is considered to be immunosuppressive, stimulating the innate immune system towards a tissue repair response which promotes tumor recurrence and progression [59][60][61][62]. Macrophages have historically been noted to be relatively radioresistant, and are considered to be activated and recruited to play central roles as both the tumor-resident population of phagocytes and the central cells directing wound healing and tissue repair in tumoral tissues following radiation [63][64][65]. The macrophages which survive the radiation, and the recruited macrophages after, display a pro-tumoral M2 macrophage phenotype with enhanced pro-survival and pro-angiogenic activities often leading to tumor recurrence and treatment failure [66].
Interestingly, this immunomodulatory effect has been detected in distant tumors outside the fi eld of the radiation treatment and is referred to as the abscopal effect, and highlights the importance of considering the immunological effects of ionizing radiation [67]. Notably, irradiation alone does not directly affect the production of effector molecules or cytokines in M1 or M2-activated macrophages but rather acts as an enhancer or inhibitor of infl ammatory mediators [68,69].
Another important aspect regarding irradiation effi cacy is hypoxia which can be compounded by TAMs as they can help modulate tumoral metabolism involved in aerobic glycolysis thus hindering the effi cacy of radiotherapy [70]. depending on the dose fractionation, the total dose, and the cancer type.
Higher dose irradiation (> 10 Gy) causes the release of damage-associated molecular patterns (DAMPs) which induce the expression of pro-infl ammatory cytokines, chemokines, and effector molecules activating the ceramide pathway which triggers apoptosis via acid sphingomyelinase [75,76]. However, an increas e in the number of M2-like TAMs has been observed for in vitro and model murine prostate, oral, and pancreatic cancer when exposed to > 10 Gy doses of radiotherapy [77][78][79].  have been applied mainly to mice as this radiation regime is not applicable to human patients [65].
Notably, the full molecular mechanisms of exactly how TAMs promote therapeutic resistance is beyond the scope of this mini-review and the authors highly recommend more indepth reviews [87][88][89].

Role of TAMs in immunotherapy resistance
Although this mini-review is mainly focused on TAMs resistance to chemo-, hormone-and radiotherapy, the importance of TAMs in other treatment strategies, such as immunotherapies, is also relevant. Immunotherapy strategies are a highly promising approach to cancer treatment. Several Indeed, the interaction between PD-L1 and PD-1, expressed by T cells, induces T cell inactivation and decrease proliferation [93,94]. The immune checkpoint blockade (ICB) strategy aims to inhibit this PD-L1/PD-1 interaction with monoclonal antibody anti-PD-L1 or anti-PD-1 to avoid T cell inhibition [95,96]. Unfortunately, although this strategy seems effi cient for several cancer types, recent evidence has indicated that macrophage-derived granulin dampens CD8 + T cell infi ltration into metastatic pancreatic tumors and drives resistance against anti-PD-1 therapy. The inhibition of granulin, produced by macrophages, promotes CD8 + T cell infi ltration and enhances ICB therapy [97]. Additionally, macrophages also express the V-Domain Ig Suppressor Of T Cell Activation (VISTA) protein [98]. VISTA shares a homology with PD-L1, and can also play a role in the dampening T cell activation [99]. The upregulation of VISTA expression by macrophages, after ICB treatment in prostate and melanoma cancer, has been hypothesized to represent a compensatory pathway implicated in ICB resistance [100].
Thus, collectively, these studies indicate the major impact of TAMs on cancer cells and chemo-hormone-,radio-and immunotherapy resistance.

Macrophages targeting therapeutic strategies to improve current treatment
The TME is known to polarize TAMs toward the M2 phenotype and the phenomenon is now commonly associated the M1 phenotype or the reduction of M2 TAMs to skew the M1/M2 population towards a pro-infl ammatory ratio will be discussed.

Specifi c depletion of pro-tumorigenic TAMs
Melittin, a major compound of bee venom, has been observed to target pro-tumorigenic M2 TAMs in a Lewis lung carcinoma mouse model, and to reduce the M2 popu lation without affecting pro-infl ammatory M1 TAMs [101]. The mechanisms associated with this decrease in M2 TAMs are not currently well known but a decrease of angiogenesis was observed in the tumor stroma of mice injected with melittin.
Melittin has also been coupled with other peptides, such as the pro-apoptotic pept ide d-(KLAKLAK) 2 , to target M2 TAMs and induce cell death by mitochondrial-dependent apoptosis thus leading to decreased angiogenesis, tumor growth rates, and tumor weight [102]. The same melittin-d(KLAKLAK) 2 compound has also been associated with enhanced anti-tumor effects of immunotherapy in breast cancer models [103].
Another study demonstrated the specifi c depletion of CD163 + TAMs using CD163 antibodies conjugated with cytotoxic lipid nanoparticles loaded with doxorubicin. The CD163+ TAMs depletion induced an infi ltration of activated T cells in tumors thus leading to tumor regression in melanoma mice models [104].

Re-education of TAMs toward anti-tumorigenic macrophages
In

Other therapeutic strategies with macrophages targeting
A few other therapeutic strategies have also been developed for macrophage targeting, although interestingly their aim is not to impact M1/M2 ratio unlike the previous strategies, but rather to prevent tumors from escaping the immune system.