Proteolytic chemokine cleavage as a regulator of lymphocytic infiltration in solid tumors

Although it was postulated more than 100 years ago that the immune system can fight established solid tumors, only in the past two decades substantial progress has been made towards a better understanding of the interaction between the different subtypes of immune cells with tumor cells and their environment [1, 2]. Starting point for many of these studies was the clinical observation that the number of tumor-infiltrating lymphocytes (TILs) is a strong and robust prognostic marker across various tumor entities, such as breast, colon, or ovarian cancer [3‐5]. Further studies have dissected the different subpopulations of TILs delineating either their tumor-suppressive (e.g., cytotoxic T cells, natural killer cells) or tumor-promoting functions (e.g., regulatory T cells) [6, 7]. Deciphering the immune cell-tumor cell interactions has prompted the development of new cancer immunotherapies, such as monoclonal antibody therapy against tumor antigens, immune checkpoint inhibition, adoptive T cell transfer, or various vaccination strategies [8]. Especially in highly immunogenic tumors, these immunotherapies have led to unprecedented improvements in survival and quality of life of cancer patients. Examples include the HER2-directed therapies in breast cancer or the immune checkpoint inhibitors in melanoma or non-small cell lung cancer [9, 10]. Moreover, there is ample evidence that classic chemotherapies also work via stimulation of an anti-tumor immune response in addition to their cytotoxic effects on the individual cancer cells [11].

However, a fundamental prerequisite for the success of all of these approaches is sufficient trafficking of the respective immune effector cells into the tumor microenvironment [12, 13]. This renders TILs or TIL subpopulations not only prognostic, but also feasible predictive biomarkers for the response to these therapies [7, 14]. In addition, it raises one of the most urgent questions in contemporary cancer immuno-oncology: how can these immune cells be efficiently recruited into the tumor? The ultimate goal is to transform a “cold” into a “hot” tumor [15].

The recruitment of immune cells into solid tumors is mediated by chemokines, a family of about 50 small proteins capable of chemotactically facilitating leukocyte migration [16]. Different chemokines can either attract tumor-suppressive or tumor-promoting leukocytes, and, thus, the intratumoral chemokine milieu is a strong determinant of the intratumoral immune milieu [17]. Besides their chemotactic function, chemokines also participate in activation or inactivation of immune cells.

Many chemokines can be posttranslationally modified by proteolytic cleavage which either activates or destroys their chemotactic function [18]. Expression of the corresponding proteases may thus significantly influence the modulation of the immune milieu and the anti-tumor immune response. Therefore, proteolytic inactivation of tumor-suppressive chemokines represents a potent immune escape mechanism of solid tumors [19]. Conversely, inhibition of these proteases might be an attractive adjuvant to immunotherapies, such as immune checkpoint inhibitors, whose function depends on the activity and presence of specialized chemokines [20].

In the following, the concept of chemokine cleavage as a modulator of the anti-tumor response will be discussed. The focus will be on the chemokines that are most notably known for their ability to recruit tumor-suppressive lymphocytes such as cytotoxic T cells (CTLs) or natural killer (NK) cells into solid tumors (Fig. 1). These chemokines are the CXCR3 receptor ligands CXCL9, CXCL10, and CXCL11 as well as the CX3CR1 ligand CX3CL1, also named fractalkine [17, 21].

Chemokines or chemotactic cytokines make up the largest family of cytokines. With 48 members expressed in human tissue, they represent a structurally and functionally related group of low molecular weight proteins, which controls chemotactic recruitment of specific lymphocyte subtypes in a tissue- and time-dependent manner [22]. The distribution of immune cells not only plays a crucial role in the innate and adaptive immune response in case of inflammation or associated disease [23‐27], but also supports tissue homeostasis [28] and angiogenesis [29, 30]. According to their major function chemokines are classified into two groups [25]: Homeostatic chemokines are constitutively expressed in order to initiate a proper immune response and to define the composition and organization of tissue-resident, blood-derived cells such as macrophages and dendritic cells [16]. On the contrary, inflammatory chemokines are mainly inducible and are strongly upregulated upon demand [31]. It is of note that this classification is not strict as there are chemokines that can contribute to both groups.

The conventional chemokine signaling is thought to be transduced through a complementary receptor network consisting of transmembrane G protein-coupled receptors [32]. However, also receptor-independent modulation of signaling has been proposed. Factors that can influence chemokine signaling are non-canonical receptors such as the atypical chemokine receptors 1-4, mRNA instability, alternative splicing, or glycosaminoglycan (GAG) binding [33‐35]. Furthermore, regulation is even more complex due to considerable promiscuity between chemokines and their receptors, i.e., several ligands may bind to the same receptor and vice versa a single ligand can activate different receptors [16]. Moreover, chemokine receptors are involved in several signaling cascades which can be differentially activated by either distinct ligands or via oligomerization of the same ligand [36, 37].

The structure of chemokines is conserved across subfamilies, in most cases built around four conserved cysteine residues, except the XC chemokines which lack two of these four cysteines [38]. The organization of the first cysteine residues in the N-terminal part of the chemokines divides them into four different groups: CXC, CC, XC, and CX3C, with X representing any amino acid [39]. The cysteines form one or two disulfide bridges, which stabilize a central three-stranded anti-parallel ß-sheet with a rigid loop structure (Fig. 2). This central core is preceded by a flexible unstructured N-terminus. Both the flexible N-terminus and the following rigid N-loop, which often is C-terminally limited by a 3₁₀-helix, within the globular core are functional domains of the chemokine in terms of receptor binding and activation. Originally, a two-site/two-step model had been proposed, in which firstly the N-terminus of the receptor interacts with the N-loop of the chemokine and, subsequently, the unstructured N-terminus of the chemokine can enter the binding pocket of the receptor resulting in receptor activation and transmembrane signaling [40]. Recent results indicate that this model may have to be extended to a three step model, in which step 1 of the original model, binding of the chemokine to the receptor, is divided into an initial low-affinity, rather unspecific binding which is then followed by the specific high-affinity binding of the chemokine to the receptor [41]. C-terminal of the chemokine core, an α-helix is present which packs onto the ß-sheet structure. The glycosaminoglycan (GAG) binding site of the chemokines is located within the β-sheet and C-terminus [42].

The CXC chemokines can be further divided according to the presence or absence of a highly conserved Glu-Leu-Arg (ELR) motif, which is shared by CXCL1-3 and CXCL5-8. Among the chemokines that lack the ELR motif (CXCL4, CXCL4L1, and CXCL9-14), most of them interact with the CXCR3 receptor [43]. The CXCR3 receptor is a 368 amino acid (aa) seven-transmembrane G protein coupled receptor with three isoforms. The originally identified, canonical CXCR3 receptor, named CXCR3-A, is bound by the ligands CXCL9, CXCL10 and CXCL11 and, upon activation, promotes chemotaxis, invasion, proliferation, and cell survival [44]. The first identified splice variant of CXCR3 was called CXCR3-B and interacts with CXCL4 and CXCL4L1 in addition to the classical ligands. This receptor isoform does not trigger chemotaxis but instead growth suppression, angiostasis, and apoptosis. The most recently discovered CXCR3 isoform, CXCR3-alt, only accepts CXCL11 as ligand [44, 45].

CXCR3 receptor expressing cells include, among others, regulatory T cells, CD4⁺ and CD8⁺ T cells, dendritic cells, NK cells, and NKT cells [21, 46]. CXCR3 ligand expression can be detected in endothelial cells, keratinocytes, and fibroblasts. Also, immune cells like T cells and monocytes are capable of secreting those chemokines. Especially CXCL9 and CXCL11 are secreted by peripheral blood monocytes and macrophages [22].

CXCL4 and CXCL4L1 are described as platelet-related agonists, whereas CXCL9, CXCL10, and CXCL11 expression is strongly inducible by interferons (IFN). All of the latter cytokines can be induced by IFN-γ, but CXCL11 is the only one being also induced by IFN-α [47]. CXCL11 show the highest binding affinity towards CXCR3 followed by CXCL10 and CXCL9. Among these three chemokines, CXCL9 shows the weakest activation upon receptor binding [22]. Interestingly, competition with CXCL11 receptor binding by CXCL9 or CXCL10 is always incomplete. Furthermore, CXCL11—in contrast to CXCL10—does also bind to CXCR3, when the receptor is uncoupled from G protein-dependent signaling [48]. Receptor activation by its three ligands also results in different effects. CXCL11 was described to be the most potent inducer of receptor internalization, whereas CXCL9 and CXCL10 mainly induce chemotaxis as well as Ca²⁺ influx [49].

Besides induction of migration and Ca²⁺ influx, ligand/receptor interaction can also lead to downstream phosphorylation of target proteins, e.g., transcription factors of the STAT family. CXCL9 and CXCL10 lead to the phosphorylation of STAT1, STAT4, and STAT5 and subsequently to an activation of T-bet and RORyT, two differentiation regulators, resulting in polarization of CD4-positive T cells towards the Th1 and Th17 effector lineage. Contrariwise, CXCL11 binding induces phosphorylation of STAT3 and STAT6, which leads to a regulatory phenotype of CD4-positive T cells (Th2 or Tr1) [50‐52].

Thus, together with the varying susceptibility to post-translational modification, differences concerning ligand-receptor interaction and GAG binding, and the fact that CXCL9, CXCL10, and CXCL11 display not completely overlapping functions, a complex regulatory network is formed affecting diverse biological functions.

Cells expressing the seven-transmembrane receptor CX3CR1 (355 aa) are monocytes, macrophages, NK cells, lymphocytes, and dendritic cells [53]. Its ligand, CX3CL1, also named fractalkine, is the only member of the CX3C chemokine family. It is expressed as a membrane-bound protein, but is also found in soluble forms [54, 55]. The 317 aa long extracellular domain encompasses a stalk formed by mucin-like domains with a chemokine domain on top. This N-terminal extracellular part is followed by a transmembrane domain and a short C-terminal cytoplasmic tail (34 aa). The soluble forms of CX3CL1 carrying the mucin-like stalk [56] and possibly also the cytokine domain only [57] provide chemotactic recruitment of CX3CR1-positive cells. As a membrane-bound protein, CX3CL1 provides adhesive capacity and helps to stabilize the interaction between CX3CL1-expressing cells and cells expressing the corresponding receptor CX3CR1, respectively [58]. CX3CL1/CX3CR1-mediated adhesion can be further enhanced by synergistic interaction with other adhesive molecules like integrins [59]. Due to functions of CX3CL1 as chemo-attractant and adhesion molecule, it plays an important role in the process of recruiting cells from the blood stream to the site of action in an integrin-independent manner, e.g., the extravasation of CX3CR1-positive leukocytes [60]. CX3CL1 expression is mainly found in endothelial cells and neurons, but based on the involvement in inflammatory processes it is also expressed in tissue showing rather dense immune cell populations like the CNS, lung, cardiac muscles, liver, small bowel, colon, and pancreas [61].

The role of the CXCR3 chemokine system in cancer biology may be a double-edged sword. On the one hand, the CXCR3 chemokines, especially CXCL9 and CXCL10, facilitate the recruitment of CXCR3-positive Th1, NK, and NKT cells as well as cytotoxic T lymphocytes to the tumor microenvironment, which trigger the development of a tumor-suppressive immune milieu [46, 62, 63]. On the other hand, tumor cells can exploit the CXCR3 receptor to escape from the primary tumor and to metastasize to niches with high CXCR3 ligand concentrations, e.g., to the lymph nodes or to the lungs [64‐69]. Moreover, all three IFN-inducible CXCR3 ligands are anti-angiogenic in vitro and in vivo, which may result in undersupplied, stagnating tumors, but also in more aggressive tumor cells with metastasizing potential [70, 71].

Raising the intratumoral concentration of intact and functional CXCR3 chemokines, e.g., by inhibition of their proteolytic inactivation, might thus kill two birds with one stone: tumor-suppressive immune cells would be attracted to the tumor site, and CXCR3-positive tumor cells would be chemotactically prevented from escaping the primary cancer. This idea is supported both by preclinical and clinical findings. Overexpression of CXCR3 ligands in murine cancer models of ovarian, breast, skin, or colon cancer caused an enhanced Th1 and NK cell infiltration and less metastatic spread [72‐75]. Moreover, in human cancers, overexpression of CXCL9 and CXCL10 is associated with a higher number of tumor-infiltrating lymphocytes and improved survival, e.g., in breast, ovarian, colon, lung, and several other cancers [67, 76‐83]. Although CXCR3 expression by tumor cells is associated with worse prognosis in these cancers [67, 72, 84, 85] and CXCR3 ligands may also attract tumor-promoting regulatory T cells [86], the net effect of CXCR3 chemokine overexpression seems to favor tumor suppression. These results are confirmed by large-panel gene expression analyses of breast and ovarian cancer, in which CXCR3 chemokines represented the most upregulated genes in the tumors of those patients exhibiting the best prognosis [76, 87, 88].

However, CXCR3 chemokines do not only cause a tumor-suppressive milieu per se, they also contribute to the effect of multiple current cancer therapeutics. Immune checkpoint inhibitors of the PD-1/PD-L1 axis rely on immune cell attraction and on the intratumoral T cell activation by CXCR3 chemokines [20, 89‐91]. Moreover, an increase in CXCR3 chemokine serum concentrations under therapy with checkpoint inhibitors is predictive for therapy response [92]. CDK4/6 inhibitors, which have emerged as a new cornerstone in the treatment of advanced estrogen receptor positive breast cancer, recruit cytotoxic T cells via induction of CXCR3 chemokines, which is indispensable for their therapeutic effect in vivo [93]. Inhibition of the poly[ADP-ribose] polymerase 1 (PARP1), which is now an established therapy in recurrent ovarian and metastatic BRCA-mutated breast cancer, also induces CXCR3 chemokines via the STING (stimulator of interferon genes) pathway in tumor cells, whereby the subsequent attraction of immune cells is critical for their function [94‐99]. In all of these therapies, proteolytic inactivation of CXCR3 chemokines, thus, represents a new resistance mechanism, which renders inhibitors of CXCR3-chemokine cleaving proteases feasible adjuvants to all of these therapies.

CX3CL1 is also capable of recruiting tumor-suppressive immune cells that express the CX3CR1 receptor such as NK cells and cytotoxic T lymphocytes (CTLs) [21]. However, as described for the CXCR3 system, the CX3CR1 receptor is also able to facilitate tumor cell migration and, thereby, metastasis in CX3CL1-rich tissues, e.g. the bone or the brain [100, 101]. Moreover, expression of the transmembrane form of CX3CL1 in neurons, endothelial cells or peritoneal cells promotes tumor cell adhesion and site-specific metastasis of CX3CR1-expression prostate, ovarian, or pancreatic tumor cells [102‐106].

Preclinical studies confirm the tumor-suppressive effect of CX3CL1 in several cancer models [107‐112]. However, there are also studies in support of tumor-promoting effects of the CX3CL1-system: one study attributed a pro-metastatic function to the CX3CR1 receptor; however, the authors did not use an immuno-competent mouse model and, thereby, excluded the influence of immune modulatory effects [101]. Another study shows that CX3CL1 promotes the development of tumors, but not metastasis, in HER2 transgenic mice via transactivation of the EGF pathway [113]. This direct effect on tumor cells may relate to the fact that the tumor cells themselves express CX3CR1, whose activation directly triggers proliferation and migration [100, 101]. So far, it has not been satisfactorily clarified yet to what extent the two forms of CX3CL1 (membrane-bound vs. soluble) contribute to these different effects.

Taken together, CXCR3 and CX3CR1 chemokines are part of a complex regulatory network which orchestrates a broad variety of physiologic functions that can modulate the anti-tumoral immune response. On the one hand, chemokines help to shape the tumor-microenvironment by regulating the amount and type of infiltrating lymphocytes. On the other hand, they are enhancing cell biological processes such as proliferation, invasion, and angiogenesis and, thereby, promote tumor aggressiveness and metastatic potential. Posttranslational modifications of these chemokines by tumor cells, such as proteolytic cleavage, can exert a strong regulatory impact resulting in a shift towards a tumor-promoting environment. Proteolytic cleavage of the tumor-suppressive CXCR3 and CX3CR1 chemokines impairs their functions and, on top of this, in feedback loops, the chemokines may even lead to increased expression of the proteases targeting themselves. Thus, we suggest that cleavage of the ligands of the CXCR3 and CX3CR1 chemokine systems represents a potent immune escape mechanism in cancer. Deeper knowledge of the mechanisms behind this also provides a panel of interesting novel target structures to support existing therapies or develop new ones.