Mast cell-derived TSLP triggers an allergic response to induce autophagy, thereby inhibiting the development of melanoma

Mast cell-derived TSLP triggers an allergic response to induce autophagy, thereby inhibiting the development of melanoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mast cell-derived TSLP triggers an allergic response to induce autophagy, thereby inhibiting the development of melanoma Hyun-Ja Jeong, Hee-Yun Kim, Ho-Geun Kang, So-Young Rah, Hyung-Min Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3070760/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract An allergic reaction is a hypersensitive reaction that suppresses cancer development and metastasis. Patients with allergic disorders have lower cancer prevalence. In the present study, we aimed to investigate into the relationship between the development of melanoma and the mast cell-mediated allergic response. Here we revealed that mast cell-mediated allergic reaction caused autophagy and apoptosis of melanoma via raising thymic stromal lymphopoietin (TSLP) levels, resulting to improved survival of tumor control mice. Single-cell RNA sequencing of mouse biopsy samples revealed that exogenous TSLP raised levels of mast cell-derived allergy-promoting factors. Moreover, TSLP suppressed melanoma development by enhancing allergic reactions in immunodeficient mice. Patients with melanoma had lower serum levels of TSLP than healthy individuals. Furthermore, in vitro stimulation of melanocytes with TSLP prompted apoptosis of melanoma by inducing the autophagy. Therefore, our findings suggest that TSLP directly/indirectly suppressed the development of melanoma through triggering allergic responses. Health sciences/Oncology/Cancer/Skin cancer/Melanoma Biological sciences/Immunology/Immunotherapy/Immunization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Melanoma, the most dangerous and frequent type of skin cancer, has a poor prognosis, and ultraviolet is the primary contributing factor. 1 Over the past few decades, the prevalence of melanoma has increased as a result of ozone layer depletion brought on by environmental pollution. If melanoma is suspected, resection is performed through early diagnosis, or radiotherapy, systemic chemotherapy, and immunotherapy are performed, but these methods are accompanied by adverse effects. Since melanoma has a high chance of being curable when it first manifests, it is critical to establish a novel anti-cancer therapy strategy that prevents the cancer development through early diagnosis. Although there has been a lot of controversy over the role of allergic reactions in cancer development over the past decades, allergic reactions can be effective in detecting and removing cancer cells by inducing hypersensitivity reactions to improve immune surveillance. 2 Also, the epidemiologic studies of the relationship between allergic reactions and various cancers showed that allergic diseases reduce the risk of pancreatic cancer, glioma, oral cancer, uterine body cancer, stomach cancer, and colorectal cancer. 3 Allergic reactions are triggered by the activation of mast cells that release histamine and cytokines. 4 Especially, mast cell-derived thymic stromal lymphopoietin (TSLP) accelerates allergic reactions. 5 TSLP promotes the activation of mast cells, B cells, dendritic cells, T cells, epithelial cells, basophils, and eosinophils. 5-7 Moreover, TSLP plays a key role in innate and adaptive immune responses. 8,9 Autophagy is involved in inflammatory responses, survival, proliferation, and apoptosis. 10-12 Autophagy-induced apoptosis is caused through activating caspases and inducing autophagy-related proteins including Janus kinases (JAKs), signal transducers and activators of transcription 5 (STAT5), class III phosphoinositide-3 kinase (PI3K), beclin-1, light chain 3 (LC3), Atg5, lysosome-associated membrane protein 1 (LAMP1), and LAMP2. 12,13 Immunotherapy is designed to activate the own immune system to fight cancer. 14 An allergic reaction is a hypersensitive reaction to various antigens. Recently, AllergoOncology using an allergic response has been actively conducted to reveal the function of an IgE-mediated immune response against cancer. 15 Melanoma is treated using a variety of techniques, but all treatments come with negative effects. Particularly, inflammatory response and hyperglycemia have been noted as side effects of immunotherapy utilizing immune checkpoint inhibitors. 16 In the present study, B16F10 melanoma-bearing animal models and in vitro models were used to examine the function and precise mechanism of mast cell-mediated allergic reactions in the suppression of melanoma development. Results Mast cell-mediated allergic reaction inhibits development of melanoma by inducing autophagy in tumor control mice As an initial approach to determine the role of mast cell-mediated allergic reaction on the development of melanoma, we implanted murine melanoma cells and injected with mast cell degranulators such as compound 48/80, colistin, and substance P. Mast cell degranulators significantly promoted survival rate of tumor control group (Fig. 1 a and Extended Data Fig. 1 a, P < 0.05). In comparison to colistin and substance P groups, compound 48/80 group had the highest survival rate, and this effect was shown to be comparable to that of the cisplatin group (Fig. 1 a and Extended Data Fig. 1 a). Tumor volume was significantly reduced in the compound 48/80 group compared to the tumor control group (Fig. 1 b, P < 0.05). qRT-PCR analysis revealed that the expression of melanoma-specific genes (Dct2 and Gp100) was significantly reduced in the compound 48/80 group compared to the tumor control group (Fig. 1 c, P < 0.05). The activities of caspase-3, 8, and 9 in the tumor control group were remarkably reduced compared to the naïve group, whereas the catalytic activities of caspase-3, 8, and 9 were all significantly increased in the compound 48/80 group compared to the tumor control group (Fig. 1 d, P < 0.05). It has been shown that apoptosis and autophagy frequently take place in the same cell, usually in a sequence where autophagy comes first. 12 Immunoblotting analysis revealed that the conversion of LC3-I into LC3-II and the expression of beclin-1 were higher in the compound 48/80 group than in the tumor control group (Fig. 1 e). Additionally, the compound 48/80 group had more melan-A/LC3 double-stained cells than the tumor control group (Fig. 1 f). TEM analysis revealed that the autophagosome was more prevalent in the compound 48/80 group than in the tumor control group (Fig. 1 g). Mast cell-mediated allergic reaction enhances levels of immunostimulatory factors in tumor control mice Histamine secreted by an allergic reaction induces cellular senescence and reduces the proliferation of melanoma cells. 17 As expected, histamine was significantly increased in the compound 48/80 group compared to the tumor control group (Fig. 2 a and Extended Data Fig. 2 a, P < 0.05). TSLP was originally connected to allergic reaction because it primarily fosters a Th2 microenvironment. 6 ELISA analysis revealed that compound 48/80 significantly increased the levels of TSLP compared to the tumor control mice (Fig. 2 b and Extended Data Fig. 2 b, P < 0.05). Colistin and substance P elevated the levels of histamine but had no impact on the levels of TSLP (Extended Data Fig. 1 b and c). Next, we analyzed the levels of IgE, Th1/Th2 cytokines, and vascular endothelial growth factor (VEGF). As shown in Figs. 2 c-f and Extended Data Figs. 2 c-f, compound 48/80 drastically increased the levels of IgE and Th1/Th2 cytokines while significantly reducing the levels of VEGF (P < 0.05). In addition, more mast cells were detected in the compound 48/80 group compared to the tumor control group (Fig. 2 g). To determine if compound 48/80 affects the immune checkpoint, we analyzed the influence of the compound 48/80 on the inhibition of PD-1/PD-L1 using competition ELISA. However, the immune checkpoint inhibitor activity of compound 48/80 was negligible (data not shown). Blood biochemical parameter levels were measured in order to examine the side effects of compound 48/80. Serum levels of ALT, AST, LDH, BUN, CK, glucose, and CRP in the tumor control group were significantly higher than those the naïve group, whereas compound 48/80 significantly reduced the serum contents of these factors compared to the tumor control group (Extended Data Table 2, P < 0.05). TSLP deficiency attenuates induction of autophagy by decreasing mast cell-mediated allergic reaction. In the above data, the survival rate and histamine secretion were both increased by all mast cell degranulators, but when compared to the colistin and substance P groups, the compound 48/80 group significantly increased the TSLP production and had the highest survival rate. Therefore, the development of melanoma was observed by producing an allergic reaction by compound 48/80 in TSLP-deficient mice in order to research the role of TSLP in the suppression of melanoma development. Injection of TSLP siRNA almost completely decreased the TSLP levels (Extended Data Fig. 3 a, P < 0.05). As shown in Fig. 3 a, tumor control mice with control siRNA showed a survival rate of 30%, and compound 48/80 injection in tumor control mice with control siRNA significantly increased the survival rate up to 55% (Fig. 3 a, P < 0.05). However, compound 48/80 only marginally improved survival in TSLP-deficient mice (Fig. 3 a). Tumor volume and melanoma-specific gene expression were significantly increased in the compound 48/80 with TSLP siRNA group compared to compound 48/80 with control siRNA group (Figs. 3 b and c, P < 0.05). In the TSLP-deficient mice, compound 48/80 drastically reduced the levels of IgE, histamine, and Th1/Th2 cytokines compared to compound 48/80 with control siRNA group (Figs. 3 d-e and Extended Data Figs. 3 b-e, P < 0.05). Moreover, TSLP deficiency significantly reduced the caspases activities and autophagy induction (Figs. 3 f and g, P < 0.05). TSLP suppresses development of melanoma via increasing the allergic reaction We next determined whether exogenous TSLP could modulate the development of melanoma. Treatment with TSLP greatly improved the survival of tumor control mice, and it also considerably reduced the tumor volume (Figs. 4 a and b, P < 0.05). qRT-PCR analysis revealed that the expression of Dct2 and Gp100 genes was maximal in the tumor control mice, but the expression of these genes was significantly reduced in TSLP-treated mice (Fig. 4 c, P < 0.05). As shown in Figs. 4 d-f and Extended Data Table 3, TSLP-treated group showed a significant increase in the levels of IgE, histamine, and Th1/Th2 cytokines compared to the tumor control group (P < 0.05). Additionally, TSLP dramatically boosted the activities of caspases and induction of autophagy (Figs. 4 g and h, P < 0.05). According to TEM examination, TSLP-treated mice had more autophagosomes in their cytoplasm than tumor control mice (Fig. 4 i). Serum levels of ALT, AST, LDH, BUN, CK, glucose, and CRP in the tumor control group were significantly higher than those the naïve group, whereas TSLP significantly reduced the serum contents of these factors compared to the tumor control group (Extended Data Table 4, P < 0.05). IgE plays a significant role in AllergoOncology, and increased serum IgE levels are linked to allergic reactions. 15 In the Fig. 4 D, TSLP significantly increased the serum levels of IgE. Therefore, it was investigated if TSLP stimulates the B cells to produce IgE. ELISA analysis revealed that TSLP significantly increased the production of IgE from U266 cells, whereas the increased TSLP levels were significantly reduced by Anti-TSLP but not by IgG (Extended Data Fig. 4 a, P < 0.05). Additionally, IgE injection into the tumor control mice also markedly improved survival rates and diminished tumor volume (Extended Data Figs. 4 b and c, P < 0.05). TSLP reduces proportion of melanocytes and increases proportion of T cell as well as increasing mast cell-derived allergy-promoting factors To investigate the role of TSLP on cellular diversity and molecular signatures of melanoma tissues, we performed single-cell RNA sequencing analysis on melanoma tissues. As shown in Figs. 5 a and b, these cells were categorized into 10 different cell clusters using known makers. Using mouse homologous genes, we identified the cell type of each cluster via the mouse transcriptome sequencing data set in WTA library, and the heatmap showed the expression of marker genes in each cell cluster (Fig. 5 b). The expression levels of the top 3 marker genes showed that the expression of these genes in each cluster was distinctive (Fig. 5 b). The altered arrangement of cell clusters between the tumor control group and the TSLP-treated group is shown in Fig. 5 c. There was a large reduction in the proportion of melanocyte cells and an increase in the proportion of T cells in the TSLP-treated group (Fig. 5 d). The differentially expressed genes, as shown in the t-SNE plots, confirmed the accuracy of cell identity (Fig. 5 e). In the TSLP-treated group, cluster 0 (T cell), 2 (macrophage), 3 (fibroblast), 4 (mast cell), 8 (neutrophil), and 9 (dendritic cell) were characterized by high expression levels of cytokines (Figs. 5 e and f). Interestingly, cluster 4 mast cell displayed high expression levels of allergy-promoting factors including TSLP, histamine, and TNF-α (Fig. 5 e). TSLP suppresses development of melanoma in immunodeficient mice lacking T cell In the single-cell RNA sequencing analysis, we confirmed that TSLP treatment increased the proportion of T cells compared to the tumor control group. Therefore, we attempted to investigate the role of TSLP in the development of melanoma in immunodeficient mice lacking T cell to determine whether the anti-cancer property of TSLP is driven by induction of T cell-mediated response. In the tumor control mice, treatment with TSLP dramatically improved the survival rate while lowering tumor volume and weight (Figs. 6 a-c, P < 0.05). Next, FST was carried out on day 26 to assess the immune-enhancing effect of TSLP. TSLP-treated mice significantly decreased the immobility times compared to the tumor control mice (Fig. 6 d, P < 0.05). In addition, treatment with TSLP showed a significant increase in the levels of allergy-promoting factors and Th1/Th2 cytokines compared to the tumor control group (Figs. 6 e-g, P < 0.05). Furthermore, more mast cells were detected in the TSLP-treated group compared to the tumor control group (Fig. 6 h). Mast cell-derived TSLP is down-regulated in melanoma patients and tumor control mice The above findings indicated that TSLP plays a significant role in melanoma development, so we examined the levels of TSLP in the serum of melanoma patients. ELISA analysis revealed that a lower protein expression of TSLP was detected in the serum of melanoma patients compared to normal subjects (Fig. 7 a, P < 0.05). In addition, serum IgE levels of melanoma patients were lower than those of normal subjects (Fig. 7 b, P < 0.05). The Fig. 2 b and Extended Data Fig. 2 b demonstrated that the tumor control mice had lower protein levels of TSLP than naïve mice. In agreement with these findings, TSLP mRNA levels of tumor control mice were significantly lower than those of naive mice (Fig. 7 c, P < 0.05). The next step was to conduct an immunohistochemistry for TSLP and tryptase (a mast cell biomarker) to determine whether TSLP is expressed in mast cells as a result of allergic responses. Immunofluorescence staining revealed that the number of mast cells expressing TSLP in the tumor control mice was lower than naïve mice, but the number of mast cells expressing TSLP was increased in the compound 48/80 group compared to the tumor control group (Fig. 7 d). Mast cell-derived TSLP decreases viability and proliferation of B16F10 melanoma cells To investigate whether mast cell-derived TSLP may affect melanoma survival and proliferation, TSLP production by mast cell stimulators was first analyzed using BMMCs. ELISA analysis revealed that mast cell stimulators (PMACI, IgE, and compound 48/80) significantly increased the production of TSLP on BMMCs, and compound 48/80 had the most an impact on these results (Extended Data Fig. 5 A, P < 0.05). Using qRT-PCR, RT-PCR, and flow cytometry, we examined the expression of the TSLP receptor (TSLPR) in B16F10 melanoma cells. As results, the TSLPR was expressed in B16F10 melanoma cells (Extended Data Fig. 5 b upper), and it was discovered that TSLP amplified this expression (Extended Data Fig. 5 b lower). Because the mast cell-mediated allergic reaction suppressed the development of melanoma based on the above data, we hypothesized that soluble TSLP released from activated mast cells would impact the survival and proliferation of melanoma. As seen in Extended Data Figs. 5 c and d, the supernatants of PMACI, IgE, or compound 48/80-stimulated BMMCs greatly decreased the viability and proliferation of B16F10 cells, and this impact was reversed by Anti-TSLP (P < 0.05). Strikingly, similar to the TSLP production result in Extended Data Fig. 5 a, compound 48/80 had the best suppressive effect on the viability and proliferation of B16F10 cells. In addition, we confirmed that viability and proliferation of B16F10 cells were greatly reduced by TSLP, and these effects were reversed by Anti-TSLP but not by IgG (Extended Data Figs. 5e and f, P < 0.05). TSLP induces apoptosis of B16F10 melanoma cells by induction of autophagy We predicted that TSLP would cause apoptosis in B16F10 cells since it prevents the survival and growth of these cells and induces the autophagy of tumor control mice. As shown in Fig. 8 a, the apoptosis rate of B16F10 cells in the blank was about 44.73%, while those of TSLP group was increased to approximately 92.96%, and these effects were reversed by Anti-TSLP but not by IgG. The similar results were obtained for caspase-3, 8, and 9 activities (Fig. 8 b). According to our earlier research, TSLP activates the JAKs/STAT5 signaling pathway, which led to the induction of autophagy. 13 Immunoblotting analysis revealed that TSLP considerably increased the expression levels of pJAK1, pJAK2, and pSTAT5 (Fig. 8 c). Next, to investigate whether TSLP is involved in autophagosome formation in melanoma, the expression of proteins related to autophagosome formation was analyzed in B16F10 cells. The expression levels of class III PI3K, beclin-1, LC3-II, Atg5, p62, LAMP1, and LAMP2 were dramatically increased in the TSLP-stimulated B16F10 cells when compared to the unstimulated group (Figs. 8 c and d). However, Anti-TSLP reduced this increase but not IgG (Figs. 8 c and d). To further delineate the relevance of TSLP and autophagy, B16F10 cells were treated with TSLP and 3-methyladenine (3-MA, autophagy inhibitor). 3-MA prevented the TSLP-induced autophagy by limiting the formation of autophagosomes, which dramatically enhanced the viability and growth of B16F10 cells (Figs. 8 e and f, P < 0.05). Moreover, activities of caspase-3, 8, and 9 were significantly decreased by 3-MA in the TSLP-treated B16F10 cells (Fig. 8 g, P < 0.05). Discussion An allergic reaction is an overreaction of your immune system. 4 Studies in the past have revealed a negative correlation between cancer and allergy illnesses. 3 , 18 On the other hand, a number of recent studies reported that allergy was linked to a lower risk of cancers. 3 , 4 , 19 However, the correlation between melanoma and allergy has been questionable up until recently. Mast cells, the causative cells of allergy, are actually multipurpose cells that are essential for both innate and adaptive immune responses. 20 , 21 It is still unclear what exactly mast cells do in cancers. In an animal model of late-stage melanoma, mast cell-derived histamine exacerbates melanoma growth. 18 Contrarily, we recently revealed that surfactin has an anti-cancer effect by triggering mast cell-mediated allergic reactions in early-stage of melanoma skin cancer. 2 Massari et al. reported that histamine increases radiosensitivity of melanoma cells, which benefits cancer radiotherapy in the treatment of melanoma. 17 IgE interactions with mast cells have strong anti-tumor effects, while IgE immunodeficiency is associated with a markedly increased risk of developing cancer. 22 , 23 In human patients with melanoma, low numbers of mast cells are linked to decreased survival and advanced tumor stages of melanoma. 24 Activated mast cells produce TSLP, and an increase in TSLP mediates the Th2 immune response by boosting the production of Th2 cytokines. 25 , 26 In the present work, we demonstrated that compound 48/80 induced the autophagy and apoptosis of melanoma by triggering allergic reactions, which improved the survival rate of tumor control mice. Further experiments revealed that TSLP deficiency reduced the levels of histamine, IgE, and Th2 cytokines as well as Th1 cytokines, partially reversing the effects of compound 48/80. Building on these findings, we suggest that mast cell-mediated allergic reactions increased the levels of TSLP, which suppressed the melanoma development in its early stage. Additionally, we suggest that the low incidence of cancer in patients with allergic disorders results from the autophagy of cancer cells brought on by allergic reactions. TSLP is crucial for preserving immunological homeostasis. 27 Although substantial research has been done on the function of TSLP in Th2 immune responses, more recent investigations have discovered that TSLP also has a growing role in allergic reactions and cancer. 27 , 28 The development of hematological tumors as well as solid tumors like breast, colon, and pancreatic cancers have been linked to TSLP, while TSLP signaling provides protection against skin cancers. 28 – 31 On the other hand, Yao et al. reported that TSLP promotes the metastasis and growth of melanoma by stimulating CD4 T cells and dendritic cells. 32 Like these, so far, the role of TSLP in the context of tumor immune responses has typically been described as both suppressive and stimulatory. 29 , 33 The reason for the different functions of TSLP in cancer models is probably because the immune response to TSLP in cancer appears differently depending on the experimental conditions (mouse type, cancer stage, Notch loss of function, increased Wnt function, H-Ras dependent tumor, and TSLP levels). In the present study, we demonstrated that TSLP promoted the survival by enhancing immunity through the increase of allergy-promoting factors in C57BL/6 and BALB/c immunodeficient melanoma mice. Therefore, these findings suggest that TSLP suppressed the development of melanoma through triggering allergic reactions. In the present study, the levels of Th1/Th2 cytokines were significantly increased in response to TSLP. Th1 cytokines are linked to a favorable prognosis for illnesses, but Th2 cytokines are linked to inflammatory responses. 6 , 18 , 26 An imbalance of Th1/Th2 cytokines is a cause of cancer development. In the present study, TSLP also reduced the level of CRP. This is presumed to be because increased Th1 cytokines regulate the amount of Th2 cytokines, preventing them from increasing above naïve levels. Therefore, these findings suggest that TSLP suppressed the inflammatory response by preserving the balance of Th1/Th2 cytokines, which in turn slows the development of melanoma. As autophagy promotes in the death of tumor cells and apoptosis reduces their survival, both processes are beneficial for suppressing tumor growth. 34 Many tumor cells display a reduced autophagic capacity when compared with normal cells, and the autophagy-related protein beclin-1 is shown to be significantly reduced in a number of human cancers. 35 – 37 These results support the notion that increasing the expression of autophagic genes can help treat various cancers, in addition to the fact that cancer cells have reduced basal autophagic activity levels. In the present study, mast cell-derived TSLP induced the apoptosis of B16F10 cells. Moreover, TSLP/TSLPR signaling pathway induced the autophagy through increasing the expression of autophagy-related proteins. Therefore, these results suggest that TSLP/TSLPR signaling pathway directly contributes to the apoptosis of B16F10 cells through the induction of autophagy. In conclusion, our findings revealed that mast cell-derived TSLP directly/indirectly suppressed the development of melanoma by triggering allergic reactions without affecting the side effects. These findings imply that TSLP-induced allergic reactions may be a novel therapeutic approach for the prevention and treatment of melanoma. Furthermore, these findings can also be employed as cancer vaccines to prevent the development of melanoma because multiple studies on melanoma-related genes have been performed and research on food allergy-based cancer vaccines is progressing significantly. However, future preclinical and clinical studies are urgently needed for the therapeutic application of melanoma according to the induction of allergic responses. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3070760","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432581143,"identity":"f44738df-82c7-4947-b212-7c1245747308","order_by":0,"name":"Hyun-Ja Jeong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACA4YDCSBazgAqwNhArBZjUrRAQOIGorWYMx54+ODjjtr07eztDx98YLCR3XCAgBbLhgPJhjPPHM/d2XPG2HAGQ5oxQS0GBw6kSfO2HcvdcCOHTZqH4XAiMVrSfwO1pBvcSH/+m4fhP1Fa0ph522oSDG4kmDHzMBwgSkuy5My2A4YbzpwxlpxhkGw8k6CWG2cSP3xsq5M3ON7+8MOHCjvZPkJaGCTOJADJwzATCCkHAf52kKl1xCgdBaNgFIyCkQoAzohPc3I67twAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7559-0373","institution":"Hoseo University","correspondingAuthor":true,"prefix":"","firstName":"Hyun-Ja","middleName":"","lastName":"Jeong","suffix":""},{"id":432581144,"identity":"f56aa2d1-3be3-42f8-85d3-327ea924c512","order_by":1,"name":"Hee-Yun Kim","email":"","orcid":"","institution":"Hoseo University","correspondingAuthor":false,"prefix":"","firstName":"Hee-Yun","middleName":"","lastName":"Kim","suffix":""},{"id":432581145,"identity":"7c62ba0f-231b-462a-94e9-2d6679da5cdd","order_by":2,"name":"Ho-Geun Kang","email":"","orcid":"","institution":"Hoseo University","correspondingAuthor":false,"prefix":"","firstName":"Ho-Geun","middleName":"","lastName":"Kang","suffix":""},{"id":432581146,"identity":"5fee4a35-ec04-487f-9e92-915f4e14b018","order_by":3,"name":"So-Young Rah","email":"","orcid":"","institution":"Chonbuk National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"So-Young","middleName":"","lastName":"Rah","suffix":""},{"id":432581147,"identity":"25fd9950-7877-4a4f-aa47-bba910a3b101","order_by":4,"name":"Hyung-Min Kim","email":"","orcid":"","institution":"Kyung Hee University","correspondingAuthor":false,"prefix":"","firstName":"Hyung-Min","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2023-06-16 06:21:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3070760/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3070760/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79259859,"identity":"f5e8f50f-1ad4-4a07-9e91-9df5d80f5aa3","added_by":"auto","created_at":"2025-03-26 09:22:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":122298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMast cell-mediated allergic reaction inhibits melanoma growth by inducing autophagy in tumor control mice.\u003c/strong\u003e C57BL/6 mice were inoculated with B16F10 cells by a s.c. injection of 3 × 10\u003csup\u003e5\u003c/sup\u003e cells to the right flank. The mice (n = 10 per group) were administrated with PBS, compound 48/80 (5 mg/kg), or cisplatin (1 mg/kg) every two days for 22 days. \u003cstrong\u003ea,\u003c/strong\u003e upper, Scheme of animal model; lower, Survival rate. \u003cstrong\u003eb,\u003c/strong\u003e Tumor volume. Arrows indicated tumors. \u003cstrong\u003ec,\u003c/strong\u003e qRT-PCR analysis of Dct2 and Gp100 \u003cstrong\u003emRNA expression in melanoma tissue. d,\u003c/strong\u003e \u003cstrong\u003eCaspase activities in melanoma tissue. e,\u003c/strong\u003e \u003cstrong\u003eWestern blot analysis of LC3 and beclin-1 expression in melanoma tissue. f,\u003c/strong\u003e \u003cstrong\u003eConfocal microscopic image \u003c/strong\u003efor melan-A (green) and LC3 (red) in melanoma tissue (magnification × 294, scale bar = 100 μm).\u003cstrong\u003e g,\u003c/strong\u003e \u003cstrong\u003eT\u003c/strong\u003eransmission\u003cstrong\u003e electron micrograph image for autophagosomes in melanoma. Arrows indicated autophagosomes. Com 48/80, compound 48/80. \u003c/strong\u003e\u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, significantly different from the naïve mice. *P \u0026lt; 0.05, significantly different from the tumor control mice.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/c483c50f880899e844e806d6.jpg"},{"id":79259861,"identity":"302fb09f-067b-4cbe-bbd6-4c45b41ccca5","added_by":"auto","created_at":"2025-03-26 09:22:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMast cell-mediated allergic reaction enhances levels of immunostimulatory factors in tumor control mice.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Serum histamine. \u003cstrong\u003eb,\u003c/strong\u003e ELISA analysis of serum TSLP, \u003cstrong\u003ec,\u003c/strong\u003e IgE, \u003cstrong\u003ed,\u003c/strong\u003e IL-4, IL-5, IL-6, and IL-13. \u003cstrong\u003ee,\u003c/strong\u003e ELISA analysis of serum IL-2, IL-12, TNF-α, and IFN-γ. \u003cstrong\u003ef,\u003c/strong\u003e ELISA analysis of serum VEGF. \u003cstrong\u003eg,\u003c/strong\u003e Immunostaining analysis for mast cells \u003cstrong\u003estained with \u003c/strong\u003ealcian blue and safranin O (magnification × 400, scale bar = 100 μm, intact mast cells, indicated by the red arrows; degranulated mast cells, indicated by the black arrows). \u003cstrong\u003eCom 48/80, compound 48/80. \u003c/strong\u003e\u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, significantly different from the naïve mice. *P \u0026lt; 0.05, significantly different from the tumor control mice.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/9e69395ebb5a83973fb170b2.jpg"},{"id":79260995,"identity":"2b5c4781-8f70-48b7-947e-518e7b8af3c6","added_by":"auto","created_at":"2025-03-26 09:30:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP deficiency attenuates autophagy induction of melanoma by decreasing allergic reaction.\u003c/strong\u003e The mice (n = 10 per group) were given a i.v. injection of\u003cstrong\u003e scramble control siRNA or TSLP-specific siRNA before \u003c/strong\u003eadministration with PBS or compound 48/80 (5 mg/kg) every two days for 22 days. \u003cstrong\u003ea,\u003c/strong\u003e upper, Scheme of animal model; lower, Survival rate. \u003cstrong\u003eb,\u003c/strong\u003e Tumor volume. \u003cstrong\u003ec,\u003c/strong\u003e qRT-PCR analysis of Dct2 and Gp100 \u003cstrong\u003emRNA expression in melanoma tissue. d,\u003c/strong\u003e ELISA analysis of serum IgE and histamine.\u003cstrong\u003e e,\u003c/strong\u003e ELISA analysis of serum IL-4, IL-5, IL-6, and IL-13. \u003cstrong\u003ef,\u003c/strong\u003e \u003cstrong\u003eCaspase activities in melanoma tissue. g,\u003c/strong\u003e \u003cstrong\u003eWestern blot analysis of LC3 and beclin-1 expression in melanoma tissue.\u003c/strong\u003e \u003cstrong\u003eCom 48/80, compound 48/80. \u003c/strong\u003e\u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05; significantly different from the control siRNA tumor control mice. \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.05; significantly different from the TSLP siRNA tumor control mice. *P \u0026lt; 0.05; significantly different from the control siRNA compound 48/80-treated mice.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/fbacfb371e80505cdcdc5f2d.jpg"},{"id":79262542,"identity":"52e82208-eccf-4d13-83b9-3190dc2652b9","added_by":"auto","created_at":"2025-03-26 09:46:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":122946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogeneous TSLP displays enhanced therapeutic efficacy against melanoma.\u003c/strong\u003e The mice (n = 10 per group) were given an i.p. injection of\u003cstrong\u003e \u003c/strong\u003ePBS or TSLP (2 μg) every two days for 22 days. \u003cstrong\u003ea,\u003c/strong\u003e upper, Scheme of animal model; lower, Survival rate. (B) Tumor volume. Arrows indicated tumors. \u003cstrong\u003ec, \u003c/strong\u003eqRT-PCR analysis of Dct2 and Gp100 \u003cstrong\u003emRNA expression in melanoma tissue. d,\u003c/strong\u003e ELISA analysis of serum IgE and Histamine.\u003cstrong\u003e e,\u003c/strong\u003e ELISA analysis of serum IL-4, IL-5, IL-6, and IL-13. \u003cstrong\u003ef,\u003c/strong\u003e ELISA analysis of serum IL-2, IL-12, TNF-α, and IFN-γ. \u003cstrong\u003eg,\u003c/strong\u003e \u003cstrong\u003eCaspase activities in melanoma tissue. h,\u003c/strong\u003e \u003cstrong\u003eWestern blot analysis of LC3 and beclin-1 expression in melanoma tissue. i,\u003c/strong\u003e \u003cstrong\u003eT\u003c/strong\u003eransmission\u003cstrong\u003e electron micrograph image for autophagosomes in melanoma. Arrows indicated autophagosomes. \u003c/strong\u003e\u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, significantly different from the naïve mice. *P \u0026lt; 0.05, significantly different from the tumor control mice.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/44c266585dca4a0585d06ea5.jpg"},{"id":79259869,"identity":"2a2a6b40-dcdb-4b0b-a39a-8c7614e4b2ab","added_by":"auto","created_at":"2025-03-26 09:22:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":135334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP reduces proportion of melanocytes and increases proportion of T cell as well as increasing mast cell-derived allergy-promoting factors.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e t-SNE plot showing the cell clusters in melanoma tissue. The 10 cell types determined using marker genes. \u003cstrong\u003eb,\u003c/strong\u003e Heatmap showing the cluster-specific marker gene expression.\u003cstrong\u003e c,\u003c/strong\u003e t-SNE plot in melanoma tissues (left panel, tumor control mice; right panel, exogeneous TSLP mice). \u003cstrong\u003ed,\u003c/strong\u003e Histogram indicating the proportion of cells in melanoma tissue of each sample. \u003cstrong\u003ee,\u003c/strong\u003e Heatmaps showing the expression of the representative genes of exogeneous TSLP mice. \u003cstrong\u003ef,\u003c/strong\u003e Violin plot showing the expression of the representative genes of exogeneous TSLP mice.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/d5511ef48649127e6a45f220.jpg"},{"id":79259877,"identity":"2f23ed5f-0ce6-44b9-ad07-74f4ea476922","added_by":"auto","created_at":"2025-03-26 09:22:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":128558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogeneous TSLP suppresses development of melanoma in immunodeficient mice lacking T cell.\u003c/strong\u003e Immunodeficient mice (n = 10 per group) were given an i.p. injection of\u003cstrong\u003e \u003c/strong\u003ePBS or TSLP (2 μg) every two days for 26 days. \u003cstrong\u003ea,\u003c/strong\u003e upper, Scheme of animal model; lower, Survival rate. \u003cstrong\u003eb,\u003c/strong\u003e Tumor volume was monitored daily. Arrows indicated tumors. \u003cstrong\u003ec,\u003c/strong\u003e Tumor weight. \u003cstrong\u003ed,\u003c/strong\u003e Immobility time (n = 10 per group). \u003cstrong\u003ee,\u003c/strong\u003e ELISA analysis of serum IgE and histamine.\u003cstrong\u003e f,\u003c/strong\u003e ELISA analysis of serum IL-4, IL-5, IL-6, and IL-13. \u003cstrong\u003eg,\u003c/strong\u003e ELISA analysis of serum IL-2, IL-12, and TNF-α. \u003cstrong\u003eh,\u003c/strong\u003e Immunostaining analysis for mast cells \u003cstrong\u003estained with \u003c/strong\u003ealcian blue and safranin O (magnification × 400, scale bar = 100 μm, intact mast cells, indicated by the red arrows; degranulated mast cells, indicated by the black arrows). \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, significantly different from the naïve mice. *P \u0026lt; 0.05, significantly different from the tumor control mice.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/fd49590c4b61d9013d461c44.jpg"},{"id":79259867,"identity":"f941b7be-052d-4378-8475-2b1a047b5d8f","added_by":"auto","created_at":"2025-03-26 09:22:16","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":103430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMast cell-derived TSLP levels are down-regulated in human patients and melanoma animal model.\u003c/strong\u003e ELISA analysis of serum \u003cstrong\u003ea,\u003c/strong\u003e TSLP and \u003cstrong\u003eb,\u003c/strong\u003e IgE in melanoma patients and normal subjects. \u003cstrong\u003ec,\u003c/strong\u003e qRT-PCR analysis of TSLP \u003cstrong\u003emRNA expression in melanoma tissue of \u003c/strong\u003etumor control mice\u003cstrong\u003e. d,\u003c/strong\u003e \u003cstrong\u003eConfocal microscopic image \u003c/strong\u003efor TSLP (green) and tryptase (red) in melanoma tissue\u003cstrong\u003e of tumor control\u003c/strong\u003e mice (magnification, × 294, scale bar = 100 μm). \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, significantly different from the naïve group.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/f69c5c97bb6353fc425225a1.jpg"},{"id":79260999,"identity":"9c5581d1-6d03-4e17-bbd5-47deba52ee9d","added_by":"auto","created_at":"2025-03-26 09:30:16","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":108457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP increases apoptosis of B16F10 cell by induction of autophagy.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e B16F10\u003cstrong\u003e cells were stimulated with TSLP (20 ng/ml), Anti-\u003c/strong\u003eTSLP (\u003cstrong\u003e20 ng/ml\u003c/strong\u003e), or IgG \u003cstrong\u003efor 48 h and stained with annexin V and PI. Flow cytometry analysis of apoptosis. b,\u003c/strong\u003e B16F10\u003cstrong\u003e cells were stimulated with TSLP (20 ng/ml), Anti-\u003c/strong\u003eTSLP (\u003cstrong\u003e20 ng/ml\u003c/strong\u003e), or IgG \u003cstrong\u003efor 24 h. \u003c/strong\u003eCaspase activities. \u003cstrong\u003ec,\u003c/strong\u003e B16F10\u003cstrong\u003e cells were stimulated with TSLP (20 ng/ml), Anti-\u003c/strong\u003eTSLP (\u003cstrong\u003e20 ng/ml\u003c/strong\u003e), or IgG \u003cstrong\u003efor 30 min or 24 h. Western blot analysis of pJAK1, pJAK2, pSTAT5, \u003c/strong\u003eclass III\u003cstrong\u003e PI3K, beclin-1, LC3, Atg5, and p62 expression in cell extracts. \u003c/strong\u003eResults are representative of three independent experiments.\u003cstrong\u003e d,\u003c/strong\u003e B16F10\u003cstrong\u003e cells were stimulated with TSLP (20 ng/ml), Anti-\u003c/strong\u003eTSLP (\u003cstrong\u003e20 ng/ml\u003c/strong\u003e), or IgG \u003cstrong\u003efor 12 h. \u003c/strong\u003eqRT-PCR analysis of LAMP1 and LAMP2 \u003cstrong\u003emRNA expression. B16F10 cells were incubated with 3-MA (5 mM) for 1 h prior to stimulation with TSLP (20 ng/ml). e,\u003c/strong\u003e Cell viability analysis of B16F10 cells using MTT assay. \u003cstrong\u003ef,\u003c/strong\u003e Cell growth analysis of B16F10 cells using BrdU assay. \u003cstrong\u003eg,\u003c/strong\u003e Caspase activities. \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, significantly different from the unstimulated cells. *P \u0026lt; 0.05, significantly different from the TSLP-stimulated cells.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/9f369bde9e86a5a7a44a38bb.jpg"},{"id":79264577,"identity":"66bbc935-d612-4c19-a1dc-a6ea849086a8","added_by":"auto","created_at":"2025-03-26 09:54:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2655849,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/1b235b47-bb77-43c1-ae2b-eb06f35acfcd.pdf"},{"id":79260992,"identity":"434e4983-7848-4d29-a322-f8a8d4b1615b","added_by":"auto","created_at":"2025-03-26 09:30:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4618089,"visible":true,"origin":"","legend":"Supplemenatry data","description":"","filename":"Supplemenatrydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-3070760/v1/1f27d6217af38578865828e6.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mast cell-derived TSLP triggers an allergic response to induce autophagy, thereby inhibiting the development of melanoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMelanoma, the most dangerous and frequent type of skin cancer, has a poor prognosis, and ultraviolet is the primary contributing factor.\u003csup\u003e1\u003c/sup\u003e Over the past few decades, the prevalence of melanoma has increased as a result of ozone layer depletion brought on by environmental pollution. If melanoma is suspected, resection is performed through early diagnosis, or radiotherapy, systemic chemotherapy, and immunotherapy are performed, but these methods are accompanied by adverse effects. Since melanoma has a high chance of being curable when it first manifests, it is critical to establish a novel anti-cancer therapy strategy that prevents the cancer development through early diagnosis.\u003c/p\u003e\n\u003cp\u003eAlthough there has been a lot of controversy over the role of allergic reactions in cancer development over the past decades, allergic reactions can be effective in detecting and removing cancer cells by inducing hypersensitivity reactions to improve immune surveillance.\u003csup\u003e2\u003c/sup\u003e Also, the epidemiologic studies of the relationship between allergic reactions and various cancers showed that allergic diseases reduce the risk of pancreatic cancer, glioma, oral cancer, uterine body cancer, stomach cancer, and colorectal cancer.\u003csup\u003e3\u003c/sup\u003e Allergic reactions are triggered by the activation of mast cells that release histamine and cytokines.\u003csup\u003e4\u003c/sup\u003e Especially, mast cell-derived thymic stromal lymphopoietin (TSLP) accelerates allergic reactions.\u003csup\u003e5\u003c/sup\u003e TSLP promotes the activation of mast cells, B cells, dendritic cells, T cells, epithelial cells, basophils, and eosinophils.\u003csup\u003e5-7\u003c/sup\u003e Moreover, TSLP plays a key role in innate and adaptive immune responses.\u003csup\u003e8,9\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAutophagy is involved in inflammatory responses, survival, proliferation, and apoptosis.\u003csup\u003e10-12\u0026nbsp;\u003c/sup\u003eAutophagy-induced apoptosis is caused through activating caspases and inducing autophagy-related proteins including Janus kinases (JAKs), signal transducers and activators of transcription 5 (STAT5), class III phosphoinositide-3 kinase (PI3K), beclin-1, light chain 3 (LC3), Atg5, lysosome-associated membrane protein 1 (LAMP1), and LAMP2.\u003csup\u003e12,13\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eImmunotherapy is designed to activate the own immune system to fight cancer.\u003csup\u003e14\u0026nbsp;\u003c/sup\u003eAn allergic reaction is a hypersensitive reaction to various antigens. Recently, AllergoOncology using an allergic response has been actively conducted to reveal the function of an IgE-mediated immune response against cancer.\u003csup\u003e15\u003c/sup\u003e Melanoma is treated using a variety of techniques, but all treatments come with negative effects. Particularly, inflammatory response and hyperglycemia have been noted as side effects of immunotherapy utilizing immune checkpoint inhibitors.\u003csup\u003e16\u003c/sup\u003e In the present study, B16F10 melanoma-bearing animal models and \u003cem\u003ein vitro\u003c/em\u003e models were used to examine the function and precise mechanism of mast cell-mediated allergic reactions in the suppression of melanoma development.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eMast cell-mediated allergic reaction inhibits development of melanoma by inducing autophagy in tumor control mice\u003c/h2\u003e \u003cp\u003eAs an initial approach to determine the role of mast cell-mediated allergic reaction on the development of melanoma, we implanted murine melanoma cells and injected with mast cell degranulators such as compound 48/80, colistin, and substance P. Mast cell degranulators significantly promoted survival rate of tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In comparison to colistin and substance P groups, compound 48/80 group had the highest survival rate, and this effect was shown to be comparable to that of the cisplatin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Tumor volume was significantly reduced in the compound 48/80 group compared to the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). qRT-PCR analysis revealed that the expression of melanoma-specific genes (Dct2 and Gp100) was significantly reduced in the compound 48/80 group compared to the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The activities of caspase-3, 8, and 9 in the tumor control group were remarkably reduced compared to the na\u0026iuml;ve group, whereas the catalytic activities of caspase-3, 8, and 9 were all significantly increased in the compound 48/80 group compared to the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It has been shown that apoptosis and autophagy frequently take place in the same cell, usually in a sequence where autophagy comes first.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Immunoblotting analysis revealed that the conversion of LC3-I into LC3-II and the expression of beclin-1 were higher in the compound 48/80 group than in the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Additionally, the compound 48/80 group had more melan-A/LC3 double-stained cells than the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). TEM analysis revealed that the autophagosome was more prevalent in the compound 48/80 group than in the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMast cell-mediated allergic reaction enhances levels of immunostimulatory factors in tumor control mice\u003c/h2\u003e \u003cp\u003eHistamine secreted by an allergic reaction induces cellular senescence and reduces the proliferation of melanoma cells.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e As expected, histamine was significantly increased in the compound 48/80 group compared to the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). TSLP was originally connected to allergic reaction because it primarily fosters a Th2 microenvironment.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e ELISA analysis revealed that compound 48/80 significantly increased the levels of TSLP compared to the tumor control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Colistin and substance P elevated the levels of histamine but had no impact on the levels of TSLP (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and c). Next, we analyzed the levels of IgE, Th1/Th2 cytokines, and vascular endothelial growth factor (VEGF). As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f and Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f, compound 48/80 drastically increased the levels of IgE and Th1/Th2 cytokines while significantly reducing the levels of VEGF (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, more mast cells were detected in the compound 48/80 group compared to the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine if compound 48/80 affects the immune checkpoint, we analyzed the influence of the compound 48/80 on the inhibition of PD-1/PD-L1 using competition ELISA. However, the immune checkpoint inhibitor activity of compound 48/80 was negligible (data not shown).\u003c/p\u003e \u003cp\u003eBlood biochemical parameter levels were measured in order to examine the side effects of compound 48/80. Serum levels of ALT, AST, LDH, BUN, CK, glucose, and CRP in the tumor control group were significantly higher than those the na\u0026iuml;ve group, whereas compound 48/80 significantly reduced the serum contents of these factors compared to the tumor control group (Extended Data Table\u0026nbsp;2, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTSLP deficiency attenuates induction of autophagy by decreasing mast cell-mediated allergic reaction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the above data, the survival rate and histamine secretion were both increased by all mast cell degranulators, but when compared to the colistin and substance P groups, the compound 48/80 group significantly increased the TSLP production and had the highest survival rate. Therefore, the development of melanoma was observed by producing an allergic reaction by compound 48/80 in TSLP-deficient mice in order to research the role of TSLP in the suppression of melanoma development. Injection of TSLP siRNA almost completely decreased the TSLP levels (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, tumor control mice with control siRNA showed a survival rate of 30%, and compound 48/80 injection in tumor control mice with control siRNA significantly increased the survival rate up to 55% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, compound 48/80 only marginally improved survival in TSLP-deficient mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Tumor volume and melanoma-specific gene expression were significantly increased in the compound 48/80 with TSLP siRNA group compared to compound 48/80 with control siRNA group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and c, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the TSLP-deficient mice, compound 48/80 drastically reduced the levels of IgE, histamine, and Th1/Th2 cytokines compared to compound 48/80 with control siRNA group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-e and Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, TSLP deficiency significantly reduced the caspases activities and autophagy induction (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and g, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTSLP suppresses development of melanoma via increasing the allergic reaction\u003c/h3\u003e\n\u003cp\u003eWe next determined whether exogenous TSLP could modulate the development of melanoma. Treatment with TSLP greatly improved the survival of tumor control mice, and it also considerably reduced the tumor volume (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). qRT-PCR analysis revealed that the expression of Dct2 and Gp100 genes was maximal in the tumor control mice, but the expression of these genes was significantly reduced in TSLP-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f and Extended Data Table\u0026nbsp;3, TSLP-treated group showed a significant increase in the levels of IgE, histamine, and Th1/Th2 cytokines compared to the tumor control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, TSLP dramatically boosted the activities of caspases and induction of autophagy (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and h, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). According to TEM examination, TSLP-treated mice had more autophagosomes in their cytoplasm than tumor control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Serum levels of ALT, AST, LDH, BUN, CK, glucose, and CRP in the tumor control group were significantly higher than those the na\u0026iuml;ve group, whereas TSLP significantly reduced the serum contents of these factors compared to the tumor control group (Extended Data Table\u0026nbsp;4, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIgE plays a significant role in AllergoOncology, and increased serum IgE levels are linked to allergic reactions.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e In the Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, TSLP significantly increased the serum levels of IgE. Therefore, it was investigated if TSLP stimulates the B cells to produce IgE. ELISA analysis revealed that TSLP significantly increased the production of IgE from U266 cells, whereas the increased TSLP levels were significantly reduced by Anti-TSLP but not by IgG (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, IgE injection into the tumor control mice also markedly improved survival rates and diminished tumor volume (Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and c, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTSLP reduces proportion of melanocytes and increases proportion of T cell as well as increasing mast cell-derived allergy-promoting factors\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the role of TSLP on cellular diversity and molecular signatures of melanoma tissues, we performed single-cell RNA sequencing analysis on melanoma tissues. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and b, these cells were categorized into 10 different cell clusters using known makers. Using mouse homologous genes, we identified the cell type of each cluster via the mouse transcriptome sequencing data set in WTA library, and the heatmap showed the expression of marker genes in each cell cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The expression levels of the top 3 marker genes showed that the expression of these genes in each cluster was distinctive (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The altered arrangement of cell clusters between the tumor control group and the TSLP-treated group is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec. There was a large reduction in the proportion of melanocyte cells and an increase in the proportion of T cells in the TSLP-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). The differentially expressed genes, as shown in the t-SNE plots, confirmed the accuracy of cell identity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). In the TSLP-treated group, cluster 0 (T cell), 2 (macrophage), 3 (fibroblast), 4 (mast cell), 8 (neutrophil), and 9 (dendritic cell) were characterized by high expression levels of cytokines (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and f). Interestingly, cluster 4 mast cell displayed high expression levels of allergy-promoting factors including TSLP, histamine, and TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eTSLP suppresses development of melanoma in immunodeficient mice lacking T cell\u003c/h3\u003e\n\u003cp\u003eIn the single-cell RNA sequencing analysis, we confirmed that TSLP treatment increased the proportion of T cells compared to the tumor control group. Therefore, we attempted to investigate the role of TSLP in the development of melanoma in immunodeficient mice lacking T cell to determine whether the anti-cancer property of TSLP is driven by induction of T cell-mediated response. In the tumor control mice, treatment with TSLP dramatically improved the survival rate while lowering tumor volume and weight (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-c, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Next, FST was carried out on day 26 to assess the immune-enhancing effect of TSLP. TSLP-treated mice significantly decreased the immobility times compared to the tumor control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, treatment with TSLP showed a significant increase in the levels of allergy-promoting factors and Th1/Th2 cytokines compared to the tumor control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-g, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, more mast cells were detected in the TSLP-treated group compared to the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMast cell-derived TSLP is down-regulated in melanoma patients and tumor control mice\u003c/h3\u003e\n\u003cp\u003eThe above findings indicated that TSLP plays a significant role in melanoma development, so we examined the levels of TSLP in the serum of melanoma patients. ELISA analysis revealed that a lower protein expression of TSLP was detected in the serum of melanoma patients compared to normal subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, serum IgE levels of melanoma patients were lower than those of normal subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb demonstrated that the tumor control mice had lower protein levels of TSLP than na\u0026iuml;ve mice. In agreement with these findings, TSLP mRNA levels of tumor control mice were significantly lower than those of naive mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The next step was to conduct an immunohistochemistry for TSLP and tryptase (a mast cell biomarker) to determine whether TSLP is expressed in mast cells as a result of allergic responses. Immunofluorescence staining revealed that the number of mast cells expressing TSLP in the tumor control mice was lower than na\u0026iuml;ve mice, but the number of mast cells expressing TSLP was increased in the compound 48/80 group compared to the tumor control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMast cell-derived TSLP decreases viability and proliferation of B16F10 melanoma cells\u003c/h3\u003e\n\u003cp\u003eTo investigate whether mast cell-derived TSLP may affect melanoma survival and proliferation, TSLP production by mast cell stimulators was first analyzed using BMMCs. ELISA analysis revealed that mast cell stimulators (PMACI, IgE, and compound 48/80) significantly increased the production of TSLP on BMMCs, and compound 48/80 had the most an impact on these results (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Using qRT-PCR, RT-PCR, and flow cytometry, we examined the expression of the TSLP receptor (TSLPR) in B16F10 melanoma cells. As results, the TSLPR was expressed in B16F10 melanoma cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb upper), and it was discovered that TSLP amplified this expression (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb lower).\u003c/p\u003e \u003cp\u003eBecause the mast cell-mediated allergic reaction suppressed the development of melanoma based on the above data, we hypothesized that soluble TSLP released from activated mast cells would impact the survival and proliferation of melanoma. As seen in Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and d, the supernatants of PMACI, IgE, or compound 48/80-stimulated BMMCs greatly decreased the viability and proliferation of B16F10 cells, and this impact was reversed by Anti-TSLP (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Strikingly, similar to the TSLP production result in Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, compound 48/80 had the best suppressive effect on the viability and proliferation of B16F10 cells. In addition, we confirmed that viability and proliferation of B16F10 cells were greatly reduced by TSLP, and these effects were reversed by Anti-TSLP but not by IgG (Extended Data Figs. 5e and f, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTSLP induces apoptosis of B16F10 melanoma cells by induction of autophagy\u003c/h2\u003e \u003cp\u003eWe predicted that TSLP would cause apoptosis in B16F10 cells since it prevents the survival and growth of these cells and induces the autophagy of tumor control mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, the apoptosis rate of B16F10 cells in the blank was about 44.73%, while those of TSLP group was increased to approximately 92.96%, and these effects were reversed by Anti-TSLP but not by IgG. The similar results were obtained for caspase-3, 8, and 9 activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to our earlier research, TSLP activates the JAKs/STAT5 signaling pathway, which led to the induction of autophagy.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Immunoblotting analysis revealed that TSLP considerably increased the expression levels of pJAK1, pJAK2, and pSTAT5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Next, to investigate whether TSLP is involved in autophagosome formation in melanoma, the expression of proteins related to autophagosome formation was analyzed in B16F10 cells. The expression levels of class III PI3K, beclin-1, LC3-II, Atg5, p62, LAMP1, and LAMP2 were dramatically increased in the TSLP-stimulated B16F10 cells when compared to the unstimulated group (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and d). However, Anti-TSLP reduced this increase but not IgG (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and d). To further delineate the relevance of TSLP and autophagy, B16F10 cells were treated with TSLP and 3-methyladenine (3-MA, autophagy inhibitor). 3-MA prevented the TSLP-induced autophagy by limiting the formation of autophagosomes, which dramatically enhanced the viability and growth of B16F10 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee and f, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, activities of caspase-3, 8, and 9 were significantly decreased by 3-MA in the TSLP-treated B16F10 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAn allergic reaction is an overreaction of your immune system.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Studies in the past have revealed a negative correlation between cancer and allergy illnesses.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e On the other hand, a number of recent studies reported that allergy was linked to a lower risk of cancers.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e However, the correlation between melanoma and allergy has been questionable up until recently. Mast cells, the causative cells of allergy, are actually multipurpose cells that are essential for both innate and adaptive immune responses.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e It is still unclear what exactly mast cells do in cancers. In an animal model of late-stage melanoma, mast cell-derived histamine exacerbates melanoma growth.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Contrarily, we recently revealed that surfactin has an anti-cancer effect by triggering mast cell-mediated allergic reactions in early-stage of melanoma skin cancer.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Massari et al. reported that histamine increases radiosensitivity of melanoma cells, which benefits cancer radiotherapy in the treatment of melanoma.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e IgE interactions with mast cells have strong anti-tumor effects, while IgE immunodeficiency is associated with a markedly increased risk of developing cancer.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e In human patients with melanoma, low numbers of mast cells are linked to decreased survival and advanced tumor stages of melanoma.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Activated mast cells produce TSLP, and an increase in TSLP mediates the Th2 immune response by boosting the production of Th2 cytokines.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In the present work, we demonstrated that compound 48/80 induced the autophagy and apoptosis of melanoma by triggering allergic reactions, which improved the survival rate of tumor control mice. Further experiments revealed that TSLP deficiency reduced the levels of histamine, IgE, and Th2 cytokines as well as Th1 cytokines, partially reversing the effects of compound 48/80. Building on these findings, we suggest that mast cell-mediated allergic reactions increased the levels of TSLP, which suppressed the melanoma development in its early stage. Additionally, we suggest that the low incidence of cancer in patients with allergic disorders results from the autophagy of cancer cells brought on by allergic reactions.\u003c/p\u003e \u003cp\u003eTSLP is crucial for preserving immunological homeostasis.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Although substantial research has been done on the function of TSLP in Th2 immune responses, more recent investigations have discovered that TSLP also has a growing role in allergic reactions and cancer.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e The development of hematological tumors as well as solid tumors like breast, colon, and pancreatic cancers have been linked to TSLP, while TSLP signaling provides protection against skin cancers.\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e On the other hand, Yao et al. reported that TSLP promotes the metastasis and growth of melanoma by stimulating CD4 T cells and dendritic cells.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Like these, so far, the role of TSLP in the context of tumor immune responses has typically been described as both suppressive and stimulatory.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e The reason for the different functions of TSLP in cancer models is probably because the immune response to TSLP in cancer appears differently depending on the experimental conditions (mouse type, cancer stage, Notch loss of function, increased Wnt function, H-Ras dependent tumor, and TSLP levels). In the present study, we demonstrated that TSLP promoted the survival by enhancing immunity through the increase of allergy-promoting factors in C57BL/6 and BALB/c immunodeficient melanoma mice. Therefore, these findings suggest that TSLP suppressed the development of melanoma through triggering allergic reactions.\u003c/p\u003e \u003cp\u003eIn the present study, the levels of Th1/Th2 cytokines were significantly increased in response to TSLP. Th1 cytokines are linked to a favorable prognosis for illnesses, but Th2 cytokines are linked to inflammatory responses.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e An imbalance of Th1/Th2 cytokines is a cause of cancer development. In the present study, TSLP also reduced the level of CRP. This is presumed to be because increased Th1 cytokines regulate the amount of Th2 cytokines, preventing them from increasing above na\u0026iuml;ve levels. Therefore, these findings suggest that TSLP suppressed the inflammatory response by preserving the balance of Th1/Th2 cytokines, which in turn slows the development of melanoma.\u003c/p\u003e \u003cp\u003eAs autophagy promotes in the death of tumor cells and apoptosis reduces their survival, both processes are beneficial for suppressing tumor growth.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Many tumor cells display a reduced autophagic capacity when compared with normal cells, and the autophagy-related protein beclin-1 is shown to be significantly reduced in a number of human cancers.\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e These results support the notion that increasing the expression of autophagic genes can help treat various cancers, in addition to the fact that cancer cells have reduced basal autophagic activity levels. In the present study, mast cell-derived TSLP induced the apoptosis of B16F10 cells. Moreover, TSLP/TSLPR signaling pathway induced the autophagy through increasing the expression of autophagy-related proteins. Therefore, these results suggest that TSLP/TSLPR signaling pathway directly contributes to the apoptosis of B16F10 cells through the induction of autophagy.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings revealed that mast cell-derived TSLP directly/indirectly suppressed the development of melanoma by triggering allergic reactions without affecting the side effects. These findings imply that TSLP-induced allergic reactions may be a novel therapeutic approach for the prevention and treatment of melanoma. Furthermore, these findings can also be employed as cancer vaccines to prevent the development of melanoma because multiple studies on melanoma-related genes have been performed and research on food allergy-based cancer vaccines is progressing significantly. However, future preclinical and clinical studies are urgently needed for the therapeutic application of melanoma according to the induction of allergic responses.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eElmas \u0026Ouml;F, Demirbaş A, D\u0026uuml;zayak S, Atasoy M, T\u0026uuml;rsen \u0026Uuml;, Lotti T (2020) Melanoma and COVID-19: A narrative review focused on treatment. Dermatol Ther 33:e14101\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HY, Jung H, Kim HM, Jeong HJ (2021) Surfactin exerts an anti-cancer effect through inducing allergic reactions in melanoma skin cancer. Int Immunopharmacol 99:107934\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerrill RM, Isakson RT, Beck RE (2007) The association between allergies and cancer: what is currently known? 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Autophagy 4:1067\u0026ndash;1068\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3070760/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3070760/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn allergic reaction is a hypersensitive reaction that suppresses cancer development and metastasis. Patients with allergic disorders have lower cancer prevalence. In the present study, we aimed to investigate into the relationship between the development of melanoma and the mast cell-mediated allergic response. Here we revealed that mast cell-mediated allergic reaction caused autophagy and apoptosis of melanoma via raising thymic stromal lymphopoietin (TSLP) levels, resulting to improved survival of tumor control mice. Single-cell RNA sequencing of mouse biopsy samples revealed that exogenous TSLP raised levels of mast cell-derived allergy-promoting factors. Moreover, TSLP suppressed melanoma development by enhancing allergic reactions in immunodeficient mice. Patients with melanoma had lower serum levels of TSLP than healthy individuals. Furthermore, \u003cem\u003ein\u003c/em\u003e \u003cem\u003evitro\u003c/em\u003e stimulation of melanocytes with TSLP prompted apoptosis of melanoma by inducing the autophagy. Therefore, our findings suggest that TSLP directly/indirectly suppressed the development of melanoma through triggering allergic responses.\u003c/p\u003e","manuscriptTitle":"Mast cell-derived TSLP triggers an allergic response to induce autophagy, thereby inhibiting the development of melanoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 09:22:11","doi":"10.21203/rs.3.rs-3070760/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6cd701d0-9744-4bcd-b699-7d51f5aa0e91","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46070865,"name":"Health sciences/Oncology/Cancer/Skin cancer/Melanoma"},{"id":46070866,"name":"Biological sciences/Immunology/Immunotherapy/Immunization"}],"tags":[],"updatedAt":"2025-03-26T09:22:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-26 09:22:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3070760","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3070760","identity":"rs-3070760","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}