RANKL/RANK signaling recruits Tregs via CCL20/CCR6 pathway and promotes stemness and metastasis in colorectal cancers

RANKL/RANK signaling recruits Tregs via CCL20/CCR6 pathway and promotes stemness and metastasis in colorectal cancers | 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 RANKL/RANK signaling recruits Tregs via CCL20/CCR6 pathway and promotes stemness and metastasis in colorectal cancers Chengming Zhu, Jing Ouyang, Shuang Hu, Qingqing Zhu, Tingting Kang, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3869046/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jun, 2024 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract TNF receptor superfamily member 11a (TNFRSF11a, RANK) and its ligand TNF superfamily member 11 (TNFRSF11, RANKL) are overexpressed in a number of malignancies. The clinical importance of RANKL/RANK in colorectal cancer (CRC) is, however, mainly unknown. We examined CRC patient samples and found that RANKL/RANK was elevated in CRC tissues as compared to nearby normal tissues. A higher RANKL/RANK expression was related with a worse survival rate. Furthermore, we found that RANKL is mostly produced by regulatory T cells (Tregs), which can promote CRC advancement. Overexpression of RANK or addition of RANKL significantly increased the stemness and migration of CRC cells. Furthermore, RANKL/RANK signaling stimulates C-C motif chemokine ligand 20 (CCL20) production by CRC cells, which leads to Treg recruitment, boosting tumor stemness and malignant progression. This recruitment process was accomplished by using CCL20-CCR6 interaction, demonstrating a connection between CRC cells and immune cells. These findings suggest that RANKL/RANK plays an important role in CRC progression and could be a potential target for CRC prevention and therapy. Health sciences/Biomarkers/Prognostic markers Biological sciences/Immunology/Cytokines/Chemokines RANKL RANK Colorectal cancer Stemness CCL20 Tregs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background Colorectal cancer (CRC) is presently recognized as one of the most prevalent forms of cancer on a global scale [ 1 ] . Despite the considerable advancements in treatment techniques, the responses of patients with CRC continue to be suboptimal. The prognosis for individuals diagnosed with metastatic CRC (mCRC) continues to be unfavorable [ 2 ] and largely resistant to treatments. Hence, it is imperative from a clinical standpoint to investigate the underlying process and identify novel targets for prevention. The tumor microenvironment (TME) in general interacts with cancer cells to decide the final fate of tumor development and migration [ 3 ] . The role of regulatory T cells (Tregs) in CRC progression is still debatable [ 4 , 5 ] . Currently, researchers are primarily interested in the role of Tregs in promoting or suppressing cancer by controlling immune response; however, the direct interaction between Tregs and CRC cells has received less attention [ 6 , 7 ] . Emerging evidence suggests that cancer stem cell (CSC) dysregulation plays an essential role in CRC growth and metastasis [ 8 ] . CSCs are a type of tumor cell with stem cell features that can self-renew and differentiate, leading to tumor development, therapy resistance, metastasis, and recurrence [ 9 ] . CSCs are typically formed as a result of numerous genetic alterations. Many unique genetic changes have been discovered as activating proto-oncogenes or inactivating tumor suppressor genes. Oncogenes and tumor suppressor genes are frequently assigned separate functions in tumor progression. However, the link between these genes and tumor stemness and advancement is still unknown. TNF superfamily members all have pro-inflammatory action, which is mediated in part by the transcription factor NF-κB [ 10 ] . Tumor necrosis factor (TNF) receptor superfamily member 11a (RANK) and its ligand TNF superfamily member 11 (TNFRSF11, RANKL) have also been linked to cancer: RANKL/RANK have been shown to cause the migration of human epithelial cancer cells and melanoma cells [ 11 ] , and have also been examined in mammary epithelial cells and prostate epithelial cells [ 12 ] . Previous research has shown that RANKL/RANK can promote CRC metastasis [ 13 ] . However, the specific mechanism by which RANKL/RANK maintains CRC stemness and promotes metastasis remains unknown. RANKL/RANK signaling promotes tumor growth by influencing multiple downstream pathways, including tumor metabolism, treatment resistance, and tumor immunity. RANKL has been linked to an increase in tumor-infiltrating lymphocytes and cancer metastasis [ 14 ] . RANKL/RANK is also important for the formation of Tregs, according to research [ 15 ] . RANKL can be produced by CD4 + CD25 + T cells [ 16 ] , and the majority of T-cells that produce RANKL [ 17 ] also express the forkhead box P3 (FOXP3), a transcription factor produced by Tregs. Furthermore, soluble RANKL released into the TME can recruit Tregs via RANKL/RANK signaling [ 18 ] . Meanwhile, in breast tumors, lack of RANK signaling promotes lymphocyte and CD8 + T cell infiltration while decreasing macrophage and neutrophil infiltration [ 19 ] . However, the specific interaction network involving RANKL/RANK, immune cells, and tumor cells has yet to be discovered. In this study, we discovered that RANKL was mostly released by Tregs and that RANKL/RANK signaling might increase the malignant development of CRC by boosting tumor stemness in this investigation. Further analysis indicated that this process is accomplished via activating the NF-κB pathway's phosphorylation of P65. At the same time, we discovered that RANKL/RANK upregulated C-C motif chemokine ligand 20 (CCL20) production via the NF-κB pathway and recruited Tregs via the CCL20-CCR6 axis, producing a "vicious cycle" in the TME. As a result, RANKL/RANK suppression is being evaluated as a potential new target for the therapy of CRC metastases. 2. Materials and methods 2.1. Clinical samples Sun Yat-Sen University's Seventh Affiliated Hospital and First Affiliated Hospital provided clinical samples. Table S1 contains patient information. The stages were carried out in accordance with UICC-TNM grading, and all samples were pathologically examined. All patients provided written informed permission in accordance with the Hospital's Institutional Review Board guidelines (ethical approval number: Science-2010-LW-1213). 2.2. Antibodies and reagents Primary antibodies used in this study included: anti-RANK (ab13918), anti-FOXP3 (ab20034), anti-CCL20 (ab106009), anti- RANKL (ab9957), and anti-CD4 (ab133616) from Abcam, UK; anti-P65 (#4764), anti-p-P65 (Ser536; #3033), anti-FLAG (#14793) and anti-IgG isotype control (#3900S) from Cell Signaling Technology, CST, USA ; anti-GAPDH (60004-1-lg), anti-CD44 (15675-1-AP), anti-CD133(66666-1-Ig), and anti-CCR6 (66801-1-Ig) from Proteintech, Wuhan, China; T-bet (SC21749), GATA-3 (SC269) and RORγ (SC365476) from Santa Cruz, USA; and anti-CD56 (GB112671) from Servicebio, Wuhan, China. The reagents included: QNZ from MCE; RANKL, EGF, and bFGF from PeproTech. 2.3. Cell lines and cell culture All cells were obtained from the American Type Culture Collection, were authenticated, and were mycoplasma-free. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. RANKL at 100 ng/ml and QNZ at 50nm were introduced. T Cell Expansion Medium (STEMCELL) was used to culture human PBMCs according to the manufacturer's recommendations. 2.4. Establishment of stable cell lines and transient transfection Plasmids were introduced into cells using Liposomal Transfection Reagent (YiSheng, Shanghai, China) according to the directions for transient transfection; for stable transfection, 293T cells were transfected with plasmids and polyethyleneimine (Polysciences, USA). Polybrene (Sigma, USA) was utilized to infect cells with virus particles. Puromycin (Sigma) was used to test plasmids for puro-resistance. Transheep (Shanghai, China) supplied all of the plasmids. Supplementary Tables S2 and S3 provide siRNA and shRNA sequences. 2.5. Real time quantitative PCR (QRT-PCR) RNA was extracted with AG RNAex Pro Reagent (Accurate Biotechnology, AG, Beijing, China). QRT-PCR was performed using 5X Evo M-MLV RT Master Mix and SYBR® Green Premix (AG). GAPDH was used as an internal reference, and specific primer sequences were shown in Table S4 . 2.6. CHIP assay The CHIP assay was performed using a CHIP assay Kit (Beyotime, Shanghai, China). Before adding cell lysates, magnetic beads were combined with anti-P65 and anti-rabbit IgG. Then DNAs were purified. QRT-PCR was used for further analyses. 2.7. ELISA The content of CCL20 in the supernatants was determined using an ELISA Kit (4 A Biotech, Beijing, China). 2.8. Western blotting (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF) RIPA Lysis Buffer (PC101, Yamei, Shanghai, China) was used for protein extraction. SDS-PAGE was used to separate protein samples, which were then transferred to a Nitrocellulose membrane (Merck Millipore, Germany). The membrane was treated with the primary antibody at 4°C overnight. Secondary antibodies were IRDye 800CW Goat anti-IgG (LI-COR, USA), which were then seen using the ChemiDocTM MP Imaging System (BIO-RAD, USA) [ 13 ] . Cells were fixed with 4% paraformaldehyde and stained with primary antibody on a sliding plate for immunofluorescence staining. A fluorescent secondary antibody was added and incubated at room temperature. Finally, the nucleus was labeled with DAPI. Tissue immunofluorescence staining was performed using the TSA PLus Kit (Servicebio). A secondary antibody was created using a DAB staining solution Kit (Gene Tech, Shanghai, China). The slices were treated overnight at 4°C with primary antibodies for immunohistochemistry [ 13 ] . A second antibody was incubated on the second day. For staining, a DAB staining solution Kit was utilized. A Leica DM4B microscope was used to examine the sections. This paper [ 21 ] was used to calculate the IHC scores. 2.8. Flow cytometry and cell sorting PBMCs were isolated from blood samples using Ficoll (TBD)-gradient centrifugation. Tumor Dissociation Kit (Miltenyi Biotec, Germany) was used for extracting lymph node cells. EasySep™ Human T Cell Enrichment Kit (STEMCELL, Cananda) was used to separate CD3 + T cells from PBMCs. Human PBMC and lymph node cells were stained for surface analysis with CD4-AF700, CD25-PE/Cy7, CCR6/PE, and RANKL-APC, and Treg intracellular analysis with FOXP3-BV421 (Biolegend, USA). Before staining FOXP3, the True-Nuclear™ Transcription Factor Buffer Set (Biolegend) was used. 2.9. Migration and Chemotaxis Assay 1 × 10 5 cells were inoculated into the upper chamber (8 µm, Corning, USA). The medium was supplemented with 10% FBS as a chemotactic agent in the inferior cavity. After 48 h, the lower chamber of membrane cells was stained. The stained cells were randomly imaged using a microscope in 5 different fields. Induced T cells were migrated and placed in the upper chamber of 24-well Transwell plates with a 3.0 µm polycarbonate membrane (SPL, Korea) (1 × 10 6 cells /well). DLD1 WT/RK cells were grown in ImmunoCult TM -XF T Cell Expansion Medium (STEMCELL), and the supernatant was collected and deposited in the bottom chamber with or without anti-CCL20. Migration was permitted to continue for 6 hours [ 22 ] . Flow cytometry was used to calculate the fraction of CD4 + FOXP3 + cells in the bottom chamber. 2.10. Tumor sphere formation assays Cells were seeded into 24-well ultralow attachment plates concentration of 5000 cells per well and re-suspended in the stem cell-conditioned medium containing DMEM-F12 (Bio-channel, Nanjing, China), 2% B-27 Supplement (PeproTech), 10 ng/ml basic fibroblast growth factor (bFGF, PeproTech), 20 ng/ml epidermal growth factor (EGF, PeproTech), and 5 µg/ml insulin (Beyotime), at 37°C with 5% CO2 and saturated humidity for 12–14 days. When spheroid diameters reached 50 µm, culture suspensions were passed every 7 days. The photographs were taken, and the number of cell spheres was tallied. 2.11. Subcutaneous xenograft implantation models and in vivo limiting dilution assay All animal research were carried out with the agreement of Sun Yat-sen University's Animal Experiment Ethics Committee. Guangdong Yaokang Biotechnology Co., LTD provided female NOD-Scid mice (4–6 weeks). For Subcutaneous xenograft assays, DLD1 cells or DLD1 RK cells (2 × 10 7 cells per mouse) were subcutaneously injected into NOD-Scid mice (n = 5). After 3 weeks, mice were sacrificed and xenograft tumors were harvested for histological study. The tumor volume was calculated according to the formula: Volume (mm 3 ) = width 2 (mm 2 ) × length (mm) × 0.4 [ 20 ] . For limiting dilution assays, DLD1 cells or DLD1 RK cells were implanted at the gradient of 1 × 10 6 , 1 × 10 7 and 2 × 10 7 cells per NOD-Scid mouse (n = 5 per group). The incidence of tumors in the two groups was analyzed. The frequency of tumor stem cells was calculated by a method described on the ELDA website (Extreme Limiting Dilution Analysis, http://bioinf.wehi.edu.au/software/elda/ ). 2.12. Statistical analysis SPSS and GraphPad Prism were used for statistical analysis. ImageJ was used to calculate the GAPDH indicator protein levels. CytExpert and Flowjo were used to evaluate flow cytometry data. The data were compared using the one-way ANOVA test. The χ 2 test was performed to investigate the connection between various biomarkers in CRC tissue slices. The survival curve was created using the Kaplan-Meier method and the log-rank test. P value < 0.05 was considered statistically significant. 2.13. Public clinical datasets Correlation analysis between gene expression in tissues of CRC patients was computed from GEPIA (Gene Expression Profiling Interactive Analysis, http://gepia.cancer-pku.cn/ ). CHIP-seq datasets were collected from the Cistrome Data Browser( http://cistrome.org/db/#/ ). Single-cell data are derived from published article [ 23 ] . 3. Results 3.1 RANKL/RANK expression is linked to the advancement of CRC malignancy. We previously found that RANK expression was elevated in CRC patient samples and was linked to a poor prognosis [ 13 ] . We concentrated on RANKL in this investigation. According to the TCGA dataset, RANKL was shown to be substantially elevated in 275 CRC tissues compared to 349 normal tissues (Fig. 1 a). Furthermore, as demonstrated in Table S1 , RANKL expression was linked to CRC clinical histology. Patients with increased RANKL protein expression had shorter overall survival (OS) and recurrence-free survival (RFS) among CRC patients having both OS and RFS data (Fig. 1 b). Immunohistochemical assays were utilized to compare RANKL protein expression in formalin-embedded tumor tissues from different TNM stages (Fig. 1 c). The findings revealed that RANKL in CRC tumor tissues was significantly ( P < 0.0001) linked with TNM staging (Fig. 1 d). WB demonstrated that RANK levels in CRC tumor tissues were higher than in neighboring normal tissues (Fig. 1 e), which was consistent with our previous findings. These findings indicated that RANKL/RANK was highly elevated in CRC patients. 3.2 RANKL is mainly generated by CD4 + CD25 + regulatory T cells in CRC We then attempted to locate the source of RANKL in CRC. Single cell sequencing data (Fig. S1 a) showed that RANKL was primarily produced from T cells in breast cancer, specifically CD4 + T cells [ 23 ] , which was similar with the findings of Wei Tan et al [ 16 ] . We used immunohistochemistry and immunofluorescent labeling (NK cells: CD56) of CRC samples to determine which subtype of T cells RANKL is primarily originated from. We discovered that RANKL expression was substantially overlapping with CD4 expression (Fig. 2 a), implying that RANKL is produced by CD4 + T cells. RANKL and FOXP3 were discovered as well to be co-localized in CRC tissues (Fig. 2 b). Flow cytometric analysis of the peripheral blood and lymph node of CRC patients revealed that membrane-RANKL (m-RANKL) was primarily expressed in CD4 + CD25 + T cells compared to CD4 + CD25 − T cells (Fig. 2 c). We concluded that Tregs expressed RANKL. 3.3 RANKL/RANK signaling enhances CRC cell stemness. To investigate whether RANKL/RANK may influence CRC stemness and metastasis, we evaluated TCGA CRC data and discovered that RANK/RANKL expression was positively linked with stemness-related genes (Fig. S2 a). We also measured the RANK mRNA levels in other CRC cell lines (Fig. S2 b) and chose HCoEpiC (normal intestinal epithelial cell line), DLD1 and Caco2 to create overexpressed cells, and LS174T to create knockdown cells. WB verified RANK overexpression cells (HCoEpiC RK, DLD1 RK, Caco2 RK) and RANK knockdown cells (LS174T sh-RK) (Fig. S2 c). Overexpression of RANK or addition of RANKL increased tumor sphere formation, whereas knocking down RANK could limit it (Fig. 3 a, S2 d). Our earlier study indicated that RANKL/RANK might accelerate CRC metastasis [ 13 ] . In this investigation, we discovered that overexpression of RANK greatly boosted cell migration while knocking down RANK inhibited migration (Fig. 3 b, S2 e). Furthermore, the inclusion of RANKL increased tumor sphere formation and CRC cell metastasis. Mechanistically, all CSCs markers were up-regulated in RANK-overexpressed cells (Fig. S3 a), with CD44 and PROMI (CD133) being the most significant. The WB data verified this conclusion (Fig. 3 c, S3 b). Clinically, patients with elevated RANKL/RANK expression similarly had increased CD44 expression in CRC (Fig. 3 d). The size and weight of the subcutaneous tumors generated by DLD1 RK cells were much larger than the control cells in in vivo tests (Fig. S2 f). To acquire a better understanding of the role of RANK in CSC stemness, we performed an in vivo limited dilution tumor transplantation investigation (Fig. 3 e). Tumor-initiating cells were found in one out of every 5.248 × 10 6 DLD1 RK cells and one out of every 4.174 × 10 7 DLD1 cells, as illustrated in Fig. 3 f. Notably, the frequency of tumor-initiating cells in DLD1 RK increased when compared to the control. Table S5 shows the detailed numbers of cell planted and tumors formed. IHC labeling with CD44 and CD133 revealed that subcutaneous tumors generated by DLD1 RK cells had a higher stemness index than control cells (Fig. 3 g). In conclusion, we found a substantial association between RANKL/RANK expression and tumor stemness. 3.4 RANKL/RANK increases the secretion of CCL20 by CRC cells. To identify critical immune-related factors for poor prognosis in CRC patients with high-RANK expression, we examined levels of chemokines. Most chemokine mRNA levels we identified were elevated in overexpressed RANK cells in vitro (Fig. 4 a), with CCL20 being the most significant, which was similar with the findings of Y. Liu et al [ 24 ] . TCGA CRC data showed that RANK/RANKL expression was strongly linked with CCL20 (Fig. S4 a). Furthermore, we discovered that CCL20 mRNA levels in CRC cell lines were higher than in normal cell lines (HCoEpiC) (Fig. S4 b). We performed immunofluorescence staining and ELISA experiments to confirm our findings and discovered that CCL20 expression was up-regulated in overexpressed RANK cells (Fig. 4 b, S4 c). Furthermore, the addition of RANKL increased the levels of CCL20 protein and mRNA (Fig. 4 c, 4 d). Clinically, CCL20 mRNA expression levels in tumor tissue were higher than in normal tissue in the majority of CRC patients (Fig. 4 e). IHC labeling with CCL20 in vivo indicated a higher expression in subcutaneous tumors generated by DLD1 RK cells than in control cells (Fig. 4 f). By combining these findings, we concluded that RANKL/RANK signaling enhanced CCL20 production in CRC cells. 3.5 Overexpression of RANK in CRC cells promotes recruitment of Tregs via the CCL20-CCR6 interaction. Numerous studies have demonstrated that the CCL20-CCR6 pathway can recruit Tregs [ 25 , 26 ] . We discovered that tumor tissues with high expression of RANK or CCL20 associated strongly with enhanced FOXP3 expression (Fig. 5 a). To demonstrate that RANK can enhance CCL20 release and thereby attract Tregs, we performed an in vitro experiment in which DLD1 WT/ RK cells were co-cultured with PBMC from CRC patients. DLD1 RK cell supernatants attracted substantially more Tregs (Fig. S5 a). To confirm the critical role of CCL20, we added anti-CCL20, which inhibited the increased recruitment of Tregs by overexpressed RANK DLD1 cells (Figs. 5 b and S5 ). To confirm that RANKL/RANK recruits Tregs via the CCL20-CCR6 pathway, we discovered that CCR6 and FOXP3 were co-localized in CRC blood (Fig. 5 c) and tumor tissue (Fig. 5 d). 3.6 RANKL/RANK increases stemness and CCL20 production by CRC cells via NF-κB signaling. Since RANK is a known activator of the NF-κB receptor, we first determined whether RANKL/RANK promoting stemness and CCL20 secretion was due to the activation of the NF-κB pathway. By WB and IF, we found that both overexpression of RANK and addition of RANKL promoted the nuclear phosphorylation of P65 (Fig. 6 a, b). We investigated the role of the NF-κB pathway in the process of RANK promoting stemness. NF-κB pathway inhibitor (QNZ) significantly inhibited the phosphorylation of P65 (Fig. S6 a), the expression of CSCs marker (Fig. S6 b), the ability of tumor sphere formation (Fig. S6 c) and metastasis (Fig. S6 d)as demonstrated by WB. To further corroborate the crucial role of P65, we discovered that CD44 was also decreased after P65 was knocked down in CRC cells by WB (Fig. 6 c) and QRT-PCR (Fig. S6 e). In addition, tumor sphere-formation and migration ability also were weakened (Fig. 6 d, e). In contrast, when we overexpressed P65 in LS174T sh-RK cells, we discovered that down-regulation of CD44 could be rescued (Fig. 6 f), along with the ability to form tumor spheres and migrate (Figs. 6 g, h). IHC staining with p-P65 confirmed a higher expression in subcutaneous tumors formed by DLD1 RK cells compared to control cells in in vivo experiments (Fig. S6 f). The suppression of P65 significantly inhibited CCL20 expression (Fig. 7 a-c). To confirm the relationship between P65 and CCL20 in CRC cells, we analyzed the Cistrome DB database and found P65 binding sites in the promoter of the CCL20 gene (Fig. 7 d). After ChIP, three primer pairs corresponding to the predicted binding sites were used for QRT-PCR (Fig. 7 e). The findings revealed that P65 interacted with the P2 (-153 to 21 bp) region of the CCL20 promoter. Collectively, our results indicated that P65 is a key target for RANK to promote CCL20 secretion in CRC. 4. Discussion RANKL/RANK has been documented to correlate closely with tumor metastasis of prostate, breast, liver cancer and melanoma [ 11 , 14 , 27 – 29 ] . Our previous research [ 13 ] has demonstrated that RANK is associated with the progression of cancer in CRC. However, the RANKL/RANK signaling mechanism in CRC remains unknown. In this study, we discovered that the RANKL/RANK pathway promotes the secretion of chemokines that CRC cells can use to recruit Tregs, thereby influencing CRC stemness, metastasis, and tumorigenesis. Although RANKL is best known for its distribution in bone [ 30 ] , it has been reported on other cell types, such as immune cells, with aberrant expression in certain tumor types [ 31 ] . Uncertain is the role of RANKL/RANK in tumor immunity. RANKL is derived from CD4 + FOXP3 + T cells in breast cancer and arthritis, according to multiple reports [ 16 , 17 ] . In this study, we demonstrate (Fig. 2 c) that RANKL is derived from CD4 + CD25 + T cells in CRC. This is the first time this result has been reported for colorectal cancer, and it is consistent with prior findings for other cancers [ 16 ] . This evidence suggests that RANKL/RANK and the TME in CRC are closely related. Previous research has linked RANKL/RANK to the metastasis of CRC [ 13 ] and other cancers [ 16 , 28 ] , and it is believed that metastasis is associated with CSCs [ 32 ] . Our research demonstrates that RANK can enhance metastasis by promoting the stemness of CRC cells (Fig. 3 a, 3 e), consistent with findings in breast cancer [ 27 , 28 ] . The screening of CSCs markers (Fig. S1 f) reveals that RANK can upregulate stemness markers. In addition, we observe a substantial increase in CD44 levels in CRC cells following RANK overexpression (Fig. 3 c). These findings suggest that the RANKL/RANK pathway influences the proliferation, metastasis, and tumorigenesis of CSCs by elevating CD44 levels. The RANKL/RANK pathway can influence TME composition. The RANKL/RANK pathway has been linked to the release of chemokines that recruit T lymphocytes in breast cancer and endometrial cancer [ 16 , 24 ] . We discovered that RANKL/RANK can enhance CCL20 production by CRC cells (Fig. 4 c), which is consistent with earlier research [ 24 ] , demonstrating that RANKL/RANK is important in the immunological microenvironment of CRC. Furthermore, CCL20 can influence the TME via immune cells such as B cells, T cells, and dendritic cells, influencing the progression of CRC [ 33 ] . Furthermore, Wang D et al. demonstrate that CCL20 generated by CRC cells can recruit Tregs to improve chemoresistance [ 34 ] . Furthermore, CCR6 expressed on the surface of Tregs as a CCL20 receptor has been investigated in tumor immunity [ 25 , 26 , 35 ] . In this study, we discovered that RANKL/RANK can enhance Tregs recruitment via the CCL20-CCR6 pathway, hence accelerating the malignant evolution of CRC. RANK, or receptor activator of nuclear factor kappa-B, activates the NF-κB pathway [ 36 ] . The NF-κB pathway is involved in cancer, epithelial-mesenchymal transition, metastasis, chemoresistance, and stemness [ 34 , 36 – 38 ] . Nonetheless, knowledge of the NF-κB pathway's significance in CRC is limited. Indeed, some studies have found that CRC cells have an active NF-κB pathway; in general, CRC growth is dependent on NF-B signaling [ 34 ] . RANK has been found in studies to activate the NF-κB pathway, promoting the malignant progression of breast cancer [ 36 ] . RANK can upregulate CCL20 (Fig. 7 a-c) and increase CRC stemness (Fig. 3 c, 6 b) via the NF-κB pathway, according to our findings. Using the CHIP-QPCR experiment, we also revealed that P65 can bind to the promoter of CCL20 (Fig. 7 d, e). All of this suggests that the NF-κB pathway is critical in the recruitment of immune cells and the advancement of CRC malignancy. In summary, we associate Tregs to CRC stemness via RANKL/RANK signaling and discover a new mechanism by which Tregs enhance CRC metastasis: Tregs trigger the RANK signal of CRC cells by secreting RANKL, promoting CRC metastasis by increasing stemness. Activation of the RANKL/RANK signaling pathway can have a positive feedback effect by recruiting more Tregs via the CCL20-CCR6 pathway, hence controlling CRC metastasis (Fig. 7 f). The RANKL/RANK pathway and the TME are known to interact throughout development and cancer, but the underlying mechanism is unknown. Finally, given the role of RANKL/RANK signaling in tumorigenesis, anti-cancer drug clinical trials in the appropriate molecular targets (such as CCL20 / CCR6) and RANKL/RANK inhibitors used in combination, or licensing effectively inhibit colorectal cancer metastasis, improve treatment effectiveness. Our findings contribute to a better understanding of the RANKL-RANK signaling pathway as it relates to the immunological microenvironment, stemness, and metastasis of CRC. Declarations Competing interests The authors declare that they have no competing interests. Authors’ contributions J.O.: Methodology, Investigation, and Writing – Original Draft. S.H., Q.Z., T.K., Y.W., Y.L., C.L., Y.Y and Y.S.: Formal analysis and Investigation. Y.L., W.X. and J.Q.: Resources. S.H., C.Z.: Writing - Original Draft, and Writing - Review & Editing. Q.L., C.Z.: Conceptualization, Supervision. T.G., L.Y. and Y.P.: Supervision. Acknowledgements This work was supported by the Research Start-up Fund of the Seventh Affiliated Hospital, Sun Yat-sen University [grant number ZSQYBRJH0003]; and the National Natural Science Foundation Youth Project of China [grant number 82203807]. Shenzhen Science and Technology Project, sustainable development project [grant number KCXFZ202002011010593]. The sponsors had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Availability of data and material The authors declare that all the data supporting the findings in this study are available in this study and are available from the corresponding author through reasonable request. References Quintana JM, Gonzalez N, Anton-Ladislao A, Redondo M, Bare M, Fernandez de Larrea N, et al. Colorectal cancer health services research study protocol: the CCR-CARESS observational prospective cohort project. BMC Cancer 2016, 16: 435. Merhi M, Ahmad F, Taib N, Inchakalody V, Uddin S, Shablak A, et al. The complex network of transcription factors, immune checkpoint inhibitors and stemness features in colorectal cancer: A recent update. Semin Cancer Biol 2023, 89. Tian S, Chu Y, Hu J, Ding X, Liu Z, Fu D, et al. 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Chang Y-W, Chiu C-F, Lee K-Y, Hong C-C, Wang Y-Y, Cheng C-C, et al. CARMA3 Represses Metastasis Suppressor NME2 to Promote Lung Cancer Stemness and Metastasis. Am J Respir Crit Care Med 2015, 192(1): 64–75. Lin S-C, Liao Y-C, Chen P-M, Yang Y-Y, Wang Y-H, Tung S-L, et al. Periostin promotes ovarian cancer metastasis by enhancing M2 macrophages and cancer-associated fibroblasts via integrin-mediated NF-κB and TGF-β2 signaling. J Biomed Sci 2022, 29(1): 109. Additional Declarations (Not answered) Supplementary Files Table.S1.xlsx Table S1 Correlation between RANKL expression and clinicopathologic features of 183 CRC patients. Table.S2.xlsx Table S2. Target sequences of siRNA Table.S3.xlsx Table S3. Target sequences of shRNA Table.S4.xlsx Table S4. Sequences of primers Table.S5.xlsx Table S5: CSCs frequency calculated by ELDA in Limiting dilution assay. figureS1.tif RANKL is mainly derived from CD4+ T cells. (a) The origin of RANKL(TNFSF11) was analyzed through a single cell database. (b) IHC co-stain RANKL with multiple markers of immune cells (CD4, CD8, CD56) in CRC patients. (100×, 200×) (c) Colorectal tumor tissues subjected to double immunofluorescence for CD4 (green), CD56 (white), RANKL (red), and DAPI (blue) (200 ×). Scales bars = 200 μm (100×), 100 μm (200×) figureS2.tif RANK promotes malignant progression of CRC. (a) The mRNA level correlation between RANK/RANKL and CD44/CD133 in CRC tissues was detected by analyzing the dataset GEPIA. (b) Log2 fold changes in related RANK mRNA expression of CRC cell lines compared to the normal cell (HCoEpiC). (c) RANK stable overexpression or knockdown efficiency was confirmed by western blotting in CRC cells. (d) RANK overexpression influenced the sphere formation and (e) the migration of Caco2 cells. And the addition of 100 ng/ml RANKL moderately increased the migration of Caco2 cells. (Sphere formation: 400×, Migration: 200×) (f) The subcutaneous tumors formed by control and DLD1 RK cells were obtained from NOD/Scid mice. Both the volume and weight of subcutaneous tumors were shown in the right panel (mean ± SD, n = 5). Scales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P < 0.05, **P < 0.01, ***P < 0.001. figureS3.tif RANK promotes stemness of CRC. (a) Relative expression of related stemness markers (CD44, CD133, NANOG, LGR5, OCT4, SOX2, NOTCH3) in RANK overexpression or knockdown cells was analyzed by q RT-PCR. (b) Western blotting of CD133 protein expressions in RANK overexpression Caco2 cells and the addition of 100 ng/ml RANKL. Scales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P < 0.05, **P < 0.01, ***P < 0.001. figureS4.tif RANK is associated with CCL20 in CRC. (a) The mRNA level correlation between RANK/RANKL and CCL20 in CRC tissues was detected by analyzing the dataset GEPIA. (b) Log2 fold changes in related CCL20 mRNA expression of CRC cell lines compared to the normal cell (HCoEpiC). (c)ELISA of CCL20 protein expressions in RANK overexpression cells. *P < 0.05, **P < 0.01, ***P < 0.001. figureS5.tif Overexpressed RANK in CRC cells can recruit more CD4 + FOXP3 + T cells. (a) Migration of PBMC from CRC patients co-cultured with the supernatants of DLD1 cells or RANK-overexpressing DLD1 cells was analyzed by transwell assay and flow cytometry, (b) and the addition of anti-CCL20 antibody. (The illustration created with BioRender.com) figureS6.tif NF-κB inhibitors/ siRNA of P65 can inhibit the stemness of CRC. (a) Western blotting of p-P65/P65 protein or (b) CD44 protein expressions in RANK-overexpressing DLD1 cells (DLD1 RK cells) and the addition of 50nm QNZ. (c) RANK overexpression or addition of 50nm QNZ influenced the sphere formation and (d) migration of DLD1 cells. (Sphere formation: 400×, Migration: 200×) (e) Changes of P65 mRNA levels in DLD1 RK cells add P65 siRNA treatment. (f) IHC staining was used to detect the expression of RANK and p-P65 in indicated subcutaneous tumors of NOD/Scid mice. (200×) Scales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P < 0.05, **P < 0.01, ***P < 0.001. 1rawimages.tif 3rawimages.tif 6rawimages.tif S2rawimages.tif S6rawimages.tif Cite Share Download PDF Status: Published Journal Publication published 20 Jun, 2024 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 11 Mar, 2024 Review # 2 received at journal 09 Mar, 2024 Review # 1 received at journal 26 Feb, 2024 Reviewer # 2 agreed at journal 24 Feb, 2024 Reviewer # 1 agreed at journal 18 Feb, 2024 Reviewers invited by journal 16 Feb, 2024 Submission checks completed at journal 16 Jan, 2024 First submitted to journal 16 Jan, 2024 Editor assigned by journal 16 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-3869046","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":273328449,"identity":"568e457a-b5fc-4158-aae7-bec0d6f7ca59","order_by":0,"name":"Chengming 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University","correspondingAuthor":false,"prefix":"","firstName":"Yihang","middleName":"","lastName":"Pan","suffix":""}],"badges":[],"createdAt":"2024-01-16 07:00:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3869046/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3869046/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-024-06806-3","type":"published","date":"2024-06-20T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51393201,"identity":"0b034655-d565-4838-b8b6-1dab223f07dc","added_by":"auto","created_at":"2024-02-20 18:51:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":105598,"visible":true,"origin":"","legend":"\u003cp\u003eRANKL/RANK expression is associated with the malignant progression of CRC.\u003c/p\u003e\n\u003cp\u003e(a) The mRNA levels of RANK and RANKL in CRC and normal colorectal tissues were determined by analyzing the GEPIA dataset.\u003c/p\u003e\n\u003cp\u003e(b) Kaplan–Meier analysis of overall survival (OS) and recurrence-free survival (RFS) in CRC patients.\u003c/p\u003e\n\u003cp\u003e(c) RANKL immunohistochemistry staining in CRC tissues (stage I, stage II, stage III, and stage IV) (100x, 400x).\u003c/p\u003e\n\u003cp\u003e(d) Percentage of RANKL expression in CRC tumors within individual TNM stage (P \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e(e) RANK protein expression was measured using WB in six matched tumor and normal tissue samples. RANK tumor/normal ratios were calculated using ImageJ software. GAPDH was used to standardize expression levels.\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/8c005013d8e1c75dd6dd73c3.png"},{"id":51393645,"identity":"11c60f51-9d78-4b6e-8ad7-8053d8de5086","added_by":"auto","created_at":"2024-02-20 18:59:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":219105,"visible":true,"origin":"","legend":"\u003cp\u003eRANKL is mainly derived from CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e regulatory T cells in CRC.\u003c/p\u003e\n\u003cp\u003e(a) IHC co-stains RANKL with numerous immune cell markers (RORγ, GATA3, T-bet, FOXP3, CD4, RANKL) in CRC patients. (100×, 400×)\u003c/p\u003e\n\u003cp\u003e(b) Colorectal tumor tissues subjected to immunofluorescence for FOXP3 (green), RANKL (red), and DAPI (blue). Two representative micrographs are shown (200×, 400×).\u003c/p\u003e\n\u003cp\u003e(c) The expression of RANKL in CD4\u003csup\u003e+ \u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e cells and CD4\u003csup\u003e+ \u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003e cells were detected by flow cytometry in PBMC and Lymph nodes of CRC patients (The illustration created with BioRender.com).\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×).\u003c/p\u003e","description":"","filename":"Onlinefigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/45041901d52331932c363ad1.png"},{"id":51392617,"identity":"abf7ba56-a04e-4593-8a18-63ee12333bc9","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":311677,"visible":true,"origin":"","legend":"\u003cp\u003eRANKL/RANK signaling promotes the stemness of CRC cells.\u003c/p\u003e\n\u003cp\u003e(a) RANK overexpression or knockdown influenced the sphere formation and (b) migration of HCoEpiC, DLD1, and LS174T cells. And the addition of 100 ng/ml RANKL moderately increased the migration of HCoEpiC, DLD1, and LS174T cells. (Sphere formation: 400×, Migration: 200×)\u003c/p\u003e\n\u003cp\u003e(c) WB of CD44 protein expression in RANK overexpression or knockdown cells, with 100 ng/ml RANKL added.\u003c/p\u003e\n\u003cp\u003e(d) The relationship between the expression of RANKL, RANK, and CD44 in CRC tissues was detected by IHC (200×; P1: low expression, P2: high expression).\u003c/p\u003e\n\u003cp\u003e(e) Tumor-initiating cell frequency was tested by in vivo limiting dilution assay in NOD/Scid mice. n = 5.\u003c/p\u003e\n\u003cp\u003e(f) A graph of the log-fraction for the limiting dilution assay. DLD1 RK is represented by black circles, while control is represented by red circles. The dashed lines represent the confidence interval at 95%.\u003c/p\u003e\n\u003cp\u003e(g) NOD/Scid mice with the specified subcutaneous tumors were stained with IHC to detect the expression of RANK, CD44, and CD133. (200×)\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/5088238d29c585da7372ef8b.png"},{"id":51392616,"identity":"8093b57e-d2ad-4e52-998d-fb5344c14e26","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":176530,"visible":true,"origin":"","legend":"\u003cp\u003eRANKL/RANK promotes the secretion of CCL20 by CRC cells.\u003c/p\u003e\n\u003cp\u003e(a) QRT-PCR was used to analyze the relative expression of related chemokines in RANK-overexpressing cells.\u003c/p\u003e\n\u003cp\u003e(b) CCL20 protein expression via immunofluorescence in RANK overexpression cells. (200×)\u003c/p\u003e\n\u003cp\u003e(c)ELISA and (d) QRT-PCR of CCL20 protein expressions in RANK overexpression cells, and the addition of 100 ng/ml RANKL.\u003c/p\u003e\n\u003cp\u003e(e) CCL20 mRNA expressions in eight paired tumor and normal tissue samples. Expression levels were normalized with GAPDH. T human CRC tissues, N paired normal colorectal tissues.\u003c/p\u003e\n\u003cp\u003e(f) IHC staining was used to detect the expression of RANK and CCL20 in indicated subcutaneous tumors of NOD/Scid mice. (200×)\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/ca9bdd90e146edb2e75e2ad7.png"},{"id":51392620,"identity":"a47fe317-5384-47b8-a483-74afd699fcfe","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":161436,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of RANK in CRC cells promotes recruitment of Tregs via the CCL20-CCR6 interaction.\u003c/p\u003e\n\u003cp\u003e(a) The relationship between the expression of RANKL, RANK, and CD44 in CRC tissues was detected by IHC (200 ×; P1: low expression, P2: high expression).\u003c/p\u003e\n\u003cp\u003e(b) Migration of PBMC from CRC patients co-cultured with the supernatants of DLD1 cells or RANK-overexpressing DLD1 cells was analyzed by flow cytometry, and the addition of anti-CCL20 inhibitor.\u003c/p\u003e\n\u003cp\u003e(c) The proportion of CD4\u003csup\u003e+\u003c/sup\u003e FOXP3\u003csup\u003e+\u003c/sup\u003e cells in CCR6\u003csup\u003e+\u003c/sup\u003e cells were detected by flow cytometry in PBMC of CRC patients. (P1 and P2 are two different samples from CRC patients)\u003c/p\u003e\n\u003cp\u003e(d) Colorectal tumor tissues subjected to three immunofluorescences for FOXP3 (green), CCL20 (red), CCR6 (orange), and DAPI (blue). Two representative micrographs are shown (200×,400×; P1: low expression, P2: high expression).\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×).\u003c/p\u003e","description":"","filename":"Onlinefigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/1667bfb77a350c7f1d5dc60e.png"},{"id":51392618,"identity":"2b2c2382-a991-4b6c-9878-0025ec201e69","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103335,"visible":true,"origin":"","legend":"\u003cp\u003eRANKL-RANK promotes the stemness via NF-κB signaling.\u003c/p\u003e\n\u003cp\u003e(a) Immunofluorescence of p-P65 protein expressions in RANK overexpression or knockdown cells (400×).\u003c/p\u003e\n\u003cp\u003e(b) WB of p-P65/P65 protein expressions in RANK overexpression or knockdown cells and the addition of 100 ng/ml RANKL.\u003c/p\u003e\n\u003cp\u003e(c) Changes of p-P65/P65 protein and CD44 protein levels in DLD1 RK cells add P65 siRNA treatment.\u003c/p\u003e\n\u003cp\u003e(d) RANK overexpression or P65 siRNA treatment influenced the sphere formation and (e) migration of DLD1 cells. (Sphere formation: 400×, Migration: 200×)\u003c/p\u003e\n\u003cp\u003e(f) WB of p-P65/P65 and CD44 protein expressions in RANK knockdown cells and overexpressed P65.\u003c/p\u003e\n\u003cp\u003e(g) RANK knockdown or overexpressed P65 influenced the sphere formation and (h) migration of LS174T cells. (Sphere formation: 400×, Migration: 200×)\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/83f38bee8c4a06e9cedd56cf.png"},{"id":51392632,"identity":"5772c732-2ebe-4322-b553-5cda4b2e6874","added_by":"auto","created_at":"2024-02-20 18:43:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":94721,"visible":true,"origin":"","legend":"\u003cp\u003eRANKL-RANK promotes the secretion of CCL20 by CRC cells via NF-κB signaling.\u003c/p\u003e\n\u003cp\u003e(a) Changes of CCL20 mRNA (QRT-PCR), (b, c) CCL20 protein (ELISA/immunofluorescence) in DLD1 RK cells add P65 siRNA treatment (200×).\u003c/p\u003e\n\u003cp\u003e(d) The schematic structures of P65 putative binding sites in the CCL20 promoter (Created with BioRender.com), and (e) ChIP analysis of P65 binding sites to the CCL20 promoter.\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e(f) The schematic illustration of working hypothesis (Created with BioRender.com). Tregs-derived RANKL activated RANK signaling pathway and upregulated the expression of downstream target genes CCL20, CD44 by activating the NF-κB pathway to recruit Tregs and maintain the CRC cancer stem cell characteristics and promotes metastasis.\u003c/p\u003e","description":"","filename":"Onlinefigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/677014f7cdca468bcc9c8458.png"},{"id":58790010,"identity":"14ecfacf-63f2-4b85-b2aa-56a988eaabae","added_by":"auto","created_at":"2024-06-21 07:08:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2281474,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/0de8d5bb-3037-4e7a-b62b-b6f1128e007f.pdf"},{"id":51392615,"identity":"d6671981-a9c8-45e4-927f-321dfda7a84c","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e \u003cstrong\u003eCorrelation between RANKL expression and clinicopathologic features of 183 CRC patients.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table.S1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/3f882ffe0a90f8d0b483923a.xlsx"},{"id":51393199,"identity":"d8796307-2683-4e9c-aa2d-01e3226f201e","added_by":"auto","created_at":"2024-02-20 18:51:01","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2.\u003c/strong\u003e \u003cstrong\u003eTarget sequences of siRNA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table.S2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/e9acc4a15638f60479860d02.xlsx"},{"id":51392610,"identity":"7b130607-65be-4b61-88a8-9b842aeafd68","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3.\u003c/strong\u003e \u003cstrong\u003eTarget sequences of shRNA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table.S3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/c6c47fde89d336666a41279c.xlsx"},{"id":51392612,"identity":"e9581e1e-1b01-44c6-ae2d-f0ce5cd4d819","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4.\u003c/strong\u003e \u003cstrong\u003eSequences of primers\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table.S4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/054587076b67805c98d91b8b.xlsx"},{"id":51393202,"identity":"6616414b-b67c-4ac7-b8bc-75a9a26e4e3e","added_by":"auto","created_at":"2024-02-20 18:51:01","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":9676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S5: CSCs frequency calculated by ELDA in Limiting dilution assay.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table.S5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/90c2ad4b87532102ccc2912d.xlsx"},{"id":51392624,"identity":"68fa884b-f9d6-4f61-9f7f-aa355c204207","added_by":"auto","created_at":"2024-02-20 18:43:02","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":7428304,"visible":true,"origin":"","legend":"\u003cp\u003eRANKL is mainly derived from CD4+ T cells.\u003c/p\u003e\n\u003cp\u003e(a) The origin of RANKL(TNFSF11) was analyzed through a single cell database.\u003c/p\u003e\n\u003cp\u003e(b) IHC co-stain RANKL with multiple markers of immune cells (CD4, CD8, CD56) in CRC patients. (100×, 200×)\u003c/p\u003e\n\u003cp\u003e(c) Colorectal tumor tissues subjected to double immunofluorescence for CD4 (green), CD56 (white), RANKL (red), and DAPI (blue) (200 ×).\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×)\u003c/p\u003e","description":"","filename":"figureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/1268e4cbc9b82b1348e208b5.tif"},{"id":51392630,"identity":"483e269c-6010-4d85-9162-00cccdd9da1e","added_by":"auto","created_at":"2024-02-20 18:43:02","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":5449092,"visible":true,"origin":"","legend":"\u003cp\u003eRANK promotes malignant progression of CRC.\u003c/p\u003e\n\u003cp\u003e(a) The mRNA level correlation between RANK/RANKL and CD44/CD133 in CRC tissues was detected by analyzing the dataset GEPIA.\u003c/p\u003e\n\u003cp\u003e(b) Log2 fold changes in related RANK mRNA expression of CRC cell lines compared to the normal cell (HCoEpiC).\u003c/p\u003e\n\u003cp\u003e(c) RANK stable overexpression or knockdown efficiency was confirmed by western blotting in CRC cells.\u003c/p\u003e\n\u003cp\u003e(d) RANK overexpression influenced the sphere formation and (e) the migration of Caco2 cells. And the addition of 100 ng/ml RANKL moderately increased the migration of Caco2 cells. (Sphere formation: 400×, Migration: 200×)\u003c/p\u003e\n\u003cp\u003e(f) The subcutaneous tumors formed by control and DLD1 RK cells were obtained from NOD/Scid mice. Both the volume and weight of subcutaneous tumors were shown in the right panel (mean ± SD, n = 5).\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/ecc14aa4e0ca26df77259f67.tif"},{"id":51392629,"identity":"8d31cc36-b832-4a4b-a261-b781d127775f","added_by":"auto","created_at":"2024-02-20 18:43:02","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":3334720,"visible":true,"origin":"","legend":"\u003cp\u003eRANK promotes stemness of CRC.\u003c/p\u003e\n\u003cp\u003e(a) Relative expression of related stemness markers (CD44, CD133, NANOG, LGR5, OCT4, SOX2, NOTCH3) in RANK overexpression or knockdown cells was analyzed by q RT-PCR.\u003c/p\u003e\n\u003cp\u003e(b) Western blotting of CD133 protein expressions in RANK overexpression Caco2 cells and the addition of 100 ng/ml RANKL.\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/3f21933008f6a9ae1fdab5f2.tif"},{"id":51392623,"identity":"51f3aa16-382b-4ba9-84d6-c2d2cc370896","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1683784,"visible":true,"origin":"","legend":"\u003cp\u003eRANK is associated with CCL20 in CRC.\u003c/p\u003e\n\u003cp\u003e(a) The mRNA level correlation between RANK/RANKL and CCL20 in CRC tissues was detected by analyzing the dataset GEPIA.\u003c/p\u003e\n\u003cp\u003e(b) Log2 fold changes in related CCL20 mRNA expression of CRC cell lines compared to the normal cell (HCoEpiC).\u003c/p\u003e\n\u003cp\u003e(c)ELISA of CCL20 protein expressions in RANK overexpression cells.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/834304609f100a9024be40d6.tif"},{"id":51393203,"identity":"36216d5f-c923-40ff-9791-cc1700c4f8df","added_by":"auto","created_at":"2024-02-20 18:51:02","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":4796324,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpressed RANK in CRC cells can recruit more CD4\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003eT cells.\u003c/p\u003e\n\u003cp\u003e(a) Migration of PBMC from CRC patients co-cultured with the supernatants of DLD1 cells or RANK-overexpressing DLD1 cells was analyzed by transwell assay and flow cytometry, (b) and the addition of anti-CCL20 antibody. (The illustration created with BioRender.com)\u003c/p\u003e","description":"","filename":"figureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/b7beb2ca90c72a9f851b016e.tif"},{"id":51393204,"identity":"752802e2-f6a5-4883-81fc-78520bbfbe84","added_by":"auto","created_at":"2024-02-20 18:51:02","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":4191996,"visible":true,"origin":"","legend":"\u003cp\u003eNF-κB inhibitors/ siRNA of P65 can inhibit the stemness of CRC.\u003c/p\u003e\n\u003cp\u003e(a) Western blotting of p-P65/P65 protein or (b) CD44 protein expressions in RANK-overexpressing DLD1 cells (DLD1 RK cells) and the addition of 50nm QNZ.\u003c/p\u003e\n\u003cp\u003e(c) RANK overexpression or addition of 50nm QNZ influenced the sphere formation and (d) migration of DLD1 cells. (Sphere formation: 400×, Migration: 200×)\u003c/p\u003e\n\u003cp\u003e(e) Changes of P65 mRNA levels in DLD1 RK cells add P65 siRNA treatment.\u003c/p\u003e\n\u003cp\u003e(f) IHC staining was used to detect the expression of RANK and p-P65 in indicated subcutaneous tumors of NOD/Scid mice. (200×)\u003c/p\u003e\n\u003cp\u003eScales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/dabb825c7f3d59e76945a374.tif"},{"id":51393205,"identity":"94e94b5e-d593-451c-a9b7-bf28164f7feb","added_by":"auto","created_at":"2024-02-20 18:51:02","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":2779612,"visible":true,"origin":"","legend":"","description":"","filename":"1rawimages.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/c313602769a7f964a3a120ff.tif"},{"id":51392622,"identity":"8c9b1004-0002-4247-9678-eedfa906eb41","added_by":"auto","created_at":"2024-02-20 18:43:01","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":1469992,"visible":true,"origin":"","legend":"","description":"","filename":"3rawimages.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/f001bf52178cfd724727a76f.tif"},{"id":51392633,"identity":"11bb1c9b-d361-4425-acff-5f21594d4141","added_by":"auto","created_at":"2024-02-20 18:43:02","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":3336808,"visible":true,"origin":"","legend":"","description":"","filename":"6rawimages.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/a4457ae9670b3c7de8c04f78.tif"},{"id":51392628,"identity":"642e7d49-e994-4f75-ab90-deaa19cc241d","added_by":"auto","created_at":"2024-02-20 18:43:02","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":800584,"visible":true,"origin":"","legend":"","description":"","filename":"S2rawimages.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/5bdc5a085d35fd5d5ae6f197.tif"},{"id":51393206,"identity":"d4fb17e2-cf57-47ce-b2e8-533409c90977","added_by":"auto","created_at":"2024-02-20 18:51:02","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":2227008,"visible":true,"origin":"","legend":"","description":"","filename":"S6rawimages.tif","url":"https://assets-eu.researchsquare.com/files/rs-3869046/v1/32d73d94dc2434a7c64ddeeb.tif"}],"financialInterests":"(Not answered)","formattedTitle":"RANKL/RANK signaling recruits Tregs via CCL20/CCR6 pathway and promotes stemness and metastasis in colorectal cancers","fulltext":[{"header":"1. Background","content":"\u003cp\u003eColorectal cancer (CRC) is presently recognized as one of the most prevalent forms of cancer on a global scale\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Despite the considerable advancements in treatment techniques, the responses of patients with CRC continue to be suboptimal. The prognosis for individuals diagnosed with metastatic CRC (mCRC) continues to be unfavorable\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e and largely resistant to treatments. Hence, it is imperative from a clinical standpoint to investigate the underlying process and identify novel targets for prevention.\u003c/p\u003e \u003cp\u003eThe tumor microenvironment (TME) in general interacts with cancer cells to decide the final fate of tumor development and migration\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The role of regulatory T cells (Tregs) in CRC progression is still debatable \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Currently, researchers are primarily interested in the role of Tregs in promoting or suppressing cancer by controlling immune response; however, the direct interaction between Tregs and CRC cells has received less attention\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEmerging evidence suggests that cancer stem cell (CSC) dysregulation plays an essential role in CRC growth and metastasis\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. CSCs are a type of tumor cell with stem cell features that can self-renew and differentiate, leading to tumor development, therapy resistance, metastasis, and recurrence\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. CSCs are typically formed as a result of numerous genetic alterations. Many unique genetic changes have been discovered as activating proto-oncogenes or inactivating tumor suppressor genes. Oncogenes and tumor suppressor genes are frequently assigned separate functions in tumor progression. However, the link between these genes and tumor stemness and advancement is still unknown.\u003c/p\u003e \u003cp\u003eTNF superfamily members all have pro-inflammatory action, which is mediated in part by the transcription factor NF-κB\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Tumor necrosis factor (TNF) receptor superfamily member 11a (RANK) and its ligand TNF superfamily member 11 (TNFRSF11, RANKL) have also been linked to cancer: RANKL/RANK have been shown to cause the migration of human epithelial cancer cells and melanoma cells\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, and have also been examined in mammary epithelial cells and prostate epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Previous research has shown that RANKL/RANK can promote CRC metastasis\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, the specific mechanism by which RANKL/RANK maintains CRC stemness and promotes metastasis remains unknown.\u003c/p\u003e \u003cp\u003eRANKL/RANK signaling promotes tumor growth by influencing multiple downstream pathways, including tumor metabolism, treatment resistance, and tumor immunity. RANKL has been linked to an increase in tumor-infiltrating lymphocytes and cancer metastasis\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. RANKL/RANK is also important for the formation of Tregs, according to research\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. RANKL can be produced by CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, and the majority of T-cells that produce RANKL\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e also express the forkhead box P3 (FOXP3), a transcription factor produced by Tregs. Furthermore, soluble RANKL released into the TME can recruit Tregs via RANKL/RANK signaling\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, in breast tumors, lack of RANK signaling promotes lymphocyte and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration while decreasing macrophage and neutrophil infiltration\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. However, the specific interaction network involving RANKL/RANK, immune cells, and tumor cells has yet to be discovered.\u003c/p\u003e \u003cp\u003eIn this study, we discovered that RANKL was mostly released by Tregs and that RANKL/RANK signaling might increase the malignant development of CRC by boosting tumor stemness in this investigation. Further analysis indicated that this process is accomplished via activating the NF-κB pathway's phosphorylation of P65. At the same time, we discovered that RANKL/RANK upregulated C-C motif chemokine ligand 20 (CCL20) production via the NF-κB pathway and recruited Tregs via the CCL20-CCR6 axis, producing a \"vicious cycle\" in the TME. As a result, RANKL/RANK suppression is being evaluated as a potential new target for the therapy of CRC metastases.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Clinical samples\u003c/h2\u003e \u003cp\u003eSun Yat-Sen University's Seventh Affiliated Hospital and First Affiliated Hospital provided clinical samples. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e contains patient information. The stages were carried out in accordance with UICC-TNM grading, and all samples were pathologically examined. All patients provided written informed permission in accordance with the Hospital's Institutional Review Board guidelines (ethical approval number: Science-2010-LW-1213).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Antibodies and reagents\u003c/h2\u003e \u003cp\u003ePrimary antibodies used in this study included: anti-RANK (ab13918), anti-FOXP3 (ab20034), anti-CCL20 (ab106009), anti- RANKL (ab9957), and anti-CD4 (ab133616) from Abcam, UK; anti-P65 (#4764), anti-p-P65 (Ser536; #3033), anti-FLAG (#14793) and anti-IgG isotype control (#3900S) from Cell Signaling Technology, CST, USA ; anti-GAPDH (60004-1-lg), anti-CD44 (15675-1-AP), anti-CD133(66666-1-Ig), and anti-CCR6 (66801-1-Ig) from Proteintech, Wuhan, China; T-bet (SC21749), GATA-3 (SC269) and RORγ (SC365476) from Santa Cruz, USA; and anti-CD56 (GB112671) from Servicebio, Wuhan, China. The reagents included: QNZ from MCE; RANKL, EGF, and bFGF from PeproTech.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell lines and cell culture\u003c/h2\u003e \u003cp\u003eAll cells were obtained from the American Type Culture Collection, were authenticated, and were mycoplasma-free. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37\u0026deg;C with 5% CO2. RANKL at 100 ng/ml and QNZ at 50nm were introduced. T Cell Expansion Medium (STEMCELL) was used to culture human PBMCs according to the manufacturer's recommendations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Establishment of stable cell lines and transient transfection\u003c/h2\u003e \u003cp\u003ePlasmids were introduced into cells using Liposomal Transfection Reagent (YiSheng, Shanghai, China) according to the directions for transient transfection; for stable transfection, 293T cells were transfected with plasmids and polyethyleneimine (Polysciences, USA). Polybrene (Sigma, USA) was utilized to infect cells with virus particles. Puromycin (Sigma) was used to test plasmids for puro-resistance. Transheep (Shanghai, China) supplied all of the plasmids. Supplementary Tables S2 and S3 provide siRNA and shRNA sequences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Real time quantitative PCR (QRT-PCR)\u003c/h2\u003e \u003cp\u003eRNA was extracted with AG RNAex Pro Reagent (Accurate Biotechnology, AG, Beijing, China). QRT-PCR was performed using 5X Evo M-MLV RT Master Mix and SYBR\u0026reg; Green Premix (AG). GAPDH was used as an internal reference, and specific primer sequences were shown in Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. CHIP assay\u003c/h2\u003e \u003cp\u003eThe CHIP assay was performed using a CHIP assay Kit (Beyotime, Shanghai, China). Before adding cell lysates, magnetic beads were combined with anti-P65 and anti-rabbit IgG. Then DNAs were purified. QRT-PCR was used for further analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. ELISA\u003c/h2\u003e \u003cp\u003eThe content of CCL20 in the supernatants was determined using an ELISA Kit (4 A Biotech, Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Western blotting (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF)\u003c/h2\u003e \u003cp\u003eRIPA Lysis Buffer (PC101, Yamei, Shanghai, China) was used for protein extraction. SDS-PAGE was used to separate protein samples, which were then transferred to a Nitrocellulose membrane (Merck Millipore, Germany). The membrane was treated with the primary antibody at 4\u0026deg;C overnight. Secondary antibodies were IRDye 800CW Goat anti-IgG (LI-COR, USA), which were then seen using the ChemiDocTM MP Imaging System (BIO-RAD, USA)\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCells were fixed with 4% paraformaldehyde and stained with primary antibody on a sliding plate for immunofluorescence staining. A fluorescent secondary antibody was added and incubated at room temperature. Finally, the nucleus was labeled with DAPI. Tissue immunofluorescence staining was performed using the TSA PLus Kit (Servicebio). A secondary antibody was created using a DAB staining solution Kit (Gene Tech, Shanghai, China).\u003c/p\u003e \u003cp\u003eThe slices were treated overnight at 4\u0026deg;C with primary antibodies for immunohistochemistry\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. A second antibody was incubated on the second day. For staining, a DAB staining solution Kit was utilized. A Leica DM4B microscope was used to examine the sections. This paper\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e was used to calculate the IHC scores.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Flow cytometry and cell sorting\u003c/h2\u003e \u003cp\u003ePBMCs were isolated from blood samples using Ficoll (TBD)-gradient centrifugation. Tumor Dissociation Kit (Miltenyi Biotec, Germany) was used for extracting lymph node cells. EasySep\u0026trade; Human T Cell Enrichment Kit (STEMCELL, Cananda) was used to separate CD3\u003csup\u003e+\u003c/sup\u003eT cells from PBMCs.\u003c/p\u003e \u003cp\u003eHuman PBMC and lymph node cells were stained for surface analysis with CD4-AF700, CD25-PE/Cy7, CCR6/PE, and RANKL-APC, and Treg intracellular analysis with FOXP3-BV421 (Biolegend, USA). Before staining FOXP3, the True-Nuclear\u0026trade; Transcription Factor Buffer Set (Biolegend) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Migration and Chemotaxis Assay\u003c/h2\u003e \u003cp\u003e1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were inoculated into the upper chamber (8 \u0026micro;m, Corning, USA). The medium was supplemented with 10% FBS as a chemotactic agent in the inferior cavity. After 48 h, the lower chamber of membrane cells was stained. The stained cells were randomly imaged using a microscope in 5 different fields.\u003c/p\u003e \u003cp\u003eInduced T cells were migrated and placed in the upper chamber of 24-well Transwell plates with a 3.0 \u0026micro;m polycarbonate membrane (SPL, Korea) (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells /well). DLD1 WT/RK cells were grown in ImmunoCult\u003csup\u003eTM\u003c/sup\u003e-XF T Cell Expansion Medium (STEMCELL), and the supernatant was collected and deposited in the bottom chamber with or without anti-CCL20. Migration was permitted to continue for 6 hours\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Flow cytometry was used to calculate the fraction of CD4\u003csup\u003e+\u003c/sup\u003e FOXP3\u003csup\u003e+\u003c/sup\u003e cells in the bottom chamber.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Tumor sphere formation assays\u003c/h2\u003e \u003cp\u003eCells were seeded into 24-well ultralow attachment plates concentration of 5000 cells per well and re-suspended in the stem cell-conditioned medium containing DMEM-F12 (Bio-channel, Nanjing, China), 2% B-27 Supplement (PeproTech), 10 ng/ml basic fibroblast growth factor (bFGF, PeproTech), 20 ng/ml epidermal growth factor (EGF, PeproTech), and 5 \u0026micro;g/ml insulin (Beyotime), at 37\u0026deg;C with 5% CO2 and saturated humidity for 12\u0026ndash;14 days. When spheroid diameters reached 50 \u0026micro;m, culture suspensions were passed every 7 days. The photographs were taken, and the number of cell spheres was tallied.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Subcutaneous xenograft implantation models and in vivo limiting dilution assay\u003c/h2\u003e \u003cp\u003e All animal research were carried out with the agreement of Sun Yat-sen University's Animal Experiment Ethics Committee. Guangdong Yaokang Biotechnology Co., LTD provided female NOD-Scid mice (4\u0026ndash;6 weeks).\u003c/p\u003e \u003cp\u003eFor Subcutaneous xenograft assays, DLD1 cells or DLD1 RK cells (2 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells per mouse) were subcutaneously injected into NOD-Scid mice (n\u0026thinsp;=\u0026thinsp;5). After 3 weeks, mice were sacrificed and xenograft tumors were harvested for histological study. The tumor volume was calculated according to the formula: Volume (mm\u003csup\u003e3\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;width\u003csup\u003e2\u003c/sup\u003e (mm\u003csup\u003e2\u003c/sup\u003e) \u0026times; length (mm) \u0026times; 0.4\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor limiting dilution assays, DLD1 cells or DLD1 RK cells were implanted at the gradient of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e and 2 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells per NOD-Scid mouse (n\u0026thinsp;=\u0026thinsp;5 per group). The incidence of tumors in the two groups was analyzed. The frequency of tumor stem cells was calculated by a method described on the ELDA website (Extreme Limiting Dilution Analysis, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.wehi.edu.au/software/elda/\u003c/span\u003e\u003cspan address=\"http://bioinf.wehi.edu.au/software/elda/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e \u003cp\u003eSPSS and GraphPad Prism were used for statistical analysis. ImageJ was used to calculate the GAPDH indicator protein levels. CytExpert and Flowjo were used to evaluate flow cytometry data. The data were compared using the one-way ANOVA test. The χ\u003csup\u003e2\u003c/sup\u003e test was performed to investigate the connection between various biomarkers in CRC tissue slices. The survival curve was created using the Kaplan-Meier method and the log-rank test. P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Public clinical datasets\u003c/h2\u003e \u003cp\u003eCorrelation analysis between gene expression in tissues of CRC patients was computed from GEPIA (Gene Expression Profiling Interactive Analysis, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). CHIP-seq datasets were collected from the Cistrome Data Browser(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cistrome.org/db/#/\u003c/span\u003e\u003cspan address=\"http://cistrome.org/db/#/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Single-cell data are derived from published article\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 RANKL/RANK expression is linked to the advancement of CRC malignancy.\u003c/h2\u003e \u003cp\u003eWe previously found that RANK expression was elevated in CRC patient samples and was linked to a poor prognosis\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. We concentrated on RANKL in this investigation. According to the TCGA dataset, RANKL was shown to be substantially elevated in 275 CRC tissues compared to 349 normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Furthermore, as demonstrated in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, RANKL expression was linked to CRC clinical histology. Patients with increased RANKL protein expression had shorter overall survival (OS) and recurrence-free survival (RFS) among CRC patients having both OS and RFS data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Immunohistochemical assays were utilized to compare RANKL protein expression in formalin-embedded tumor tissues from different TNM stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The findings revealed that RANKL in CRC tumor tissues was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) linked with TNM staging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). WB demonstrated that RANK levels in CRC tumor tissues were higher than in neighboring normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), which was consistent with our previous findings. These findings indicated that RANKL/RANK was highly elevated in CRC patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 RANKL is mainly generated by CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e regulatory T cells in CRC\u003c/h2\u003e \u003cp\u003eWe then attempted to locate the source of RANKL in CRC. Single cell sequencing data (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea) showed that RANKL was primarily produced from T cells in breast cancer, specifically CD4\u0026thinsp;+\u0026thinsp;T cells\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, which was similar with the findings of Wei Tan et al\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. We used immunohistochemistry and immunofluorescent labeling (NK cells: CD56) of CRC samples to determine which subtype of T cells RANKL is primarily originated from. We discovered that RANKL expression was substantially overlapping with CD4 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), implying that RANKL is produced by CD4\u003csup\u003e+\u003c/sup\u003e T cells. RANKL and FOXP3 were discovered as well to be co-localized in CRC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Flow cytometric analysis of the peripheral blood and lymph node of CRC patients revealed that membrane-RANKL (m-RANKL) was primarily expressed in CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e T cells compared to CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). We concluded that Tregs expressed RANKL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 RANKL/RANK signaling enhances CRC cell stemness.\u003c/h2\u003e \u003cp\u003eTo investigate whether RANKL/RANK may influence CRC stemness and metastasis, we evaluated TCGA CRC data and discovered that RANK/RANKL expression was positively linked with stemness-related genes (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also measured the RANK mRNA levels in other CRC cell lines (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb) and chose HCoEpiC (normal intestinal epithelial cell line), DLD1 and Caco2 to create overexpressed cells, and LS174T to create knockdown cells. WB verified RANK overexpression cells (HCoEpiC RK, DLD1 RK, Caco2 RK) and RANK knockdown cells (LS174T sh-RK) (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ec). Overexpression of RANK or addition of RANKL increased tumor sphere formation, whereas knocking down RANK could limit it (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS2\u003c/span\u003ed). Our earlier study indicated that RANKL/RANK might accelerate CRC metastasis\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In this investigation, we discovered that overexpression of RANK greatly boosted cell migration while knocking down RANK inhibited migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS2\u003c/span\u003ee). Furthermore, the inclusion of RANKL increased tumor sphere formation and CRC cell metastasis. Mechanistically, all CSCs markers were up-regulated in RANK-overexpressed cells (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea), with CD44 and PROMI (CD133) being the most significant. The WB data verified this conclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS3\u003c/span\u003eb). Clinically, patients with elevated RANKL/RANK expression similarly had increased CD44 expression in CRC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The size and weight of the subcutaneous tumors generated by DLD1 RK cells were much larger than the control cells in in vivo tests (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ef). To acquire a better understanding of the role of RANK in CSC stemness, we performed an in vivo limited dilution tumor transplantation investigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Tumor-initiating cells were found in one out of every 5.248 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e DLD1 RK cells and one out of every 4.174 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e DLD1 cells, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ef. Notably, the frequency of tumor-initiating cells in DLD1 RK increased when compared to the control. Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e shows the detailed numbers of cell planted and tumors formed. IHC labeling with CD44 and CD133 revealed that subcutaneous tumors generated by DLD1 RK cells had a higher stemness index than control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). In conclusion, we found a substantial association between RANKL/RANK expression and tumor stemness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 RANKL/RANK increases the secretion of CCL20 by CRC cells.\u003c/h2\u003e \u003cp\u003eTo identify critical immune-related factors for poor prognosis in CRC patients with high-RANK expression, we examined levels of chemokines. Most chemokine mRNA levels we identified were elevated in overexpressed RANK cells in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), with CCL20 being the most significant, which was similar with the findings of Y. Liu et al\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. TCGA CRC data showed that RANK/RANKL expression was strongly linked with CCL20 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ea). Furthermore, we discovered that CCL20 mRNA levels in CRC cell lines were higher than in normal cell lines (HCoEpiC) (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe performed immunofluorescence staining and ELISA experiments to confirm our findings and discovered that CCL20 expression was up-regulated in overexpressed RANK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS4\u003c/span\u003ec). Furthermore, the addition of RANKL increased the levels of CCL20 protein and mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Clinically, CCL20 mRNA expression levels in tumor tissue were higher than in normal tissue in the majority of CRC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). IHC labeling with CCL20 in vivo indicated a higher expression in subcutaneous tumors generated by DLD1 RK cells than in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). By combining these findings, we concluded that RANKL/RANK signaling enhanced CCL20 production in CRC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Overexpression of RANK in CRC cells promotes recruitment of Tregs via the CCL20-CCR6 interaction.\u003c/h2\u003e \u003cp\u003eNumerous studies have demonstrated that the CCL20-CCR6 pathway can recruit Tregs\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. We discovered that tumor tissues with high expression of RANK or CCL20 associated strongly with enhanced FOXP3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). To demonstrate that RANK can enhance CCL20 release and thereby attract Tregs, we performed an in vitro experiment in which DLD1 WT/ RK cells were co-cultured with PBMC from CRC patients. DLD1 RK cell supernatants attracted substantially more Tregs (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003ea). To confirm the critical role of CCL20, we added anti-CCL20, which inhibited the increased recruitment of Tregs by overexpressed RANK DLD1 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). To confirm that RANKL/RANK recruits Tregs via the CCL20-CCR6 pathway, we discovered that CCR6 and FOXP3 were co-localized in CRC blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) and tumor tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 RANKL/RANK increases stemness and CCL20 production by CRC cells via NF-κB signaling.\u003c/h2\u003e \u003cp\u003eSince RANK is a known activator of the NF-κB receptor, we first determined whether RANKL/RANK promoting stemness and CCL20 secretion was due to the activation of the NF-κB pathway. By WB and IF, we found that both overexpression of RANK and addition of RANKL promoted the nuclear phosphorylation of P65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). We investigated the role of the NF-κB pathway in the process of RANK promoting stemness. NF-κB pathway inhibitor (QNZ) significantly inhibited the phosphorylation of P65 (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ea), the expression of CSCs marker (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eb), the ability of tumor sphere formation (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ec) and metastasis (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ed)as demonstrated by WB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further corroborate the crucial role of P65, we discovered that CD44 was also decreased after P65 was knocked down in CRC cells by WB (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) and QRT-PCR (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ee). In addition, tumor sphere-formation and migration ability also were weakened (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). In contrast, when we overexpressed P65 in LS174T sh-RK cells, we discovered that down-regulation of CD44 could be rescued (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), along with the ability to form tumor spheres and migrate (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h). IHC staining with p-P65 confirmed a higher expression in subcutaneous tumors formed by DLD1 RK cells compared to control cells in in vivo experiments (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eThe suppression of P65 significantly inhibited CCL20 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c). To confirm the relationship between P65 and CCL20 in CRC cells, we analyzed the Cistrome DB database and found P65 binding sites in the promoter of the CCL20 gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). After ChIP, three primer pairs corresponding to the predicted binding sites were used for QRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). The findings revealed that P65 interacted with the P2 (-153 to 21 bp) region of the CCL20 promoter. Collectively, our results indicated that P65 is a key target for RANK to promote CCL20 secretion in CRC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRANKL/RANK has been documented to correlate closely with tumor metastasis of prostate, breast, liver cancer and melanoma\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Our previous research\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e has demonstrated that RANK is associated with the progression of cancer in CRC. However, the RANKL/RANK signaling mechanism in CRC remains unknown. In this study, we discovered that the RANKL/RANK pathway promotes the secretion of chemokines that CRC cells can use to recruit Tregs, thereby influencing CRC stemness, metastasis, and tumorigenesis.\u003c/p\u003e \u003cp\u003eAlthough RANKL is best known for its distribution in bone\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, it has been reported on other cell types, such as immune cells, with aberrant expression in certain tumor types\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Uncertain is the role of RANKL/RANK in tumor immunity. RANKL is derived from CD4\u003csup\u003e+\u003c/sup\u003e FOXP3\u003csup\u003e+\u003c/sup\u003e T cells in breast cancer and arthritis, according to multiple reports \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In this study, we demonstrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) that RANKL is derived from CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e T cells in CRC. This is the first time this result has been reported for colorectal cancer, and it is consistent with prior findings for other cancers\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. This evidence suggests that RANKL/RANK and the TME in CRC are closely related.\u003c/p\u003e \u003cp\u003ePrevious research has linked RANKL/RANK to the metastasis of CRC\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e and other cancers\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and it is believed that metastasis is associated with CSCs\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Our research demonstrates that RANK can enhance metastasis by promoting the stemness of CRC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), consistent with findings in breast 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 screening of CSCs markers (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ef) reveals that RANK can upregulate stemness markers. In addition, we observe a substantial increase in CD44 levels in CRC cells following RANK overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These findings suggest that the RANKL/RANK pathway influences the proliferation, metastasis, and tumorigenesis of CSCs by elevating CD44 levels.\u003c/p\u003e \u003cp\u003eThe RANKL/RANK pathway can influence TME composition. The RANKL/RANK pathway has been linked to the release of chemokines that recruit T lymphocytes in breast cancer and endometrial cancer\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. We discovered that RANKL/RANK can enhance CCL20 production by CRC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), which is consistent with earlier research\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, demonstrating that RANKL/RANK is important in the immunological microenvironment of CRC. Furthermore, CCL20 can influence the TME via immune cells such as B cells, T cells, and dendritic cells, influencing the progression of CRC \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Furthermore, Wang D et al. demonstrate that CCL20 generated by CRC cells can recruit Tregs to improve chemoresistance\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Furthermore, CCR6 expressed on the surface of Tregs as a CCL20 receptor has been investigated in tumor immunity\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. In this study, we discovered that RANKL/RANK can enhance Tregs recruitment via the CCL20-CCR6 pathway, hence accelerating the malignant evolution of CRC.\u003c/p\u003e \u003cp\u003eRANK, or receptor activator of nuclear factor kappa-B, activates the NF-κB pathway\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The NF-κB pathway is involved in cancer, epithelial-mesenchymal transition, metastasis, chemoresistance, and stemness\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Nonetheless, knowledge of the NF-κB pathway's significance in CRC is limited. Indeed, some studies have found that CRC cells have an active NF-κB pathway; in general, CRC growth is dependent on NF-B signaling\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. RANK has been found in studies to activate the NF-κB pathway, promoting the malignant progression of breast cancer\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. RANK can upregulate CCL20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c) and increase CRC stemness (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) via the NF-κB pathway, according to our findings. Using the CHIP-QPCR experiment, we also revealed that P65 can bind to the promoter of CCL20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, e). All of this suggests that the NF-κB pathway is critical in the recruitment of immune cells and the advancement of CRC malignancy.\u003c/p\u003e \u003cp\u003eIn summary, we associate Tregs to CRC stemness via RANKL/RANK signaling and discover a new mechanism by which Tregs enhance CRC metastasis: Tregs trigger the RANK signal of CRC cells by secreting RANKL, promoting CRC metastasis by increasing stemness. Activation of the RANKL/RANK signaling pathway can have a positive feedback effect by recruiting more Tregs via the CCL20-CCR6 pathway, hence controlling CRC metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). The RANKL/RANK pathway and the TME are known to interact throughout development and cancer, but the underlying mechanism is unknown. Finally, given the role of RANKL/RANK signaling in tumorigenesis, anti-cancer drug clinical trials in the appropriate molecular targets (such as CCL20 / CCR6) and RANKL/RANK inhibitors used in combination, or licensing effectively inhibit colorectal cancer metastasis, improve treatment effectiveness. Our findings contribute to a better understanding of the RANKL-RANK signaling pathway as it relates to the immunological microenvironment, stemness, and metastasis of CRC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e \u003cp\u003eJ.O.: Methodology, Investigation, and Writing \u0026ndash; Original Draft. S.H., Q.Z., T.K., Y.W., Y.L., C.L., Y.Y and Y.S.: Formal analysis and Investigation. Y.L., W.X. and J.Q.: Resources. S.H., C.Z.: Writing - Original Draft, and Writing - Review \u0026amp; Editing. Q.L., C.Z.: Conceptualization, Supervision. T.G., L.Y. and Y.P.: Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the Research Start-up Fund of the Seventh Affiliated Hospital, Sun Yat-sen University [grant number ZSQYBRJH0003]; and the National Natural Science Foundation Youth Project of China [grant number 82203807]. Shenzhen Science and Technology Project, sustainable development project [grant number KCXFZ202002011010593]. The sponsors had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eThe authors declare that all the data supporting the findings in this study are available in this study and are available from the corresponding author through reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQuintana JM, Gonzalez N, Anton-Ladislao A, Redondo M, Bare M, Fernandez de Larrea N, \u003cem\u003eet al.\u003c/em\u003e Colorectal cancer health services research study protocol: the CCR-CARESS observational prospective cohort project. 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J Biomed Sci 2022, 29(1): 109.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RANKL, RANK, Colorectal cancer, Stemness, CCL20, Tregs ","lastPublishedDoi":"10.21203/rs.3.rs-3869046/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3869046/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTNF receptor superfamily member 11a (TNFRSF11a, RANK) and its ligand TNF superfamily member 11 (TNFRSF11, RANKL) are overexpressed in a number of malignancies. The clinical importance of RANKL/RANK in colorectal cancer (CRC) is, however, mainly unknown. We examined CRC patient samples and found that RANKL/RANK was elevated in CRC tissues as compared to nearby normal tissues. A higher RANKL/RANK expression was related with a worse survival rate. Furthermore, we found that RANKL is mostly produced by regulatory T cells (Tregs), which can promote CRC advancement. Overexpression of RANK or addition of RANKL significantly increased the stemness and migration of CRC cells. Furthermore, RANKL/RANK signaling stimulates C-C motif chemokine ligand 20 (CCL20) production by CRC cells, which leads to Treg recruitment, boosting tumor stemness and malignant progression. This recruitment process was accomplished by using CCL20-CCR6 interaction, demonstrating a connection between CRC cells and immune cells. These findings suggest that RANKL/RANK plays an important role in CRC progression and could be a potential target for CRC prevention and therapy.\u003c/p\u003e","manuscriptTitle":"RANKL/RANK signaling recruits Tregs via CCL20/CCR6 pathway and promotes stemness and metastasis in colorectal cancers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-20 18:42:56","doi":"10.21203/rs.3.rs-3869046/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-03-11T16:04:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-09T15:47:24+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-02-26T15:09:54+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-24T08:04:58+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-19T01:51:26+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-02-17T04:28:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-16T11:47:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-01-16T06:56:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-16T06:56:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"966e54af-0c0d-4520-812a-569339411c6d","owner":[],"postedDate":"February 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28814021,"name":"Health sciences/Biomarkers/Prognostic markers"},{"id":28814022,"name":"Biological sciences/Immunology/Cytokines/Chemokines"}],"tags":[],"updatedAt":"2024-06-21T07:08:12+00:00","versionOfRecord":{"articleIdentity":"rs-3869046","link":"https://doi.org/10.1038/s41419-024-06806-3","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2024-06-20 04:00:00","publishedOnDateReadable":"June 20th, 2024"},"versionCreatedAt":"2024-02-20 18:42:56","video":"","vorDoi":"10.1038/s41419-024-06806-3","vorDoiUrl":"https://doi.org/10.1038/s41419-024-06806-3","workflowStages":[]},"version":"v1","identity":"rs-3869046","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3869046","identity":"rs-3869046","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}