The leukocyte infiltrate is a key component of the cancer stromal compartment. Within the tumor, the wide range of chemokines produced by both malignant and stromal cells can affect the composition and the phenotype of the cell infiltrate, and influence tumor growth, survival and metastasis (1–5). Chemokines that regulate leukocyte migration and play key roles in both physiology and pathological conditions (6–8), are small proteins of 8–12 KDa, which can be divided into 4 groups (CCL-, CXCL-, CX3CL1, and XCLs chemokines) according to the position of two conserved cysteine residues within their structure. The chemokine system is characterized by a set of almost 50 ligands, which engage in a promiscuous fashion a panel of more than 20 chemokine receptors, including conventional and atypical receptors, expressed by immune cells, endothelial cells and cancer cells (9–12). The promiscuous pattern of interaction, together with the large number of ligands and receptors, enables the chemokine system to mediate a variety of cell functions. This is of particular relevance in tumors, since chemokines can influence angiogenesis, cell-adhesion, cell extravasation, and survival (7). Different chemokines can also interact together showing antagonistic or synergistic activity on specific chemokine receptors. They can trigger simultaneously different receptors, resulting either in the inhibition or in the enhancement of the intracellular cell signaling (13, 14), or a single receptor can be activated by a heterocomplex formed between two chemokines, resulting in a stronger cellular response (13, 15). Additionally, chemokines can also interact with inflammatory mediators released in the microenvironment, amplifying cellular responses induced by chemokine receptors (16, 17).

While the multiple roles of heterocomplexes in the early stage of inflammation and in regeneration have been clearly dissected (13, 18, 19), little is known about their functions in tumors (20), and further studies are necessary to define their significance. Von Hundelshausen and colleagues have performed a thorough study mapping the chemokine heterocomplexes by bidirectional immunoligand blotting (21). This study opens the debate on the in vivo relevance of the multitude of heterocomplexes found in vitro. In the present review, we discuss examples on how the concomitant expression of several chemokines with either anti- or pro-tumor functions could favor heterocomplexes formation in the tumor microenvironment (TME), thus adding an additional feature to be considered in tumor immunity.

Several studies in the last decade have described the heterodimerization between chemokines as a regulatory mechanism that governs their activity under inflammatory conditions. In the TME, chemokines play crucial roles either favoring immune responses against the tumor or promoting cancer progression and metastasis. Of note, similar chemokine expression profiles can result in a different tumor-specific leukocyte infiltrate. This phenomenon suggests that additional regulatory mechanisms might be involved, including the release of proteins able to modify chemokine activity. It is now well established that a chemokine receptor can be triggered by a low concentration of its selective agonist when a synergy-inducing chemokine, not selective for the receptor but able to form a complex with the agonist, is concomitantly present (17, 22, 23) (Table 1). The first evidence of the synergism induced by the presence of two chemokines was provided by Struyf and colleagues (24), who described the synergy between Regakine-1 and CXCL8, and between Regakine-1 and CCL7. Few years later, the same group has shown that CXCL8 can enhance CXCL12 responses and this enhancement is CXCR4 mediated (25). In 2005, three groups described the formation of heterocomplexes between chemokines, able to enhance the activity of CCR7, CCR4, CCR5, and CXCR2 (23, 26, 29, 30). In particular, CXCL13 forms a complex with CCL19 and CCL21, leading to CCR7 activation at lower agonist concentrations (23). The CXCR3- and CCR4-agonists, CXCL10 and CCL22, co-expressed in the inflamed skin, synergistically interact together, through the first β-strand of CCL22, enhancing CCR4-mediated chemotaxis of T cells, independently from CXCR3 or GAGs binding (29). Other studies showed that the heterocomplex between CCL5 and CXCL4, formed through heterophilic interactions, plays a crucial role in triggering monocyte arrest on the endothelium (30). In this case, the authors demonstrated the requirement of GAGs on the cell surface, and that the CCL5/CXCL4 complex showed paired N-terminus, resembling a CC-type heteromer that promoted a more efficient receptor activation (30, 31). NMR spectroscopy and molecular modeling, followed by in vitro analysis, have shown the structure of a heterocomplex between CXCL8 and CXCL4. This complex was shown to enhance the anti-proliferative effect of PF4 on endothelial cells, and the CXCL8-induced migration of CXCR2 transfected cells (26).

Later on, the CCR7-agonists, CCL19 and CCL21, were described as enhancer of monocytes recruitment by forming heterocomplexes with CCL7 and CCL2, resulting in an augmented CCR2 response, and preventing CCL7 and CCL2 degradation by ACKR2 (32).

A study directly supporting the idea that the activity of heterocomplexes can be relevant also in cancer was performed in our laboratory, showing the role of the CXCL9/CXCL12 heterocomplex in primary central nervous system lymphoma (PCNSL). In this work, it was shown that CXCL9 and CXCL12 are co-expressed in the perivascular area of the tumor, and can form a complex enhancing CXCR4-mediated recruitment of tumor-infiltrating lymphocytes and malignant B cells. This synergism might serve as regulator of the recruitment of CD8+/CXCR4+/CXCR3+ T cells and CXCR4+/CXCR3- malignant B cells in the perivascular cuffs, forming the typical lesions of these tumors (20).

A synergism, mediated by the heterocomplex formed between CXCL12 and the DAMP protein HMGB1 has been shown to be relevant in monocyte recruitment (16, 33) and in tissue regeneration (19, 34). However, its involvement in modulating tumor progression and metastasis has never been assessed. Nonetheless, both CXCL12 and HMGB1 are key players in the TME, where they orchestrate a variety of functions that sustain cancer progression. Indeed, the CXCL12/CXCR4 axis is hyper-activated in lymphomas and in many solid tumors. Their activity is central in the promotion of tumor progression and metastasis to the lungs, brain and bone (35, 36). HMGB1 plays a variety of functions based on its cellular location: in the nucleus, is essential for nucleosomes dynamics and chromosomal stability; in the cytosol or mitochondria, modulates autophagy and apoptosis and regulates mitochondrial morphology and functions; on the cell surface of neurons, promotes axon sprouting and neurite outgrowth (37). Stressed and cancer cells release HMGB1 in the extracellular space, where it activates different receptors in a redox-sensitive manner. The disulphide-HMGB1 promotes TLR-4 activation and mediates production of inflammatory cytokines and chemokines, whereas the reduced-HMGB1 triggers RAGE to promote autophagy and CXCL12 secretion. The reduced form is also able to complex with CXCL12 mediating CXCR4-dependent chemotaxis (33, 38). The CXCL12/HMGB1 heterocomplex acts as an enhancer of CXCR4-mediated signaling, potentiating ERK activation, calcium rise and chemotaxis, both in vitro and in vivo (17). The effect can be blocked by glycyrrhizin and by anti-CXCL12 antibodies, which prevent the formation of the heterocomplex, or by AMD3100, proving the sole involvement of CXCR4 (16, 39). Moreover, the heterocomplex induces a rearrangement of the N-terminus of CXCL12 and conformational changes in the CXCR4-dimers (16) that might suggest a different mode of receptor triggering.

Recently an important role of the CXCL12/HMGB1 heterocomplex has been described in tissue regeneration. Fully reduced HMGB1 promotes liver and muscle regeneration through CXCR4, by acting on muscle stem cells, hepatocytes, and infiltrating cells (18). In a similar study, HMGB1 was detected after fracture both in humans and in animal models, and the heterocomplex acting via CXCR4 promotes in vivo skeletal, hematopoietic and muscle regeneration (19).

During the different phases of cancer progression, many types of inflammatory cells that exhibit either anti- or pro-tumoral functions are recruited from the blood stream by specific chemotactic cues. The leukocyte infiltrate includes neutrophils with different phenotypes (40), macrophages (41), natural killer cells (NK) (42), myeloid-derived suppressor cells (MDSC) (43, 44), dendritic cells (DCs) (45), T and B lymphocytes (46, 47). Several chemokines have been shown to be expressed in tumors, guiding leukocyte recruitment and positioning, and to support tumor spread at distal organs (7). Below we provide some examples in which different cell types present in tumors can be recruited in the TME thanks to the activity of chemokines, and possibly to the presence of heterocomplexes.

Many chemokines are abundantly and concomitantly expressed in the TME and orchestrate a variety of functions that sustain cancer progression or suppression. While the activity of chemokine heterocomplexes has been deeply investigated in inflammatory conditions, and in models of tissue regeneration, a direct prove that a heterocomplex can enhance the responses of tumor cells to chemokines has been demonstrated only for the CXCL12/CXCL9 heterocomplex in PCNSL (20). The concepts covered in the present review suggest that the nature and function of tumor infiltrating immune cells might not be the simple result of the interaction occurring between a chemokine agonist and its specific receptor, but, could be mediated by chemokine heterocomplexes that can differently modulate the activation of a variety of chemokine receptors regulating cell recruitment, positioning, and the switch in the components of the cellular infiltrate in different tumor stages.

The mapping of the possible chemokine-chemokine interactions by bidirectional immunoligand blotting suggests that the synergism might preferentially be mediated by CC-type heterodimers, whereas the CXC-types might promote inhibitory effects (21). Additional studies are required to determine whether this distinction can be applied to the whole chemokine system, and in particular if the heterocomplexes identified are relevant in the TME.

As testified by the diverse expression of chemokine receptors in tumors and by the multiple activities of the heterocomplexes studied so far, we might expect different responses to the same heterocomplex according to the distinctive features of each TME. A deeper understanding of the modulation of the chemokine system in TME, will tell us the relevance of the heterocomplexes, and their possible involvement in shaping the activity of the microenvironment.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.