The tumor microenvironment (TME) promotes tumor development via complex intercellular signaling, aiding tumor growth and suppressing immunity. Eph receptors (Eph) and their ephrin ligands control cell interactions during normal development, and reemerge in tumors and the TME, where they are implicated in invasion, metastasis, and angiogenesis. Recent studies also indicate roles for Ephs in suppressing immune responses by controlling tumor interactions with innate and adaptive immune cells within the TME. Accordingly, inhibiting these functions can promote immune response and efficacy of immune checkpoint inhibition. This research highlights Ephs as potential targets to enhance efficacy of immune-based therapies in patients with cancer.
Eph receptors (Eph) make up the largest family of receptor tyrosine kinases (RTK), with 14 members distinguished into two types, A and B, dependent on sequence similarity and their preferential binding to type A and type B ephrin ligands, respectively. Ephrin subtypes are defined by their mode of membrane anchoring: ephrin-As are inserted into the outer lipid bilayer via a glycophosphatidylinositol anchor, whereas ephrin-Bs have transmembrane and intracellular domains, capable of signaling via protein–protein interactions. Binding of ligand and receptor across cell–cell boundaries can thus initiate bidirectional signaling from both receptor (“forward signaling”) and ligand (“reverse signaling”). Eph–ephrin interactions lead to a myriad of signaling outcomes via phosphotyrosine-dependent and -independent protein–protein interactions. As reviewed previously, Ephs can regulate cell differentiation, proliferation, and survival, and most prominently control cell–cell adhesion and migration via cytoskeletal signaling, where strength of tyrosine kinase activity modulates dichotomous outcomes of adhesion and deadhesion/repulsion (1).
The functions of Ephs are most prominent during embryonic development, where they guide cell movement and attachment during gastrulation, somitogenesis, tissue and organ boundary formation, axon pathfinding, cardiovascular development, and other developmental processes. Ephs are generally less active in adult tissues, but continue to play important roles in organ maintenance and regeneration, synaptic plasticity, nerve and vascular remodeling, and response to injury (2). They also regulate cell fate, and are associated with stem cell maintenance, migration, differentiation, and proliferation, which are deregulated during tumor progression (2, 3). Ephs are important for maintenance and migration of tumor “stem” or initiator cells in glioblastoma multiforme (GBM; ref. 4), colon (5), melanoma, and lung cancer (6). Both overexpression and loss of expression can occur in different tumor types, and even in the same tumor at different stages, as is well established for EphB2 and B4 in colon cancer (7), reflecting the complexity of Eph functions in tumor development.
In addition, Ephs are important players in the immune system, in both innate and adaptive immune cells, where they are thought to play multiple roles during development, trafficking, and activation (8, 9). Indeed, both A- and B-type Ephs are widely expressed on diverse immune cell types, including platelets, monocytes, macrophages, and dendritic cells (DC), where they regulate adhesion, extravasation, and trafficking; and on B and T lymphocytes, where they have reported roles in adhesion, migration, and activation (9). EphB2/4 and ephrin-B2 mediate interactions between hematopoietic stem cells (HSC) and stromal cells in the bone marrow, and regulate HSC mobilization and differentiation (10, 11). The diverse functions of Ephs in immune cells have importance not only in normal development, but also in immune-based diseases, such as atherosclerosis and fibrosis, and in cancer. Indeed, recent studies point to important roles of Ephs in tumor immunity, through regulating recruitment of normal cells to the tumor microenvironment (TME), which can support tumor survival. These cells include mesenchymal stromal cells (MSC) and innate immune cells, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). Collectively, these cell types can be coopted by tumors to take on immunosuppressive roles, inhibiting the influx and activity of cytotoxic T cells (12). While roles of Ephs in immune cells and in tumor development have been extensively reviewed previously (1–3, 6, 8, 9), this review highlights recent work showing emerging roles of Ephs and ephrins in influencing immune suppression in the TME.
Eph Interactions in the Immune TME
Tumor intrinsic Eph expression
In terms of tumor immunology, Ephs have received most attention as tumor neoantigens, owing to their generally low expression in adult tissues and upregulated expression in many malignancies. EphA3 was the first receptor identified as a tumor antigen, in lymphoblastic leukemia cells, and independently in melanoma cells from a patient with an EphA3-reactive T-cell immune response. Subsequently, EphA2 has received most attention as a tumor antigen, as it is more widely expressed on tumor cells (reviewed in ref. 8). EphA2 peptides produced CD8+ T-cell immune reactivity in vitro, or when used for DC-based vaccines in mice, where they decreased tumor growth. Although spontaneous EphA2 T-cell reactivity has not been reported in humans, CD4+ and CD8+ T cells from patients with EphA2-expressing renal cell carcinoma show increased immune reactivity to EphA2 peptides compared with T cells from normal donors (13). EphA2 peptides are being explored along with other tumor antigens as DC vaccines in melanoma (14) and GBM (15).
In contrast, a number of studies link Ephs expression on tumor cells or tumor-infiltrating cells with suppression of antitumor immune response. An important study from the Weinberg laboratory linked EphA4 receptor expression on cancer stem cells (CSC) with the innate immune system, through mediating cell–cell interactions of CSCs and TAMs (Fig. 1A; ref. 16). The authors identified EphA4 as a key protein upregulated during epithelial–mesenchymal transition of mammary epithelial cells, a process that confers stem cell–like properties on tumor cells, and is associated with increased malignancy, invasiveness, and resistance to therapy. These observations are consistent with other work showing mesenchymal expression of Ephs during normal and oncogenic development (2), including in GBM, where EphA3 expression on CSCs is primarily associated with mesenchymal tumor subtypes (4). In mammary CSCs, Lu and colleagues (16) found preferential expression of CD90, a protein that interacts with integrins on adjacent cells, and is found on fibroblasts, mesenchymal, and progenitor cells, including CSCs and MSCs. The CD90/EphA4-high cells were localized around the rim of breast tumors in mouse models, and associated with a high density of infiltrating TAMs. The latter expressed markers indicative of so-called M2-polarized TAMs, known to correlate with poor prognosis and mediate suppression of antitumor immune responses (17). Indeed, coinjection of purified TAMs with CD90-expressing tumor cells led to increased tumor growth, and induced cytokine production in the CSC compartment via activation of EphA4, possibly resulting from EphA4 binding to A-type ephrins expressed on the TAMs (16). This led to increased tumor cell proliferation, via activation of Src kinase, phospholipase Cγ1, protein kinase C, and NF-κB, and production of IL6, IL8, and GM-CSF. In comparison, CD90-negative tumors were necrotic and fluid-filled, suggesting CD90/EphA4-mediated CSC–TAM interactions also inhibited antitumor immune responses, although this was not investigated (16).
More recently, Markosyan and colleagues showed that tumor intrinsic EphA2 expression in pancreatic cancer cells controls immune suppression, by maintaining exclusion of T cells (Fig. 1B; ref. 18). The authors found that EphA2 is the highest expressed Eph in pancreatic tumors, where expression correlated with low T-cell infiltration. Knockout (KO) of the EphA2 gene in pancreatic cancer cells in a genetic (KPCY) mouse model resulted in increased CD8+ and CD4+ T-cell abundance in the tumors, while simultaneously reducing MDSCs and TAMs. Tumors comprised of EphA2KO cancer cells also showed increased sensitivity to a combination of chemo- and immunotherapy. Transcriptional profiling of EphA2KO tumors revealed increased IFN response and inflammatory pathways, including STAT1, underpinning the elevated immune response. The TGFβ/SMAD signaling pathway, and its downstream target Ptgs2/cyclooxygenase-2 (COX-2), were reduced in EphA2KO tumors. Furthermore, TGFβ treatment induced EphA2 expression in tumor cells, indicating a positive feedback loop. This observation supports previous reports of high COX-2 expression in pancreatic cancer cells and stromal cells, and its regulation by TGFβ signaling (19, 20). Ptgs2 deletion from tumor cells resulted in slower growing tumors and correlated with decreased EphA2 expression, and increased infiltration of CD8+ T cells. Conversely, Ptgs2 overexpression decreased T-cell infiltration and activation, and increased myeloid cell infiltrates and resistance to immunotherapy. This is consistent with the known role of COX-2 in production of prostaglandins, such as PGE2, which drive MDSC proliferation and recruitment (20). Pharmacologic inhibition of COX-2 decreased tumor growth, enabled tumor infiltration of T cells, and sensitized tumors to immunotherapy. While high EphA2 expression was clearly associated with increased TGFβ/Ptgs2 signaling and immune suppression, EphA2 kinase activity, or a role for ephrins on tumor-infiltrating cells, remained unexplored. Indeed, ephrin-dependent signaling may not necessarily be required, as increased Eph expression can promote tumor growth in the absence of significant ligand expression or tyrosine kinase activity (2).
Cell–cell contact–dependent expression of EphA2, EphA4, and EphA10 in breast cancer cells in culture also coincided with increased expression of the immunosuppressive protein PD-L1, which binds to programmed cell death protein 1 (PD-1) on T cells, to inhibit inflammatory activity and promote tumor tolerance (Fig. 1C; ref. 21). Surprisingly, an RTK array suggested all three Ephs were tyrosine phosphorylated, including EphA10, which is kinase dead, indicating possible association with another phosphoprotein, and/or the previously described cross-activation by another Eph (1). Individual KO or knockdown of EphA2, EphA4, or EphA10 from breast cancer cells suggested that only EphA10 was required for PD-L1 upregulation. Consistent with this, EphA10 deletion from mouse 4T1 breast cancer cells resulted in reduced growth as syngeneic mammary tumors, coinciding with significantly increased CD8+ T cells, T-cell activation, and tumor cell apoptosis. The decreased growth of EphA10KO tumors was dependent on immune response, as it was not observed in immune-deficient mice (21). Importantly, expression of EphA10 and PD-L1 also positively correlated in breast tumor samples. The molecular mechanism(s) linking EphA10 and PD-L1 expression is yet to be defined, but warrants further investigation, as would approaches to block this function of EphA10 as a potential therapy.
Interestingly, EphA3 was recently identified as a binding partner for PD-L1 in a high-throughput screen for transmembrane receptors binding to proteins of the immunoglobulin superfamily (22). This interaction was verified by binding of PD-L1 to cells overexpressing EphA3, and by protein–protein binding in vitro, showing moderate binding affinity, which was competed by the therapeutic anti-PD-L1 antibody, atezolizumab. While the interaction or its functional relevance was not directly shown in tumors, gene expression analysis of urothelial cancer specimens showed EphA3/PD-L1 coexpression was associated with a CD8 T effector cell signature, as was overall PD-L1 expression. Aside from PD-L1, overall EphA3 expression was high in immune-excluded tumors, and its coexpression with ephrin-Bs correlated negatively with a T effector cell signature and patient survival, as did coexpression of ephrin-Bs with EphB3, 4, and 6, while ephrin-B2/EphA10 coexpression indicated good prognosis (22). This suggests Eph expression can have both positive and negative effects on antitumor immune responses, depending on the context and the expression of binding partners, and highlights the need for determining their relative expression in cell subtypes in tumors and the TME.
Ephs and tumor-infiltrating cells
In addition to tumor cells, Ephs have also been identified on stromal and myeloid cells in tumors (summarized in Fig. 1D). These untransformed cells of the TME can be coopted by tumors to promote proliferation, angiogenesis, metastasis, and immune evasion (12). MSCs are multipotent and share many features in common with cancer-associated fibroblasts, stromal cell types known for their capacity to promote tumor progression (23). MSCs are often found in perivascular regions, and support blood vessel formation by secretion of angiogenic factors (24, 25). EphA3 was shown to be expressed on stromal fibroblasts and perivascular cells in many human solid malignancies (26). This was also evident in prostate and colon xenografts in mice, where EphA3-expressing MSCs trafficked to tumors from the bone marrow. Coinjection of EphA3-positive MSCs with tumor cells promoted tumor growth, and treatment with an anti-EphA3 antibody inhibited growth, and disrupted the stroma and vasculature, establishing an important role for EphA3 in the TME (26). Similarly, EphA2 is overexpressed in stromal cells associated with gastric tumors, and is identified as an independent prognostic factor for relapse in patients (27). Proteomic and PCR analysis of these cells also showed overexpression of EphA1/3/5 and EphB2/4, suggesting roles for multiple Ephs in tumor stromal cells. In addition to supporting angiogenesis, MSCs can promote tumor growth by regulating infiltration and/or activation of innate and adaptive immune cells, including TAMs, MDSCs, neutrophils, and T cells (28, 29). Indeed, EphB2 and ephrin-B2 on human bone marrow–derived MSCs can bind ephrin-B1 and EphB4, respectively, on T cells, and inhibit T-cell proliferation, by inducing production of immunosuppressive factors and reducing activating cytokines (30). Whether these insights gained from in vitro models also have implications in the TME remain to be established.
Ephs are also found on innate immune cell types in tumors. EphA3 has been detected on TAMs in prostate and colon models (26), and in GBM (31), although its functional role has yet to be demonstrated. One possible function for Ephs in these cells is for their mobilization from the bone marrow into the circulation. This was shown for trafficking of hematopoietic stem and progenitor cells (HSPC) and other myeloid cells in murine models of melanoma and mammary cancer, where interaction between EphB4 in bone marrow sinusoids and ephrin-B2 in HSPCs controls mobilization of the HSPCs into the circulation (10). HSPC trafficking from bone marrow to tumor could be inhibited by blocking the ephrin–Eph interaction with peptides or antibodies, or silencing of EphB4 expression, leading to inhibition of tumor growth (10). Interestingly, expression of Ephs on TAMs, as well as on tumor cells and infiltrating lymphocytes was also detectable in ascites from patients with ovarian cancer, where elevated EphA1/2 and EphB3 correlated with worse prognosis (32).
EphA4 expression in the TME is also important for growth and metastasis of syngeneic mouse mammary 4T1 tumors. As distinct from the study described earlier showing an important role for EphA4 on tumor cells, delayed development of 4T1 breast tumors grown in EphA4-deficient mice indicate an important role for EphA4 also in the TME (33). 4T1 tumors have a large myeloid cell infiltrate and produce G-CSF, a cytokine that stimulates myeloproliferation, and can induce extramedullary hematopoiesis and splenomegaly, and ultimately results in tumor infiltration of MDSCs. EphA4 deletion reduced production of G-CSF and its effects, including on MDSCs. The authors had previously shown that EphA4 can enhance IGF1 production via JAK-STAT activation (34) and, consistent with this, IGF1 levels were lower in EphA4KO mice, and IGF1 treatment overcame the effects of EphA4 deletion. Further details of EphA4 function, and the TME cell type/s on which it is expressed, were not determined, but warrant further investigation.
EphB4 also regulates both innate and adaptive components of the immune system in head and neck cancers. A recent study identified EphB4 and ephrin-B2 both on tumor cells and some stromal cells, with higher expression of EphB4 particularly on TAMs and FoxP3-expressing regulatory T cells (Treg), which like M2-TAMs can have an immunosuppressive role in tumors (35). Using a murine orthotopic head and neck squamous cell carcinoma model, inhibition of EphB4–ephrin-B2 interaction with a blocking peptide reduced recruitment of Tregs and M2-TAMs to tumors. In contrast, the number of proinflammatory M1-TAMs significantly increased following EphB4 blockade, as did activation of both CD4+ and CD8+ T cells. Accordingly, blocking EphB4 on CD4+ T cells in vitro resulted in decreased STAT3 activation and proliferative markers in Tregs. Tumor growth was significantly inhibited by EphB4 blockade, alone or in combination with radiation. Interestingly, combining EphB4 blockade with radiotherapy significantly decreased the levels of macrophage colony-stimulating factor, and increased both GM-CSF and IFNγ, consistent with promoting TAM polarization toward a proinflammatory, antitumor phenotype.
Ephs have long been recognized as tumor neoantigens, regulating tumor cell stemness, invasion, and angiogenesis, reflecting their roles during normal development. Emerging evidence now also indicates their roles in tumor–microenvironment interactions extend to regulation of tumor immunity. While studies show a range of mechanisms for Ephs expressed both on tumor cells and on “host” cells in the TME, a common theme lies in their regulation of innate immune cell infiltrate, often favoring an immunosuppressive TME, and inhibiting cytotoxic T-cell responses. Targeting of Ephs, for example, by small-molecule- or antibody-based therapies (2, 36), may thus provide a novel approach to modulate tumor immunity. Given the clinical success of immune checkpoint inhibitors, but only in a limited proportion of patients, further investigation of the roles of Ephs in regulating cancer immune suppression may provide additional approaches to enhance immune-based cancer therapy.
P.W. Janes reports grants from Australian National Health and Medical Research Council and Cancer Council of Victoria during the conduct of the study. A.M. Scott reports grants from National Health and Medical Research Council and Cancer Council Victoria, other funding from KaloBios, Humanigen, and Daiichi Sankyo during the conduct of the study, and AbbVie, EMD Serono, ITM, Telix, Cyclotek, Medimmune, AVID, AdAlta, and Theramyc, personal fees from Life Science Pharmaceuticals and Imagion Bio outside the submitted work, as well as has a patent for EphA3 licensed to Humanigen. No disclosures were reported by the other authors.