Cadherin adhesion complexes direct cell aggregation in the epithelial transition of Wnt-induced nephron progenitor cells.

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a ß-catenin (Ctnnb1)-driven, transcriptional nephrogenic program and the mesenchymal to epithelial transition (MET) of NPCs. Using an in vitro mouse NPC culture model, we observed that activation of the Wnt pathway results in the aggregation of induced NPCs, which is an initiating step in the MET program. Genetic removal showed aggregation was dependent on ß-catenin. Modulating extracellular Ca2+ levels showed cell-cell contacts were Ca2+ dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2, Cdh4 and Cdh11 in NPCs, and the ß-catenin directed upregulation of Cdh3 and Cdh4 accompanying the MET of induced NPCs. Mutational analysis of ß-catenin supported a role for a Lef/Tcf-ß-catenin-mediated transcriptional response in the cell aggregation process. Genetic removal of all four cadherins, and independent removal of α-catenin or of ß-catenin-α-catenin interactions, abolished aggregation, but not the inductive response to Wnt pathway activation. These findings, and data in an accompanying article highlight the role of ß-catenin in linking transcriptional programs to the morphogenesis of NPCs in mammalian nephrogenesis.

Combinatorial Wnt signaling landscape during brachiopod anteroposterior patterning.

BACKGROUND: Wnt signaling pathways play crucial roles in animal development. They establish embryonic axes, specify cell fates, and regulate tissue morphogenesis from the early embryo to organogenesis. It is becoming increasingly recognized that these distinct developmental outcomes depend upon dynamic interactions between multiple ligands, receptors, antagonists, and other pathway modulators, consolidating the view that a combinatorial "code" controls the output of Wnt signaling. However, due to the lack of comprehensive analyses of Wnt components in several animal groups, it remains unclear if specific combinations always give rise to specific outcomes, and if these combinatorial patterns are conserved throughout evolution. RESULTS: In this work, we investigate the combinatorial expression of Wnt signaling components during the axial patterning of the brachiopod Terebratalia transversa. We find that T. transversa has a conserved repertoire of ligands, receptors, and antagonists. These genes are expressed throughout embryogenesis but undergo significant upregulation during axial elongation. At this stage, Frizzled domains occupy broad regions across the body while Wnt domains are narrower and distributed in partially overlapping patches; antagonists are mostly restricted to the anterior end. Based on their combinatorial expression, we identify a series of unique transcriptional subregions along the anteroposterior axis that coincide with the different morphological subdivisions of the brachiopod larval body. When comparing these data across the animal phylogeny, we find that the expression of Frizzled genes is relatively conserved, whereas the expression of Wnt genes is more variable. CONCLUSIONS: Our results suggest that the differential activation of Wnt signaling pathways may play a role in regionalizing the anteroposterior axis of brachiopod larvae. More generally, our analyses suggest that changes in the receptor context of Wnt ligands may act as a mechanism for the evolution and diversification of the metazoan body axis.

NOTUM-mediated WNT silencing drives extravillous trophoblast cell lineage development.

Trophoblast stem (TS) cells have the unique capacity to differentiate into specialized cell types, including extravillous trophoblast (EVT) cells. EVT cells invade into and transform the uterus where they act to remodel the vasculature facilitating the redirection of maternal nutrients to the developing fetus. Disruptions in EVT cell development and function are at the core of pregnancy-related disease. WNT-activated signal transduction is a conserved regulator of morphogenesis of many organ systems, including the placenta. In human TS cells, activation of canonical WNT signaling is critical for maintenance of the TS cell stem state and its downregulation accompanies EVT cell differentiation. We show that aberrant WNT signaling undermines EVT cell differentiation. Notum, palmitoleoyl-protein carboxylesterase (NOTUM), a negative regulator of canonical WNT signaling, was prominently expressed in first-trimester EVT cells developing in situ and up-regulated in EVT cells derived from human TS cells. Furthermore, NOTUM was required for optimal human TS cell differentiation to EVT cells. Activation of NOTUM in EVT cells is driven, at least in part, by endothelial Per-Arnt-Sim (PAS) domain 1 (also called hypoxia-inducible factor 2 alpha). Collectively, our findings indicate that canonical Wingless-related integration site (WNT) signaling is essential for maintenance of human trophoblast cell stemness and regulation of human TS cell differentiation. Downregulation of canonical WNT signaling via the actions of NOTUM is required for optimal EVT cell differentiation.

Identification of a pathogenic variant and pre-implantation genetic testing for a Chinese family affected with split-hand/foot malformation.

Split-hand/foot malformation is a serious congenital limb malformation characterized by syndactyly and underdevelopment of the phalanges and metatarsals. In this study, we reported a case of a fetus with hand-foot cleft deformity. Whole exome and Sanger sequencing were used to filter out candidate gene mutation sites and provide pre-implantation genetic testing(PGT) for family members. Genetic testing results showed that there was a homozygous mutation c.786G>A (p.Trp262*) in the fetal WNT10B, and both parents were carriers of heterozygous mutations. PGT results showed that out of the two blastocysts, one was a heterozygous mutant and the other was a homozygous mutant. All the embryos had diploid chromosomes. The heterozygous embryo was transferred, and a singleton pregnancy was successfully achieved. This study suggests that homozygous mutations in WNT10B are the likely cause of hand-foot clefts in this family. For families with monogenic diseases, preimplantation genetic testing can effectively prevent the birth of an affected child only after identifying the pathogenic mutation.

Wnt7B enhances the osteogenic differentiation of lipopolysaccharide-stimulated human periodontal ligament stem cells and inhibits the M1 polarization of macrophages by binding FZD4.

Periodontitis, a common oral disease characterized by the progressive infiltration of bacteria, is a leading cause of adult tooth loss. Periodontal stem cells (PDLSCs) possess good self­renewal and multi­potential differentiation abilities to maintain the integrity of periodontal support structure and repair defects. The present study aimed to analyze the roles of Wnt7B and frizzled4 (FZD4) in the osteogenic differentiation and macrophage polarization during periodontitis using an in vitro cell model. First, Wnt7B expression in the periodontitis­affected gingival tissue of patients and lipopolysaccharide (LPS)­stimulated PDLSCs was assessed using the GSE23586 dataset and western blot analysis, respectively. In Wnt7B­overexpressing PDLSCs exposed to LPS, the capacity of osteogenic differentiation was evaluated by detecting alkaline phosphatase activity, the level of Alizarin Red S staining and the expression of genes related to osteogenic differentiation. Subsequently, conditioned medium from PDLSCs overexpressing Wnt7B was used for M0 macrophage culture. The expression of CD86 and INOS was examined using immunofluorescence staining and western blot analysis. In addition, reverse transcription­quantitative PCR was employed to examine the expression of TNF­α, IL­6 and IL­1ß in macrophages. The binding between Wnt7B and FZD4 was estimated using co­immunoprecipitation. In addition, FZD4 was silenced to perform the rescue experiments to elucidate the regulatory mechanism between Wnt7B and FZD4. The results demonstrated a decreased expression of Wnt7B in periodontitis­affected gingival tissue and in LPS­exposed PDLSCs. Wnt7B overexpression promoted the osteogenic differentiation of LPS­exposed PDLSCs and suppressed the M1 polarization of macrophages. Additionally, Wnt7B bound to FZD4 and upregulated FZD4 expression. FZD4 silencing reversed the effects of Wnt7B overexpression on the osteogenic differentiation in LPS­exposed PDLSCs and the M1 polarization of macrophages. In summary, Wnt7B plays an anti­periodontitis role by binding FZD4 to strengthen the osteogenic differentiation of LPS­stimulated PDLSCs and suppress the M1 polarization of macrophages.

Lineage tracing using Wnt2b-2A-CreERT2 knock-in mice reveals the contributions of Wnt2b-expressing cells to novel subpopulations of mesothelial/epicardial cell lineages during mouse development.

Mesothelial and epicardial cells give rise to various types of mesenchymal cells via epithelial (mesothelial)-to-mesenchymal transition during development. However, the genes controlling the differentiation and diversification of mesothelial/epicardial cells remain unclear. Here, we examined Wnt2b expression in the embryonic mesothelium and epicardium and performed lineage tracing of Wnt2b-expressing cells by using novel Wnt2b-2A-CreERT2 knock-in and LacZ-reporter mice. Wnt2b was expressed in mesothelial cells covering visceral organs, but the expression was restricted in their subpopulations. Wnt2b-expressing cells labeled at embryonic day (E) 10.5 were distributed to the mesothelium and mesenchyme in the lungs, abdominal wall, stomach, and spleen in Wnt2b2A-CreERT2/+;R26RLacZ/+ mice at E13.0. Wnt2b was initially expressed in the proepicardial organ (PEO) at E9.5 and then in the epicardium after E10.0. Wnt2b-expressing PEO cells labeled at E9.5 differentiated into a small fraction of cardiac fibroblasts and preferentially localized at the left side of the postnatal heart. LacZ+ epicardium-derived cells labeled at E10.5 differentiated into a small fraction of fibroblasts and smooth muscle cells in the postnatal heart. Taken together, our results reveal novel subpopulations of PEO and mesothelial/epicardial cells that are distinguishable by Wnt2b expression and elucidate the unique contribution of Wnt2b-expressing PEO and epicardial cells to the postnatal heart.

Wnt7b acts in concert with Wnt5a to regulate tissue elongation and planar cell polarity via noncanonical Wnt signaling.

Intercellular signaling mediated by evolutionarily conserved planar cell polarity (PCP) proteins aligns cell polarity along the tissue plane and drives polarized cell behaviors during tissue morphogenesis. Accumulating evidence indicates that the vertebrate PCP pathway is regulated by noncanonical, ß-catenin-independent Wnt signaling; however, the signaling components and mechanisms are incompletely understood. In the mouse hearing organ, both PCP and noncanonical Wnt (ncWnt) signaling are required in the developing auditory sensory epithelium to control cochlear duct elongation and planar polarity of resident sensory hair cells (HCs), including the shape and orientation of the stereociliary hair bundle essential for sound detection. We have recently discovered a Wnt/G-protein/PI3K pathway that coordinates HC planar polarity and intercellular PCP signaling. Here, we identify Wnt7b as a ncWnt ligand acting in concert with Wnt5a to promote tissue elongation in diverse developmental processes. In the cochlea, Wnt5a and Wnt7b are redundantly required for cochlear duct coiling and elongation, HC planar polarity, and asymmetric localization of core PCP proteins Fzd6 and Dvl2. Mechanistically, Wnt5a/Wnt7b-mediated ncWnt signaling promotes membrane recruitment of Daple, a nonreceptor guanine nucleotide exchange factor for Gαi, and activates PI3K/AKT and ERK signaling, which promote asymmetric Fzd6 localization. Thus, ncWnt and PCP signaling pathways have distinct mutant phenotypes and signaling components, suggesting that they act as separate, parallel pathways with nonoverlapping functions in cochlear morphogenesis. NcWnt signaling drives tissue elongation and reinforces intercellular PCP signaling by regulating the trafficking of PCP-specific Frizzled receptors.

Investigating the WNT and TGF-beta pathways alterations and tumor mutant burden in young-onset colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer in the United States. Recent epidemiological evidence demonstrates an increasing incidence of young-onset CRC cases, defined as CRC cases in individuals 50 years old or younger. Studies have established that alterations in both the WNT and TGF-Beta signaling pathways have contributed to CRC development. While this is well understood, the comprehensive analysis of WNT and TGF-Beta pathway alterations in young-onset CRC cases has yet to be investigated. Here, we conducted a comprehensive bioinformatics analysis of mutations associated with each of the WNT and TGF-Beta signaling pathways according to age (≤ 50 years old versus > 50 years old) utilizing published genomic data from the cBioPortal. Chi-square results demonstrated no significant difference in WNT alterations between young-onset CRC and those > 50 years old. However, across all age groups, WNT alterations were frequently found in rectal cancers. We also found that WNT alterations were associated with better outcomes. The mutations associated with TGF-beta were observed at a higher rate in older CRC patients when compared to those ≤ 50 years old. Additionally, these mutations were found more frequently in colon primaries.

WNT2B high­expressed fibroblasts induce the fibrosis of IBD by promoting NK cells secreting IL-33.

Fibrosis is an important pathological change in inflammatory bowel disease (IBD), but the mechanism has yet to be elucidated. WNT2B high­expressed fibroblasts are enriched in IBD intestinal tissues, although the precise function of this group of fibroblasts remains unclear. This study investigated whether WNT2B high­expressed fibroblasts aggravated intestinal tissue damage and fibrosis. Our study provides evidence that WNT2B high­expressed fibroblasts and NK cells were enriched in colitis tissue of patients with IBD. WNT2B high­expressed fibroblasts secreted wnt2b, which bound to FZD4 on NK cells and activated the NF-κB and STAT3 pathways to enhance IL-33 expression. TCF4, a downstream component of the WNT/ß-catenin pathway, bound to p65 and promoted binding to IL-33 promoter. Furthermore, Salinomycin, an inhibitor of the WNT/ß-catenin pathway, inhibited IL-33 secretion in colitis, thereby reducing intestinal inflammation.Knocking down WNT2B reduces NK cell infiltration and IL-33 secretion in colitis, and reduce intestinal inflammation and fibrosis. In conclusion, WNT2B high­expressed fibroblasts activate NK cells by secreting wnt2b, which activates the WNT/ß-catenin and NF-κB pathways to promote IL-33 expression and secretion, potentially culminating in the induction of colonic fibrosis in IBD. KEY MESSAGES: WNT2B high-expressed fibroblasts and NK cells are enriched in colitis tissue, promoting NK cells secreting IL-33. Wnt2b activates NF-κB and STAT3 pathways promotes IL-33 expression by activating p65 and not STAT3. syndrome TCF4 binds to p65 and upregulates the NF- κB pathway. Salinomycin reduces NK cell infiltration and IL-33 secretion in colitis. Knocking down WNT2B mitigates inflammation and fibrosis in chronic colitis.

The expression pattern of Wnt6, Wnt10A, and HOXA13 during regenerating tails of Gekko Japonicus.

Wnt signal is crucial to correctly regenerate tissues along the original axis in many animals. Lizards are able to regenerate their tails spontaneously, while the anterior-posterior axis information required for the successful regeneration is still elusive. In this study, we investigated the expression pattern of Wnt ligands and HOX genes during regeneration. The results of in situ hybridization revealed that Wnt6 and Wnt10A mRNA levels are higher in wound epithelium (WE) than that in blastema during regeneration. In addition, we showed that Wnt agonist positively regulated the expression of HOXA13 in cultured blastema cells, while did not show similar effect on that of HOXB13, HOXC13 and HOXD13. Finally, we found that HOXA13 showed a gradient level along the anterior-posterior axis of regenerated blastema, with higher level at the caudal end. These data proposed that Wnt6, Wnt10A and HOXA13 might play an important role in establishing distal position for regeneration.

Positive cooperativity in synergistic activation of Wnt proteins.

BACKGROUND: Wnt proteins are crucial for embryonic development, stem cell growth, and tissue regeneration. Wnt signaling pathway is activated when Wnt proteins bind to cell membrane receptors. METHODS AND RESULTS: We employed a luciferase reporter assay in HEK293STF cells to measure Wnt protein-induced signaling. We observed that Wnt3a uniquely promotes the Wnt/ß-catenin pathway through positive cooperativity. Additionally, MFH-ND, a molecular mimic of Wnt ligands, markedly increased Wnt3a-induced signaling in a dose-responsive manner. This suggests that various Wnt ligands can synergistically enhance Wnt pathway activation. CONCLUSIONS: The study suggests the likelihood of various Wnt ligands coexisting in a single signalosome on the cell membrane, providing new insights into the complexities of Wnt signaling mechanisms.

Caenorhabditis elegans SEL-5/AAK1 regulates cell migration and cell outgrowth independently of its kinase activity.

During Caenorhabditis elegans development, multiple cells migrate long distances or extend processes to reach their final position and/or attain proper shape. The Wnt signalling pathway stands out as one of the major coordinators of cell migration or cell outgrowth along the anterior-posterior body axis. The outcome of Wnt signalling is fine-tuned by various mechanisms including endocytosis. In this study, we show that SEL-5, the C. elegans orthologue of mammalian AP2-associated kinase AAK1, acts together with the retromer complex as a positive regulator of EGL-20/Wnt signalling during the migration of QL neuroblast daughter cells. At the same time, SEL-5 in cooperation with the retromer complex is also required during excretory canal cell outgrowth. Importantly, SEL-5 kinase activity is not required for its role in neuronal migration or excretory cell outgrowth, and neither of these processes is dependent on DPY-23/AP2M1 phosphorylation. We further establish that the Wnt proteins CWN-1 and CWN-2, together with the Frizzled receptor CFZ-2, positively regulate excretory cell outgrowth, while LIN-44/Wnt and LIN-17/Frizzled together generate a stop signal inhibiting its extension.

Her4.3+ radial glial cells maintain the brain vascular network through activation of Wnt signaling.

During vascular development, radial glial cells (RGCs) regulate vascular patterning in the trunk and contribute to the early differentiation of the blood-brain barrier. Ablation of RGCs results in excessive sprouting vessels or the absence of bilateral vertebral arteries. However, interactions of RGCs with later brain vascular networks after pattern formation remain unknown. Here, we generated a her4.3 transgenic line to label RGCs and applied the metronidazole/nitroreductase system to ablate her4.3+ RGCs. The ablation of her4.3+ RGCs led to the collapse of the cerebral vascular network, disruption of the blood-brain barrier, and downregulation of Wnt signaling. The inhibition of Wnt signaling resulted in the collapse of cerebral vasculature, similar to that caused by her4.3+ RGC ablation. The defects in the maintenance of brain vasculature resulting from the absence of her4.3+ RGCs were partially rescued by the activation of Wnt signaling or overexpression of Wnt7aa or Wnt7bb. Together, our study suggests that her4.3+ RGCs maintain the cerebral vascular network through Wnt signaling.

Molecular signaling directing neural plate border formation.

During embryonic development, the vertebrate embryonic epiblast is divided into two parts including neural and superficial ectoderm. The neural plate border (NPB) is a narrow transitional area which locates between these parts and contains multipotent progenitor cells. Despite its small size, the cellular heterogeneity in this region produces specific differentiated cells. Signaling pathways, transcription factors, and the expression/repression of certain genes are directly involved in these differentiation processes. Different factors such as the Wnt signaling cascade, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) signaling, and Notch, which are involved in various stages of the growth, proliferation, and differentiation of embryonic cells, are also involved in the determination and differentiation of neural plate border stem cells. Therefore, it is essential to consider the interactions and temporospatial coordination related to cells, tissues, and adjacent structures. This review examines our present knowledge of the formation of the neural plate border and emphasizes the requirement for interaction between different signaling pathways, including the BMP and Wnt cascades, the expression of its special target genes and their regulations, and the precise tissue crosstalk which defines the neural crest fate in the ectoderm at the early human embryonic stages.

Dorsomorphin inhibits AMPK, upregulates Wnt and Foxo genes and promotes the activation of dormant follicles.

AMPK is a well-known energy sensor regulating cellular metabolism. Metabolic disorders such as obesity and diabetes are considered detrimental factors that reduce fecundity. Here, we show that pharmacologically induced in vitro activation (by metformin) or inhibition (by dorsomorphin) of the AMPK pathway inhibits or promotes activation of ovarian primordial follicles in cultured murine ovaries and human ovarian cortical chips. In mice, activation of primordial follicles in dorsomorphin in vitro-treated ovaries reduces AMPK activation and upregulates Wnt and FOXO genes, which, interestingly, is associated with decreased phosphorylation of ß-catenin. The dorsomorphin-treated ovaries remain of high quality, with no detectable difference in reactive oxygen species production, apoptosis or mitochondrial cytochrome c oxidase activity, suggesting safe activation. Subsequent maturation of in vitro-treated follicles, using a 3D alginate cell culture system, results in mature metaphase eggs with protruding polar bodies. These findings demonstrate that the AMPK pathway can safely regulate primordial follicles by modulating Wnt and FOXO genes, and reduce ß-catenin phosphorylation.

AR loss in prostate cancer stroma mediated by NF-κB and p38-MAPK signaling disrupts stromal morphogen production.

Androgen Receptor (AR) activity in prostate stroma is required to maintain prostate homeostasis. This is mediated through androgen-dependent induction and secretion of morphogenic factors that drive epithelial cell differentiation. However, stromal AR expression is lost in aggressive prostate cancer. The mechanisms leading to stromal AR loss and morphogen production are unknown. We identified TGFß1 and TNFα as tumor-secreted factors capable of suppressing AR mRNA and protein expression in prostate stromal fibroblasts. Pharmacological and RNAi approaches identified NF-κB as the major signaling pathway involved in suppressing AR expression by TNFα. In addition, p38α- and p38δ-MAPK were identified as suppressors of AR expression independent of TNFα. Two regions of the AR promoter were responsible for AR suppression through TNFα. FGF10 and Wnt16 were identified as androgen-induced morphogens, whose expression was lost upon TNFα treatment and enhanced upon p38-MAPK inhibition. Wnt16, through non-canonical Jnk signaling, was required for prostate basal epithelial cell survival. These findings indicate that stromal AR loss is mediated by secreted factors within the TME. We identified TNFα/TGFß as two possible factors, with TNFα mediating its effects through NF-κB or p38-MAPK to suppress AR mRNA transcription. This leads to loss of androgen-regulated stromal morphogens necessary to maintain normal epithelial homeostasis.

Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling.

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion.

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral to calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. Time-lapse light-sheet imaging of mouse embryos revealed calvarial progenitors intercalate in 3D in the CM above the eye, and exhibit protrusive and crawling activity more apically. CM cells express non-canonical Wnt/planar cell polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand Wnt5a-/- mutants have less dynamic cell rearrangements and protrusive activity. Loss of CM-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of Osx+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

WNT5B drives osteosarcoma stemness, chemoresistance and metastasis.

BACKGROUND: Treatment for osteosarcoma, a paediatric bone cancer with no therapeutic advances in over three decades, is limited by a lack of targeted therapies. Osteosarcoma frequently metastasises to the lungs, and only 20% of patients survive 5 years after the diagnosis of metastatic disease. We found that WNT5B is the most abundant WNT expressed in osteosarcoma tumours and its expression correlates with metastasis, histologic subtype and reduced survival. METHODS: Using tumor-spheroids to model cancer stem-like cells, we performed qPCR, immunoblotting, and immunofluorescence to monitor changes in gene and protein expression. Additionally, we measured sphere size, migration and forming efficiency to monitor phenotypic changes. Therefore, we characterised WNT5B's relevance to cancer stem-like cells, metastasis, and chemoresistance and evaluated its potential as a therapeutic target. RESULTS: In osteosarcoma cell lines and patient-derived spheres, WNT5B is enriched in stem cells and induces the expression of the stemness gene SOX2. WNT5B promotes sphere size, sphere-forming efficiency, and cell proliferation, migration, and chemoresistance to methotrexate (but not cisplatin or doxorubicin) in spheres formed from conventional cell lines and patient-derived xenografts. In vivo, WNT5B increased osteosarcoma lung and liver metastasis and inhibited the glycosaminoglycan hyaluronic acid via upregulation of hyaluronidase 1 (HYAL1), leading to changes in the tumour microenvironment. Further, we identified that WNT5B mRNA and protein correlate with the receptor ROR1 in primary tumours. Targeting WNT5B through inhibition of WNT/ROR1 signalling with an antibody to ROR1 reduced stemness properties, including chemoresistance, sphere size and SOX2 expression. CONCLUSIONS: Together, these data define WNT5B's role in driving osteosarcoma cancer stem cell expansion and methotrexate resistance and provide evidence that the WNT5B pathway is a promising candidate for treating osteosarcoma patients. KEY POINTS: WNT5B expression is high in osteosarcoma stem cells leading to increased stem cell proliferation and migration through SOX2. WNT5B expression in stem cells increases rates of osteosarcoma metastasis to the lungs and liver in vivo. The hyaluronic acid degradation enzyme HYAL1 is regulated by WNT5B in osteosarcoma contributing to metastasis. Inhibition of WNT5B with a ROR1 antibody decreases osteosarcoma stemness.