Abstract

Introduction

Macrophages are represented in all tissues by functionally and phenotypically distinct resident populations that are critical for development and homeostasis (Wynn et al., 2013). Under non-pathological conditions, most resident macrophage populations derive from embryonic progenitors and are maintained through local proliferation (Epelman et al., 2014). Exceptions to this include intestinal, dermal and alveolar macrophages at barrier sites (Bain et al., 2014; McGovern et al., 2014; Perdiguero et al., 2014; Yona et al., 2013), and macrophages in the adult heart that are replaced by circulating bone marrow-derived Ly6C⁺ inflammatory monocytes over a time scale of several weeks (Molawi et al., 2014). Under pathological conditions, there is evidence for both local proliferation and recruitment, with differences observed by tissue location and type of inflammatory insult (Epelman et al., 2014).

Solid tumors appear to be unique; preclinical studies indicate absence of macrophage proliferation and shorter half-lives as compared to resident macrophages in counterpart homeostatic tissues, measurable in days to weeks (Movahedi et al., 2010; Strachan et al., 2013). That said, proliferating CD68⁺ cells, also positive for proliferating cell nuclear antigen (PCNA) expression, have been observed in breast cancers where they are associated with poor clinical outcome (Campbell et al., 2011). Whether macrophage life span in this context is reflecting diminished tissue integrity, extent of damage/inflammation, or instead represents an adaptive process engaged by tumors to support growth is unclear, but production of the C-C chemokine ligand 2 (CCL2) and/or colony stimulating factor-1 (CSF-1) are necessary to sustain their numbers (Noy and Pollard, 2014). With the critical role for CCL2 and CSF-1 in recruiting macrophages to neoplastic tissue there is growing interest in therapeutics targeting these ligands and/or their respective receptors in an effort to ablate pro-tumorigenic properties of macrophages. This therapeutic approach has led to improved outcomes in a range of pre-clinical models — particularly for agents targeting CSF-1 or the CSF-1 receptor (CSF-1R) — results of which have spurred several clinical trials (Table 1).

As monotherapy, CSF-1R inhibition alone impedes growth of orthotopically implanted pancreatic ductal adenocarcinoma (PDAC) cell lines (Mitchem et al., 2013), prevents cervical carcinogenesis (Strachan et al., 2013), and induces regression of glioblastoma multiforme (GBM) (Pyonteck et al., 2013). In other tumor models, CSF-1R inhibition is without consequence as monotherapy; however, synergism with other modalities, including chemotherapy (DeNardo et al., 2011; Mitchem et al., 2013; Paulus et al., 2006; Ruffell et al., 2014), radiation therapy (Shiao et al., 2015; Xu et al., 2013), angiogenic inhibitors (Priceman et al., 2010), adoptive cell transfer (Mok et al., 2014), and immune checkpoint blockade (Zhu et al., 2014) have been revealed. Together, these findings implicate macrophages in regulating therapeutic responses, and indicate that durable responses may be more likely by augmenting standard-of-care or emerging therapies with “macrophage antagonists”. This review will focus on the mechanisms underpinning these observations, and conclude with a discussion of targeting approaches that extend beyond inhibiting macrophage recruitment.

Clinical Significance of Macrophages

For many solid tumor types, high densities of cells expressing macrophage-associated markers have generally been found to associate with poor clinical outcome (Figure 1) (Komohara et al., 2014; Zhang et al., 2012). There is conflicting data for lung, stomach, prostate and bone, where both positive and negative outcome associations have been reported (Zhang et al., 2012), possibly related to the type/stage of cancer evaluated, (e.g., Ewings sarcoma versus osteosarcoma) (Buddingh et al., 2011; Fujiwara et al., 2011), or to the type of analysis performed (e.g. quantitation of stromal versus intratumoral macrophages). Some discrepancy may also reflect use of different macrophage markers. CD68, a glycoprotein predominantly resident in intracellular granules, represents a fairly specific marker for murine macrophages, and in combination with F4/80, identifies a majority of tumor-associated macrophages. In humans however, CD68 expression is widespread, and includes granulocytes, dendritic cells, fibroblasts, endothelial cells, and some lymphoid subsets (Gottfried et al., 2008; Hameed et al., 1994; Ruffell et al., 2012b); use of CD68 for association studies in this context is thus of variable utility. A clear example of this is non-small cell lung cancer, where detection of macrophage scavenger receptors CD163 and CD204, but not CD68, yielded clear correlations with negative outcome (Chung et al., 2012; Hirayama et al., 2012; Ohri et al., 2011; Quatromoni and Eruslanov, 2012).

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Figure 1

Clinical implications of macrophage density

Organs where tumor progression and/or clinical outcome are negatively associated with increased macrophage density are shown in red. Green indicates a positive association. Organs where the implications of macrophages density are unclear or unknown are shown in gray and black, respectively. Image created by Tarot Walker.

In addition to potentially representing more selective macrophage biomarkers, both CD163 and CD204 are associated with activation of macrophages towards an alternative or tumor-promoting and immunosuppressive phenotype, and accordingly, significant correlations between CD163/CD204 and negative outcomes have been reported across multiple tumor types (Komohara et al., 2014). This correlation may indicate that macrophage polarization can direct clinical outcome, also supported by the positive association between presence of CD68⁺ cells and survival in colorectal adenocarcinoma (Roxburgh and McMillan, 2012). Unlike most populations of tumor-associated macrophages that possess pro-tumor and immunosuppressive properties (Biswas and Mantovani, 2010), macrophages in human colorectal cancer have been found to be functionally (and phenotypically) anti-tumor (Edin et al., 2012; Ong et al., 2012; Zhang et al., 2014). Together, these data collectively support the tenet that repolarizing macrophages towards an anti-tumor phenotypic state, either by impeding activities/signals that drive pro-tumor polarization, or delivering exogenous signals that enhance anti-tumor polarization, could act as an alternative and perhaps more efficacious approach to blocking macrophage recruitment, even though these activities and responses are all dynamically regulated in vivo. Indeed, an agonist monoclonal antibody against CD40, a co-stimulatory protein found on professional antigen-presenting cells, has demonstrated efficacy in mouse models of pancreatic ductal adenocarcinoma (PDAC) (Beatty et al., 2011) and patients with PDAC (Beatty et al., 2013) when delivered in combination with the chemotherapeutic gemcitabine, ostensibly via the anti-tumor activities of macrophages and CD8⁺ T cells (Vonderheide et al., 2013). In addition to use of an anti-CSF-1R depleting antibody in diffuse-type giant cell tumors (Ries et al., 2014), these are the first clinical studies to demonstrate potential efficacy of macrophage-targeted agents.

Polarization and Macrophage Function

Macrophages produce an array of cytokines, chemokines, polypeptide growth factors, hormones, matrix remodeling proteases and metabolites, many of which possess tumor-promoting activities (De Palma and Lewis, 2013; Noy and Pollard, 2014; Ruffell et al., 2012a). A caveat to some of these reported activities is that many findings originate from cell culture studies utilizing neoplastic myeloid cell lines or bone marrow-derived macrophages, and thus cannot account for the complex milieu of polarization signals that macrophages would be exposed to in vivo (Figure 2). This includes the aforementioned CSF-1 and CCL2, prostaglandin E₂ (PGE₂), and damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 protein (HMGB1), extracellular ATP, and degraded extracellular matrix components (Ruffell et al., 2012a; Zelenay and Reis e Sousa, 2013).

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Figure 2

Macrophage polarization as a dynamic system

The integration of multiple signals emanating from the tumor microenvironment (outer circle) dictates the functional role of macrophages (inner circle). Integrins and toll-like receptors (TLRs) will be engaged by multiple ligands.

Stabilization of hypoxia inducible factor (HIF)-1α and -2α are also important in mediating the pro-tumor properties of macrophages, as evidenced from the use of LysM-cre mice to induce myeloid-specific loss of either factor (Doedens et al., 2010; Imtiyaz et al., 2010). As might be expected, hypoxic conditions drive an angiogenic phenotype in macrophages, and in vivo this occurs specifically in a subpopulation of macrophages found within hypoxic regions of tumors that express low levels of major histocompatability complex (MHC) II (Laoui et al., 2014; Movahedi et al., 2010). The recruitment of macrophages (presumably MHCII^LO) into hypoxic regions through Neuropilin-1 also supports an immunosuppressive phenotype (Casazza et al., 2013) that is likely dependent upon HIF-1α (Doedens et al., 2010). Surprisingly however, while hypoxia can induce HIF-1α-dependent expression of arginase-1 in macrophages (Doedens et al., 2010), neither improving tumor oxygenation, nor preventing macrophage recruitment into hypoxic areas alters arginase-1 expression (Casazza et al., 2013; Laoui et al., 2014). This discrepancy might be explained by the recent finding that lactic acid promotes arginase-1 expression by macrophages in a HIF-1α-dependent manner (Colegio et al., 2014).

Finally, the use of immune competent murine models has firmly established that macrophage polarization and function within tumors is strongly influenced by lymphocytes through the production of multiple factors including interleukin (IL)-4, IL-10, IL-13, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and immunoglobulins (Andreu et al., 2010; DeNardo et al., 2009; Gocheva et al., 2010; Guiducci et al., 2005; Kang et al., 2011). These activities include mediating responses to therapy, as B cells coated in anti-CD20 antibody can suppress their phagocytic removal from circulation by Kupffer cells through the secretion of IL-10 (Horikawa et al., 2011), while B cell production of immunoglobulins (Affara et al., 2014) and CD4⁺ T cell expression of IL-4 (Shiao et al., 2015) suppress responses to cytotoxic therapy by altering macrophage polarization. Even the efficacy of CSF-1R inhibition depends upon altered macrophage polarization, rather than depletion, in certain models (Pyonteck et al., 2013).

Macrophage Function and Therapeutic Resistance

Macrophages as Therapeutic Targets

Based on compelling preclinical data from numerous laboratories indicating that macrophage presence and/or activity are malleable in vivo (Figure 3), clinical studies are now ongoing in several solid tumor types wherein macrophages are being targeted via CSF-1R inhibitors or blocking monoclonal antibodies (Table 1). While a goal of these clinical studies is to reduce the presence of tumor-associated macrophages, based on preclinical and clinical studies, we anticipate that not all macrophages will be eradicated. The hope is however, that those remaining will be reprogramed towards an anti-tumor phenotypic state wherein they would support T cell responses, and together with cytotoxic therapy, limit ongoing tumor growth. CSF-1R antagonists appear well tolerated as single agents in both pre-clinical and clinical studies (Radi et al., 2011; Ries et al., 2014; Ruffell et al., 2014), but with that significant macrophage depletion in the colon and liver is observed in nonhuman primates, toxicity is a significant concern for combinatorial studies moving forward. It should also be noted that while increased CSF-1 serum concentrations resulting from the use of CSF-1R antagonists (Ries et al., 2014) provides an excellent biomarker to evaluate on target efficacy, recent findings with CCL2 inhibition (Bonapace et al., 2014) indicate that recurrence or exacerbation of disease is a possibility after therapy cessation. These potential issues will need to be incorporated into the design of clinical studies for appropriate drug combinations and patient monitoring.

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Figure 3

Macrophage function in the tumor microenvironment

(a) Macrophage expression of IL-6 and TNF-α promotes survival signaling in neoplastic cells and resistance to chemotherapy and targeted agents. Expression of survival factors is dependent upon the protease activity of cathepsin B and/or S. (b) Neoplastic cell invasion of ectopic tissue can be promoted through directed release of cytokines/chemokines such as EGF and CCL18, or through protease-dependent ECM remodeling that may directly affect neoplastic migration or increase chemoattractant bioavailability. EGF expression is driven by signaling through the CSF-1R via neoplastic cell production of CSF-1, as well as T cell-derived IL-4 (not shown). (c) Macrophages directly promote angiogenesis via production of VEGFA and other angiogenic factors, and can enhance VEGFA expression by endothelial cells through WNT7B. A subset of macrophages expressing the Tie2 receptor are recruited to the vasculature by mural cell/pericyte expression of ANG2 and are important in regulating vascular structure. (d) Direct suppression of a cytotoxic T cell (CTL) response can occur via expression of B7 family ligands (PD-L1, B7-H4). Indirect suppression may occur through release of IL-10 or recruitment of IL-10-expressing regulatory T cells (T_Reg) via CCL22, whereby IL-10 suppresses the capacity of dendritic cells to produce IL-12 and promote a T_H1/CTL anti-tumor immune response.

Blocking macrophage recruitment into tumors (DeNardo et al., 2011; Shiao et al., 2015), pro-tumor polarization (Affara et al., 2014; Pyonteck et al., 2013; Shiao et al., 2015) effector function (Ruffell et al., 2014; Shree et al., 2011), or directly promoting macrophage activation (Beatty et al., 2011), have all been used successfully in preclinical models to enhance response to cytotoxic therapy. The question remains, which of these approaches (Table 1) will be most efficacious when combined with cytotoxic, targeted, or immune checkpoint blockade therapy. At least in a murine model of squamous cell carcinogenesis, repolarizing macrophages was more effective than blocking recruitment, and in fact, repolarized macrophages were necessary for recruitment of CD8⁺ T cells via CCR5 during paclitaxel chemotherapy (Affara et al., 2014). For this reason, going forward it will be critical to understand if depletion, or instead repolarization is the “best” therapeutic approach to accompany combination therapy, for which tumor types, and at which stage of tumor progression (primary or metastatic disease). Multiple agents targeting T_H2 cytokines and their receptors have gone beyond Phase II clinical trials with demonstrated efficacy in autoimmune disorders and acceptable safety profiles (Beck et al., 2014; Corren et al., 2011; Danese et al., 2015). Although none of these compounds have been re-directed towards therapy in solid tumors, we have recently found that targeting this pathway (IL-4, IL-13, IL-4Rα) improves response to cytotoxic therapy (Shiao et al., 2015).

One lesson learned from results comparing efficacy of immunotherapy in mice bearing orthotopic versus subcutaneously implanted tumors is that the later exhibit enhanced sensitivity (Devaud et al., 2014), indicating that context matters. In light of these findings, and given the dearth of late-stage metastatic studies, it will be important to be mindful of the fact that a one size-fits-all approach, while attractive, may not be realistic as we strive to translate preclinical findings to the clinic. A major question to address from these preclinical and clinical studies with either macrophage depletion or reprogramming approaches will be to evaluate the durability of resultant anti-tumor immune responses formed against the tumor. Along these lines, evaluating patients longitudinally for indicators of T cell function could reveal an important diagnostic opportunity whereby monitoring circulating T cell receptor diversity becomes a routine diagnostic strategy to indicate when addition of combination therapy, e.g., immune checkpoint or anti-tumor vaccine, might be beneficial to the patient.

Acknowledgments

Footnotes

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