Cutaneous melanoma is considered as the deadliest form of skin cancer because of its high tendency to metastatic spread. While in situ lesions can be effectively cured by surgical removal, the treatment of the metastatic disease is still challenging. Until the last decade, the standard therapy for metastatic melanoma was chemotherapy, based on the usage of three classes of drugs: alkylating agents, mitotic inhibitors and alkylating-like drugs (1).

The alkylating agents dacarbazine (DTIC) and temozolomide (TMZ) were the most widely used and effective chemotherapeutic agents used for the treatment of metastatic melanoma, with a general good tolerance and limited side effects which include modest bone marrow suppression (1). However, the outcome was poor, with ORRs around 20%, response duration of 4-6 months and complete responses observed only in 5% of treated patients (2, 3). The mitotic inhibitors paclitaxel (PTX) and docetaxel (DTX) belong to taxanes and were both associated with ORRs and survival rates similar to those observed with DTIC. However, they caused more severe side effects, including myelosuppression, hypersensitivity reactions and peripheral neuropathy, thus they were generally used as second-line therapy (1, 2). Along with PTX, the activity of the alkylating-like agent cisplatin as a first-line single therapy showed modest results (2). Furthermore, important side effects and several mechanisms of drug resistance development were associated to cisplatin usage, thus the drug was generally used in combinatorial regimens (1).

Overall, the effects of chemotherapy in metastatic melanoma were modest, mainly palliative and poorly effective in terms of survival. The picture has been dramatically changed by the introduction of immunotherapy, particularly by the immune checkpoints inhibitors (ICIs). The first ICI approved by FDA in 2011 was Ipilimumab, a monoclonal antibody (mAb) targeting the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), which increased ORR to 19% and 5-years survival to 20% (4). The introduction of Nivolumab and Pembrolizumab, targeting the Programmed cell death protein 1 (PD-1), further increased ORRs and 5-year survival rates to 40% and 30-40%, respectively (5–7). Even more striking results were reached combining Nivolumab and Ipilimumab, whose subsequential administration in metastatic melanoma patients gave 45% and 60% of ORRs and 5-year survival, respectively (7, 8). However, a large portion of metastatic melanoma patients still does not respond to current treatments, highlighting the need to develop further therapeutical approaches. It has been proposed that this lack of effectiveness may be due to the strong immunosuppression exerted in the tumor microenvironment (TME) by both cancer and stromal cells. Indeed, immunosuppression is nowadays considered as a hallmark of cancer (9). Thus, it is likely that strategies able to counteract such immune suppression and to generate a supportive TME would represent valuable approaches to improve ICIs effectiveness.

In this context, several lines of evidence showed that, together with the well-known cytotoxic activities, chemotherapeutic drugs also display the capability to affect immune responses against tumors. These immunomodulatory properties mainly rely on five mechanisms: 1) shrinkage of the cancer mass, which reduces the systemic immune suppression induced by the tumor, 2) enhancement of the expression and/or presentation of tumor antigens, which increases cancer cell antigenicity, 3) emission of danger signals by damaged cells, which augments cancer cell immunogenicity, 4) induction of stress signals on cell surface, which increase the susceptibility of cancer cells to be killed by immune cells, 5) direct effects on immune and stromal cells within the TME (10). In addition, even the lympho- and mielodepletion, generally regarded as an undesired collateral effect of chemotherapy, may offer a therapeutic advantage by resetting the immune system and favoring the appearance and/or expansion of immune cell subsets with anti-tumor activities. It has been proposed to occur through homeostatic proliferation, that is the peripheral expansion of specific immune cell clones induced by targeted stimuli such as cytokines and/or antigens (11, 12). Thus, the combination with chemotherapy may be a promising approach to improve the outcome of metastatic melanoma patients treated with ICIs. Indeed, recent reports indicated that the standard chemotherapeutics used in metastatic melanoma can both cooperate with ICIs, increasing response rates compared to single-agent mAbs, and sensitize patients that do not respond when ICIs are administrated as first-line therapies (13–17). However, the molecular mechanisms underlying this synergism are still not fully elucidated.

Although the main target of current immunotherapies has been T cells, increasing attention is being given to Natural Killer (NK) cells because of their alternative mechanism to recognize cancer cells. However, their usage in clinical settings is limited by the same issues observed for T cells and needs to be implemented, possibly by combinatorial approaches. Here, we discuss the immunomodulatory properties of anti-melanoma chemotherapeutic drugs that could be exploited to enhance NK cells anti-tumoral functions within melanoma TME. Furthermore, we discuss the possibility to further improve such properties by the usage of nanocarriers for the targeted release of chemotherapeutic drugs.

NK cells belong to Innate Lymphoid Cells (ILCs), a family of innate lymphocytes largely contributing to tissue homeostasis. Particularly, NK cells are considered the cytotoxic arm of ILCs and, more in general, of innate immunity, thus playing a pivotal role in mounting early defenses against stressed, viral-infected and tumor-transformed cells (18). Phenotypically, NK cells are characterized by the lack of T cell lineage marker CD3 and the expression of CD56, which further distinguishes NK cells in two main subpopulations. CD56^bright NK cells are poorly cytolytic and mainly immunoregulatory, while CD56^dim NK cells display strong cytotoxicity but low secretory capabilities (19).

Peripheral blood NK cells have been shown to be widely affected by melanoma, as indicated by the association between NK cell modifications and melanoma progression and/or response to therapy (20). In melanoma, NK cells are recruited by the inflammatory chemokines CCL5 and CXCL9-11, expressed within the TME, by the engagement of the cognate receptors CCR5 and CXCR3, respectively (21). However, infiltrating NK cells are usually poor and clusterized around the stroma, not in direct contact with tumor cells (22, 23). Indeed, NK cells have been proposed to contribute to anti-melanoma responses mainly by the elimination of tumor cells spreading throughout blood circulation, particularly cancer stem cells, thus limiting metastasization (20, 24, 25). Still, the presence of NK cells in melanoma TME has been associated with tumor regression and good prognosis (22).

As part of the innate immune system, NK cells recognize cells to eliminate through germline-encoded receptors that engage poorly polymorphic molecules/determinants expressed on target cells. Such ligands can display either inhibitory or activating effects and are indeed recognized by inhibitory or activating receptors, respectively. Thus, the activation and killing of NK cells will depend on the balance between the signals delivered by the different receptors, as stated by the “missing-self hypothesis”. Accordingly, the expression of inhibitory molecules will spare cells from killing, while inhibitory ligands down-regulation and/or activating ligands over-expression will induce NK cell cytotoxicity (26) ( Figure 1 ).

The main inhibitory molecules for NK cells are classical Major Histocompatibility Complex (also known as Human Leucocyte Antigen, HLA, in humans) class I (MHC-I) molecules recognized by the Killer-cell Immunoglobulin-like Receptors (KIRs), which represent the most important inhibitory receptors expressed by NK cells. Of notice, KIRs interact with common determinants shared by HLA-A, -B, and -C molecules. While MHC-I molecules are expressed by the majority of healthy cells, they are usually down-regulated in infected or transformed cells, avoiding the engagement of KIRs and thus inducing NK cells activation (27). Indeed, down-regulation of MHC-I molecules has been widely described in melanoma and it is regarded as one of the major mechanisms determining NK cell-mediated killing of melanoma cells (28) ( Figure 1 ).

Another important NK cell inhibitory receptor is CD94/Natural Killer Group (NKG) 2A, belonging to the heterodimeric C-type lectin NKG2 receptor family. CD94/NKG2A binds to HLA-E, a poorly polymorphic non-classical MHC-I molecule loaded with peptides deriving from the leader sequence of the other MHC-I molecules. Thus, CD94/NKG2A allows NK cells to sense the overall expression levels of MHC-I molecules on target cells (29) ( Figure 1 ).

While inhibitory signals for NK cells are primarily mediated by MHC-I molecules, activating ligands are more heterogeneous and induced by cellular stress. The main class of NK cells activating receptors are the Natural Cytotoxicity Receptors (NCRs), consisting of three Ig-like proteins whose ligands are still poorly defined. NK cells constitutively express two NCRs, NKp30 and NKp46, while the third member of the family, NKp44, can be found on NK cell surface only after activation. Collectively, NCRs are the most important receptors triggering NK cell-mediated killing of cancer cells. Indeed, their expression correlates with the magnitude of the NK cell cytolytic activity (30) ( Figure 1 ).

Another fundamental receptor involved in NK cell activation is NKG2D. While belonging to the NKG2 family, NKG2D stands out for being a monomeric receptor. Ligands recognized by NKG2D include two types of MHC-like molecules, MHC class I chain-related protein (MIC) A and B and unique long 16-binding proteins (ULBPs). Both the families of ligands are generally not found on healthy cells but are induced by stresses such as malignant transformation (31, 32). The DNAX accessory molecule-1 (DNAM-1) is a co-receptor enhancing NK cell activation triggered by the other activating receptors. DNAM-1 recognizes two ligands, CD155 (poliovirus receptor, PVR) and CD112 (Nectin-2), that are widely expressed by cancer cells (33). Moreover, NK cells can be activated through the engagement of CD16, which binds the Fc portion of IgG antibodies and thus allows NK cells to mediate the antibody-dependent cell cytotoxicity (ADCC) against opsonized target cells (34) ( Figure 1 ).

The analysis of large panels of melanoma cells showed that ligands for NKG2D (MICA/B, ULBPs) and DNAM-1 (PVR, Nectin-2) are widely expressed by melanoma cells. Of them, MICA/B were more frequently observed compared to ULPBs (35, 36) and PVR was widely found, while Nectin-2 was scarcely expressed (35). The expression of NCR ligands has been also reported, though to a lesser extent (37, 38).

Either an over-expression of activating ligands or a loss of inhibitory signals triggers the main cytotoxic pathway of NK cells, that is the release of cytolytic granules, specialized secretory organelles containing perforin and granzymes inducing apoptotic cell death. Specifically, granzymes are serine proteases able to cleave and activate several intracellular proteins involved in apoptosis induction, while perforin generates pores on eucaryotic cell membrane allowing granzymes entry within the target cell (39).

Though the balance between inhibitory and activating signals represents the main factor determining target recognition and cytotoxicity, both the processes are strengthened by accessory molecules involved in cell adhesion. Among them, the β2-integrin Leucocyte Functional Antigen (LFA)-1, recognizing the Intercellular Adhesion Molecule (ICAM)-1, is found to be expressed on melanoma cells (40, 41), where it plays a central role in mediating the firm adhesion between NK and target cells as well as the polarized delivery of cytotoxic granules (42). Additionally, NK cells can eliminate targets by inducing the extrinsic apoptotic pathway. Indeed, resting NK cells largely express Fas ligand (FasL), while activated NK cells also express Tumor Necrosis Factor-related Apoptosis-Inducing Ligand (TRAIL), which engage death receptors expressed on the surface of target cells (43, 44). In melanoma cells, NK cell-mediated activation of the apoptotic extrinsic pathway is induced by the engagement of Fas (45, 46) ( Figure 1 ).

Activated NK cells also release cytokines, particularly Interferon (IFN)γ and Tumor Necrosis Factor (TNF)α, exerting both direct and indirect effects contributing to target cells clearance (19). IFNγ is able to directly inhibit cell cycle progression and to promote apoptosis in infected and/or transformed cells as well as to increase their immunogenicity by enhancing antigen presentation (47). IFNγ also dampens proliferation and survival of endothelial cells, thus counteracting angiogenesis (48). Moreover, IFNγ broadly affects the immune system by stimulating the recruitment, differentiation and activation of several immune cell subsets involved in anti-viral and anti-tumoral responses (47). Similar effects have been observed for TNFα, albeit the activities of the two cytokines are not completely overlapping. Indeed, TNFα is considered to be poorly cytotoxic/cytostatic against tumor cells, while it is particularly effective in inducing inflammation and vasculature destruction (49).

The proper activation of NK cells is also supported by several cytokines produced by different immune cell subsets. Interleukin (IL)-2, IL-15, IL-12 and IL-18 as well as type I IFNs are known to promote several aspects of NK cell functioning, including proliferation, maturation, survival, cytokine secretion and up-regulation of activating receptors and cytotoxic molecules (50). Moreover, IFNγ can act as autocrine factor to stimulate NK cell cytotoxicity by up-regulating the expression of perforin, granzymes and FasL. Of notice, while most of the processes can be induced by individual cytokines, they represent a weak stimulus for IFNγ secretion, which instead requires the cooperation between IL-12 and the other cytokines (50).

Solid tumors consist of cancer cells and the surrounding tissue cells that constitute TME, which include several types of cells such as fibroblasts, endothelial and immune cells as well as extracellular matrix (ECM). In melanoma, the elevated mutational burden makes the tumor highly immunogenic. In addition, oncogenes commonly mutated in melanoma induce the expression of cytokines, chemokines, enzymes and growth factors recruiting and regulating immune cells (51). As a consequence, melanoma lesions usually show a substantial immune infiltrate composed by several distinct populations exerting specific, and even opposed, functions towards cancer cells. Moreover, the same subset can display different activities based on its state of maturation/polarization (52). The dynamic interactions between tumor and immune cells largely contribute to determine tumor progression. This complex interplay can be described through the immunoediting paradigm, that postulates an elimination phase in which the immune system efficiently eliminates cancer cells, an equilibrium phase in which immune response acts as an evolutive pressure selecting resistant clones, and an escape phase in which the tumor overcomes the immune response and successfully progresses (53). The immune escaping of melanoma cells can occur through two not mutually exclusive mechanisms: intrinsic phenomena reducing the capability of tumor cells to be recognized and killed by immune cells and extrinsic processes involving the active production of molecules generating, directly or indirectly, an immunosuppressive TME (54). Moreover, the capability of the TME to support or limit melanoma progression is affected by the relative proportion and contribution of the different immune cell subsets (52) ( Figure 2 ). Many of these processes can be, at least partially, counteracted by standard chemotherapeutics, which provides the rationale for their combination with ICIs.

Nanomedicine represents an alternative strategy to deliver anti-neoplastic agents. Nanotechnology-based drug delivery systems act to improve effectiveness of chemotherapeutics in terms of bio-distribution, water solubility, targeting capability and therapeutic index. Many different types of nanosystems have emerged in the last years, empathizing their important role in the treatment of solid tumors, especially melanoma (174, 175). To facilitate their accumulation within TME, drugs can be encapsulated, adsorbed or covalently attached on nanocarriers. Furthermore, in the past decades, nanocarriers have been used to target TME in order to inhibit it suppressive capability by modulating immune cells, tumor stroma, cytokines and enzymes (176).

In the clinical practice, the introduction of ICIs reactivating T cell-mediated immune responses against metastatic melanoma has dramatically changed patients’ outcome. However, the failure of a fraction of patients to respond to therapy spurred the investigation of novel approaches in order to improve the success rate. In this context, NK cells gained increasing attention because of their alternative and complementary capability to kill tumor cells compared to T cells. Most of the ICIs that are currently used or under development target inhibitory receptors also expressed by NK cells. Indeed, it has been shown that their blockade is able to reactivate NK cell-mediated cytotoxicity and that NK cells are essential for ICIs anti-tumoral activity (212). Based on these evidences, ICIs targeting NK cell inhibitory receptors such as NKG2A and KIRs have been developed and tested in clinical trials (213). An alternative strategy to improve NK cell-mediated killing is the usage of bi- and trispecific Killer Cell Engagers, which engage activating receptors on NK cells and specific antigens on tumor cells, thus favoring immune synapse formation and NK cell degranulation (212).

In addition, NK cells have been evaluated for adoptive cell transfer both in autologous and allogenic settings. Of notice, NK cells to transfer can be modified to express a Chimeric Antigen Receptor (CAR). Initially developed for T cells to allow them to recognize naïve antigens, CAR technology can be applied also to NK cells to redirect them against specific targets. Compared to CAR-T cells, CAR-NK cells are not antigen-restricted and maintain their capability to recognize the target through multiple pathways, ensuring effective activation. Moreover, CAR-NK cells are short-living cells that do not generate memory, thus displaying a very low risk of adverse reactions and a high safety profile (214). However, when used in solid tumors, these approaches suffer from the same limits observed for T cells, that is the presence of an immune suppressive TME that limits NK cell trafficking within the tumor and/or blunts their cytotoxic activities.

The usage of chemotherapeutics to treat cancer relied on their capability to counteract uncontrolled tumor cells proliferation. However, their action is not cell-specific, which means that all the cell types with high rates of replication, including immune cells, are negatively affected by chemotherapy. Indeed, immune suppression is one of the most important collateral effect of standard-dosage chemotherapy and thus was regarded as the major mechanism making chemotherapy incompatible with immunotherapy. However, more recent evidence challenged this assumption, showing that sub-toxic concentrations of chemotherapeutics exert immunomodulatory activities able to reactivate immune responses. Additionally, it has been shown that suppressive immune subsets display a higher sensitivity to chemotherapy-mediated cytotoxicity, making low doses of chemotherapeutics a potential strategy to deplete specific immune cell populations (85). Thus, the usage of chemotherapy is nowadays gaining attention as therapeutic strategy to improve effectiveness rates of current immunotherapy.

The evidence discussed here indicate that metastatic melanoma chemotherapy affects not only tumor cells but also the TME, thus having the potential to unleash the anti-tumor activities of the immune system. However, the combination between chemo- and immunotherapy has recently begun to be investigated and there is still not a consensus about the schedule to be used. Indeed, while some reports suggested that chemotherapy should be administrated before immunotherapy (11–13), other studies demonstrated that the survival benefit could be achieved also by the concomitant administration of the two kinds of drugs (14, 15). Moreover, some case reports indicated that chemotherapy could be effectively used after immunotherapy in non-responding patients (16, 17). Although in theory initial chemotherapy would represent the best strategy to reactivate the immune system and sensitize patients to immunotherapy, the lack of biomarkers predicting immunotherapy response makes the subsequent usage of chemotherapy a more feasible approach. Overall, additional studies are needed in order to define the best therapeutic strategy to combine chemo- and immunotherapy as well as to identify those patients actually needing the combinatorial therapy.

NK cells within melanoma TME can directly kill tumor cells and/or promote anti-tumoral activities by other immune subpopulations. However, cell-intrinsic pathways in melanoma cells as well as their capability to recruit and activate suppressive subsets can profoundly impair NK cells cytolytic and anti-proliferative functions. The evidences reported here clearly suggest that chemotherapeutic drugs may support NK cell-mediated elimination of melanoma cells by four different, complementary mechanisms: 1) increase of melanoma cells immunogenicity, mainly occurring through the up-regulation of NKG2D ligands but also involving other activating molecules specifically induced by the different drugs, thus boosting the natural cytotoxic activity of NK cells; 2) induction of NK cells infiltration within TME, which would contribute to overcome an important limitation in the usage of autologous, allogenic as well as CAR-NK cells; 3) promotion of myeloid cells activation and differentiation towards anti-cancer phenotypes that largely contribute to develop and support NK cells functions; 4) specific impairment and depletion of immune suppressive cells, which play the major role in generating and maintaining the suppressive conditions blunting NK cells cytotoxicity within TME.

Moreover, these effects could be further enhanced by nano-based drug delivery systems designed to improve pharmacokinetics behaviors and to increase drugs stability in vivo, thus allowing the effective usage of sub-toxic amounts of drugs that keep the immunomodulatory properties avoiding at the same time the immune suppression commonly associated to high-dosage of chemotherapeutics (215).

Overall, the association of immunotherapies with low dosages of chemotherapeutics possibly delivered by nanosystems could restore NK cells anti-tumor properties thus representing an effective alternative therapeutic strategy for those melanoma patients that fail to respond to the current immunotherapeutic treatments.

CMC conceived the work. CG and CMC reviewed the relevant literature. CG, CDM and CMC wrote the paper. CG, CDM, and CMC generated the figures. All the authors critically read and revised the manuscript, contributed to the article and approved the submitted version.

This work was supported by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) PRIN 2017 2017M8YMR8_002.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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