Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Seminars in Cancer Biology

journal homepage: www.elsevier.com/locate/semcancer

Review

Unraveling the crosstalk between melanoma and immune cells in the tumor
microenvironment
⁎
Monica Marzagallia, Nancy D. Ebeltb, Edwin R. Manuelb,
a
Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italy
b
Department of Immuno-Oncology, Beckman Research Institute of City of Hope, Duarte, California, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Cutaneous melanoma is the most common skin cancer with an incidence that has been rapidly increasing in the
Cutaneous melanoma past decades. Melanomas are among the most immunogenic tumors and, as such, have the greatest potential to
Tumor microenvironment respond favorably to immunotherapy. However, like many cancers, melanomas acquire various suppressive
Innate and adaptive immunity mechanisms, which generally act in concert, to escape innate and adaptive immune detection and destruction.
Immunotherapy
Intense research into the cellular and molecular events associated with melanomagenesis, which ultimately lead
Immune escape
to immune suppression, has resulted in the discovery of new therapeutic targets and synergistic combinations of
Suppression
Metastasis immunotherapy, targeted therapy and chemotherapy. Tremendous effort to determine efficacy of single and
Checkpoint inhibition combination therapies in pre-clinical and clinical phase I-III trials has led to FDA-approval of several im-
Cytokine therapy munotherapeutic agents that could potentially be beneficial for aggressive, highly refractory, advanced and
Vaccination metastatic melanomas. The increasing availability of approved combination therapies for melanoma and more
microRNA rapid assessment of patient tumors has increased the feasibility of personalized treatment to overcome patient
Metabolites and tumor heterogeneity and to achieve greater clinical benefit. Here, we review the evolution of the immune
Treg
system during melanomagenesis, mechanisms exploited by melanoma to suppress anti-tumor immunity and
MDSC
methods that have been developed to restore immunity. We emphasize that an effective therapeutic strategy will
NK
DC require coordinate activation of tumor-specific immunity as well as increased recognition and accessibility of
T cells melanoma cells in primary tumors and distal metastases. This review integrates available knowledge on mela-
noma-specific immunity, molecular signaling pathways and molecular targeting strategies that could be utilized
to envision therapeutics with broader application and greater efficacy for early stage and advanced metastatic
melanoma.

1. Introduction management of this aggressive cancer, and doubling the median sur-
vival for metastatic disease [6,7]. (Table 1)
The recent rise of immunotherapies has led to an old, but revolu- Malignant melanoma represents one of the most immunogenic tu-
tionary, concept of cancer treatment based on the improved activation mors due to it’s incredibly high genomic mutational load, and has the
of the endogenous immune system against cancer cells [1–3]. The re- highest potential to elicit specific adaptive antitumor immune re-
levance of the field is well represented by the joint contribution of the sponses. It serves as an excellent model for the evaluation of innovative
2018 Nobel Prize recipients for Physiology or Medicine James P. Allison immunotherapies such as checkpoint inhibitors as well as anticancer
and Tasuku Honjo for their discovery of cancer therapy by inhibition of vaccines and engineered chimeric antigen receptor T cells (CAR T cells)
negative immune checkpoint regulation [4,5]. The first immune- [8–10]. Moreover, melanoma may be vulnerable to a newer cohort of
checkpoint inhibitors, antibodies that specifically target the im- checkpoint inhibitors targeting B- and T-lymphocyte attenuator (BTLA),
munoregulatory molecules cytotoxic T-lymphocyte-associated protein 4 T-cell immunoglobulin and mucin domain-3 (TIM-3) and lymphocyte-
(anti-CTLA-4, Ipilimumab) and programmed cell death protein 1 (anti- activation gene 3 (LAG-3) that continue to be areas of intense research
PD-1, Nivolumab), were approved by the US Food and Drug Adminis- [11]. Despite these major advances in cancer immunotherapy, a large
tration (FDA) in 2011 and 2014, respectively, for the treatment of un- subset of melanoma patients do not respond or relapse due to primary
resectable or metastatic melanoma, thus enormously improving the or acquired resistance, resulting in 40–65% treatment failure for

⁎
Corresponding author at: City of Hope, 1500 E. Duarte Rd, Duarte, California, 91010, USA.
E-mail address: emanuel@coh.org (E.R. Manuel).

https://doi.org/10.1016/j.semcancer.2019.08.002
Received 29 March 2019; Received in revised form 10 July 2019; Accepted 4 August 2019
1044-579X/ Published by Elsevier Ltd.

Please cite this article as: Monica Marzagalli, Nancy D. Ebelt and Edwin R. Manuel, Seminars in Cancer Biology,
https://doi.org/10.1016/j.semcancer.2019.08.002
M. Marzagalli, et al. Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Table 1
Immunotherapies for cutaneous melanoma treatment.
Target Effects Treatment FDA Approved

CTLA-4 Bypass immune checkpoint Ipilimumab Yes

PD-1 Bypass immune checkpoint Nivolumab, Pembrolizumab Yes
PD-L1/B7-H1 Bypass immune checkpoint Atezolizumab, Avelumab, Durvalumab, In Trials: NCT02535078, NCT02027961, NCT03212404, NCT03167177,
CK-301 NCT03138889, NCT03178851
DCs/T cells Stimulate anti-tumor IFN alpha-2b Yes
immunity
DCs/T cells Stimulate anti-tumor Interleukin-2 Yes
immunity
DCs/T cells Stimulate anti-tumor Sylatron (PEG-Intron) Yes
immunity
TLR7/8 Stimulate anti-tumor Resiquimob In Trials: NCT02650635, NCT00960752, NCT02320305
immunity
Tregs Depletion of Tregs Ontak Yes
T cells Stimulate anti-tumor gp100 Vaccine In Trials: NCT01744171, NCT02889861, NCT00960752, NCT00470015,
immunity NCT01176461, NCT01176474, NCT02535078
Melanoma cells Direct cytotoxicity CAR T cell In Trials: NCT02107963, NCT03060356, NCT02830724
Melanoma cells Direct lysis of tumor cells Talimogene laherparepvec Yes

Abbreviations: CTLA-4cytotoxic T-lymphocyte antigen 4; PD-1programmed death 1; PD-L1programmed death ligand 1; IFNinterferon; TLRtoll-like receptor;
DCdendritic cell; Tregsregulatory T cells; gp100glycoprotein 100; PEG-Intronpeginterferon alfa-2b; CARchimeric antigen receptor.

patients treated with anti-PD-1, and treatment failure in over 70% of task. However, melanoma tends to be incredibly immunogenic, gen-
patients treated with anti-CTLA-4 [12]. erating neoantigens through chromosomal rearrangements or genetic
The plasticity of melanoma cells leads to a phenomenon called polymorphisms that can mimic “foreign” infection and thus potentially
“immune escape”, whereby cancer cells acquire a less immunogenic elicit cytotoxic responses [27–31]. Many of the key innate and adaptive
phenotype and the ability to suppress anti-tumor immune cells within subsets capable of anti-tumor activity have been identified in in-
the tumor microenvironment (TME) [13,14]. While many factors con- dependent studies of melanoma and other solid malignancies [23,32].
tributing to immune escape have been elucidated, a therapeutic The most effective therapeutic strategy should integrate as many of
strategy to completely re-instill curative anti-tumor immunity has not these immune subsets, without causing significant toxicity, for the
yet been realized. This review will describe the immune landscape greatest clinical benefit [33]. Here, we focus on the inherent anti-tumor
participating in the initial control of pre-malignant cells and then will functions of both innate and adaptive immune subsets that are postu-
highlight the molecular and cellular “crosstalk” exploited by melanoma lated to control pre-malignant cells during early stages of melanoma-
to reprogram immune cells and the TME to cause immune escape and genesis.
progression to advanced disease. Finally, this review will shed light on
the innovative immunotherapies that are currently under investigation
2.2. Innate immunity
with the aim to rescue anti-tumor immunity.
The fast and non-specific anti-tumor responses elicited by innate
2. Immune surveillance in melanoma immunity are not only critical in preventing and controlling early stages
of melanoma, but also in priming robust adaptive immunity to provide
2.1. Immunity and melanoma long-term, tumor-specific immune surveillance. Several therapeutic
strategies to inhibit melanoma growth have specifically focused on the
The immune system is generally thought to keep the body in a state activation of the anti-tumor activities of naive or differentiated innate
of homeostasis by defending against infection and disease caused by subsets found within tumors, which includes, but are not limited to,
bacteria, viruses, fungi, and parasites. However, it is now generally macrophages, polymorphonuclear neutrophils (PMN), natural killer
accepted that the immune system also functions to constantly survey (NK) cells and dendritic cells (DC) [33,34]. These innate cells comprise
and eliminate pre-cancerous cells to prevent progression to melanoma an immune system within the skin known as the skin-associated lym-
[15–19]. In most cases, intracellular check points are engaged within a phoid tissue (SALT) [35]. It is important to note that innate cells are
malfunctioning cell that leads to a process of self-destruction, or incredibly plastic and can acquire both pro- and/or anti-tumor func-
apoptosis, negating the need for immunity. However, in instances when tions depending on cell-cell or tumor-cell engagement and soluble
pre-malignant cells do not properly undergo apoptosis, the immune factors present in the microenvironment [36,37]. Here, we first discuss
system must quickly act to prevent further transformation and the po- the anti-tumor activities that can be exerted by innate immune cells.
tential for immune escape [20–22]. Tumor-resident macrophages, PMN, NK and DC are among the first
As in infection, both the innate and adaptive arms of immunity must to contribute to immediate, non-specific cytotoxicity against melanoma
work together to eliminate both pre-malignant and early stages of cells. Through activating NK receptors (NKG2D, NKp30, NKp46,
melanoma, and to provide long-term protection from potential relapse DNAM-1) and agonists present on the surface of melanoma cells, NK are
[23–26]. For this to occur, the innate compartment must quickly independently activated to eliminate melanoma cells that have sig-
eliminate tumor cells and act to recruit adaptive immune cells, present nificantly downregulated their MHC class I molecules [23,38]. Indeed,
tumor antigens through major histocompatibility complexes (MHC) and previous studies have found that IL-15 stimulation of NK cells is suffi-
provide the proper co-stimulatory signaling through surface receptors cient to cause regression of MHC class I low melanomas in mice. It has
and/or cytokines to generate a long-term, tumor-specific memory po- been demonstrated that the expression of NK receptor ligands by mel-
pulation. Cytokines involved in this process include interleukins (IL), anoma cells can be both dependent on tumor progression (acute vs. late
interferons (IFN) and colony stimulating factors (CSF), which can be recognition) and localization (primary vs. metastatic and in different
activating or suppressive in nature. Orchestrating an effective, anti-tu- metastatic sites). For example, NKp44 and NKp46 are expressed on
moral response, especially after negative selection in the thymus that lymph node metastases but to a lesser extent in skin metastasis. The
leads to self-tolerance of tumor cells, seems to be a nearly impossible expression of DNAM-1 ligands, such as nectin-2 (CD112) and PVR

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(poliovirus receptor), is independent of the anatomical site and tumor transferred M1- and N1-polarized cells generated in vivo with GM-CSF
stage, and the disruption of their interaction with DNAM-1 is re- (granulocyte-macrophage colony stimulating factor) treatment have
sponsible of the loss of cytotoxicity and of failed tumor rejection. also been shown to eliminate melanoma in recipient mice [66–70].
DNAM-1+ NK cells showed higher cytotoxicity with respect to DNAM- Overall, the innate immune system plays a critical role in first-line
1− NK cells, despite the two populations sharing the same positivity for defense against melanoma and will likely be indispensable for gen-
both NKp46 and NKG2D, suggesting the relevance of DNAM-1 signaling erating effective anti-tumor immunity using immunotherapy.
in activating NK cells against melanoma, at least during early re-
cognition and lymph node metastasis [38]. 2.3. Adaptive immunity
In addition to direct tumor interactions, NK cytolytic activity can be
induced through DCs activated by soluble antigenic peptides present in Long-term memory responses critical for life-long melanoma re-
the TME [23,39]. In general, NK cells poorly infiltrate primary cuta- mission involve the activation and expansion of adaptive immune cells,
neous melanomas and mostly accumulate in the peritumoral space, namely helper CD4 + T cells and cytotoxic or memory CD8 + T cells.
however, during regression, they can be observed more dispersed As previously mentioned, DC, and to some extent macrophages, are the
throughout the tumor tissue [40–42]. Intratumoral NK cells (activated) most capable in priming adaptive immunity to incite cytotoxicity of
can then indirectly contribute to recruitment and maturation of an- CD8+ effector T cells and also mediate generation of memory immune
tigen-presenting cells through the secretion of cytokines such as CXCL1 populations involved in long-term remission. DC are initially activated
(CXC-motif ligand 1) and CCL5 (CC-motif ligand 5). Macrophages, PMN in the tumor bed in the presence of cytosolic melanoma DNA through
and DC that are recruited to tumor tissue can then phagocytize apop- the cGAS-STING pathway [71–74]. Soluble tumor antigens from ne-
totic or dead melanoma cells or debris and cross-present tumor antigens crotic melanoma cells are engulfed by DC and macrophages and pro-
that drive secondary adaptive immune responses involving CD4 helper teolytically processed for direct presentation or cross presentation to
and CD8 effector T cells (CD4 Th and CD8 Teff cells, respectively) naïve T cells in tumor-draining lymph nodes (TDLNs) via MHC class I
[43–45]. Interestingly, several studies in pre-clinical melanoma models and II molecules. In some instances, neutrophils have also been shown
have shown that soluble factors secreted by activated T cells in turn to present antigen in TDLN, although their efficiency to elicit T cell
induce anti-tumor activity of innate immune cells (macrophages and responses is incredibly low [75]. Cytokines, such as interferon-γ (IFN-γ)
granulocytes) to assist in primary tumor growth control and minimize and tumor necrosis factor-α (TNF-α), play pivotal roles in activation of
lung metastases [46]. melanoma-specific T cells during TCR-MHC interactions and are ex-
As professional antigen-presenting cells (APCs), DCs are among the pressed by DCs and macrophages of the M1 phenotype.
most efficient in eliciting cytotoxic T cell responses against infection While neo-antigens expressed by melanoma cells would be the most
and malignancy. DCs circulate and survey various tissues throughout immunogenic, i.e. most likely to expand cytotoxic T cells, self-antigens
the entire body, ultimately migrating to lymph nodes where interac- expressed at high levels could potentially break tolerance and activate
tions with naive or memory T cells occur [39]. Mature DCs express a low-affinity CD8 + T cells. It is becoming more apparent, however, that
plethora of co-stimulatory markers, including CD80 and CD86 (cluster more than just overexpression of wildtype peptide sequences from
of differentiation 80 and 86, respectively), which are essential for ac- melanoma cells is needed to induce effective anti-tumor responses from
tivation of melanoma-specific T cells [47,48]. The T cell receptor tolerant or low-affinity T cells. Hence, the use of checkpoint inhibitors,
(TCR)-MHC class I interaction, co-stimulatory markers and proper cy- such as anti-PD1/PD-L1 and anti-CTLA-4, and adjuvants in combination
tokines (IL-12, IFN-γ) produced by DCs and helper T cells are requisite with self-antigen cancer vaccines is almost requisite [76,77]. Currently,
for the proper development of melanoma-specific, cytotoxic T cells identification of neo-antigens broadly expressed by melanomas is an
[49,50]. Ultimately, functional effector T cells must be recruited to area of intense research. Indeed, vaccines encoding neo-antigens ex-
melanomas through a chemokine gradient (CXCL9, CXCL10, CXCL11) pressed by a single melanoma can be therapeutically beneficial as de-
generated by DCs or tumor-associated stroma [51,52]. Inefficiency in termined in pre-clinical models of melanoma utilizing a single mela-
any or all of these steps can lead to compounding deficiencies in noma cell line or graft. However, neo-antigens are not identical from
adaptive tumor-specific immunity. Overall, DCs exert a protective role patient to patient or even from distal melanomas within the same in-
against melanoma tumors as evidenced by high frequency of DCs in dividual, thus requiring development of highly personalized vaccines,
tumor-negative sentinel lymph nodes [53–55]. which can be both cost- and labor-intensive with no guarantee of suc-
Anti-tumor macrophages and neutrophils, designated M1 and N1, cess [78,79].
respectively, have been studied extensively for their potential use as Once CD8 + T cells are sufficiently primed and activated, naturally
immunotherapy for melanoma [56–61]. These innate subsets exert anti- or through vaccination, they begin to seek out and induce apoptosis of
tumoral effects through phagocytosis, secretion of tumoricidal agents melanoma cells through the release of perforin and granules, which
(reactive oxygen species, nitric oxide, IFN-γ, Fas ligand/FasL) or as- then provides additional antigens for presentation and expansion of
semble other tumor-specific immune cells through secretion of che- melanoma-specific T cells [80]. Life-long remission can then be
motactic factors. Interestingly, while macrophages can stimulate achieved with this repeating cycle (Fig. 1), but in many cases, a select
adaptive T cell responses, a reciprocal relationship also exists whereby population of resistant melanomas subvert immune recognition and
activated Th1 cells generate tumor-killing macrophages through the destruction by downregulating antigen presentation machinery, direct
expression of IFN-γ, CD40 ligand and lymphotoxin-alpha. Use of mi- suppression of both innate and adaptive immune cells or expansion of
crobial agents (Bacillus Calmette-Guerin (BCG) and vaccinia virus) have immune-suppressive subsets.
been shown to be effective against melanoma by inducing the anti-
microbial, cytotoxic functions of macrophages. Topical agents, such as 3. Melanoma cross-talk leading to immune escape
ingenol-3-angelate, are known to recruit neutrophils in cutaneous
melanomas and induce their N1 anti-tumor functions [62,63]. Simi- 3.1. Hallmark of cancer: immune escape
larly, we found that the primary immune population mediating tumor
growth control of subcutaneous melanomas, following intravenous The existing relationship between cancer cells and immune cells can
administration of a tumor-colonizing, Salmonella-based therapy, were be described following the 3 E’s rule (Elimination, Equilibrium,
cytotoxic PMN responding to the bacterial vector [64,65]. In both cases Escape). During early phases, transformed cells are actively eliminated
of neutrophil-mediated killing, cytotoxic activity was contained within by immune cells, thus impeding tumor initiation. Due to the high
tumor tissue, minimizing adverse effects to healthy tissue that would plasticity of tumor cells, and the eventual development of favorable
normally be observed with radiotherapy or chemotherapy. Adoptively mutations, a subset of transformed cells can acquire properties that lead

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Fig. 1. Melanoma Clearance by Functioning Immune Cells. In the innate arm of immunity, natural killer (NK) cells bind tumor cells via receptor/ligand interactions
and release cytolytic molecules causing tumor cell death. Phagocytes, such as polymorphonuclear neutrophils (PMN), macrophages (Mφ), and dendritic cells (DC)
take up dead tumor cells and process and present tumor associated antigens (TAA). DCs are actively recruited by cytokines secreted from activated NK cells.
Recruitment of T- and B-lymphocytes by chemokine gradients and presentation of TAAs to T- and B-cells activates the adaptive arm of immunity. Tumor specific
CD8 + T-cells bind tumor cells presenting TAA on MHC molecules via engagement of the T-cell receptor, leading to release of cytotoxic granules into tumor cells.
Tumor specific CD4 + T-cells engage B-lymphocytes via TAAs presented by MHC molecules leading to release of antibodies specific for TAAs whose binding causes
tumor cell death through various mechanisms including NK cell killing. Adaptive immune cells also re-activate innate immunity through receptor/ligand interaction
as well as cytokine release, and tumor cell killing by adaptive immune cells releases further TAAs to be endocytosed and processed by APCs.

to immune evasion. During “equilibrium”, tumor initiation is achieved cancer cells;

by selection and expansion of the “immune-resistant” clones, but the 3 Neoantigens, antigens originating from somatic mutations.
host’s immune system is able to control tumor outgrowth through
continuous elimination of “immune-sensitive” clones. The last phase is Melanoma cells can escape T cell recognition through: i) the
characterized by immune escape: cells that are most fit to evade im- downregulation of TAAs, ii) defects/deletions in antigen processing
munity are free to proliferate leading to tumor progression, analogous machinery that may include proteasome subunits or transporters asso-
to Darwinian selection [9,81]. ciated with antigen processing (TAP) and/or iii) the downregulation of
Escape mechanisms are easily developed by cells with high plasti- MHC molecules, typically through β2-microglobulin mutations. By re-
city. Malignant melanoma represents one of the most immunogenic ducing antigen presentation alone, melanoma cells are capable of be-
tumors due to its high mutational burden, but plasticity of melanoma coming virtually “invisible” to the immune system [9,83–85]. On the
cells allows them to adapt to a hostile immune microenvironment. In other hand, melanoma cells are not only restricted to a single avenue of
this context, tumor cells can acquire different immunogenic features, as immune escape: the high antigen load within the tumor micro-
well as the ability to produce immunomodulatory molecules that can, in environment can contribute itself to T cell exhaustion and failed tumor
turn, affect immune cell activation or the composition of the immune control, thus adding to the complexity, and difficulty, of curative
infiltrate within the tumor [82]. The immunogenicity of melanoma cells treatments (Fig. 2). It is important to note that melanoma cells are able
is due to the expression of tumor-associated antigens (TAAs), highly to affect the behavior of stromal cells, namely cancer-associated fibro-
immunogenic proteins that can be divided into three classes: blasts (CAFs), in order to promote the recruitment of pro-tumor im-
mune cells [86,87]. CAFs are known to promote cancer cell prolifera-
1 Lineage-specific markers that are overexpressed in melanoma cells, tion and invasion [88], and other findings demonstrated that these cells
such as MART-1 (Melanoma Antigen Recognized by T cells), tyr- can participate to the evolution of an immunosuppressive environment,
osinase and gp100 (glycoprotein 100); both within the tumor bulk and in metastatic niches, through the re-
2 Cancer-testis (CT) antigens, physiologically expressed (low levels) lease of immune-modulatory factors. It has been recently demonstrated
by adult germ cells and placenta, but aberrantly over-expressed by that melanoma-derived extracellular vesicles can induce a pro-

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Fig. 2. Mechanisms of Immune Escape in Melanoma. Melanoma cells express IDO which inhibits the natural cytotoxicity of NK cells against tumor cells through the
conversion of available tryptophan to suppressive kynurenine. Melanoma-associated fibroblasts (MAFs) further inhibit NK cells through secretion of Prostaglandin E2
(PGE2). Melanoma cells also secrete factors such as VEGF and TGF-β to inhibit recruitment and function of APCs such as DCs. Immunosuppressive regulatory T-cells
(Treg) are recruited by melanoma cells through chemokine secretion and these cells further inhibit APCs through engagement of CD86 by CTLA-4 expressed on the
Treg surface. Treg also release inhibitory cytokines to activated effector T-cells (Teff) which prohibits their melanoma-directed cytotoxicity, as well as express
membrane-bound TGF-β which inhibits NK cell action. Melanoma cells directly inhibit Teff action through expression of the PD-L1 ligand which when binding the PD-
1 receptor on T-cells induces anergy. Secretion of apoptosis –inducing factors such as Fas ligand within exosomes leads to apoptosis of Teff. Melanoma cells also
recruit and convert myeloid-derived suppressor cells (MDSC) through secretion of GM-CSF or IL-6 as well as delivery of exosomes loaded with micro RNAs (miRNA).
MDSC inhibit Teff through multiple mechanisms such as expression of IDO. Finally, melanoma cells deplete glucose and amino acids such as glutamine and arginine
from the tumor microenvironment resulting in immune cell starvation.

inflammatory signature in lung fibroblasts (upregulation of cytokines cell antigen presentation; if only this signal is present, T cells are not
and chemokines, including IL-1α, IL-1β, CXCL10, CXCL1, CCL2, CCL3 completely stimulated and become anergic. The complimentary signal
and CCL5), thus enhancing the recruitment of myeloid-derived cells is the expression of co-stimulatory molecules on T cells (e.g. CD28), that
such as neutrophils [86]. Moreover, pro-inflammatory pathways, bind to their cognate receptors on antigen presenting cells (e.g. CD80,
eventually associated with BRAF mutations, can in turn induce the CD86). Similarly, inhibitory receptors (e.g. CTLA-4, PD-1, PD-L1, B7-
expression of PD-1 ligands and COX-2 on CAFs, contributing to the H2, B7-H3) trigger negative stimuli that lead to T cell anergy [94]. It
immune suppression [89]. CAFs can favor immune escape through the has been demonstrated that melanoma suppresses T cell activation
secretion of MMPs and of Prostaglandin E2 (PGE2), decreasing NK- through upregulation of co-inhibitory molecules such as PD-L1 [95,96]
mediated lysis of melanoma cells [90] mediated by a decreased surface and that the overexpression of PD-1 in T cells continuously exposed to
expression of the activating receptor NKp44 [91]. T cell functions can cancer antigens leads to T cell anergy [97]. Indeed, it has been de-
be also affected by CAFs: the secretion of cytokines (i.e., CXCL5) can monstrated that high antigen load is involved in CD8+ cell dysfunction
induce the expression of PD-1 on cancer cells [92], and the CAF- and exhaustion, so despite high immunogenicity of melanomas, T cells
mediated metabolic stress of CD8 + T cells limits their functions against are not able to restrain tumor growth. Dysfunctional T cells are char-
tumor cells [93]. The following section will highlight the mechanisms acterized by high levels of clonal expansion together with the over-
exploited by melanoma cells to communicate with cells of the tumor expression of checkpoint molecules (i.e., PD-1, TIM-3, LAG-3): thus,
microenvironment in order to promote an immunosuppressive setting. exhaustion might be induced by antigen-driven interactions with mel-
anoma cells [98,99]. From a functional point of view, dysfunctional T
cells lose their effector functions (i.e., cytotoxicity) while maintaining
3.2. Cell-cell contact the proliferative capacity, at least early during the acquisition of the
exhausted phenotype [100]. In an elegant study, Schietinger A. et al
In order to elicit an efficient antitumor immune response, T cells were able to follow the activation state of T cells during tumor initiation
must be fully activated by two co-stimulatory signals. The first one is and progression. The authors demonstrated that chronic antigen
MHC-T cell receptor (TCR) interactions and depends on DC or tumor

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presentation, eventually by non-professional presenting cells (such as Physiologically, Treg functions are required for maintaining self-toler-
tumor cells), in a non-inflammatory context, is responsible for the in- ance, thus providing suppressive control over antigen-specific Teff cells.
duction of an alternative, dysfunctional differentiation program in T Thus, it is not surprising that their recruitment by melanoma cells re-
cells. This program is triggered early during tumor initiation, even in presents a strategy to evade elimination. Treg produce im-
pre-malignant lesions, evolving to a “fixed” dysfunctional state as munosuppressive cytokines and chemokines, such as IL-10, IL-35 and
tumor progresses. In this context, a comparative whole-genome tran- TGF-β, and can also engage DC directly through CTLA-4 in order to
scriptomic analysis showed that “early” and “late” dysfunctional T cells inhibit antitumor immune responses [113,114]. Furthermore, they can
share a signature related to the inhibition of their effector functions inhibit effector functions of NK cells through expression of membrane
(e.g. Tbx21, Eomes, Id2, Gzmk, Ccr5, Cxcr3), with early phase T cells bound TGF-β which is responsible for NK cell downregulation of the
specifically characterized by the downregulation of genes involved in Natural Killer Group 2D receptor (NKG2D) [115].
activating T cell functions (e.g., Foxo1, Foxp1, Tcf7, Klf2) and the late The ratio of Teff/Treg within the tumor has a predictive value in
phase by the upregulation of genes involved in reducing immune immunotherapeutic responses [116]. A study by Shabaneh et al. de-
function (e.g., Egr1, Batf, Blimp, Lag3, Ctla4) [101]. T cell dysfunction monstrated that Treg cells have an important role not only in late phases
seems to be reversible during early phases, but becomes irreversible of anti-tumor suppression, but also in early phases of tumor develop-
over time. The transition from the dynamic and the fixed states is ment. In this study, an inducible PTEN/BRAF melanoma mouse model
epigenetically imprinted: the two conditions are characterized by dif- was utilized to demonstrate the importance of BRAF oncogenic sig-
ferent chromatin assets, leading to a differential gene expression. For naling in the recruitment of regulatory cells, driven by CCL2, CCL17
example, in early dysfunction, T cells show low expression of CD38, and CCL22 [117]. Moreover, defective production of cytokines and
CD101, CD30 L and high expression of CD5, while late dysfunction is chemokines involved in T cell homing was observed, contributing to an
characterized by the opposite pattern [102]. Otherwise, other studies imbalance between Teff and Treg cells. Among these molecules, IFN-γ
demonstrated that PD-1-expressing T cells chronically stimulated by expression contributed to both a tumor-supportive and a tumor-sup-
tumor antigens undergo fast proliferation followed by apoptosis due to pressive immune environment. Aberrant IFN-γ signaling has been as-
microenvironment-driven DNA damage. In this way, equilibrium is sociated with the downregulation of the Jak1/2 pathway in tumor cells
reached between proliferation and death, impeding T cell expansion and subsequent reduced production of CXCL9 and CXCL10 within the
and leading to failed control of melanoma outgrowth [103]. The ex- TME, two important chemotactic molecules responsible of T cell mi-
pression of immune-checkpoint molecules by melanoma cells, or by gration and infiltration [118]. Epigenetic silencing of genes encoding
tumor-associated immune or stromal cells, formed the rationale for the chemotactic molecules is one mechanism that has been described in
development of the immune-checkpoint inhibitor therapies (i.e., PD-1/ melanoma [85].
PD-L1/CTLA-4 axis inhibitors), that have been approved for the treat- Melanoma cells recruit and modify the function of macrophages (M)
ment of unresectable, metastatic melanoma. However, most patients and neutrophils (N) within the TME. These inflammatory, phagocytic
show primary or acquired resistance and, eventually, tumor relapse. cells can behave in an anti-tumor (M1 and N1) or in a pro-tumor (M2
Unfortunately, melanoma cells are known to exploit alternative me- and N2) fashion, depending on the signals received within the TME.
chanisms to suppress the immune system such as upregulation of TIM-3, Macrophages are recruited by secretion of CCL2 (MCP-1, monocyte-
LAG-3 and BTLA which can compensate for PD-1 or CTLA-4 axis in- chemoattractant protein-1) and it has been reported that the expression
hibition, thus sustaining immunosuppression [104–108]. levels of this chemokine is determinant for the induction of a pro-tumor
or an anti-tumor setting, with high levels favoring tumor rejection and
3.3. Pro-tumor cytokines and chemokines low-to-intermediate levels sustaining tumor growth [119]. A similar
biphasic effect is achieved by VEGF-C, involved in macrophage re-
Cancer cells are able to shape the local immune landscape through cruitment in addition to pro-angiogenic processes [120]. Tumor-asso-
the recruitment of pro-tumor, and the suppression or exclusion of anti- ciated macrophages (TAM) are frequently polarized toward a M2 phe-
tumor, immune subsets. In this context, the secretion of cytokines and notype because of the secretion of TGF-β [121]. IL-10 plays a pivotal
chemokines by melanoma cells is often mediated through the hyper- role in the ability of macrophage to modulate immune responses, which
activation of NFκB signaling pathways [109]. Pro-inflammatory events functions to downregulate MHC class II antigens and upregulate the
initiated by melanomas result in the recruitment of innate immune costimulatory molecule B7, leading to poor antigen-presentation and
cells, which include neutrophils, macrophages and DCs. As previously the inhibition of T cells [121]. Other M2 tumor-supportive features are
discussed, DCs can process tumor antigens and migrate to regional related to the production of matrix metalloproteases (MMPs), involved
lymph nodes where they can prime effector T cells (Teff) [85], thus in tumor invasion, and the production of proangiogenic molecules such
representing the functional bridge between innate and adaptive im- as VEGF to mediate extravasation of tumor cells [122].
munity. Melanoma cells, however, impair DC recruitment and ma- Together with DCs, neutrophils are among the first to respond to
turation through vascular endothelial growth factor (VEGF) and tumor-mediated inflammatory signals. Malignant melanomas produce
transforming growth factor (TGF)-β secretion, which impedes T cell chemokines that lead to neutrophil infiltration during tumor initiation
targeting of tumor cells [107,110]. Moreover, dysregulation of the and progression. Particularly, the mobilization of neutrophils is
Wnt/β-catenin signaling pathway in melanoma cells, often related to achieved by molecules that bind to CXCR2, which includes CXCL1,
aggressive cancer cell subsets (i.e., cancer stem cells), leads to defective CXCL2, CXCL3, CXCL5 and CXCL8 [123]. Interestingly, UVB radiation
CCL4 production, which in turn impairs DC and T cell recruitment exposure, one of the well-known causes of melanoma, induces the
while also inducing resistance to anti-PD-1 therapies [107,111]. IL-37b production of CXCL1 and CXCL8 and the recruitment of anti-tumor
is another factor involved in anti-tumor suppression that mediates the neutrophils [124]. Like macrophages, neutrophils can be polarized to
downregulation of costimulatory molecules CD80 and CD86 on APCs become predominantly anti-tumor (N1) or pro-tumor (N2). The neu-
[12], resulting in suboptimal activation and dramatic impairment of Teff trophils role in tumor initiation and progression has been matter of
cells. debate: it seems that tumor initiation is characterized by the presence of
In addition to the impairment of DC-mediated T cell recruitment, N1 neutrophils that mediate melanoma cell killing, while in late stages
melanoma cells can redirect the production of cytokines to favor re- N2 neutrophils are the most abundant phenotype and have a role in
cruitment of pro-tumor T cells while rejecting Teff cells. Melanoma tumor progression [125,126]. To date, emerging clinical evidence
growth and progression have been correlated with the presence of support the finding that a high neutrophil-to-lymphocyte ratio (NLR)
CD4+CD25+Foxp3+ regulatory T cells (Treg) within the tumor in represents poor prognostic outcomes and is a negative predictive in-
B16F10 tumor-bearing mice and in melanoma patients [112]. dicator of immune checkpoint inhibitor therapy success [127,128]. The

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M. Marzagalli, et al. Seminars in Cancer Biology xxx (xxxx) xxx–xxx

specific mechanisms that drive neutrophil phenotypic switching are not Amino acid availability within the TME is crucial for T cells that, similar
fully understood, but may be related to the complex network of soluble to other immune cells, are unable to synthesize amino acids (i.e.,
mediators within the TME. Tumor-derived IFN-β has been demon- tryptophan, glutamine, arginine). Malignant melanoma has been de-
strated to be involved in the induction of the N1 phenotype [59], thus monstrated to be highly dependent on glutamine metabolism that fuels
limiting the pro-angiogenic and pro-invasive properties of tumor-asso- oxidative phosphorylation by entering the TCA (tricarboxylic acid
ciated neutrophils [129]. Moreover, it has been demonstrated that cycle) after conversion to glutamate and then α-ketoglutarate [139].
circulating tumor cells (CTCs) promote the establishment of metastasis The glutamine addiction of melanoma cells restricts glutamine avail-
through the secretion of G-CSF (granulocyte colony stimulating factor) ability for T cells, preventing proper T cell activation [140].
and CXCL6, and subsequent recruitment of N2 neutrophils [130]. Pro- L-arginine metabolism in melanoma has been associated with im-
tumor neutrophils can contribute to immune evasion orchestrated by munosuppression [141]. Arginine uptake is crucial because its en-
melanoma cells through the expression of immune checkpoint proteins dogenous synthesis rate is not sufficient to sustain highly proliferative
(i.e., PD-L1), the overexpression of other immunosuppressive mole- cells, and studies have demonstrated that downregulation of argino-
cules, such as IDO (indoleamine 2,3-dioxygenase) and iNOS (inducible succinate synthetase in melanoma cells renders them unable to generate
nitric oxide synthase), or the secretion of molecules involved in the arginine [142,143]. High arginine uptake by cancer cells leaves little
recruitment of Treg (i.e. IL-17) [131,132]. for T cells, leading to reduced proliferation and survival [141,144].
Melanoma is also known to recruit myeloid-derived suppressor cells Importantly, L-arginine is the precursor for NO synthesis. NO is a cru-
(MDSC). MDSC are comprised of immature precursors of DCs, macro- cial immunomodulatory factor that exerts its suppressive effects though
phages and neutrophils, usually retained within the bone marrow, but inhibition of T cell proliferation and function. Moreover, reactive ni-
that can be mobilized upon appropriate stimuli. Their expansion and trogen species, such as peroxynitrite, are known to induce apoptosis of
migration can be induced by inflammatory molecules produced during T cells [145,146].
chronic inflammation and cancer progression, such as GM-CSF, IL-6, IL- In contrast to glutamine and arginine, which are directly consumed
10, IFN-γ and VEGF [103] and a central role seems to be played by by tumor cells, tryptophan deficiency within the TME is due to the
CCR5 ligands in melanomas: CCL3, CCL4 and CCL5. Importantly, an upregulation of the catabolic enzyme IDO in tumor cells and MDSC
enriched CCR5+MDSC infiltrate within the melanoma microenviron- [147,148]. Physiologically, this enzyme is involved in tolerance during
ment has been observed in mouse models, and the administration of pregnancy to prevent rejection of the fetus and it can thus be exploited
CCR5-Ig fusion protein leads to melanoma growth inhibition associated by tumor cells as a mechanism of immune escape. In fact, it is fre-
with impaired MDSC trafficking [104]. The inhibitory role of MDSCs on quently overexpressed by cancer cells and its upregulation has been
anti-tumor immunity is due to: 1) the production of NO (nitric oxide) associated with tumor progression and poor prognosis [149]. Regarding
and Arg-1 (arginase 1), inducing T cell apoptosis and cell cycle arrest, melanoma, its expression has been correlated with Breslow thickness
2) high expression of PD-L1, inducing T cell exhaustion, 3) upregulation and PD-L1 expression, and it negatively correlates with progression-free
of IDO, leading to T cell anergy and 4) secretion of IL-10 and TGF-β, survival [150,151]. Mechanistically, local tryptophan deprivation is
suppressing T cell trafficking [103]. The presence of MDSC in cancer signaled through glucokinase, an amino acid-sensing kinase that in turn
tissue is another potential prognostic indicator for immune-checkpoint triggers downstream pathways such as mTORC1 (mammalian target of
inhibitor therapy treatment, and has been reported as a predictive rapamycin complex 1) inhibition and the consequent activation of the
marker of response to ipilimumab in melanoma patients [105]. autophagic process, leading to T cell anergy [152]. Another mechanism
implicated in T cell anergy due to tryptophan deficiency is the activa-
3.4. Metabolic mediators tion of stress sensors such as GCN2 (general control nonderepressible
2), which senses the lack of tryptophan-charged tRNAs and induces a
Cancer cells can escape immunosurveillance or evade im- stress response that limits protein translation. Moreover, GCN2 acti-
munotherapies via metabolic reprogramming. Metabolic mediators can vation promotes Treg differentiation and activation [153]. However,
drive immunosuppressive signaling and essential substrate consump- recent evidence has demonstrated that this sensor does not affect im-
tion by cancer cells can cause metabolic depletion leading to anergy of munity in B16 melanoma tumors, and this could be due to the main-
immune cells. Cancer cells require high nutrient consumption in order tenance of adequate tryptophan levels within the TME despite its cat-
to support tumor growth. Their plastic phenotype allows for rapid re- abolism through IDO [154].
programming of cell metabolism for survival in hostile conditions such IDO catalyzes the conversion of tryptophan to kynurenine which
as hypoxia, as well as the ability to activate “unconventional” metabolic acts as an immunosuppressive molecule. Increased kynurenine in the
pathways, such as glycolysis, even in the presence of normal oxygen TME directly inhibits NK cell cytolytic activity through the down-
levels (Warburg effect). The increase of glucose consumption by mel- regulation of activating receptors (NKp44, NKp30, and NKG2D) [155].
anoma cells leads to glucose deprivation for cells within the TME whose In other cell types, kynurenine binds to the aryl hydrocarbon receptor
metabolism is strikingly glycolytic. In this context, glucose deprivation (AhR), a ligand-dependent transcription factor that, once activated,
inhibits T cell proliferation and activation, dampening anti-tumor im- promotes the differentiation of Treg cells, reduces the immunogenicity
mune responses. CD28 is involved in multiple pathways related to T cell of APCs and induces the upregulation of PD-1 expression on Teff cells
activation, such as the upregulation of the glucose transporter GLUT1, [156]. Another metabolic product that acts as an immunosuppressive
which when silenced, significantly impairs T cell functions [133]. It has mediator is adenosine obtained from ATP through the activity of the
been demonstrated that the production of IFN-γ, cytolytic activity and ectonucleotidases CD39 and CD73. Specifically, the release of in-
cell cycle progression of T cells are regulated by glucose consumption tracellular ATP is followed by its conversion to AMP by CD39, and
[134]. Moreover, recent studies show that the increase of oxidative subsequently AMP undergoes dephosphorylation by CD73. Hypoxic
metabolism that can occur in melanoma cells consumes oxygen from conditions, as well as extracellular stresses, represent the driving
the TME leading to oxygen deprivation for T cells [135]. events: the induction of the transcription factor HIF1α (hypoxia in-
Lactate derived from cancer cell glycolysis represents another me- ducible factor 1) in response to low oxygen levels promotes the ex-
tabolite involved in immune cell suppression. In particular, it has been pression of CD39 and CD73 both on cancerous and non-cancerous cells
involved in the reduction of antigen-presenting efficiency of DCs [136]. (i.e., endothelial cells and lymphocytes) [157,158]. The A2A high-af-
Furthermore, as a consequence of excessive amounts of lactate, extra- finity adenosine receptor has been implicated in the im-
cellular acidosis causes the inhibition of NK and T cells in mouse models munosuppressive effect of this molecule because of its high expression
of melanoma [137], and the neutralization of acidic conditions im- levels on immune cells. Specifically, adenosine has been shown to in-
proves response to immune-checkpoint inhibitor therapies [138]. hibit NK infiltration and function, to impair macrophage activation and

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to favor Treg cell maturation, while impairing Teff cell priming, pro- They are secreted into extracellular spaces and are exploited by tumor
liferation and cytokine release [159–161]. Furthermore, increased cells to deliver signals to the surrounding cells, allowing crosstalk with
production of adenosine has been observed in melanoma progression other tumor cells or cells of the TME.
during immunotherapy (i.e., during adoptive T cell transfer or immune- T cell function is frequently affected by tumor-derived exosomes.
checkpoint blockade), and it has been attributed to the phenotype They have been implicated in the expansion of Treg and in the promo-
switch of melanoma cells. Thus, adenosine can be implicated both in tion of their function [174], thus reinforcing immune evasion. Mela-
immune escape and in mechanisms leading to adaptive resistance noma exosomes can also deliver membrane-bound ligands, such as PD-
[162]. L1, that find their cognate receptors on T cells providing inhibitory
signals [175]. Moreover, exosomes may carry soluble factors such as
3.5. MicroRNAs (miRNAs) Fas and TRAIL (TNF-related apoptosis-inducing ligand) which can in-
duce Teff cell apoptosis [176]. As mentioned above, melanoma-derived
MicroRNAs are small, 20–25 nucleotide-long, non-coding RNAs that exosomes can carry miRNAs that silence anti-apoptotic Bcl (B-cell
are involved in the attenuation or complete inhibition of protein lymphoma) family proteins proteins, such as Bcl-2, Bcl-xl, and Bcl-w, to
translation. Their binding to a specific RNA is due to their compli- induce mitochondrial-mediated apoptosis in CD4+ T cells [177]. In
mentary nucleotide sequence: a fully complimentary sequence leads to some cases, however, the exposure of MHC-I and melanoma-associated
mRNA degradation and inhibition of protein expression, whereas a antigens (MART-1, gp100, tyrosinase) on exosome membranes can
partial complementarity is responsible for the attenuation of protein mimic antigen presentation processes, leading to T cell activation
expression [163]. Several miRNAs have been implicated in cancer [178].
progression and both oncogenic and tumor-suppressor miRNAs have Several studies demonstrated the important role of tumor-derived
been recognized. The overexpression of oncogenic miRNAs by cancer exosomes in the development of metastatic niches. An elegant study
cells often leads to the inhibition tumor suppressor proteins (i.e., performed by Peinado H. et al. demonstrated that melanoma-derived
apoptotic proteins, proteins involved in cell differentiation or in cell exosomes induce the acquisition of pro-angiogenic and pro-metastatic
cycle regulation), and the downregulation of tumor-suppressor miRNAs phenotypes by bone marrow cells leading to accelerated tumor growth
leads to aberrant expression of proteins involved in cancer progression and increased metastasis. This is due, at least partially, to the exosomal-
(i.e., oncogenes, anti-apoptotic proteins or proteins involved in cell mediated transfer of the Met receptor to bone marrow progenitor cells,
proliferation) [164]. MiRNAs can modulate intracellular processes, as inducing their mobilization via S6 induction and ERK phosphorylation
well as be transferred to nearby cells for cross-talk within the TME. In [179].
order to overcome degradation by RNAses in the extracellular space, The two-fold nature of melanoma-derived exosomes is further de-
miRNAs are carried by transporters, such as proteins (argonaute, ARG), monstrated in DCs where melanoma exosomes may carry TAAs to DCs
high-density lipoproteins (HDL), or extracellular vesicles (exosomes) to promote the activation and expansion of cytotoxic T cells, but they
(163). may also inhibit differentiation of DCs from monocytes due to high IL-6
miRNAs are involved in the modulation of the immune micro- content [178].
environment in malignant melanoma. The miR-30b/-30d cluster, up-
regulated in melanoma cells, has been associated with GalNAc trans-
ferase 7 (polypeptide N-acetylgalactosaminyltransferase 7) 4. Augmenting or rescuing immunity in melanoma
downregulation, which impairs recruitment of Teff cells and increases
infiltration of Treg following increased IL-10 secretion [165]. Recently, 4.1. The era of cancer immunotherapy
a panel of miRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e,
miR-125a, miR-146b, miR-99b), carried from tumor cells by extra- In recent years, several treatments have been approved by the
cellular vesicles, have been implicated in monocyte conversion to United States Food and Drug Administration (FDA) for melanoma.
MDSC and in immune-checkpoint inhibitor resistance in melanoma Application of each treatment is dependent on the features of the cancer
patients [166]. Among other miRNAs, miR-210 is upregulated by hy- (stage, location and genetics) and can include combinations of surgery,
poxic conditions in melanoma cells and impairs the susceptibility of photodynamic therapy, chemo/radiotherapy, targeted therapy or im-
tumor cells to T cell-mediated lysis [167]. MiR-21 and -29a are known munotherapy. Most melanomas of stage I-III are removed by surgery
to target anti-angiogenic pathways, thus promoting tumor angiogen- followed by adjuvant therapy (targeted or immunotherapy) [180].
esis, and genes involved in M1 macrophage polarization [168]. The role Metastases are treated with a combination of surgery (if solitary/loca-
of miR-155 is more controversial because it can exert both immune- lized) and adjuvant chemotherapy as well as radiotherapy for advanced
promoting and immune-suppressive effects [169]. It has been im- metastases of the skin, bone and brain [181]. Metastases are the main
plicated in macrophage polarization toward the M1 phenotype to pro- cause of death, thus requiring more effective strategies to target distal
mote anti-tumor immunity, but in tumors with increased IL-1β sig- metastases with greater efficacy and significantly less toxicity. To date,
naling, it mediates the induction MDSCs [170,171]. Moreover, tumor infiltrating lymphocytes (TILs) have been associated with posi-
transgenic mice lacking miR-155 showed defective T cells and increased tive outcome and improved survival in patients with malignant mela-
B16F10 melanoma growth [172]. nomas [182]. Thus, immunotherapeutic strategies have been a focal
Other studies have demonstrated that melanoma-derived miRNA point for treatment of advanced stage, metastatic melanoma, with some
can affect response to immune-checkpoint inhibitor therapies. MiR- having shown incredible success in a select cohort of melanoma pa-
146a has been implicated in supporting immune suppression during tients.
melanoma growth wherein mice lacking its expression showed lower The prognostic factors that determine the efficacy of a particular
metastasis and increased survival. Targeting miR-146a with a specific immunotherapy are still largely unknown. Despite successes, cancer
antagomir acted synergistically with anti-PD-1 to enhancing the anti- relapse and variable response rates among patients of all stages are
tumor immune response [173]. observed. This is likely exacerbated by the ability of melanoma to
quickly adapt multiple immune suppressive pathways to escape im-
3.6. Exosomes mune attack as previously discussed. A better understanding of these
escape mechanisms has led to predictions that targeting multiple sup-
Exosomes are small vesicles originating in the cytosol and derived pressive pathways will be more efficacious than single immunotherapy
from endosomal compartments. They are delimited by a phospholipid treatment, and will be critical for maintaining long-term tumor sur-
bilayer and can carry various molecules (proteins, lipids, nucleic acids). veillance.

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4.2. Administration of cytokines CTLA-4−/− knockout mice, lethal hyperproliferative lymphocyte ex-
pansion occurs early in life and prevents survival past three weeks
IFNs are cytokines normally secreted by leukocytes during infection [202].
and are instrumental in the development of anti-proliferative and anti- Given the role that CTLA-4 plays as a negative regulator of T cell
angiogenic activities against melanoma [183]. IFNs act as agonists for activation, it was hypothesized that blocking engagement with CTLA-4
the anti-tumor activity of both adaptive and innate immune cells and could boost immunity against tumor cells [203]. Ipilimumab, FDA ap-
are antagonistic towards suppressive immune subsets such as MDSCs proved in 2011 for melanoma, is an antibody which binds and prevents
and Treg. IFN-α and IL-2 are cytokines generally given in combination signaling through CTLA-4 [197,204]. Anti-CTLA-4 antibodies act as
with surgery and chemotherapy, radiotherapy or targeted therapy. IFN- antagonists, blocking inhibitory signaling, and increasing the potential
α was FDA approved in 1995 as adjuvant therapy for resected stage IIB/ for cytotoxic T cells in melanomas to become activated and expand.
III melanoma [184]. IFN-α has been shown to induce upregulation of Ipilimumab has been combined with cytokine therapies, as discussed
MHC class I on melanoma cells and immune cells to cause increases in previously, with overall response rates of ˜40% and median progres-
cancer cell death and extension of survival [185]. Only a small per- sion-free survival (PFS) of 6 months [205]. In Phase III trials, ipili-
centage of patients, however, respond to IFN-α adjuvant treatment, mumab monotherapy has been shown to be more effective than cancer
with ulceration of the primary melanoma being a predictive indicator of vaccines alone (gp100 vaccine) in increasing median overall survival
IFN sensitivity [186]. A pegylated form of IFN-α, Peg-IFN has also been (OS) of metastatic melanoma patients (10.1 months vs. 6.4 months,
approved for stage III melanomas, with effects mimicking those of un- respectively). Combination therapy did not increase median OS, sug-
pegylated IFN-α [187], but with a longer half-life in circulation leading gesting that in situ vaccination may be occurring [206].
to increased efficacy. However, additional care must be taken to Another checkpoint receptor found on the surface of T cells, PD-1,
minimize adverse events. FDA-approved in 1998 for metastatic mela- binds its agonist PD-1 ligand (PD-L1) to also suppress T cell activation.
noma, IL-2 is known to act directly on T cells, which includes effector PD-L1 is expressed by melanoma cells or tumor-associated stroma, and
CD8 and regulatory CD4 cells [188]. Due to potentially dangerous ad- this expression is strongly correlated with efficacy of anti-PD-1 im-
verse events such as tachycardia and multisystem organ failure, patients munotherapy [207]. PD-1 is also expressed on B and NK cells, and thus
are first screened for biomarkers such as VEGF and fibronectin [189]. therapeutic blockade could potentially affect these immune subsets as
well [95]. Nivolumab was the first anti-PD-1 antibody to be approved
4.3. Expanding melanoma-specific T cells and targeting suppressive immune by the FDA in 2014 for the treatment of patients with metastatic mel-
subsets anoma. Nivolumab binds PD-1 to prevent the interaction between PD-1
receptor and its ligands in the TME, favoring a more active anti-tumor
Gp100 is a glycoprotein that is overexpressed by melanoma cells phenotype. Nivolumab treatment significantly increases median PFS to
and has negligible expression in healthy tissues, making it an ideal 6.9 months, compared to 2.9 and 2.2 for ipilimumab and chemotherapy
melanoma-specific antigen for vaccine development. Monotherapy with monotherapies, respectively [205,208]. However, more impressive is
gp100 peptides has shown little efficacy in preclinical melanoma the median PFS of 11.5 months with combination nivolumab/ipili-
models, however, combination treatment with gp100 peptides and IL-2 mumab. Another anti-PD-1 antibody, pembrolizumab, was FDA-ap-
showed a dramatic increase in median progression-free survival (PFS) proved in 2015, for advanced melanomas [209,210]. Like nivolumab,
and complete responses of 5% [190]. Most importantly, gp100 peptides pembrolizumab prolongs PFS and OS with less toxicity than ipili-
are capable of inducing T cell responses in patients with advanced mumab. Currently, there are numerous clinical trials utilizing check-
melanoma [191,192]. Gp100 is currently being evaluated in several point inhibitors alone or in combination with chemotherapy, radio-
clinical trials as monotherapy (NCT01744171, NCT0-117,647) or in therapy and other immunotherapies (NCT01103635, NCT0253078,
combination with immunotherapies (NCT00960752, NCT01176461, NCT02643303, NCT03086174, NCT02608268). Similar studies are also
NCT02535078). being performed using anti-PD-L1 antibodies such as durvalumib,
Tregs suppress effector T cell responses and can be found circulating avelumab and atezolizumab (NCT02535078, NCT03167177,
or infiltrating tumors in melanoma patients, ultimately contributing to NCT03138889).
poor clinical outcome [193]. Strategies to target Tregs and increase T
cell immunity are limited. Ontak, which was FDA-approved in 1999, is
an IL-2 protein fused to diphtheria toxin, and is designed to target 5. Concluding remarks
peripheral blood Tregs through their IL-2 receptor [194]. A Phase II trial
in late stage melanoma patients (stage IV) showed 17% partial re- Fully unraveling the crosstalk between the immune system and
sponse, 15% mixed responses and 5% stable disease [195]. However, in melanoma that causes loss or suppression of anti-tumor responses will
another clinical study, no objective response, survival benefit, or de- be critical for the development of more effective and less toxic treat-
pletion of Tregs was observed [196]. Thus, while Treg depletion could ments. The identification of cytokines, immunosuppressive immune
provide great benefit alone or combined with other immunotherapeutic subsets and checkpoint pathways that are critical for melanoma pro-
treatments, more effective strategies must be developed. gression has led to the development and FDA approval of numerous
immunotherapeutic agents. This is important since the heterogeneity of
4.4. Checkpoint inhibitors advanced melanomas will undoubtedly require a combination of these
agents to establish durable, life-long immunity. Also, one must take into
Current FDA-approved checkpoint inhibitors consist of antibodies consideration the optimal doses required to establish durable tumor
which bind checkpoint proteins or receptors to prevent signaling which control while minimizing adverse events. Since each patient would be
causes T cell anergy. CTLA-4, also known as CD152, is an important unique in the features of their melanoma, it will be necessary to de-
negative regulator (checkpoint) in T lymphocyte activation [197,198]. termine those features prior to treatment in order to select a balanced
CTLA-4 is upregulated on the surface of tumor-associated T cells and, in combination that will maximize PFS and minimize toxicity.
contrast to CD28, transmits an inhibitory signal when bound to co-sti-
mulatory molecules B7-1 (CD80) or B7-2 (CD86) found on antigen
presenting cells [199]. CTLA-4 binds with much greater affinity and Declaration of competing interest
avidity to co-stimulatory molecules compared to CD28, thus favoring a
suppressive phenotype in the TME [200]. Furthermore, CTLA-4 en- The authors declare that there are no conflicts of interest.
gagement inhibits T cell cytokine production and proliferation [201]. In

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