Melanoma-derived mediators can foster the pre-metastatic niche: crossroad to lymphatic metastasis

Abstract

The natural history of advanced malignant melanoma demonstrates that, in most cases, widespread tumor dissemination is preceded by regional metastases involving tumor-draining lymph nodes (sentinel lymph nodes). Under physiologic conditions, lymph nodes play a central role in immunosurveillance to non-self-antigens to which they are exposed via afferent lymph. The dysfunctional immunity in sentinel lymph nodes is mediated by tumor secretory factors that allow the survival of metastatic melanoma cells within the lymph node by creating a premetastatic niche. Recent studies outline the altered microenvironment of lymph nodes shaped by melanoma mediators. Here, we discuss tumor secretory factors involved in subverting tumor immunity and remodeling lymph nodes and highlight emerging therapeutic strategies to reinvigorate antitumoral immunity in sentinel lymph nodes.

Melanoma metastasis: the impact of disrupted lymph nodes

Human cutaneous melanoma is an aggressive form of skin cancer that has continuously increased in incidence over the past 50 years, and If current rates continue (analyzed by GLOBOCAN 2020 data) (gco.iarc.fr), cases of malignant melanoma are estimated to increase by 50% in incidence and 68% in mortality by 2040 [1]. Melanoma spreads rapidly to distant sites, but a regional tumor-draining or sentinel lymph node (SLN) (see Glossary) is almost always the first site of metastasis. Hence, an SLN biopsy is crucial for the prognosis of cutaneous malignant melanoma and is recommended by the National Comprehensive Cancer Network [2]. Histopathologic assessment of SLN for the presence or absence of metastatic cells is essential for melanoma staging and a key predictor of disease outcome [3]. Indeed, conventional hematoxylin and eosin assessment and immunostaining for melanoma-specific antigens are often used to reliably identify melanoma metastases within the SLN. A recent study showed that SOX10 and pS100 could be specific protein markers for detecting melanoma metastasis in SLN [4]. Notably, SLNs also undergo structural remodeling before cancer metastases – a process mediated by tumor-derived extracellular mediators such as soluble factors and extracellular vesicles (Box 1).

Box 1. Evolution of the tumor niche by metastatic melanoma-derived secreted factors.

Melanoma cells secrete many factors in the tumor microenvironment (TME) that are associated with altering surrounding tissues to support tumor spread. For instance, certain secretory factors can weaken endothelial barrier integrity and allow tumor cells to metastasize [5–7]. The extracellular matrix (ECM) is an important aspect of the migratory behavior of tissue-infiltrating cancer cells because it is constantly modified inside the tumor with the activity of several cell types found within the TME, such as tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and cancer cells themselves [8,9].

Cancer cells infiltrate the epithelial basement membrane and proliferate invasively into the underlying vascularized tissue, which is crucial for the metastasis of melanoma tumors. Tumor-vascular interfaces can be established by generating peri- or intratumoral angiogenesis and lymphangiogenesis simultaneously [10]. Cancer cells within the invasive vicinity of the primary tumor mass collectively infiltrate the surrounding normal tissue [8]. Some melanoma patients also develop regional cutaneous or subcutaneous metastases, known as “in-transit metastases ” that have lesions developed in dermal and subdermal epithelial layers before reaching the lymph nodes which are associated with poor prognoses [11].

Tumor dissemination through the lymphatic system occurs by high amounts of lymphangiogenic factors secreted by the tumor [12]. In melanoma, lymphangiogenesis is mediated by lymphangiogenic factors produced by the cancer cell itself; TAMs, CAFs, and/or activated platelets play a crucial role in tumor growth [13,14]. The dynamic production of VEGF-C and VEGF-D by tumors is connected to the amount of tumor lymphangiogenesis and LN metastasis in melanoma [15].

Extracellular mediators make profound changes in the SLN through increased lymphangiogenesis, remodeling of the blood vessel, and elevated secretion of immunosuppressive cytokines or chemokines which help develop a pre-metastatic niche within the tumor-draining LNs (e.g., SLN). Tumor-derived factors such as extracellular vesicles, VEGF, TGF-β, prostaglandin, and IL-6 are involved in tumor-draining lymph node alterations such as stromal cell proliferation, lymphangiogenesis, and ECM remodeling [6,16].

These secretory factors released from the tumor do not only contribute to metastasis but can also help in tumor cells’ survival and proliferation [17]. For example, melanoma or melanoma-associated cells secrete VEGF, TGF-β, IL-1β, TNFα, IL-8, and MCP-1 which can act as metastatic mediators [5]. In the primary tumor site, these tumor secretory soluble factors and vesicles are responsible for the interaction of melanoma cells, macrophages, and endothelial cells and can further mediate melanoma-endothelial interactions at the secondary tumor site [15,18,19]. Additionally, a study also showed that VEGFR1⁺ cells expressing VLA-4 or integrin α4β1 upregulated resident fibroblast by tumor conditioned media in the permissive niche, supporting the incoming tumor [20].

The immunosuppressive microenvironment within the SLN favors the development of a premetastatic niche (PMN) that helps the metastatic colonization of malignant cells. We and others are leveraging the multiplex immunofluorescence imaging (MxIF) platform to explore early changes in SLN that can promote metastasis. For instance, exploring lymph node (LN) immunity with and without melanoma metastasis will be crucial for developing suitable candidate targets that can prevent early nodal metastasis. Mounting evidence in recent literature suggests that tumor secretory factors can mediate major immunologic or lymphovascular events in the SLN in melanoma, leading to LN metastasis. In this review, we overview the determinants of SLN remodeling by characterizing some of the mediators of metastatic melanoma and their interactions with the SLN’s cellular machinery to initiate PMN development. We also highlight current technologies used to establish tools for prognoses and therapies against immunosuppressive and lymphangiogenic pathways activated in the PMN.

Melanoma-derived secretory factors contribute to creating the PMN.

Melanoma cells secrete crucial mediators that suppress immune cells in the tumor microenvironment (TME) that flow into the SLN and can modify the structure and/or function of the latter, ahead of cancer metastases. These melanoma secretory factors are released in the tumor’s interstitial fluid (lymph), which flows into tumor-draining LNs via afferent lymphatic vessels. A study shows that melanoma-secretory factors are highly enriched in the lymphatic exudate compared with blood plasma [21], which might help explain the development of the PMN in the SLN. Melanoma-secretory factors include key mediators such as VEGF, TGF-β, bFGF, galectins, Th2 cytokines, interleukins (e.g. IL-1β, IL-6, IL-8, IL-10, and IL-15), chemokines (e.g. CXCL 1, CXCL 2, CXCL 3, CXCL 8, CXCL 10, CCL2, and CCL5), and exosomes, which favor the metastatic colonization at SLNs by suppressing immune activity [6,14,22–28] (Table 1). In one study using immunohistochemical staining, our laboratory group found no significant differences in CD8⁺ T-cells (cytotoxic T lymphocytes; CTLs), antigen-presenting cells (APCs), costimulatory surface markers of APCs (CD40, CD86), and vascular markers of melanoma SLNs, with or without metastasis; however, decreased numbers of CTL were reported in SLN with metastasis compared with no metastasis 4% vs. 35.7%, as well as decreased expression of CD86 or VEGFR3 with metastasis: 5.2% vs. 23.8%, and 10.3% vs. 36.7%, respectively, compared to dormant LN (collected from a breast cancer patient who presented with no active cancer and had undergone prophylactic contralateral mastectomy with LN biopsy) [29]. This suggested that altered regional immunity in the SLN might precede LN metastasis [29]. From another angle, metabolic adaptation in the SLN is also relevant in enabling LN metastasis [30]. For example, high lipid metabolism in SLNs has revealed acidification of the LNs with breast cancer cells; these can provide a real-time diagnosis of a cancerous state using LN metastatic lymph diagnoser (MMLD) [31]. Moreover, and acidic niche in LNs has been shown to inhibit CTL effector function present in the extracellular space of the paracortical zone in LNs in a B16 melanoma mouse model, as measured by the seahorse assay[32]. These SLN alterations are inexorably related to the tumor of origin because of the secretion of tumor-promoting soluble molecules and extracellular vesicles (EVs) that not only support tumor growth in situ but can also mediate LN remodeling (Fig.1, Table 1 and Supplementary Tables 1 & 2). Examples of such modulators are described below.

Table:1.

Selected human melanoma-derived soluble factors implicated in the development of PMNs in human and mouse models.

Secretory factors	Reported effects	Model(s)/clinical samples	Refs
Amyloid β	Preventing phagocytosis of melanoma cells;involved in altering immune cells toward PMN development	Human melanoma short term cultures (STCs), melanoma cell lines and Rat	[107]
APO2L/TRAIL and FASL	Defending against activated effector immune cells (CTL and NK cells) through engagement of FasL and APO2L/TRAIL-sorted microvesicles with effector cells	Human melanoma cell line (MelJuSo)	[108]
Adrenomedullin	Involved in angiogenesis and lymphangiogenesis promoting melanomas	Human melanoma cell lines (A375, SK-MEL-28, and MeWo)	[109]
Galectin 1	InducingVEGF-like signaling that contributes to angiogenesis; vaccination against Galectin-1 in mice is immunogenic	B16 melanoma cells and C57BL/6 J mice	[110]
Galectin 9	Modulating tumor immunity by promoting Th2/M2-like macrophage differentiation	Melanoma patient samples	[28]
IL-1	Inhibiting JAK1-STAT3 mediated actomyosin contractility of LN FRCs; decreasing expression of MITF-M and melanocyte differentiation antigens	Human LN-fibroblast, human melanoma STCs and C57BL/6 J mice	[111]
IL-6	Inducing CCR5 expression and immunosuppressive activity of MDSC in RET transgenic melanoma mouse model	Human RET transgenic mice and MSC-2, myeloid suppressor cell lines	[112]
IL-8	Involved in FKBP51-induced melanoma growth.	Human melanoma cell lines, A375P and A375SM	[113]
IL-10	Directing M2-like monocytic differentiation	Human foreskin (healthy donors) and melanoma derived tissue	[114]
IL-15	Increasing TIGIT and CD226 gene expression via IL-15 in tumor-infiltrating NK cells	Healthy donor blood, melanoma patient blood, and tissue biopsies	[115]
Melanoma-secreted lysosomes	Triggering monocyte-derived DC apoptosis	Transgenic mice (CD11C-DTR, NLRP3 knockout and B6.Cg-Ptprca Tg (UBC-PA-GFP)1Mnz/J), murine melanoma cell line (B16F10 and YUMM1.7)	[116]
Melano-transferrin	Located on the surface of exosomes; influencing iron metabolism and cellular differentiation	Human melanoma cell lines (A-375 and SK-MEL-28) and human blood plasma	[117]
MIA	Regulating cellular senescence in melanocytes in transgenic (grm1) mouse model	Melanoma mouse model Tg(Grm1)/MIA−/− and human cell lines (NHEM and HEK293T)	[118]
MIDKINE	Involved in lymphangiogenesis and metastasis in an engineered mouse model	Multiple transgenic mouse models, including Vegfr3Luc nu/nu; egfr3Luc; BrafV600E and Vegfr3Luc; BrafV600E and PTENlox/lox. HLEC and other multiple melanoma cell lines	[119]
MMP-1	Promoting endothelial cell activation by targeting endothelial proteinase-activated receptor 1	Human melanoma and colon cancer cells (A375, WM9, A7, and HT-29) and human endothelial cells (HUVEC and HDMEC).	[120]
MMP-3	Stimulating proteolytic activity of keratinocytes	HaCaT keratinocytes	[121]
miR-155-5p	Activating SOCS1/JAK2/STAT3 axis to promote the expression of proangiogenic factors in recipient fibroblasts	NIH/3T3, B16 and AG490 cells	[122]
miR-378a-5p	Facilitating vasculogenesis for melanoma cells; regulating melanoma progression	M14, SBCL1 and A375 human melanoma cell lines	[123]
miR-146a	Controlling immune responses to support the melanoma microenvironment	Human melanoma tissue, and miR146a−/− transgenic mouse model	[124]
PDGF	Pericyte recruitment for vascular mimicry, which could be prevented by PDGF inhibition	Human cutaneous (c81-61, C8161) and uveal (OCM-1) melanoma cell lines and xenograft model in Swiss/nude mice	[125]
S100A8/A9	Acting as a potent chemoattractant, growth factor, and immunosuppressant, fostering premetastatic cancer sites in mouse models	Mouse malignant (B16-BL6) cells and orthotopic mouse model	[126]
TGF-β1	Pronounced effect on barrier integrity of brain endothelial cells	NZM human melanoma cell line (NZM7, NZM48 and NZM74) and cerebral microvascular endothelial cells (hCMVECs).	[5]
TGF-β1	Reducing epidermal integrity by loss of epidermal AMBRA	HUVEC cell line and keratinocytes from human foreskin	[127]
THBS1	Acting as a mediator for YAP-induced melanoma cell invasion	Multiple human metastatic melanoma cell lines and melanoma mouse xenograft	[128]
YB-1	Extracellular YB-1 stimulating melanoma cell migration	Multiple human melanoma cell lines	[129]

Abbreviations: CCR, C-C chemokine receptor; IL, interleukin; MIA, melanoma inhibitory activity; MMP, matrix metalloproteinase; PMN, premetastatic niche; PDGF, platelet-derived growth factor; TGF, transforming growth factor; THBS1, Thrombospondin1; VEGF, vascular endothelial growth factor; YB-1, Y-box binding protein 1.

Figure 1. Melanoma-derived secretory factors in the tumor microenvironment can augment tumor growth and elicit immunosuppression at SLNs.

(A). The melanoma tumor microenvironment (TME) is a complex ecosystem of various cells encompassing the tumor through intercellular signaling governed by melanoma secretory factors such as cytokines, chemokines, growth factors, metabolites, and other tumor-promoting factors. These factors are carried away from the tumor either standalone or through tumor-derived extracellular vesicles (EVs) in the interstitial fluid which channel into tumor-draining lymph nodes (LN). Many of these tumor secretory factors are crucial for developing a premetastatic niche (PMN) and play a major role in tumor cell invasion in the LN. Melanoma-secretory factors engage with immune cells to alter their immunosurveillance directly, or through various other mechanisms involving adipocytes, endothelial cells, cancer-associated fibroblasts (CAF), and other, in the tumor milieu. These factors play many tumor-supportive roles including extracellular matrix (ECM) remodeling, lymphangiogenesis, and stromal proliferation. Among these factors, VEGFs are secreted by cancer or tumor-infiltrating inflammatory cells in all melanomas which are potent lymphangiogenic factors. Many other soluble factors are associated with hypoxia-induced changes and LN remodeling to support PMN development. These tumor secretory factors eventually prepare the sentinel LN (SLN) for melanoma cell metastasis (listed in Table 1). See main text for references. This figure was created with BioRender.com.

Melanoma-secreted soluble factors help prepare the SLN for metastasis.

Melanoma cells secrete a wide range of soluble proteins at a high concentration which may cause tumor growth and LN metastasis (Table. 1). For instance, melanoma cells (NZM 7, NZM48, NZM 74) secrete TGF-β and the C-terminal fragment of ANGPTL-4 in conditioned media detected by the Luminex assay, disrupting the barrier integrity of human endothelial cells (hCMVECs) (using electric cell-substrate impedance sensing (ECIS) biosensor technology) [5]. Tumor-promoting cytokines in serum, such as VEGFA, TNFα, and IFNγ have also positively correlated with the Breslow thickness of the melanoma as analyzed by multiplex immunobead assays [33]. VEGF can also polarize helper T cells (Th) from Th1 (CD4⁺/IFNγ⁺) to Th2 (CD4⁺/IL-4⁺) functional subsets in human T-cell cultures, suggesting that melanoma-associated secretory factors might contribute to suppressing immune activity [25]. Moreover, the melanoma-derived secretome inblood samples from melanoma patients and in the human metastatic melanoma 1205Lu cell line exhibit upregulated IL37 gene expression (IL-37) in multiple lymphocytes, including regulatory T (Treg) cells, that can lead to immunosuppression [34]. Also, melanoma patients have been reported to have higher serum concentrations of IL-1RA, IFN-γ, VEGF-A, PDGF-BB, PIGF-1, TNF-α, and MIP-1α than healthy controls [33]. Our group also found increased plasma concentrations of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in patients with widely metastatic melanoma compared to melanoma patients in whom the tumor was resected [24]. One study reported that the gene expression of IL10 and IFNG in tumor-positive SLNs (n=13) were higher than non-SLN (n=9) in melanoma patients; these mediators which are also known inducers of the enzyme indoleamine 2,3-dioxygenase (IDO), were significantly higher in SLN than non-SLN in the same LN tissues [35]. Also, anti-tumorigenic cytokines were decreased in the TME compared with non-tumor tissue: for example, IL7, known to stimulate CTLs through CD127 signaling, was reported to be reduced in the plasma of melanoma patients (55.22 ± 13.55 pg/mL) compared with healthy subjects (80.80 ± 11.74 pg/mL) [36]. Moreover, using a proteomic approach, secretome analysis of a paired set of fibroblasts ((Hs 895.T (metastatic site), 895.Sk (skin)) and malignant melanoma cells (A375 and SH4) showed that the occurrence of functionally related proteins in which MMP-2 was enriched in the secretome as well as activated in a paired set of fibroblast and A375 cells (as evidenced by gelatin zymography analysis)[37]. The study also suggested that MMP-2 might be involved in the extracellular matrix (ECM) remodeling via cancer-associated fibroblast (CAF) through interaction with a known partner such as αvβ3 integrin in melanoma cells [37]. In addition, murine CAFs were shown to produce CXCL5 which promoted the expression of immune checkpoint molecule PD-L1 on mouse melanoma cells [38], and CXCL5 enhanced neutrophil infiltration, facilitating LN metastasis in a mouse melanoma xenograft study [39]. Melanoma cells also secrete a variety of noncoding RNAs that can promote tumor metastasis such as miR-192-5p, which is highly induced in the hypoxic conditions of the melanoma, and which suppress CTL activity [40]. Several other examples of soluble secretory factors of primary tumors are briefly included in Table 1. These factors are secreted in the form of metabolites, proteins, or RNAs that are required to sustain the tumorigenic potential and support a developing PMN for establishing metastasis to new tumor sites.

Melanoma-derived extracellular vesicles (EV) can contribute to the regulation of PMN development.

Melanoma-derived EVs are key factors that help build the PMN at the SLNs by modulating immune activity. These EVs can target lymphatic endothelial cells (LECs) and other immune cells (e.g., macrophages, dendritic cells (DC), NK cells) to develop the PMN [6,16,41,42] (Fig-2). Also, EV’s tetraspanins are crucial for selective biomolecule recruitment, target selection, cell-specific entrance, angiogenesis, and vasculogenesis [43]. A plethora of evidence shows that EVs carry proteins, mRNA, miRNAs, lnRNAs, metabolites, enzymes, and several other factors that help induce the PMN in SLNs in melanoma tumors [44] (supplementary table 1). Moreover, we showed that EVs collected from the lymphatic channel of SLN melanoma patients harbor several protein and peptide cargo molecules that possess immunomodulatory functions (proteomics analysis of lymphatic EVs with data mining using NCBI and STRING webservers) [45]. In this study, lymphatic trafficking of S100A9 was analyzed in EVs from afferent lymphatic fluid and further validated in the lymphatic channel and primary tumors in the same patients [45]. An In vitro study showed that addition of recombinant S100A8/A9 in DC culture media decreased the expression of DC maturation markers compared to controls [42]. Also, B16F10 melanoma cells secreted EVs that could transform 3T3 fibroblasts into CAFs via Gm26809 transfer, as shown by the increased expression of CAF makers (α-SMA and fibroblast activation protein-α (FAP)) in treated fibroblasts [46]. Nerve growth factor receptors (NGFR, p75NTR) from melanoma-derived EVs also enhanced lymphangiogenesis, as demonstrated via B16F10-derived EVs in Prox1-GFP and Vegfr3-EGFP-Luc reporter mice [7]. Of note, EVs produced by melanoma cells can transform healthy myeloid cells into myeloid-derived suppressor cells (MDSCs) by upregulating PD-L1; via this signaling pathway, they can dampen CTL-mediated antitumor immune responses [47]. Further, miRNAs of melanoma EVs that have the potential to differentiate monocytes into MDSCs are elevated in tumor samples, plasma, and circulating CD14⁺ monocytes [48]. Several examples from recent research suggest that EVs produced by melanoma cells may play a substantial part in determining the establishment of the PMN environment with a potential impact on treatment responses in cancer [6,49] (supplementary table 1 & 2). Therefore, developing strategies that target tumor-derived EVs might be crucial in preventing PMN formation in the SLN, although this remains to be further investigated.

Figure 2. Melanoma-derived EVs can mediate immunosuppression and support PMN development in SLN.

Melanoma-derived EVs are among the crucial tumor secretory factors that enter the SLN via afferent tubules in the LN where these EVs first interact with LEC and SCS macrophages (SSM or SCS Mφ) in the subscapular sinus (SCS), triggering mechanisms that contribute to developing the PMN. EVs are associated with the alteration of various immune cells present in LNs through factors carried by EVs as shown, including 1) inhibition of NK cell adaptive immunity (HSP70, MICA) caused by upregulation of NK inhibitory receptor (CD94-NKG2A) and downregulation of homodimer activating receptor NKG2D as well as NK cell death due to apoptosis; 2) loss of DC maturation (S100A8/A9 and TGF-β) caused by reduced expression of T cell activation markers such as CD80, CD83, CD86, and others that render SLN’s DCs tolerogenic; 3) loss of cytotoxic T lymphocytes (CTLs) as well as their effector function (Tumor antigen1, PD-L1, and PTPN11); 4) proliferation of Treg in the SLN (e.g. CD300a) suppressing NK cell and CTL activities; 5) proliferation of immunosuppressive (so-called M2-like) TAMs (miR-125b-5p and miR-135-5p) and impaired proinflammatory so-called M1-like macrophage maturation; 6) myeloid-derived suppressor cells (MDSCs) are increased in the cortical region in SLN (eg. HSP86); and 7) B cells regulate tumor promoting humoral immunity; however, the direct role of EVs cargoes has yet to be established (supplementary table 2). See main text for references. This figure was created with BioRender.com.

Subversion of LN immunity in the PMN.

In general, cancer cells metastasize to LNs with of the aid of tumors that secrete immunosuppressive cytokines, chemokines, and other tumor-promoting factors [50]. Melanoma-derived EVs are mostly taken up by LN-resident LECs, and subcapsular-sinus macrophages (SSM) to perform LN remodeling [6,51]. Melanoma-EVs transport tumor antigens to LN-LECs for cross-presentation on MHC-I, causing antigen-specific CD8⁺ T-cells to undergo apoptosis [6]. Melanoma-bearing SLNs provide an immunotolerant environment due to impaired NK cell numbers, as well as increased CD4⁺PD1⁺, CD8⁺PD1⁺, and CD8⁺CD57⁺PD1⁺ cells in melanoma patient SNLs, compared to non-melanoma bearing SLNs [52]; it has been speculated that this might be due to the draining of melanoma-secretory factors in LNs, which remains to be further addressed.

T-lymphocyte responses

The magnitude of T cell responses is a crucial prognostic factor in SLN metastasis in melanoma [53,54]. CTLs are important immune cells due to their tumor cell-killing activity, whereas Tregs induce regional tolerance in SLN [55]. Recent data have revealed an increased immunotolerant environment in melanoma-bearing SLNs compared to non-melanoma-bearing SLNs [52]. For instance, the CTL numbers in tumor-centric cellular neighborhoods are decreased in the SLNs of melanoma patients who recurred, compared with melanoma patients who did not recur following immunotherapy [56]. T-cell malfunctions in the SLN have been thought to be in part due to tumor-derived EVs, posing a significant obstacle to the effectiveness of immunotherapy [57]. However, further work is necessary in this area. Nevertheless, most factors that might contribute to immune escape and tumor development in melanoma-EVs are abundantly present in patient plasma and melanoma exosomes isolated from patient blood plasma inhibited CD69 expression, CTL proliferation, and NKG2D induction on primary human cells [58]; this suggested that melanoma-derived EVs might also promote a certain level of systemic immunosuppression in melanoma patients, warranting further investigation.

Of note, CTL-induced tumor cell death releases tumor antigens that have been shown to cross-prime with LECs in an IFNγ-dependent manner to inhibit metastasis in murine studies [59]. In addition, decreased numbers of NK cells but increased numbers of CD8⁺ +CD57⁺PD-1⁺ cells with i) low CD8⁺CD69⁺ T cell/Treg cell ratios, (ii) high PD-1 expression on CD4⁺ and CD8⁺ T cells, and (iii) high CTLA-4 expression on γδ T-cells have been associated with low melanoma killing abilities in patient melanoma-bearing SLNs relative to non-melanoma-bearing SLNs [52]; this has been linking to the possibility that melanoma-secretory factors can elicit immunotolerance in the SLN [52]. A recent study mouse melanoma model showed that tumor-specific resident-memory CD8⁺T-cells (TRM) could restrict metastasis of melanoma cells in SLNs, and these TRMs exhibited enriched expression of IL-7R and CD69 relative to controls [53]. Of note, TRMs are distinct from circulatory CD8⁺ T memory cells (TCIRM), and the study reported that TRM, but not TCIRM, might offer long-term protection by preventing metastasis into the subcapsular-sinuses (SCS) of the SNL [53]; this in turn suggested that perhaps implanting memory T cells across the SLN might be explored as a putative therapeutic approach for preventing LN metastasis, pending robust investigation. From another angle, high stem-like TCF1⁺CD8⁺ T-cells in the LN have been reported to be more prevalent in nonmetastatic LN relative to metastatic LN in lung cancer patients, representing an interesting area of future in melanoma models [60]. Overall, the results discussed here suggest that the absence or reduced function of various populations of local CTLs in the SLNs of melanoma patients might lead to failed prevention of nodal metastases, presumably, and at least in part, because the loss of mature DCs and upregulated inhibitory co-receptors on CTLs might exert an inhibitory effect on metastasis [56,60,61]. Thus, understanding the mechanisms of CTL dysfunction resulting from tumor-derived factors in the SLN might provide an effective way to reinvigorate immunity in the SLN, although this remains to be further assessed.

Dendritic Cell responses

DCs are important for T-cell responses in LNs; however, impaired matured DCs fail to present melanoma antigens to activate T-cells in the SLN [62]. The release of immunosuppressive factors from the primary melanoma can cause localized paralysis of the paracortical area along with interdigitating DC (IDC) in the SLN, preventing the recognition of immunogenic melanoma by DCs ; indeed, IDC density in the SLN has been reported to be significantly reduced compared to non-SLN of melanoma patients [63]. Moreover, melanoma-derived EVs have been shown in vitro to reduce the expression of the DC maturation markers CD83 and CD86, as well as the Th1 polarizing chemokines FLT3L and IL15, and the migratory chemokine MIP-1, when compared to liposome controls, suggesting that melanoma EVs might provide a potential mechanism for suppressing DC maturation [42]. Also, in this study, melanoma-derived EVs from patients contained protein cargoes of S100A8 and S100A9, and the latter were found to impede DC maturation in vitro [42]. S100A8/A9 act as vascular permeability factors that are associated with CCR2-CCL2 signaling that is activated during PMN development, as evidenced by lung permeability assays in Ccl2^−/− and Ccr2^−/− mouse models [64]. Moreover, a spatial analysis of SLNs revealed that patients with metastatic melanoma LNs who did not experience recurrence with anti-PD1 antobody therapy exhibited enrichment for DCs and CTL, in addition to their interactions in tumor cell-centric neighborhoods (p<0.0001) compared with patients who recurred; this suggested that a metastatic TME can provide a risk of recurrence following immunotherapy [56].

In general, 4 conventional DC subsets have been identified in tumor-negative human SLNs: among them two subsets are skin-derived migratory CD1a⁺ and the other 2 are CD1a⁻, which could be part of CD14⁻ (BDCA3 ^high) and CD14⁺ (BDCA3^low) subsets [65]. The migratory subsets and CD1a⁻BDCA3⁺ have been demonstrated as having a powerful cross-priming ability with CTL for the generation of antitumor immunity against melanoma in ex vivo systems using human melanoma patient samples [66]. The sequential suppression of migratory DCs involves local spread, whereas LN resident DCs are involved in a regional spread in melanoma patients [67]. Langerhans cells (LCs) are the most common DC subset with CD1a⁺ Langerin⁺ found in melanoma SLNs [68]. LCs can act as regulators for antitumor immunity [69] but are phenotypically and functionally altered in the SLN which can favor immune tolerance by overexpressing IDO, although this remains to be further demonstrated [68]. Accordingly, IDO is involved in the immunosuppressive TME and tumor progression in melanoma, which has also positively correlated with the presence of Foxp3⁺ Tregs [70]. These findings might help explain, at least in part, why certain tumors, such as melanoma, do not optimally respond to immunotherapy in SLN when there is abnormal DC differentiation and suppressed DC function [67].

Macrophages

LN-macrophages play crucial functions in antitumor immunity in melanoma. For example, CD169⁺ macrophages or SSMs are predominant in naïve LNs and are directly in contact with the lymphatic fluid; they are also vitally important in protecting against LN metastasis and increasing the overall survival of melanoma patients [51,71]. LN macrophages are mostly sinus-based, such as SSMs that line the SCS floor lying over lymphoid follicles, and medullary sinus macrophages (MSMs), which are associated with the medullary sinus. Macrophages present within the medullary cords are called medullary cord macrophages (MCMs) and those SSMs that penetrate from SCS to the interfollicular region have been termed interfollicular macrophages. Melanoma-derived factors originate in the skin and flow into the SLN where these factors interact with SSMs that can limit tumor-derived vesicle-B cell interactions and prevent melanoma metastasis [51]. An in vivo study showed that melanoma-derived EVs were strongly taken up by SSMs but not by MSM or MCM [6] which might suggest that SSMs can play an important role in PMN development in the SLN. SSMs serve as the first layer of immune cells to embrace tumor antigens; however, their functional disruption and protective nature can be lost, promoting cancer growth [72]. Further, SSMs have promoted melanoma metastasis to SLNs by producing pro-tumoral IL1α-mediated STAT3 signaling in mouse allografts, suggesting a new putative targeting pathway for blocking LN metastasis [73]. Another study also showed that Siglec⁺-expressing SSMs and MSMs were associated with metastatic colonization of melanoma cells by interacting with α–2,3-linked sialylated melanoma cells, supporting the colonization of metastatic cells in SLNs [74]. Of note, melanoma-derived EVs have the potential to induce changes in macrophages by delivering miR-125b-5p to macrophages and generate the tumor-associated macrophage (TAM) phenotype to aid tumor growth, as evidenced from co-culture assays of induced THP1 macrophages with MV3 melanoma-derived exosomes, as well as transfection-based studies [75]. Another in vitro study also reported that melanoma EVs (B16F10) induced mixed ‘M1-like’/’M2-like’ protumor activation phenotypes on primary mouse bone marrow derived macrophages [76]. Hence, macrophages might be considered to play significant roles in the development of PMN within regional LNs, although further studies are necessary to dissect their possible contribution to PMNs.

Natural-killer (NK) cells

The SLN has demonstrates a significant downregulation of APCs in the paracortex [63]. Melanoma-bearing SLNs have shown a marked immunotolerant environment, as seen by diminished NK cells that exhibit low melanoma-killing potential [52,77]. Human peripheral blood NK cells have traditionally been used to study melanoma cell recognition and anti-tumor immunity. For instance, highly cytotoxic KIR⁺CD57⁺ NK cells have been documented in SLNs and a high proportion of CD57⁺CD56^dim noted in infiltrated SLNs; these have been linked to better patient survival in melanoma than SNL CD57⁺CD56^bright cells in stage III patients [78]. Also, the NKG2D and NKp46-activating receptors on memory like NK-cells can moderately control antitumor responses against melanoma, as evidenced by the adoptive transfer of control and melanoma NK cells in NSG mice [79]. NK cell activity has also been associated with Treg cells, and Treg:NK cell ratios have been reported as increased in tumor-draining LN in breast cancer patients compared to healthy controls; in a breast cancer mouse model, NK cell inhibition was reported to be mediated by Tregs, which facilitated LN metastasis but not lung metastasis [55]. These findings suggest that NK cells may paly a significant role in regulating anticancer responses and help to prevent tumor cell invasion in SLNs.

Melanoma-derived factors can modify SLN functional anatomy.

Melanoma SLNs are profoundly altered relative to normal LN in terms of the distribution of lymphoid cells and stromal cells (SCs), modifying functional anatomy. The remodeling of SLNs is observed with the influence of the primary melanoma [6]. For instance, the size and cellularity of SLN can be significantly increased because of leukocyte influx and local proliferation of resident macrophages and LECs, initiating the expansion of sinuses in LNs, as reported in breast cancer and melanoma murine models [18]. The alteration in SLNs is accompanied by the proliferation of LECs and lymphatic enlargement that occurs through lymphatic hyperplasia [16] (shown in Fig-3 for illustrative purposes, Table-2). Moreover, the high endothelial venule (HEV) and fibroblastic reticular cells (FRCs) are a key part of stromal cell infrastructure, generating a conduit system of ECM microfibers by channeling fluids ridden of antigens and inflammatory mediators [80]. The FRCs of LNs participate in molecular and cellular mobility and have been implicated in the compartmentalization of B and T-cells by producing fibroreticular fibers and stellate cell–cell contacts [16]. Additionally, in a murine melanoma model, FRCs of tumor-derived LN were transcriptionally altered by tumor-derived factors, in particular the downregulation of CCL21 and IL-7, which are involved in leukocyte migration and activation [81]. Furthermore, CD90⁺SC proliferation and the ECM showed expansion in fibrotic LNs of melanoma samples relative to controls; among CD90⁺SC, three subpopulations were noted, namely, CD34⁺SCs, FRCs (CD34⁻PDPN⁺ (podoplanin)), and pericytes (CD34⁻PDPN⁻CD146⁺), which exhibited upregulated FAP (based on flow cytometry-based methods on SLN samples from melanoma patients [9]. Overall, interstitial fluxes in lymphatic capillaries can enable the tumor to survive and metastasize, with SLNs becoming dysfunctional, and facilitating tumor colonization in the LNs [14,82].

Table: 2.

Stromal subsets of LNs implicated in generating PMN and lymph node metastasis

LNs components	Putative mechanism of LN remodeling	Biological model/human samples	Refs
Lymphatic endothelial cells (LEC) Gp38+CD157− CD31+LYVE+	• Increased inflammatory response in LECs and upregulated CD41, associated with peri-lymphatic fibrin deposition.	B16F10 melanoma mouse model	[18]
	• LECs stimulated melanoma metastasis in mouse LNs; dependent on MMP14, Notch3, and β1-integrin activation.	In vitro (WM852), SCID mice zebrafish model (roy−/−; mitfa−/−)	[19]
	• LECs upregulated integrin αIIb-mediated LEC adhesion to fibrinogen,associated with lymphatic remodeling.	B16F10 melanoma mouse model	[18]
	• LECs expressed PPP2R1A that facilitated tumor cell-LEC interactions	Orthotopic C8161 tumor xenografts in nude mice	[130]
	• LECs upregulated VCAM-1, increasing lymphatic permeability by interacting with tumor-derived SPARC.	Athymic (nu/nu) mice (Harlan)	[131]
Fibroblastic reticular cells (FRCs) Gp38+CD31−ER−TR7+	• FRCs controlled the elasticity of LNs; however, the contractility of FRCs was decreased by IL-1, secreted by differentiated melanoma cells • The relaxation of FRCs was dependent on the inhibition of constitutive JAK1/STAT3 and YAP/TAZ signaling	Human melanoma cell lines, athymic nude mice and immunocompetent C57BL/6 J mice	[111]
	• FRCs played a role in LN expansion by controlling matrix production.	CD11cΔCLEC-2 mice and Cre-negative control littermates	[132]
Marginal reticular cells (MRCs) Gp38+CD31−CXCL13+	• MRCs: subset of CXCL13-producing stroma located underneath the subcapsular sinus (SCS) and at the surface of the outer follicle: control the functionality of lymphoid tissue organizer in the LN	Mouse model (aly/aly), mouse cell line (BLS4, BLS12)	[133]
	• MRC controlled IGA2b+ LECs in murine LN through receptor activator of NF-κB ligand (RANKL) signaling.	Multiple transgenic mice (Itga2b−/−, ACKR4-eGFP, RANKLΔCcl19)	[134]
Integrin α7 pericytes (IAPs), Gp38−CD31−ITGA7+	• IAPs, gp38-CD31− (double negative cells) of stromal subsets in the cortex and medulla of mouse and human LNs; immunologically relevant	Mouse and human LN	[135]
	• Integrin α7 correlated with advanced disease; however more detailed studies are needed to determine IAP roles in the SLN.	Rectal cancer patients	[136]
Follicular DCs (FDCs), Gp38+CD31−ER−TR7−, CD35+	• FDCs located in the follicular area serve as APCs for naïve B cells; play an important role in the maintenance of the germinal center. • Imaging studies revealed that melanoma antigens led to accumulated FDS; however, these were reduced in the tumor-draining LN.	Multiple transgenic mice (Igh-Jtm1Cgn B cell-deficient (JHT), C57BL/6-Tg (CAG-EGFP)1Osb/J, Tg(IghelMD4)4Ccg BCR-, Rag−/−-Tg CX3CR1tm1Litt, and CX3CR1+/gfp)	[137]
HEV endothelial cells (HEV-EC) Gp38−CD31+	• BMP-4 was reduced in HEV-EC which changed from cuboidal to flattened shape.	Human LN (breast cancer associated LNs and control non-tumor LNs)	[138]
	• HEVs limited malignant melanoma development through both naïve and effector T-lymphocyte recruitment and activation.	Human melanoma tumor tissue	[88]
	• DC controlled lymphocyte entry in the LN through HEVs using lymphotoxin-β-receptor- dependent signaling.	Transgenic mouse models C57BL/6: (Cd11c-DTR, C57BL/6:Lt−/−, Cd45.1, Rag2−/−, and Ccr7−/−)	[139]
	• HEVs density was altered in a LAMP-DC+ dependent manner in melanoma	Human melanoma tumor tissue	[88]
Adipose tissue	• LNs were embedded in adipose tissue; adipocytes were anatomically associated with LNs harboring fatty acid composition and controlling of lipolysis.	Rats	[140]
	• Obesity increased two-fold the likelihood of presenting LN metastasis in melanoma patients	Case-control human samples	[141]
	• Secretome analysis of adipose tissue suggested a mechanism of adipocyte contribution to melanoma progression that included VEGF, adiponectin, leptin, resistin, and MCP1, etc .	3T3-L1 adipocyte cell line and B16-F10 and MeWo melanoma cell lines	[142]
	• Tumor-positive SLNs enhanced leptin expression compared to tumor-negative SLNs.	Human melanoma SLN tissues	[143]

Abbreviations: APCs, antigen-presenting cells; BMP4, bone morphogenetic protein 4; CXCL13, chemokine ligand 13; DC, dendritic cells; IL, interleukin; JAK1/STAT3, Janus kinase 1/ signal transducer and activator of transcription 3; LAMP, lysosomal associated membrane protein; LN, lymph node; NK, natural killer; PPP2R1A, protein phosphatase 2 scaffold subunit Aalpha; RORY, retinoic acid-related orphan receptor γ; SPARC, secreted protein acidic and rich in cysteine; SLN, sentinel lymph node; VCAM, vascular cell adhesion molecule 1; YAP/TAZ, yes-associated protein/transcriptional coactivator with PDZ-binding motif.

Lymphangiogenesis

Extensive lymphangiogenesis can be observed in SLNs prior to the appearance of metastasis. LECs line lymphatic vessels and are crucial for lymphangiogenesis [14]. Primary tumors start preparing the location of their metastatic spread by secreting lymphangiogenic molecules. For instance, the high expression of VEGF-C and its specific receptor VEGFR-3 have been reported to play a significant role in melanoma during metastasis [83]. Apart from VEGF-C, the role of LYVE-1, podoplanin, and PROX-1 have been well reported in the induction of lymphangiogenesis in various cancers including melanoma [14]. The role of LYVE-1, a cell surface receptor on LEC that can be employed as a lymphatic endothelial cell marker, is being debated. PROX1 is a transcription factor that promotes LEC development, and podoplanin, which is abundant in LECs, can interact with and immobilize the chemokine CCL21 in the lymphatic endothelium [13,14]. The angiogenic process in endothelial cells is also regulated by CD147, a transmembrane glycoprotein overexpressed in melanoma LN that promotes lymphangiogenesis in human melanoma [84]. Tumor-secreted EVs also induce lymphangiogenesis through an NGFR-dependent mechanism and trigger ERK, NF-κB activation, and ICAM-1 overexpression in LECs, facilitating tumor cell adhesion in melanoma [7]. Further, LECs secrete CCL21, which promotes the chemotactic behavior of CCR7-expressing metastatic melanoma cells to migrate to regions with higher lymphatic density, as evidenced by a chemotactic metastasis study in nude mice which suggested that the upregulation of CCR7 in tumor cells might be a mechanism of tumor metastasis in LNs [85].

HEVs remodeling

HEVs are specialized postcapillary venules for lymphocyte extravasation into lymphoid tissues; they are composed of cuboidal blood endothelial cells with a prominent glycocalyx and perivascular layer of FRC that allow blood to transmigrate into LN as well as other secondary lymphoid organs; tumors can grow around CD3⁺ T cell-enriched regions [86]. HEVs can limit malignant melanoma progression via both naïve and effector T-lymphocyte recruitment and activation in LNs [86]. HEVs exhibit high expression of PNAd, CCL21, and CD105, which are important for the selective recruitment of lymphocytes to LNs [87]. HEVs in melanoma tumors are associated with high numbers of lymphocyte infiltration [88]. Moreover, imaging analysis of immunostained tumor-draining LNs in breast cancer patients show that HEV length and branching patterns can alter blood flow and lymphocyte trafficking which are linked to the loss of CCL21-binding heparan sulfate in FRCs associated with HEVs remodeling [89].

Stromal cells and remodeling in the SLN

LN-SC subpopulations are important players in the invasion of malignant cells. Major subtypes of SC in LN are blood endothelial cells, LEC, FRC, and double-negative cells (Gp38⁻CD31⁻) [90]. The proliferation and modification of SCs are crucial in the remodeling of SLN and different sets of SC subpopulations have been reported in melanoma-infiltrated-LNs compared to healthy LNs [9]. LN-SCs play a crucial role in preserving immunologic homeostasis and regulating T-cell immunity during tumor growth [90]. Single cell-transcriptomic analysis has indicated that several distinct subpopulations of LECs exist with different roles in skin-draining LNs in mouse models [91]. Human LN lymphatics comprise 6 majors transcriptionally distinct LEC subtypes located in different anatomical sites; the analysis of non-SLN axillary LN of breast cancers has indicated that LECs of the SCS floor and medulla express chemoattracts that support neutrophil adhesion [92]. Also, LECs in the SCS area play a role in the scavenging of modified low-density lipoprotein, and another cortical LEC subtype is involved in rapid lymphocyte egress from LNs [91], suggesting that LEC modification in melanoma SLN might affect the distribution of lymph and antigen. These findings might help explain in part the role of SCs in SLN remodeling and immunomodulation in melanoma and provide numerous options for investigating reversal mechanisms.

SLN prognosis and therapy in melanoma.

In the clinic, the status of the SLN is crucial for determining clinical outcomes in melanoma. For instance, the distribution of micrometastatic LN (SLN or non-SLN) is important for the clinical recommendation of adjuvant therapy for stage IIIA melanoma patients because micrometastases at non-SLN are at higher risk of recurrence [93]. Also, SLN topography analyses indicate that stage I-III cutaneous melanoma patients exhibit progressive immune dysfunction as evidenced from the distinct expression pattern of T cell, NK cell, and innate immune cell signatures (using mass cytometry, flow cytometry, and T-cell receptor sequencing) linked to micrometastases [94]. Moreover, comprehensive analysis of immune infiltrates and spatial-immune networks using the MxIF platform (supplementary fig 1) in the metastatic TME in SLN’s tissue has been reported to help predict recurrence risk after immunotherapy in melanoma patients [56]. For example, high expression of CD3 (T cells) and CD86, HLA-DR, and OX40L (well-known indicators of DC activation) on CD11c cells within SLN’s tissue have been associated with better overall survival in melanoma patients [95]. The genetically manipulated murine B16 melanoma cells secreting recombinant IL-4 has increased the pre-effector T-cell response in the tumor-draining LN in murine models [96]. Adipocytes in the TME also support melanoma progression in many ways. For instance, these are known to recruit monocytes in the TME that differentiate into TAMs, aiding the growth of tumors. Of note, recent drug agents include the lipid-conjugated TLR7/8 agonist (telratolimod) which can target adipocytes inducing M1-like polarization to increase phagocytosis in the TME and suppress tumor growth, as evidenced from mouse melanoma models [97]. In addition, a study using a melanoma mouse model similarly demonstrated that - vaccination resulted in powerful tumor-specific T-cell immunity, effective tumor suppression, and longterm immunological memory [98].

Leveraging a deeper understanding of SLNs can reveal novel strategies to invigorate anti-cancer immunity within the SLN. Several preclinical investigations have provided a better understanding of altered states of immunity in melanomas, along with innovative putative therapeutic approaches to be eventually tested in melanoma patients (Box 2 and supplementary table 3). Unfortunately, interventional trials specifically directed to modify abnormalities in melanoma SNLs, to our knowledge, have not yet been conducted.

Box 2: Leveraging the PMN development archetype for developing putative melanoma therapies.

Certain melanoma secretory factors that mediate PMNs might be targeted to revive anti-tumor immunity. Indeed, novel strategies of reprogramming SLN to reactivate immunity at LNs are considered important tools for targeting melanoma metastasis [87]. For instance, synthetic cytokines such as PEGylated IL-2 (NKTR-214), PEGylated IL-10 (AM0010), and IL-15 superagonist (ALT-803) that can target the TME by producing an anti-tumor effect have been designed and shown encouraging clinical activity and safety profiles in melanoma patients [99]. In a clinical phase 1 trial (NCT02869295)ⁱⁱ, 28 melanoma patients were treated with NKTR-214 which had no immune-related adverse effect (IrAE) and were tolerated. Pegilodecakin (pegylated IL-10) is currently being tested in a phase 1 clinical trial (NCT02009449)ⁱⁱⁱ with multiple combinations of chemotherapeutic and immunotherapeutic drugs which currently have 350 participants enrolled that include melanoma patients. Another phase 1 (NCT01946789)^iv clinical trial utilizing ALT-803 against advanced solid tumors including melanoma, enrolled 26 participants but no result has been posted yet.

Further, EV-based prognoses and treatments are also under development in melanoma. A preclinical study using human melanoma cell lines revealed the heterogeneity of EV phenotypes with response to treatment using multiplex EV phenotype analyzer chip (EPAC) and also established specific profiles during the process of development of drug resistance in melanoma patient blood plasma; these might serve as candidate biomarkers to monitor the development of resistance against drug responses [100]. Further, EVs from B16F10 irradiated murine cells enhanced antitumor immunity against tumor-engrafted mice; these EVs injections in mice increased DC maturation markers (CD86, CD80) and correlated with IFNγ-producing NK cells (confirmed by flow cytometry) [101]. The innate immune receptor retinoic acid-inducible gene I (RIG-I) is activated by its particular ligand 5’-triphosphate-RNA in human naïve NK cells to kill human melanoma cells in a TRAIL-dependent manner [102]. Additionally, Hsp70-enriched EVs from B16 melanoma cells exhibited potent anti-cancer effects by increasing serum anti-tumor cytokine titers (IFN-γ and TNFα) as a result of improved CTL responses in B16 engrafted mice [103].

The ability of CTLs to recognize antigenic peptides is essential in killing cancer cells and has thus become a prominent focus of cancer therapy. In this context, the peptide ligandome of EVs displays cell surface HLA-I molecules on cancer cells and peptides originating from tumor-associated antigenic proteins to trigger T-cell responses [104]. Certain reactive T-cells originating from melanomas seem to respond to cryptic or subdominant self-epitopes in melanosomal proteins, suggesting that modification of these epitopes might be leveraged to ideally improve immunotherapy against melanoma [105]. A variety of techniques are used to identify novel melanoma/melanocyte antigens recognized by T-cells and antibodies, especially by employing genetic engineering approaches to screen recombinant cDNA libraries to improve adoptive T-cell therapies [105].

Abbreviations: IL, interleukin; LN, lymph node; MCP, monocyte chemoattractant protein; SLN, sentinel lymph node; TGF, tumor growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

Concluding remarks

Melanoma secretes many factors that can influence tumor immunity, and these not only locally support tumor growth but also stimulate lymphatic remodeling. LN immunomodulation in melanomas is in part, carried out by immune interactions between T cells/APCs and tumor antigens or other melanoma-derived secretory factors. The SLNs are the most common site of early metastases due to their dramatic loss of anti-tumor immunity capabilities [29].

In addition, massive remodeling of the LN and the loss of functional immunity are crucial for PMN development in SLNs. One of the research aims has been aiming to activate APCs and other cytotoxic cells to induce anticancer immune responses, including the priming of T-cells in the SLN. Also, certain encouraging outcomes in human clinical trials of melanoma are promising (listed in supplementary table 3), such as a phase 3 clinical trial (NCT02278887)ⁱ reporting that patients with advanced metastatic melanoma refractory against anti-PD1 antibody exhibited better progression-free survival with adoptive T-cell therapy compared to ipilimumab (anti-CTLA antibody) [106]. In addition, a recent preclinical study in a mouse melanoma model reported that LN functionally-impaired T-cells could be sensitized to become immunogenic against implanted tumors, as proposed by a 2-stage activation approach through which tumor specific CTL were activated by adoptive transfer of lymphocytic choriomeningitis virus (LCMV)-specific (Armstrong from acute infection) CD8⁺ T cells in tumor-draining LN, and subsequent costimulation with anti-CD3/CD28 antibodies for effector program acquisition [54]. Indeed, activating antigen-specific T-cells seems to be crucial for achieving better efficacy of immunotherapy in melanoma patients. Additionally, the development of novel putative DC vaccines to stimulate the immune system in melanoma patients have the potential to diminish lymphatic metastasis but which remain to robustly tested.

We posit that the dynamic interaction between different immune cell subsets within the tumor-immune milieu through successful migration of immunomodulators to the LNs might be crucial for developing innovative therapies against PMN, and these remain to be explored (see outstanding questions). However, due to the limitation of appropriate technologies, it is currently challenging to appreciate how melanoma mediators can influence the SLN’s three-dimensional architecture and how its spatial evolution ensues as melanoma develops. Likewise, current therapeutic regimens lack the ability to directly treat tumor draining LN and reverse their immunosuppressive environment. We are excited by the possibility that future therapy strategies involving the use of cytokines/EV-based approaches to target certain melanoma mediators in combination with cytotoxic regimens might be promising in reinvigorating LN immunity in melanoma, but these remain to be thoroughly explored.

Outstanding questions.

• Secretory components produced from primary tumors support the TME and are involved in altering LN immunity to establish the premetastatic niche (PMN), yet the question that remains unanswered is: to what extent do melanoma-derived secreting factors govern immune escape mechanisms?

• Mounting evidence from spatial biology indicates that LNs undergo massive remodeling that alters LN functionality, and melanoma-derived secretory factors are key stimuli to induce such remodeling, establishing a favorable niche for metastatic cancer cells. How do melanoma mediators induce lymphatic spatial remodeling to support metastatic colonization in sentinel lymph nodes (SNL)?

• The expansion of LNs is mediated by extracellular vesicles (EV) and soluble factors secreted by the tumor that involve cellular signaling crosstalk. The evolution of secreted factors during tumor growth can also trigger the failure of lymphatic homeostasis. Do melanoma mediators act in a stepwise manner to carry out the expansion of the lymphatic sinus that ultimately enlarges SNLs?

• Certain ongoing clinical studies aim to activate LN immunosurveillance and prevent nodal metastasis in cancer. Can melanoma-derived factors that modulate PMN development be reversed to prevent melanoma nodal metastasis?

Figure 3. Reported structural remodeling of SLN in melanoma.

(A) The remodeling of SLN favors lymph node (LN) metastasis in melanoma. The factors secreted by melanoma alter stromal cells and immune cells in SLN. The immune cells present in the SLN are significantly altered in their nature and consequentially fail to destroy tumor antigens. This is mainly because the immune cells are reduced in SLN or exhibit disturbed cellular physiology under stimulation of tumor secretory factors. The LEC, FRC, and blood endothelial cells BEC are prominently altered to change the structure and functions of the SLN, which is mostly involved in the expansion of SLN, increasing the flow rate through the lymphatic sinuses (B). For illustrative purposes, using the MxIF platform, SLN histological sections can be analyzed to elucidate the structural remodeling and altered immunophenotypes. Representative examples are shown of SLN (+) or SLN (−) tissue sections of melanoma patients who recurred and did not recur following immunotherapy. The depicted micrograph MxIF images (100 μm) acquired by INCell Analyzer 2500HS scanner suggested that the stromal and fibroblast proliferation (markers used: ASMA, FAP, Tenascin C) diminished the effect of immunotherapy, also representing common indicators of early development of PMN in SLNs (Supplementary table 4). This figure was created with BioRender.com.

Abbreviations: cLEC (ceiling lymphatic endothelial cells), FDC (follicular dendritic cells), fLEC (floor lymphatic endothelial cells), FRC (fibroblastic reticular cells), MCM (medullary cord macrophages LEC (lymphatic endothelial cells), MRC (marginal reticular cells), MSM (medullary sinus macrophages), SCS (subcapsular sinus), SSM (subcapsular sinus macrophages).

Box: Clinician’s Corner.

• Melanoma secretory factors that direct the remodeling of lymph nodes (LN) might be targeted to revive antitumor immunity in sentinel lymph nodes (SNL) [99]. Also, a better understanding of the altered molecular landscape in a premetastatic niche can inform novel putative therapeutic strategies to enhance antitumor immunity within SNLs.

• New neoadjuvant therapy concepts can be inspired by the evidence of tumor-mediator-induced LN dysfunction. The immune profile of SNLs is extensively modified in malignant melanoma patients, in contrast to nodes that are not sentinel. Immune indicators (tumor infiltration by lymphocytes) in the primary tumor might inform on patient prognosis.

• Increased peri-tumoral and intra-tumoral lymphatic density has been linked to shorter survival in melanoma patients with SNL metastases. Melanoma cells express lymphatic-specific ligands that locate and invade lymphatics, providing plausibility to the idea that the tumor spreads through chemotactic metastasis, which is evident inside SLN metastasis in the intra-tumorous lymphatic vascular zone.

• Understanding the nature of the premetastatic niche of SNLs can inform the development of more effective neoadjuvant treatment strategies that might ultimately obviate the need for LN surgery.

Highlights.

• The melanoma sentinel lymph node (SLN) is the first tumor-draining lymph node (LN) exposed to tumor-secretory factors that contribute to enabling cancer cell evasion of immunosurveillance.

• Melanoma-derived secreted factors can serve as immunosuppressants and lymphangiogenic factors, facilitating melanoma cells to locate and invade LNs.

• Extracellular vesicles (EV) secreted by tumor cells sequester oncogenic molecules from the tumor that are transported to SLNs which can mediate molecular dysregulation to suppress antitumor immunity and remodel the lymphatic system.

• Immunomodulators and blockers of melanoma-secreted mediators are under clinical investigation for the prevention of nodal metastasis through the interruption of premetastatic niche establishment.

Significance.

Melanoma cell-derived mediators contribute to immunological tumor escape via immune paralysis of tumor-draining lymph nodes (LN) by creating a pre-metastatic niche (PMN). This allows melanoma cells to survive in the immune environment of lymph nodes leading to significantly worsened clinical outcomes. Understanding the mechanisms of PMN formation offers an opportunity for developing early therapeutic intervention approaches and interrupting melanoma metastases, ideally impacting on clinical outcomes in high-risk melanoma.

Glossary

Breslow thickness

Measurement of the depth of the melanoma from the skin’s surface to the tumor’s deepest point.

Cryptic or subdominant epitopes

unique MHC I restricted T cell epitopes that may elicit weak (subdominant) or no (cryptic) CTL responses upon immunization with whole antigens.

Extracellular vesicles

Here, melanoma cells secreting lipid-bound vesicles into the extracellular space known as melanoma-derived EVs.

Exosomes

Here, components of EVs produced by the endosomal compartment of melanoma cells.

Lymphangiogenesis

Here, de-novo formation of lymphatic vessels, which reorganizes LNs in metastatic melanoma and increases lymph flow through lymphatic sinuses.

High endothelial venule (HEV)

specialized blood vessels in the LN harboring a thicker and rounder wall than a typical endothelial cell; enable lymphocytes to pass from the blood capillaries to LNs.

Lymph node immunity

In the LN, the antigenic substance in lymph interacts with various resident immune cells where the cortex of LNs carries follicles with enormous B cells, follicular DC, and macrophages; the paracortex possesses large, scattered T cells and DCs, and the medulla possesses antibody-secreting plasma cells that elicit antibody-mediated responses.

Lymphatic fluid (lymph)

originates from interstitial fluid, which flows through lymphatic vessels to LNs, where antigenic substances are removed by immune cells before eventually returning the fluid to subclavian veins.

Lymphatic flow

In the LN, lymph enters through multiple afferent lymphatic vessels to drain into the subcapsular sinus and then to trabecular, cortical, and medullary sinuses, and lastly through a single efferent lymphatic vessel.

M1-like macrophage

arbitrarily termed; refers to a classically activated macrophage that produces a proinflammatory immune response.

M2-like macrophage

arbitrarily termed; refers to an alternatively activated macrophage, considered immunotolerant in the TME.

Multiplex immunofluorescence imaging (MXIF) platform

offers spatially resolved multiple markers in tissue biopsies; produces high-resolution composite images using a multiplex protein imager with fluorescent cyclic imaging that provides staining details down to the single-cell level.

Myeloid-derived suppressor cells

immature myeloid cells that suppress immune responses to support tumor growth. The accumulation of MDSCs in the TME has impeded effective therapy for cancer patients.

Sentinel lymph node (SLN)

referred to as the first LN that cancer cells are most likely to disseminate to from the main tumor.

SOX10 and pS100

SRY-box transcription factor 10 (SOX10): transcription factor regulating melanocyte differentiation; S100 protein (pS100): calcium-binding protein significantly abundant in melanomas in both primary and metastatic melanomas. Both are considered specific markers in detecting melanoma metastases in the LN.

Neoadjuvant therapy

procedure used to shrink a tumor prior to primary therapy, which is frequently surgery, especially in melanoma. It includes procedures such as hormone therapy, radiation therapy, and chemotherapy.

Premetastatic niche

favorable microenvironment that facilitates colonization of disseminated tumor cells in SNLs; mediated by cytokines, chemokines, growth factors, enzymes, and EVs secreted by tumors.

Stem-like TCF 1⁺ CD8⁺ T Cells

memory-like progenitor cells that maintain long-term persistence for T cell responses and efficacy of immunotherapy.

Stellate Cells

quiescent fibroblasts that are activated by inflammatory mediators.

Tumor-associated macrophages

immune cells identified in tumors; contribute to the formation of TME and promote tumor development, invasion, and metastasis.