Harnessing tumor-associated macrophages as aids for cancer immunotherapy

One strategy for targeting TAMs is to block their recruitment or infiltration of monocytes/macrophages into tumors. TAMs replenishment in the tumor is often mediated by monocytic recruitment via the CCL-2-C-C chemokine receptor (CCR)-2 axis [12, 59]. CCL-2 released by tumor cells, macrophages, and stromal cells within the TME recruits monocytes that express the receptor CCR-2 and granulocytes (i.e. myeloid-derived suppressor cells; MDSCs) that express the receptor CCR-5 to tumors, thereby driving tumor progression. Thus, targeting the CCL-2-CCR-2 axis can reduce numbers of TAMs in the tumor milieu [12, 59]. Combination therapy of CCL-2 or CCR-2 blockade with chemotherapy, radiation therapy or immunotherapy reduces the infiltration of myeloid cells and results in improved antitumor effects in preclinical models [60, 61]. Clinical trials with several CCR-2 inhibitors (PF-04136309, MLN1202, BMS-813160, and CCX872-B) are currently ongoing for the treatment of solid tumors [24]. CCL-2 inhibition reduces tumor growth and metastasis in preclinical models, when administered in combination with chemotherapy, neutralization of CCL-2 improved the efficacy of treatment. However, it has also been observed that cessation of anti-CCL-2 treatment leads to a rebound effect, with increased release of the monocytes previously trapped within the bone marrow, thus accelerating breast cancer metastasis by promoting angiogenesis [62]. Nevertheless, a CCL-2-blocking antibody, carlumab (CNTO 888), and a CCR-2 small molecule inhibitor (PF-04136309) are the main drugs currently being tested [10, 11, 25]. PF-04136309 has shown some benefit in pancreatic cancer patients in combination with the FOLFIRINOX (a chemotherapeutic regimen of folinic acid, fluorouracil, irinotecan, and oxaliplatin for pancreatic cancer patients). Patients treated with PF-04136309 plus FOLFIRINOX did not experience worse toxicity than those receiving chemotherapy alone. Patients treated only with FOLFIRINOX did not show an objective response. By contrast, in combination group, 16 of 33 imaging-evaluated patients had an objective tumor response (49%), and 32 of those patients achieved local tumor control (97%) [60].

Other pathways also involved in TAM recruitment include the C-X-C ligand (CXCL)-12-C-X-C receptor (CXCR)-4 axis and VEGF receptor pathway. Stromal cell-derived CXCL-12 is another chemokine that facilitates the migration of macrophages through endothelial barriers and drives TAM accumulation and survival in hypoxic areas of tumors [63]. Thus, blocking CXCL-12-CXCR-4 signaling represents a promising strategy to modulate macrophage infiltration and prevent metastasis [64‐66]. Indeed, targeting CXCR-4 in several preclinical models, including breast, prostate, and ovarian cancer, was shown to significantly reduce total tumor burden and metastases [65, 66]. In addition, blockade of CXCL-12/CXCR-4 axis prevented the post-sepsis-induced tumor progression, TAM accumulation, and TAM in situ proliferation [64]. Vascular endothelial growth factor (VEGF) also functions to recruit macrophages into tumors, which requires VEGFR2 expressed by the macrophages [67]. Selective inhibition of VEGFR2 results in reduced macrophage infiltration and decreased angiogenesis in breast and pancreatic cancer models.

Colony-stimulating factor 1 (CSF1) controls proliferation, differentiation, recruitment, survival, and function of mononuclear phagocytes (e.g., macrophages, monocytes). CSF1 receptor (CSF1R) signaling in TAMs may promote their acquisition of an immunosuppressive and pro-tumorigenic, M2-like phenotype [68, 69]. Thus, the CSF1/CSF1R signaling axis is an obvious and attractive target. CSF1R belongs to the tyrosine kinase transmembrane receptor family and binding to its ligand (e.g., CSF1, IL34) induces homodimerization of the receptor and subsequent activation of receptor signaling [68]. Based on the development of a number of small-molecule and antibody antagonists to prevent receptor dimerization, current research is thereby focused on abrogating binding to CSF1 and activation of signaling, decreasing TAM infiltration and augmenting the effect of antitumor immune in the tumor [70, 71]. Macrophage recruitment can be remarkably reduced by blocking the CSF1/CSF1R axis, which has recently been reviewed elsewhere [11, 24, 25, 68]. Based on these preclinical data, several clinical trials of CSF1/CSF1R inhibitors have been completed or are ongoing. Among the small molecules, PLX3397, a small-molecule CSF1R inhibitor that can be administered orally, has the broadest clinical development, as it is being tested clinically in a variety of malignancies [72, 73]. Blockade of CSF1R with PLX3397 induced clinical regression in patients and decreased the intratumoral accumulation of immunosuppressive macrophages, thus confirming the validity of TAMs as a therapeutic target in cancer therapy [70, 71]. Several other small molecules are also being tested clinically including PLX7486, BLZ945, JNJ-40346527, and ARRY-382. Another strategy involves monoclonal antibodies designed to target either CSF1R (AMG820 and IMC-CS4) or its ligand CSF1 (MCS110 and PD-0360324) (also reviewed in [11]). The preclinical data strongly suggest that targeting the CSF1/CSF1R axis has the potential to complement conventional therapeutic strategies. While targeting monocyte/macrophage recruitment before their arrival to the tumors is effective in various preclinical cancer models, TAMs can also be directly targeted by other strategies once they invade the tumors. Strikingly, blockade of CSF1R also induces an extensive metabolic rewiring that culminates with the restoration of glycolysis to favor the maintenance of M1-like TAMs [74]. Thus, TAMs metabolism and the immunometabolic circuitries linking TAMs to other stromal cells within the TME may also be harnessed in combination with immunotherapeutic agents for cancer immunotherapy, which has recently been reviewed [75].

The selective elimination of TAMs in tumors has been explored for cancer therapy. One attractive strategy for depleting TAMs within the tumor milieu is to trigger their apoptosis, which could effectively inhibit tumor growth and restore local immune surveillance in the TME. Mechanistically, in addition to preventing TAMs recruitment into the tumors, blockade of the CSF1/CSF1R axis has been shown to reduce macrophage survival [70, 71]. Several compounds have been shown to induce apoptosis of macrophages including zoledronate, clodronate, and trabectedin as will be reviewed as the following.

Bisphosphonates, a class of anti-resorptive drugs, are taken up by phagocytosing cells and have cytotoxic effects on myeloid cells [76]. Based on their structure, bisphosphonates could be divided into two categories, non-nitrogen-containing and nitrogen-containing categories [76]. Bisphosphonates have traditionally been used in the clinic to prevent or inhibit the development of bone metastases or excessive bone resorption and for therapy of inflammatory diseases [77]. TAMs are a potential target of the bisphosphonates, which has been reviewed [78]. Moreover, bisphosphonates exert a range of direct and indirect anti-tumor effects, including inhibition of tumor cell proliferation and induction of tumor cell apoptosis, inhibition of tumor cell adhesion and invasion, anti-angiogenesis, synergism with anti-neoplastic drugs, and enhancement of immune surveillance (reviewed in [79]). Clodronate, currently used in the treatment of osteoporosis and bone metastasis, belongs to the drug family of bisphosphonates. Liposomes with encapsulation of clodronate act as an efficient reagent for selective depletion of macrophages. Indeed, these clodronate-liposomes, due to their big size, are rapidly recognized and engulfed by macrophages, resulting in the apoptosis of the host cells [80]. Notably, large, multilamellar liposomes containing clodronate have been developed and successfully applied in several cancer models, leading to the regression of tumor growth, angiogenesis and metastasis [81]. The benefits of macrophage depletion is not only seen with clodronate, but also with other bisphosphonates, of which zoledronate has been shown to be the most active [77]. Zoledronate is a third-generation nitrogen-containing bisphosphonate that has been shown to exhibit selective cytotoxicity to matrix metalloproteinase-9 (MMP9)-expressing TAMs, and to impair differentiation of myeloid cells into TAMs, which improves tumoricidal activity of macrophages [82].

Trabectedin (ET-743, Yondelis®) is a tetrahydroisoquinoline alkylating agent, originally extracted from a marine organism, the turbinate Ecteinascidia, but can now be produced synthetically [83, 84]. It has been approved in Europe and other countries as a second-line therapy for treating patients with advanced soft tissue sarcoma after failure of doxorubicin or ifosfamide and in relapsed platinum-sensitive ovarian cancers [85]. It binds the minor groove of DNA and can cause cancer cell death by inducing cell cycle arrest, blocking active transcription and inflicting DNA double-strand breaks [86]. Intriguingly, trabectedin specifically targets mononuclear phagocytes, including TAMs, by activation of the caspase 8 cascade via TNF-related apoptosis-inducing ligand (TRAIL) receptors [87]. Unlike other leukocyte subsets, monocytes that express very low levels of TRAIL decoy receptors are exquisitely sensitive to TRAIL [88]. Patients treated with trabectedin have reduced TAMs density correlated with a reduction in angiogenesis [87]. Taken together, depleting TAMs by stimulating their apoptosis may be of high therapeutic potential in limiting tumor development. Thus, macrophage destruction within the tumor is being further explored in the setting of preclinical models and clinical trials in different cancers.

Although depletion of TAMs could delay tumor progression, whether only immunosuppressive myeloid cells are targeted remain unclear. Likely, immunoprotective cells will also be depleted, causing severe adverse effects, such as bacterial infections. Hence, complete deletion approaches for myeloid cells are not feasible in the context of cancer.

In addition to directly depleting TAMs, it is also appealing to revert the activated state of the pro-tumorigenic TAMs into a quiescent state or even to induce them to acquire tumor-suppressive phenotypes. As discussed above, macrophages are functionally plastic, as the cells may respond differently to an alternation of molecules in the TME, including chemokines, cytokines, pattern recognition receptors and hormones [8]. Thus, to switch tumor-promoting M2-like TAMs to a tumoricidal M1-like phenotype by manipulating environmental stimuli is a potential strategy for tumor eradication. Tellingly, macrophages have been shown to be required for the efficacy of chemotherapy and immunotherapy [89, 90], which undermines the rational for depleting TAMs during cancer therapy. Alternatively, there are multiple strategies of reorienting rather than directly depleting TAMs for cancer therapy, which has recently been reviewed elsewhere [10, 11, 24], including targeting both the tumor cells (e.g., CD47 antibody) and the TAMs (e.g., CSF1/CSF1R blockades, TLR agonists, PI3Kγ inhibitors, CD40 agonists, and Class IIa histone deacetylase (HDAC) inhibitors). Hence, we will briefly review studies related to this strategy here (also described in Fig. 2).

TAMs express surface receptors that bind the Fc fragment of antibodies and enable them to engage in antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP), which are amenable to therapeutic strategies capitalizing on the effector function of TAMs [10]. Signal regulatory protein alpha (SIRPα) is an ITIM-bearing inhibitory receptor expressed on myeloid cells, including macrophages. It recognizes CD47, which acts physiologically as a “don’t eat me” signal and is found to be ubiquitously overexpressed on many different types of cancer. Interactions between CD47 and SIRPα prevent tumor cells from undergoing phagocytosis, allowing cancer cells to escape immune surveillance [91]. Masking of CD47 on tumor cells using monoclonal antibody or soluble SIRPα-Fc construct can trigger ADCP of tumor cells by TAMs [92, 93]. Enabling phagocytosis by macrophages in the tumor can lead to induction of effective immune responses against cancer [94]. However, enhancement of macrophage-dependent ADCP via interference with the inhibitory CD47-SIRPα pathway might involve mechanisms that lie beyond pure activation of TAMs effector function. ADCP elicited by targeting the CD47-SIRPα axis resulted in functional skewing of mouse macrophages towards an M1-like phenotype in tumor models, thus contributing to antitumor immune responses [95]. Of note, multiple therapies targeting the CD47-SIRPα axis are under preclinical and clinical investigation, including conventional antibodies, recombinant polypeptides, and bispecific molecules [96]. It should be noted, however, that engagement of macrophage-Fcγ receptors by therapeutic antibodies was shown to enhance the immunosuppressive, proangiogenic, and protumoral functions of TAMs [97]. Agents that enhance TAMs-mediated ADCP should potentially be used in combination with checkpoint blockade therapy [98], which could stimulate effective antitumor immunity.

CD40, a member of the TNF receptor family, is broadly expressed on many cell types, including APCs, DCs, B cells, macrophages, monocytes, as well as a number of nonhematopoietic cell types and some tumor cells [99]. When CD40 signaling is initiated and activated on APCs by the engagement of the ligand of CD40 (CD40L) expressed mainly on activated T helper cells, APCs release proinflammatory cytokines, and increase costimulatory molecules, such as CD80 and CD86, which may help support antitumor T cell activity [99]. To date, CD40 agonist antibodies show combinatorial efficacy in pancreatic cancer with gemcitabine, resulting in the regression of tumors by promoting antitumor macrophages that acquired antigen-presenting capabilities, re-establishing immune surveillance as well as prolonging patient survival [100, 101]. Clinical trials combining CD40 agonists and recombinant CD40Ls with chemotherapy, immunotherapy, vaccines, and angiogenic inhibitors are currently ongoing. In mouse models of cancer, CSF1R blockades synergize with the agonistic CD40 antibodies to remove inhibitory immune populations and to drive endogenous antitumor immune responses, resulting in improved tumor clearance and significantly lengthened overall survival [102]. Intriguingly, one study has suggested that TAMs depletion using CSF1R blockade diminished the antitumor activity of infused effector CD8⁺ T cells, whereas TAMs programming with agonistic CD40 antibody enhanced the accumulation and longevity of TCR-engineered cells [103]. Echoing the above observations, reprogramming TAMs to anti-tumor phenotype, rather than targeted ablation of the TAMs, may be the preferable therapeutic paradigm for cancer therapy.

Other strategies to activate TAMs for antitumor therapy include the targeting of phosphoinositide 3-kinase (PI3K)-γ, which is one of the four Class I PI3K p110 catalytic isoforms and is also activated in TAMs downstream of multiple pathways. Activation of PI3Kγ signaling selectively drives immunosuppressive transcriptional programing in macrophages that inhibits adaptive immune response and promotes myeloid cells invasion into tumors [104]. In mouse tumor models, pharmacological inhibition of PI3Kγ using IPI-549, a PI3Kγ-selective inhibitor, results in macrophage reprogramming, which reduced protumor macrophages while increasing antitumor macrophages and T cell responses [105]. Additionally, epigenetic reprogramming of macrophages via inhibition of histone deacetylases (HDACs), enzymes that regulate activity of many transcription factors, can also elicit T cell responses [106]. In mammary tumor models, TMP195, a selective Class IIa HDAC inhibitor, enabled TAMs populations within tumors to acquire antitumor phenotypes that support T cell responses [106].

The macrophage receptor with collagenous structure (MARCO), a member of the class A scavenger receptor family, is a nonopsonic phagocytic receptor mainly expressed by macrophages, dendritic cells and certain endothelial cells, wherein it is appreciated for its role in sensing and clearing pathogens though the recognition of pathogen-associated molecular patterns (PAMPs) [107]. Strikingly, mounting evidence has recently indicated that MARCO also plays important roles in regulating macrophage polarization [108, 109]. For instance, a study conducted on alveolar macrophages demonstrated that MARCO acts as an initial signaling receptor for asbestos and polarizes macrophages to a profibrotic M2 phenotype [108]. Additionally, MARCO is also expressed by a subpopulation of TAMs with an M2-like immunosuppressive gene signature in the TME of both murine tumor models and in human cancer. Treatment with antibodies targeting MARCO resulted in reduced tumor growth and inhibition of metastasis and a switch to a more proinflammatory macrophage phenotype [108]. Of note, MARCO is also a viable target for chimeric antigen receptor (CAR) T cell strategies for cancer therapy which has emerged as an important immunotherapeutic approach. CAR T cell therapy is a revolutionary milestone for the treatment hematological malignancies (such as B cell acute lymphoblastic leukemia) [110‐112]. However, its efficacy in the treatment of solid tumors still needs to be further improved. As MARCO supports proinflammatory signals, CAR T cells targeting MARCO have the potential to reinforce other immunotherapeutic modalities, including checkpoint blockade-based immunotherapy. Thus, further studies are needed to determine whether co-expression of two different CARs, one for TAMs and another for tumor cells, can enhance therapeutic effects of CAR T cell transfer therapy. All of these studies suggest that future cancer therapy should focus not only on cancer cells but also on TAMs by their reorientation or by selective depletion in the TME.

Complement is a central part of the innate immune system that acts as a first defense against pathogens and triggers release of inflammatory cytokines [113]. The physiological functions of complement are traditionally believed to defend against microbes and unwanted host molecules, to bridge innate and adaptive immunity, and to bind to immune complexes for complement-mediated lysis [114]. However, complement activation is also involved in many more inflammatory and immunological processes, including those that occur in the tumor context. Strikingly, complement has been implicated in the elimination of tumor cells by complement-fixing antibodies, paradoxically, complement-induced inflammation can also promote tumor progression [115]. The protumoral effect of the complement system is achieved through establishing an immunosuppressive microenvironment, promoting angiogenesis, modulating immune cells, and enhancing tumor cell proliferation, invasion and migration [115, 116]. Complement effectors such as C1q, anaphylatoxins C3a and C5a inhibit the antitumor immune response through the recruitment and/or activation of immunosuppressive cell subpopulations, including MDSCs, Tregs, or TAMs [115]. Macrophages express cognate receptors for both C3a and C5a on their cell surface, and specific binding of C3a and C5a affects the functional modulation and alters the TME and tumor immunity. Thus, the complement system has emerged as a target for cancer immunotherapy. In mouse model of squamous carcinogenesis, urokinase (uPA)⁺ macrophages release C5a in a C3-independent manner during premalignant progression. C5a regulates pro-tumorigenic properties of C5aR1⁺ mast cells and macrophages, resulting in immunosuppressive macrophage polarization and inhibition of CD8⁺ T cell activation [117]. While neither PMX-53 (a C5aR1 peptide antagonist) nor paclitaxel alone significantly altered tumor progression, a combination of the two synergized to effectively inhibit tumor growth by repolarizing TAMs towards the M1-like phenotype that not only affects angiogenic programs but also leads to recruitment of cytotoxic T lymphocytes [117]. Another study has also demonstrated that intracellular activation of complement C3 derived from tumor cells suppressed the infiltration and function of CD8⁺ cytotoxic T lymphocytes by promoting the accumulation and immunosuppressive activity of TAMs in a C3aR-dependent, rather than C5aR-dependent manner, resulting in activation of PI3Kγ signaling that mediates TAMs immunosuppressive activity [118]. Strikingly, blocking tumor-derived complement C3 is sufficient to enhance antitumor immunity [118]. Taken together, the complement pathway in the tumor setting might be potentially exploited as a target to reeducate TAMs for cancer therapy, but given the complex interactions of complement system in the tumor context, such therapy strategies should be developed carefully and with clearly outlined biological endpoints. Attempts to revert tumor-promoting TAMs or to alter their phenotype could provide new opportunities for the development of novel antitumor therapies.

A new generation of drug delivery methods, so called live cell-mediated drug delivery systems, consists in the use of the host cells (i.e. monocytes [119], macrophages [120], mesenchymal stem cells [121]), either as a whole or by employing selected key components of these cells, as ‘Trojan Horses’ loaded with drugs [119, 122]. Macrophages, professional phagocytic cells, are non-immunogenic, which endows them long blood-circulation times, and high phagocytic capability that enables them to extensively internalize and hold considerable drug loadings, both of which are prerequisites for drug delivery carriers [8, 22]. Moreover, macrophages are one of the most abundant types of circulating cells, which can be easily separated, loaded with drugs ex vivo, and reintroduced into the circulation. Of most importance, macrophages represent most of the leukocytes in tumors and account for up to 50% of cells in a tumor mass [8, 22]. Thus, the use of natural living macrophages as drug delivery carriers may be a potential strategy for cancer therapy.

Macrophages receive considerable interest as a drug delivery carrier due to their tropism to hypoxia and their ability to migrate and infiltrate into tumors. It is crucial that macrophages are alive in circulation to ensure their tumor-homing capacity and to unload the drug upon their arrival at tumor sites [123]. However, most antitumor drugs cannot be directly loaded into macrophages because their high cytotoxicity will quickly kill the cells. Alternatively, loading nanoparticles into macrophages may be a feasible approach. Inspired by the uptake of antitumor nanoparticles by macrophages, researchers have employed nanoparticles that can be taken by TAMs to combat cancer [124, 125]. Macrophage-mediated drug delivery systems designed with precisely controlled drug release when macrophages reach the target sites would minimize adverse effects of the loaded drug on the macrophages to reserve their functions, and maximize the drug loading content. Therefore, it is desirable to develop systems that take the advantages of both nanomedicine and macrophages in tumor-targeting [124]. Up to date, relatively few studies have assessed the in vivo antitumor efficacies of macrophage-mediated drug delivery systems. Choi et al. showed improved in vivo tumor accumulation of liposomal doxorubicin (Dox) when delivered by macrophages, exhibiting higher therapeutic efficacy in a subcutaneous tumor model than liposomal-Dox or Dox alone [120]. Because premature drug release in macrophages before their arrival at the target site will cause cell death or dysfunction, the precise control of the drug release in macrophage-mediated drug delivery system should be considered. A silica-based drug nanocapsule, consisting of a drug-silica complex filling and a solid silica sheath, may solve this issue [126]. When taken up by macrophages, the silica-based drug nanocapsule minimally release drug molecules in the early hours of cell entry, allowing macrophages to home to tumors and release drugs within tumors. Therapy with Dox-laden nanocapsule leads to efficient tumor growth suppression, while causing little systemic toxicity [126].

Although macrophages may truly carry “active” nanomedicines towards tumors, the filed is still in its infancy and many challenges remain. Of most importance, since macrophages may exhibit both protumoral and antitumoral activities in TME, it is important to fully understand the in vivo fate and functional state of these macrophages and ideally, to promote and maintain the anti-tumor functional state of the carrier macrophages to further maximize this cell-directed therapy. Further development of the technology for delivery of biologics, such as peptides or drugs that may enhance in vivo antitumor properties of these macrophages would greatly expand the utility of macrophage-mediated drug delivery system in cancer therapy. Finally, other selective drug delivery strategies using macrophages await further investigation. For instance, macrophages have been used to facilitate virus infection by providing a cell carrier for oncolytic virus delivery [127, 128]. Macrophage-based virotherapy was shown to mount efficient attack on tumors where the virus could infect tumor cells, achieving significant antitumor efficacy.