Chemogenetic modulation of sensory neurons reveals their regulating role in melanoma progression

Sensory neurons have recently emerged as components of the tumor microenvironment. Nevertheless, whether sensory neuronal activity is important for tumor progression remains unknown. Here we used Designer Receptors Exclusively Activated by a Designer Drug (DREADD) technology to inhibit or activate sensory neurons’ firing within the melanoma tumor. Melanoma growth and angiogenesis were accelerated following inhibition of sensory neurons’ activity and were reduced following overstimulation of these neurons. Sensory neuron-specific overactivation also induced a boost in the immune surveillance by increasing tumor-infiltrating anti-tumor lymphocytes, while reducing immune-suppressor cells. In humans, a retrospective in silico analysis of melanoma biopsies revealed that increased expression of sensory neurons-related genes within melanoma was associated with improved survival. These findings suggest that sensory innervations regulate melanoma progression, indicating that manipulation of sensory neurons’ activity may provide a valuable tool to improve melanoma patients’ outcomes. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01273-9.


Introduction
Melanoma represents one of the leading causes of cancer-related deaths, being the most aggressive skin cancer type worldwide [124]. It emerges from molecularly altered melanocytes, which are the producers of melanin in the skin [48]. These cancer cells are embedded within the cutaneous microenvironment where they reside and interact dynamically with its constituents during disease progression [15,54]. Understanding the interplay between the different components within the tumor microenvironment is crucial for the success of therapeutic applications, since each component can be influenced by the others, resulting in impacts on the cancer cells [9,10,45,52,88,106]. The presence of individual nerve fibers within the tumor microenvironment was ignored for many years as they are difficult to detect in classical histology. For a long time, only large nerve trunks were detected within tumors, and they were always associated with perineural invasion of cancer cells, a process in which these cells grow and migrate along native passive tissue nerves [84]. Recently, a different phenomenon was described, by which the tumor itself is infiltrated pro-actively by newly developed peripheral nerve projections [32,36,71,89,108,115,116,121,154,158].
To understand how peripheral innervations behave within the tumors, functional studies, in which Open Access *Correspondence: birbrair@icb.ufmg.br 1  intra-tumoral nerves were eliminated, have relied on the surgical or pharmacological manipulation of nerves. Each such strategy, however, has its disadvantages. Peripheral nerves contain mixtures of different nerve fiber types [41,82], and therefore, surgical denervation of a peripheral nerve leads to the disruption of all the nerve fibers present within that specific nerve [101]. Consequently, the role of particular nerve projections in the tumor cannot be isolated, as other nerve fibers are also affected. On the other hand, pharmacological drugs cause systemic reactions in several organs and indirect effects on unexpected targets. Thus, achieving the neuronal type-specificity that is needed to understand the role that specific nerve fibers perform in the tumor microenvironment is difficult with these methods, and the observed outcomes could be due to the unwanted effects on other innervations in addition to the targeted neurons. Wherefore, conclusions drawn from studies based on surgical or pharmacological denervation may be imprecise. These are some of the reasons, in addition to tumor tissue specificity, for some of the ambiguity about the roles of specific nerve fibers in cancer behavior. Accordingly, contradictory reports have been published: while some studies have claimed that certain neuronal types promote cancer progression [57,158], others concluded that they suppress tumorigenesis [32,116]. Therefore, to study the role of specific innervations, these should be directly manipulated in a nerve-fibertype-specific manner. Recently, this approach became possible with the advent of powerful genetically-based tools, that precisely allow the targeting and elimination of specific peripheral nerve fibers for studying their functions in vivo [13]. Our group showed that specific genetic depletion of sensory neurons promotes melanoma growth [108]. Nevertheless, genetic ablation of these innervations may result in the generation of a proinflammatory microenvironment, secondary to cell death in the site where the neurons were ablated (Männ et al. 2016; Christiaansen, Boggiatto, and Varga 2014; Bennett et al. 2005), which itself is strongly tumor growth promoting [49,67], and can affect cancer cells' behavior [50]. Thus, it remains unclear which facets of the sensory neuron-ablated tumor phenotype are due to the loss of sensory innervations, rather than indirect effects due to the local inflammation caused by the death of these neurons. To circumvent this issue, in the present study, we used chemogenetics, an experimental strategy that has empowered neuroscience studies [131,147], to determine the precise role of sensory neurons in the regulation of melanoma progression. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) enable the silencing or overactivation of genetically defined neuronal populations upon binding to small-molecule designer drugs [119]. This approach allowed for highly selective and non-invasive modulation of sensory neurons' activity in the tumor. Here, we revealed that silencing of sensory neurons' activity, without ablating them, is sufficient to trigger increase in melanoma growth and in intra-tumoral new blood vessel formation. In contrast, chemogenetic stimulation of sensory neurons counteracted melanoma progression, by regulating tumoral growth, angiogenesis and immunosurveillance. Our results provide unequivocal evidence of the influence of sensory neurons in cancer progression.
All animal care and experimental procedures were approved by the Ethics Animal Care and Use Committee (CEUA), in accordance with the Guide for the Care and Use of Laboratory Animals from the Federal University of Minas Gerais. All colonies were housed in

Cell culture
Murine B16-F10 melanoma is a common cell line that naturally originated in melanin-producing epithelia of C57BL6 mice. These cells were originally obtained from ATCC (USA), and were used to study melanoma development in vivo. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum/2 mM L-glutamine/100 U/ml penicillin/100 μg/ml streptomycin. The cells have been tested and found negative for mycoplasma. Cells were cultured in a humidified atmosphere of 95% air and 5% (v/v) CO2 at 37 °C.

Melanoma tumor implantation
B16-F10 cells were suspended in PBS and examined for viability using trypan blue staining. B16-F10 cells were used for transplantation only when more than 90% of cells were viable. For subcutaneous injection, the skin of all mice at an age of [8][9][10][11][12] weeks was shaved at the place of application. 1 × 10 5 cells in 100 μL were injected subcutaneously into the right flank of each mouse and the growth of the tumors was monitored until sacrifice. Growth of the tumors was assessed over time with a caliper as previously reported [12]. For determination of tumor volume, tumor-bearing animals were anesthetized with isoflurane in O2 by manually restraining the mice and placing their heads in in-house-built nose cones. Tumors were removed 16 days after transplantation and weighted. Length (L) and width (W) were calculated to measure tumor volume (V) using the formula V = 0.5 × (L × W2) [40]. Tumor area was determined using calibrated photographs of each tumor using Fiji software ® , version 1.53 (National Institute of Health, Bethesda, MD).

Capsaicin-induced spontaneous behavior
To confirm sensory neurons inhibition efficiency, following acclimation, Nav1.8-Cre-/hM4Di + and Nav1.8-Cre + /hM4Di + mice were injected with an intra-plantar subcutaneous dose of 10 μl of capsaicin (1 μg/10 μl; Sigma-Aldrich). A video recording was taken for 5 min post-capsaicin injection. The time that the animals spent performing spontaneous behaviors of licking, lifting, and flicking the paw were measured for 5 min after injection of capsaicin from these videos.

Quantification of CGRP within tumors
Tumor samples from Nav1.8-Cre + /hM4Di + and Nav1.8-Cre + /hM3Dq + animals, as well as from their respective controls (Nav1.8-Cre-/hM4Di + and Nav1.8-Cre-/hM3Dq +) were analyzed to measure the amount of CGRP using commercially available Sandwich-CGRP ELISA kit purchased from Elabscience (Catalog # E-EL-M2744). Briefly, tumor pieces were weighed and then homogenized in PBS (0.01 M, pH = 7.4) with a glass homogenizer on ice. The homogenates were centrifuged for 5 min at 5000 × g at 4 ℃ to get the supernatant. ELISA of CGRP were performed according to manufacturer's instructions. After ELISA, Optical Density (OD) was measured using Varioskan Flash (Thermo) set at 450 nm.

In silico analysis
RNA sequencing count data of 103 Skin Cutaneous Melanoma (SKCM) patients was downloaded from The Cancer Genome Atlas (TCGA) repository (https:// portal. gdc. cancer. gov/). Differential gene expression analyses were performed between samples of alive and dead patients (considering a 5-year interval) using DESeq2 [83]. We stratified patients in these two groups, alive or dead, based on their vital status in a 5-year interval of their tumor diagnosis (clinical data available at TCGA and curated by Liu et al. (2018) [85]. Genes with absolute log2(Fold-change) ≥ 1 and False Discovery Rate (FDR) adjusted P-value < 0.05 were considered differentially expressed. To identify biological processes associated with genes differentially expressed, we performed a Gene Ontology (GO) enrichment analysis using ShinyGO [42]. We used the STRING database [132] (parameters: full STRING network, considering only text-mining, databases and experiments interactions with score > 0.400, and only genes with 3 or more interactions) and Cytoscape (https:// cytos cape. org/) to construct protein-protein interactions (PPIs) among our manually curated list of 34 gene related to sensory neurons selected based on the literature [29,37,51,114,141,144]. The set of 18 genes showing at least two PPI interactions are shown. For the remaining analyses, RNA sequencing counts were first Transcripts Per Million (TPM)-normalized using a local R script. To identify a gene signature associated with SKCM cancer patient survival we used Reboot [31] with parameters "-B 100 -G 5 -P 0.3 -V 0.01". Briefly, Reboot finds genes associated with cancer patient prognosis using multivariate penalized Cox regression combined with a bootstrap approach. In the first step of Reboot, it produces regression coefficients (numerical values) that determine the contribution of each submitted gene to patients' survival. These coefficients may be positive or negative values indicating that high expression of a particular gene potentially contributes to worse or better prognosis, respectively. Once these coefficients are produced, Reboot then calculates the score of each patient (sample) as the sum of each gene coefficient multiplied by the corresponding gene expression level in that patient. Finally, when all patients' scores are calculated, we then stratify them into groups with high/low scores based on the median score of all patients to create the survival curve (Kaplan-Meier). For further information, see [31]. SCN10A box plots and survival curves were created using R (https:// www.r-proje ct. org/) scripts.

Statistical analysis
Graphs were plotted using GraphPad Prism 7 (San Diego, CA). Shapiro-Wilk normality test was performed, and unpaired t test was used to determine statistical significance.

Chemogenetic inhibition of Nav1.8 + neurons accelerates melanoma progression
We have previously demonstrated that melanomas are infiltrated by Nav1.8 + sensory innervations, and that those tumors grow slower when these neurons are pharmacologically or genetically ablated [108]. However, these investigations were performed using Nav1.8-Cre + /DTA + mice, in which a diphtheria toxin fragment A is constitutively activated in Nav1.8 + sensory neurons, resulting in the toxin induced-death of these cells. Therefore, this technique lacks temporal control of neuronal ablation, and enables compensatory effects during the development of these animals. Importantly, the approach by which specific neurons are ablated from the tissue microenvironment is also limited because of the secondary consequences, such as inflammation, caused in the tissue where sensory neurons are eliminated. Thus, it remains unclear whether these damageinduced changes in the tissue may influence the observed cancer outcomes. Here, we applied a chemogenetic approach to specifically inhibit the activity of Nav1.8expressing sensory neurons without killing these cells.
We used DREADDs to specifically control sensory neuron activity. DREADDs are derived from different types of mutated muscarinic receptors that have been engineered to lose affinity to their endogenous ligand acetylcholine [5], but to gain responsiveness to a synthetic ligand, clozapine-N-oxide (CNO). Inhibitory DREADDs (hM4Di) elicit an intracellular cascade that results in the silencing of neuronal activity [113], without changing the number of innervations as previously reported [68]. This method allows for the selective silencing of specific types of neurons in vivo without physical manipulation or destruction in the tissue. DREADDs were expressed specifically in sensory neurons, using a transgenic murine approach: mice carrying the construct encoding for Credependent expression of hM4Di were crossed to Nav1.8-Cre animals. In the resulting mice, Nav1.8-Cre + / hM4Di + , only Nav1.8 + sensory neurons expressed inhibitory DREADDs. As controls in this study, we used littermate mice carrying Cre-dependent hM4Di, but lacking the Cre gene (Nav1.8-Cre-/hM4Di +) (Fig. 1A). This allowed us to control for any potential side effects from CNO administration.
In order to ascertain that the expression of DREADD receptors was driven to intra-tumoral Nav1.8-expressing neurons in Nav1.8-Cre + /hM3Di + mice, we used the solid tumor model B16F10. We assessed tumor sections from melanoma grown in Nav1.8-Cre + /TdTomato + mice and detected Nav1.8 + neurons expressing TdTomato present within the tumor microenvironment (Fig. 1B). To validate sensory neuronal inhibition following daily CNO injection, we used a behavioral test to evaluate the sensitivity to capsaicin, confirming the silencing of sensory neurons, as previously described [2]. Indeed, Nav1.8-Cre + /hM4Di + animals spent less time (25.53 ± 2.27 s) licking their paws after intra-plantar injection of capsaicin, compared to control animals (56.53 ± 3.92 s) (Fig. 1C). To analyze the effect of sensory neurons silencing on tumor growth, we subcutaneously transplanted B16F10 cells to the lower right flank of both inhibitory DREADD-expressing mice (Nav1.8-Cre + / hM4Di +) and their controls (Nav1.8-Cre-/hM4Di +). Following cancer cell injection, we treated the animals daily with CNO to induce sensory neuronal activity inhibition (controls were also treated with CNO) (Fig. 1D). After 14 days of continuous sensory inhibition, tumor volume was significantly enhanced in the sensory neuron-silenced mice when compared to the controls (tumor volume was increased from 82.1 ± 29.6 to 319.6 ± 72.8 mm 3 ; Fig. 1E). After 16 days of repeated sensory inhibition, tumor weight was also significantly enhanced in the sensory neuron-silenced mice when compared to the controls (tumor weight was increased from 0.50 ± 0.04 to 0.98 ± 0.23 g; Fig. 1F, G). Animal weights were not affected by sensory inhibition (data not shown).
Chemogenetic activation of hM3Dq excitatory DREADD receptors in Nav1.8 + neurons promotes melanoma regression As we found that inhibition of sensory neuron activity promotes melanoma advancement, we hypothesized that increasing sensory excitability would result in the reverse: blockage of melanoma progression. To test this hypothesis, we used again chemogenetics, by which we induced the expression of excitatory hM3Dq DREADDs [127] only in Nav1.8 + sensory neurons. We crossed Nav1.8-Cre mice to a mouse line with a Cre-dependent evolved Gq protein-coupled receptor (hM3Dq) expression. In the resulting Nav1.8-Cre + /hM3Dq + mice, upon removal of loxP-stop-loxP cassette by Cre recombination, the Gq-coupled hM3Dq is expressed specifically in Nav1.8-sensory nerve fibers. Sensory neurons in those mice can thus be overactivated by the administration of CNO. It has been shown previously that Gq-DREADD activation by CNO increases neuronal activity in the targeted neurons, including sensory neurons [68,90], without changing the number of neurons [110].
To evaluate the role of sensory stimulation on tumor growth, we transplanted subcutaneously B16F10 melanoma cells to the lower right flank of both stimulatory DREADD-expressing mice (Nav1.8-Cre + /hM3Dq +) and their controls (Nav1.8-Cre-/hM3Dq +). Following the cancer cell implantation, we treated mice daily with CNO to induce Nav1.8 + sensory neuron activation (controls were also treated with CNO) (Fig. 3A, B). After repeated sensory neuron activation, melanoma development was decreased in the sensory neuron-overactivated mice when compared to the controls (at day 14, tumor volume per body weight was reduced from 3.51 ± 0.89 to 0.71 ± 0.20 mm 3 ; at day 16, tumor weight was reduced from 0.38 ± 0.07 to 0.17 ± 0.03 g; Fig. 3C-F). Animal weights were not affected by genetic stimulation of sensory neurons in melanoma-bearing mice (data not shown). Moreover, genetic overactivation of sensory neurons led to a decrease in proliferating cells within the tumor (from 3050 ± 203 to 1292 ± 367 Ki67 + cells per μm 2 , analyzed by immunohistochemistry) (Fig. 3G, H), corroborated by flow cytometry analysis of CD45-cells for Ki67 expression (the was a decrease from 8.13 ± 1.00 to 5.07 ± 0.70% of CD45-/Ki67 + cells within the population of CD45-cells) (Fig. 3I). Additionally, there was a decrease in the intra-tumoral blood vessels' area (from 0.010 ± 0.001 to 0.006 ± 0.001 µm 2 of CD31 + area / µm 2 of tumor area) (Fig. 3J, K). Our data suggest that increase in neuronal activity in sensory neurons counteracts melanoma development.

High expression of genes related to sensory neurons correlates with best prognosis in human melanoma patients
In order to investigate our findings also in human tumors, we analyzed The Cancer Genome Atlas (TCGA) samples TIL from tumor-draining lymph nodes of B16F10-inoculated Nav1.8-Cre-/hM3Dq + (n = 14) and Nav1.8-Cre + /hM3Dq + (n = 13) mice were analyzed. B Absolute number of CD4 + , CD8 + , γδ, NKT and NK cells from tumor-draining lymph nodes of B16F10-inoculated mice. C IFN-γ and IL-17 were quantified in CD4, CD8, γδ, NKT and NK cells. D Schematic representation of the collected tumor non-draining lymph nodes. TIL from tumor non-draining lymph nodes of B16F10-inoculated Nav1.8-Cre-/hM3Dq + (n = 14) and Nav1.8-Cre + /hM3Dq + (n = 13) mice were analyzed. E Absolute number of CD4 + , CD8 + , γδ, NKT and NK cells from tumor non-draining lymph nodes of B16F10-inoculated mice. F IFN-γ and IL-17 were quantified in CD4, CD8, γδ, NKT and NK cells. Cytokines levels were measured in cells from tumor-draining and tumor non-draining lymph nodes of B16F10-inoculated Nav1.8-Cre-/hM3Dq + and Nav1.8-Cre + /hM3Dq + mice. Data are shown as mean ± SEM. Unpaired t test, *.01 < P < .05; **.001 < P < .01; ****P < 0.0001 First, we stratified SKCM patients in two groups, alive or dead based on a 5-year interval. Next, we searched for differentially expressed genes between these groups. We found 112 up-regulated and 195 down-regulated genes (|log2(Fold change)|≥ 1 and adjusted P-value < 0.05; Table 2). Next, we performed a Gene Ontology (GO) analysis of Biological Processes (BP) in which these genes are involved. The up-regulated genes set are enriched for three biological processes, while the down-regulated genes are enriched to a wide variety of processes ( Fig. 8A; Table 3). Curiously, for the former (up-regulated genes), two out of three Biological Processes represent nervous system development (Fig. 8A), indicating the importance of neuronal networks in melanoma outcomes. Next, we investigated the interactions (and putative regulation) of 34 sensory neurons-related genes which were selected from the literature [29,37,51,114,141,144] (Table 4). Figure 8B shows a strong connection among 18 of these genes, suggesting that they work on the same cellular pathways or cell types. Next, using the expression levels of these 34 gene markers for sensory neurons we investigated their potential to be "a signature" associated with SKCM cancer patient survival. Figure 8C shows that high expression of these genes (lower patient scores) are associated with a better overall survival of SKCM patients. Next, we investigated the expression of these genes in the two sample sets (alive and dead patients). We found that SCN10A, which codifies Nav1.8, a key gene based on which our mouse models target sensory neurons, is more expressed in alive than in dead patients (Fig. 8D). Finally, we investigated the impact of SCN10A expression on SKCM patients' survival. Figure 8E shows that high expression of SCN10A trends to be associated with a better overall survival of SKCM patients, even without statistical support (P-value = 0.26). Taken together, these results confirm that genes related to nervous system development are enriched in samples from live Skin Cutaneous Melanoma patients. Focusing on gene markers for sensory neurons, we confirmed that these genes are strongly connected, suggesting a synergistic activity, and that the higher expression of some of these genes are associated with a higher overall survival. Strikingly, the high expression of SCN10A is potentially associated with better SKCM patient survival, indicating that the presence of sensory neurons within melanoma counteracts cancer progression. We also found that TCGA samples from tumors with a worst prognosis (dead patients) have an enrichment of genes promoting angiogenesis (Tables 3 and 5; 15 genes related to angiogenesis). We focused on this set of 15 genes related to angiogenesis and we confirmed that they are strongly connected (Additional file 2: Fig. 2), indicating a synergic function in promoting angiogenesis. Our results indicate that there is an increase in genes related with angiogenesis in tumors with worst prognosis (from dead patients) and a decrease in their expression in tumors from alive patients which show an increased expression of SCN10a, a sensory neuron marker used in this study (Fig. 8C, D, E). By using the CIBERSORT tool [102], we investigated immune infiltrated cells in the same TCGA cohort of alive vs. dead patients (Additional file 3: Fig. 3). CIBERSORT uses gene expression (RNA sequencing data) and support vector regression combined with prior knowledge of expression profiles from purified leukocyte subsets (gene signatures) to produce an estimation of the abundances of immune infiltrated cells subpopulations in a sample. In line with our data presented in this manuscript, we have checked the enrichment of immune infiltrated cells in the tumors of patients alive vs. dead (Additional file 4: Fig. 4). We found an increase of CD4 + T cells, CD8 + T cells, NK cells and dendritic cells in patients with better prognosis (alive). Thus, tumors showing a better prognosis (alive) have an increased infiltration of some key immune cells.
Additionally, microarray data evidenced a down-regulation of genes related to the Th17 immune response in melanoma patients (Additional file 5: Fig. 5). These analyses are consistent with the data generated in our mouse models: that the overactivation of sensory innervations in the tumor microenvironment was associated with suppressed melanoma progression. Albeit gene expression in tumor biopsies from human cancer patients is used as a tool to define novel biomarkers and to contribute to prognosis, the obtained data should be also validated by the quantification of sensory neuron-related proteins in human melanoma biopsies and correlation with clinical outcomes in future research.

Discussion
In the present study, we examined how melanoma progression is affected by sensory neurons activity. Our chemogenetic approach revealed that inhibition of sensory activity promotes tumor growth and intra-tumoral angiogenesis. In contrast, excitation of sensory neurons induces melanoma regression with decrease in tumor growth and in new blood vessel formation, as well as a boost in the anti-tumor immune surveillance (Fig. 9). This work indicates that induction of hyperactivation in Nav1.8-expressing sensory neurons represents a potential new therapeutic path in the battle against melanoma. Just as the role of particular genes in a specific biological process can be examined by evaluating examining the assessable consequences that result from their removal (e.g. using knockout animals), the role of neurons in the tumor microenvironment was previously assessed by eliminating them [32, 36, 71, 89, 108, 115,       To circumvent these limitations, novel powerful technologies have been created in the field of modern neuroscience which allow to manipulate the firing of specific neurons without killing them: optogenetics and chemogenetics. These methods use genetic strategies to deliver the expression of lightsensitive proteins or designer receptors exclusively activated by designer drugs, respectively, to the membrane of defined neuronal populations. Therefore, by using these techniques, manipulation of neurons by exposure to light or to designer drugs, without killing them, became feasible. As melanoma is a chronic disease, the use of optogenetics for longer periods, may not be the best approach, as the unavoidable surgical preparation with the chronic implantation of hardware for stimulation and prolonged exposure to highly energetic laser light will eventually culminate in confounding regional inflammation and tissue degradation. Therefore, we chose to use chemogenetics to examine the participation of sensory neurons in melanoma development, as it is more suitable to evaluate the long-term effects of sensory stimulations with less side-effects. Future studies may use similar approaches to explore the role of sensory neurons and other innervations in other cancers. Our findings suggest that sensory neurons' overactivation affects the immune response to melanoma. Melanoma progression is influenced by the complex interplay between cancer cells and different components of the immune system [8]. Melanoma cells may cause disruption of the organism's immunity to overrun and escape the immune system control [96,104]. The role of sensory innervations in these interactions remains completely unknown. Lymphocytes are the dominant immune elements found infiltrating the melanoma microenvironment. Their composition correlates with patients' survival [38]. While regulatory T cells play pro-tumorigenic roles; CD8 + T cells, CD4 + T cells, γδ T cells, and NK cells have been shown to act against the transformed cells, [38,43,46,47,58,64,73,118]. Conversely neutrophils and myeloid-derived suppressor cells have been associated with poor prognosis and are largely protumorigenic [22,28,69,126,129,145,155]. Our data shows that sensory overactivation induce an increase in the number of tumor-infiltrating anti-cancer lymphocytes (CD8 + T cells, CD4 + T cells, γδ T cells, and NK cells), while we did not detect changes in the number of tumor-infiltrating regulatory T cells. We also found that there is a decrease in the number of neutrophils and myeloid-derived suppressor cells within the tumor. We found that these changes were tumor-specific, as we did not detect any alterations in the number of lymphocytes in the non-draining lymph nodes. Tumor-draining lymph nodes presented an increase in some of the anti-tumor lymphocytes probably because of the tumor-priming effect previously reported [135]. Signals transmitted to T cells via PD-1 or CTLA-4 (considered markers for T cells "exhaustion") promote T cell dysfunction, thereby turning off the immune response [59,98,149]. We found that the tumor-infiltrating lymphocytes decrease their expression of PD1 and CTLA-4, possibly indicating that these cells are "less exhausted" within the melanoma microenvironment after sensory hyperactivation. The more active phenotypes of lymphocytes have been associated to the increase in the production of cytokines. A variety of lymphocytes are capable of producing IL-17 [14,20,24,53,56,77,94,103] which has presented anti-tumorigenic effects in melanoma [3,63,74,75,91,99,100]. We found an increase in tumor-infiltrating lymphocytes producing IL-17 after sensory stimulation. Thus, in light of our overall findings, we suggest that induced increase in firing of sensory innervations contributes to boosting of the immune response against melanoma. Future studies will need to explore the exact molecular mechanisms involved in the interactions of sensory neurons and immune cells in the melanoma microenvironment. A variety of cellular and molecular mechanisms may be involved in the effect of sensory neurons' modulation on melanoma behavior. For instance, it has been documented that the same drug that is used to denervate sensory neurons, resiniferatoxin (RTX), an analogue of capsaicin, also induces stress by causing hyperactivation of the sympathetic nervous system [16,62,157]. These studies also revealed that sensory nerves may tune down sympathetic nerve activity [16,62,105]. Sympathetic neurons release norepinephrine [70,123], which has been shown to strongly induce tumorigenesis [1,60,154]. It remains open the important question whether the effect of sensory innervations in the tumor microenvironment depend also on the modulation of the sympathetic tone.

Future perspectives
The present study reveals the short-term impact of chemogenetic modulation of sensory neurons on melanoma behavior. It remains to be examined what are the long-term effects of this manipulations. In the current study, the sensory neurons' activity is being continuously inhibited or overactivated. Are changes in sensory neurons' activity at specific time points sufficient to influence cancer outcomes? Also, it remains to be determined what are the changes within the tumor microenvironment at different stages of cancer progression. Are some stages more sensible to changes in the activity of sensory neurons? Moreover, this study focuses on melanoma tumors. Future studies should explore what is the role of sensory neurons in the development of other solid tumors.
A variety of factors secreted from sensory neurons may be implicated in the regulation of the melanoma microenvironment described here [18]. The overactivation of sensory nerve fibers may induce the release of neuropeptides, such as substance P, CGRP, VIP, GRP, neurokinin A, neurokinin B, neuropeptide Y (NPY) and adrenomedullin, which have been shown to interact                directly with several components within the tumor microenvironment, including cancer cells, immune cells, endothelial cells and others [4,6,17,25,26,30,35,61,80,81,93,95,117,120,142,143,148]. We detected an increase in intra-tumoral CGRP after overactivation of sensory neurons (Fig. 10). However, it remains unknown whether the decrease in the tumor size after sensory neurons overactivation is due to this increase in the intra-tumoral concentration of CGRP. Future studies will need to genetically eliminate specific receptors for this and other neuropeptides (such as receptor activity-modifying protein 1 (RAMP1) for CGRP) from different cellular components of the  tumor microenvironment to reveal whether those communications are relevant for melanoma outcomes.

Conclusion
In conclusion, this work identifies sensory neurons overactivation as a potential strategy for blocking melanoma progression and improving patient outcomes. We also anticipate that drugs that reduce sensory neurons hyperexcitability used for analgesic treatment may cause undesired effects in cancer patients and need to be carefully evaluated in pre-clinical models of cancer before using them in these patients [34,125]. Moreover, the relationship between sensory hyperexcitability and cancer outcomes is likely to inform studies of other cancers that are also infiltrated by sensory neurons.