Fig 2: RANKL/RANK expression is associated with the malignant progression of CRC.a The mRNA levels of RANK and RANKL in CRC (n = 275) and normal colorectal (n = 349) tissues were determined by analyzing the GEPIA dataset. b Kaplan–Meier analysis of overall survival (OS) and recurrence-free survival (RFS) in CRC patients (n = 183). c RANKL immunohistochemistry staining in CRC tissues (stage I, stage II, stage III, and stage IV) (100x, 400x). d Percentage of RANKL expression in CRC tumors (n = 183) within individual TNM stage (P < 0.0001). e RANK protein expression was measured using WB in six matched tumor and normal tissue samples. Scales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: RANKL-RANK promotes the secretion of CCL20 by CRC cells via NF-κB signaling.a Changes of CCL20 mRNA (QRT-PCR, n = 3), (b, c) CCL20 protein (ELISA/immunofluorescence) in DLD1 RK cells add P65 siRNA treatment (200×). For ELISA, error bar indicates mean + SD of 2 technical replicates. 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. Scales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P < 0.05, **P < 0.01, ***P < 0.001, ns no significance. 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.

Fig 4: RANKL/RANK promotes the secretion of CCL20 by CRC cells.a QRT-PCR was used to analyze the relative expression of related chemokines in RANK-overexpressing cells (n = 3). b CCL20 protein expression via immunofluorescence in RANK overexpression cells. (200×). c ELISA (error bar indicates mean + SD of 2 technical replicates) and (d) QRT-PCR (n = 3) of CCL20 protein expressions in RANK overexpression cells, and the addition of 100 ng/ml RANKL. 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 (P1–8: eight different CRC patients). f IHC staining was used to detect the expression of RANK and CCL20 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.

Fig 5: RANKL-RANK promotes the stemness via NF-κB signaling.a Immunofluorescence of p-P65 protein expressions in RANK overexpression or knockdown cells (400×). b WB of p-P65/P65 protein expressions in RANK overexpression or knockdown cells and the addition of 100 ng/ml RANKL. c Changes of p-P65/P65 protein and CD44 protein levels in DLD1 RK cells add P65 siRNA treatment. d RANK overexpression or P65 siRNA treatment influenced the sphere formation (n = 3) and (e) migration (n = 5) of DLD1 cells. (Sphere formation: 400×, Migration: 200×). f WB of p-P65/P65 and CD44 protein expressions in RANK knockdown cells and overexpressed P65. g RANK knockdown or overexpressed P65 influenced the sphere formation (n = 3) and (h) migration (n = 5) of LS174T cells. (Sphere formation: 400×, Migration: 200×). Scales bars = 200 μm (100×), 100 μm (200×), and 50 μm (400×). *P < 0.05, **P < 0.01, ***P < 0.001, ns no significance.