The Intermucosal Connection between the Mouth and Gut in Commensal Pathobiont-Driven Colitis.

The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation in vivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic T cells.

Inflammatory bowel disease and carcinogenesis.

Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer mortality worldwide. Colitis-associated colorectal cancer (CAC) is a subtype of CRC associated with inflammatory bowel disease (IBD). It is well known that individuals with IBD have a 2-3 times higher risk of developing CRC than those who do not, rendering CAC a major cause of death in this group. Although the etiology and pathogenesis of CAC are incompletely understood, animal models of chronic inflammation and human cohort data indicate that changes in the intestinal environment, including host response dysregulation and gut microbiota perturbations, may contribute to the development of CAC. Genomic alterations are a hallmark of CAC, with patterns that are distinct from those in sporadic CRC. The discovery of the biological changes that underlie the development of CAC is ongoing; however, current data suggest that chronic inflammation in IBD increases the risk of developing CAC. Therefore, a deeper understanding of the precise mechanisms by which inflammation triggers genetic alterations and disrupts intestinal homeostasis may provide insight into novel therapeutic strategies for the prevention of CAC.

The Butyrate-Producing Bacterium Clostridium butyricum Suppresses Clostridioides difficile Infection via Neutrophil- and Antimicrobial Cytokine-Dependent but GPR43/109a-Independent Mechanisms.

Short-chain fatty acids, such as butyrate, are major gut microbial metabolites that are beneficial for gastrointestinal health. Clostridium butyricum MIYAIRI588 (CBM588) is a bacterium that produces a robust amount of butyrate and therefore has been used as a live biotherapeutic probiotic in clinical settings. Clostridioides difficile causes life-threatening diarrhea and colitis. The gut resident microbiota plays a critical role in the prevention of C. difficile infection (CDI), as the disruption of the healthy microbiota by antibiotics greatly increases the risk for CDI. We report that CBM588 treatment in mice significantly improved clinical symptoms associated with CDI and increased the number of neutrophils and Th1 and Th17 cells in the colonic lamina propria in the early phase of CDI. The protective effect of CBM588 was abolished when neutrophils, IFN-γ, or IL-17A were depleted, suggesting that induction of the immune reactants is required to elicit the protective effect of the probiotic. The administration of tributyrin, which elevates the concentration of butyrate in the colon, also increased the number of neutrophils in the colonic lamina propria, indicating that butyrate is a potent booster of neutrophil activity during infection. However, GPR43 and GPR109a, two G protein-coupled receptors activated by butyrate, were dispensable for the protective effect of CBM588. These results indicate that CBM588 and butyrate suppress CDI, in part by boosting antimicrobial innate and cytokine-mediated immunity.

Quantitative proteomics identifies STEAP4 as a critical regulator of mitochondrial dysfunction linking inflammation and colon cancer.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder and is a major risk factor for colorectal cancer (CRC). Hypoxia is a feature of IBD and modulates cellular and mitochondrial metabolism. However, the role of hypoxic metabolism in IBD is unclear. Because mitochondrial dysfunction is an early hallmark of hypoxia and inflammation, an unbiased proteomics approach was used to assess the mitochondria in a mouse model of colitis. Through this analysis, we identified a ferrireductase: six-transmembrane epithelial antigen of prostate 4 (STEAP4) was highly induced in mouse models of colitis and in IBD patients. STEAP4 was regulated in a hypoxia-dependent manner that led to a dysregulation in mitochondrial iron balance, enhanced reactive oxygen species production, and increased susceptibility to mouse models of colitis. Mitochondrial iron chelation therapy improved colitis and demonstrated an essential role of mitochondrial iron dysregulation in the pathogenesis of IBD. To address if mitochondrial iron dysregulation is a key mechanism by which inflammation impacts colon tumorigenesis, STEAP4 expression, function, and mitochondrial iron chelation were assessed in a colitis-associated colon cancer model (CAC). STEAP4 was increased in human CRC and predicted poor prognosis. STEAP4 and mitochondrial iron increased tumor number and burden in a CAC model. These studies demonstrate the importance of mitochondrial iron homeostasis in IBD and CRC.

Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer.

Tumor angiogenesis research and antiangiogenic drug development make use of cultured endothelial cells (ECs) including the human microvascular ECs among others. However, it has been reported that tumor ECs (TECs) are different from normal ECs (NECs). To functionally validate antiangiogenic drugs, cultured TECs are indispensable tools, but are not commercially available. Primary human TECs are available only in small quantities from surgical specimens and have a short life span in vitro due to their cellular senescence. We established immortalized human TECs (h-imTECs) and their normal counterparts (h-imNECs) by infection with lentivirus producing simian virus 40 large T antigen and human telomerase reverse transcriptase to overcome the replication barriers. These ECs exhibited an extended life span and retained their characteristic endothelial morphology, expression of endothelial marker, and ability of tube formation. Furthermore, h-imTECs showed their specific characteristics as TECs, such as increased proliferation and upregulation of TEC markers. Treatment with bevacizumab, an antiangiogenic drug, dramatically decreased h-imTEC survival, whereas the same treatment failed to alter immortalized NEC survival. Hence, these h-imTECs could be a valuable tool for drug screening to develop novel therapeutic agents specific to TECs or functional biological assays in tumor angiogenesis research.

Increased Expression of DUOX2 Is an Epithelial Response to Mucosal Dysbiosis Required for Immune Homeostasis in Mouse Intestine.

BACKGROUND & AIMS: Dual oxidase 2 (DUOX2), a hydrogen-peroxide generator at the apical membrane of gastrointestinal epithelia, is up-regulated in patients with inflammatory bowel disease (IBD) before the onset of inflammation, but little is known about its effects. We investigated the role of DUOX2 in maintaining mucosal immune homeostasis in mice. METHODS: We analyzed the regulation of DUOX2 in intestinal tissues of germ-free vs conventional mice, mice given antibiotics or colonized with only segmented filamentous bacteria, mice associated with human microbiota, and mice with deficiencies in interleukin (IL) 23 and IL22 signaling. We performed 16S ribosomal RNA gene quantitative polymerase chain reaction of intestinal mucosa and mesenteric lymph nodes of Duoxa(-/-) mice that lack functional DUOX enzymes. Genes differentially expressed in Duoxa(-/-) mice compared with co-housed wild-type littermates were correlated with gene expression changes in early-stage IBD using gene set enrichment analysis. RESULTS: Colonization of mice with segmented filamentous bacteria up-regulated intestinal expression of DUOX2. DUOX2 regulated redox signaling within mucosa-associated microbes and restricted bacterial access to lymphatic tissues of the mice, thereby reducing microbiota-induced immune responses. Induction of Duox2 transcription by microbial colonization did not require the mucosal cytokines IL17 or IL22, although IL22 increased expression of Duox2. Dysbiotic, but not healthy human microbiota, activated a DUOX2 response in recipient germ-free mice that corresponded to abnormal colonization of the mucosa with distinct populations of microbes. In Duoxa(-/-) mice, abnormalities in ileal mucosal gene expression at homeostasis recapitulated those in patients with mucosal dysbiosis. CONCLUSIONS: DUOX2 regulates interactions between the intestinal microbiota and the mucosa to maintain immune homeostasis in mice. Mucosal dysbiosis leads to increased expression of DUOX2, which might be a marker of perturbed mucosal homeostasis in patients with early-stage IBD.

GLI2 is a novel therapeutic target for metastasis of osteosarcoma.

Aberrant activation of the Hedgehog (Hh) pathway has been reported in several malignancies. We previously demonstrated that knockdown of GLI2 inhibited proliferation of osteosarcoma cells through regulation of the cell cycle. In this study, we analyzed the function of GLI2 in the pathogenesis of osteosarcoma metastasis. Immunohistochemical studies showed that GLI2 was overexpressed in patient osteosarcoma specimens. Knockdown of GLI2 inhibited migration and invasion of osteosarcoma cells. In contrast, the forced expression of constitutively active GLI2 in mesenchymal stem cells promoted invasion. In addition, xenograft models showed that knockdown of GLI2 decreased lung metastasis of osteosarcomas. To examine clinical applications, we evaluated the efficacy of arsenic trioxide (ATO), which is a Food and Drug Administration-approved antitumor drug, on osteosarcoma cells. ATO treatment suppressed the invasiveness of osteosarcoma cells by inhibiting the transcriptional activity of GLI2. In addition, the combination of Hh inhibitors including ATO, vismodegib and GANT61 prevented migration and metastasis of osteosarcoma cells. Consequently, our findings suggested that GLI2 regulated metastasis as well as the progression of osteosarcomas. Inhibition of the GLI2 transcription may be an effective therapeutic method for preventing osteosarcoma metastasis.

Suprabasin as a novel tumor endothelial cell marker.

Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker.

Adherent-invasive E. coli - induced specific IgA limits pathobiont localization to the epithelial niche in the gut.

Background and aim: Adherent-invasive E. coli (AIEC) has been identified as a pathobiont associated with Crohn's disease (CD), that prefers to grow in inflammatory conditions. Although the colonization by AIEC is implicated in the progression of the disease and exacerbates inflammation in murine colitis models, the recognition and response of host immunity to AIEC remains elusive. Methods: Antibiotic treated female C57BL/6 mice were inoculated by commensal E. coli and LF82 AIEC strains. Luminal-IgA fractions were prepared from feces and their binding to AIEC and other strains was assessed to confirm specificity. IgA binding to isogenic mutant strains was performed to identify the functional molecules that are recognized by AIEC specific IgA. The effect of IgA on epithelial invasion of LF82 strain was confirmed using in vitro invasion assay and in vivo colonization of the colonic epithelium. Results: Persistent colonization by AIEC LF82 induced secretion of luminal IgA, while commensal E. coli strain did not. Induced anti-LF82 IgA showed specific binding to other AIEC strains but not to the commensal, non-AIEC E. coli strains. Induced IgA showed decreased binding to LF82 strains with mutated adhesin and outer membrane proteins which are involved in AIEC - epithelial cell interaction. Consistently, LF82-specific IgA limited the adhesion and invasion of LF82 in cultured epithelial cells, which seems to be required for the elimination in the colonic epithelium in mice. Conclusion: These results demonstrate that host immunity selectively recognizes pathobiont E. coli, such as AIEC, and develop specific IgA. The induced IgA specific to pathobiont E. coli, in turn, contributes to preventing the pathobionts from accessing the epithelium.

Mucolytic bacteria license pathobionts to acquire host-derived nutrients during dietary nutrient restriction.

Pathobionts employ unique metabolic adaptation mechanisms to maximize their growth in disease conditions. Adherent-invasive Escherichia coli (AIEC), a pathobiont enriched in the gut mucosa of patients with inflammatory bowel disease (IBD), utilizes diet-derived L-serine to adapt to the inflamed gut. Therefore, the restriction of dietary L-serine starves AIEC and limits its fitness advantage. Here, we find that AIEC can overcome this nutrient limitation by switching the nutrient source from the diet to the host cells in the presence of mucolytic bacteria. During diet-derived L-serine restriction, the mucolytic symbiont Akkermansia muciniphila promotes the encroachment of AIEC to the epithelial niche by degrading the mucus layer. In the epithelial niche, AIEC acquires L-serine from the colonic epithelium and thus proliferates. Our work suggests that the indirect metabolic network between pathobionts and commensal symbionts enables pathobionts to overcome nutritional restriction and thrive in the gut.

A potential pathogenic association between periodontal disease and Crohn's disease.

Oral conditions are relatively common in patients with inflammatory bowel disease (IBD). However, the contribution of oral maladies to gut inflammation remains unexplored. Here, we investigated the effect of periodontitis on disease phenotypes of patients with IBD. In all, 60 patients with IBD (42 with ulcerative colitis [UC] and 18 with Crohn's disease [CD]) and 45 healthy controls (HCs) without IBD were recruited for this clinical investigation. The effects of incipient periodontitis on the oral and gut microbiome as well as IBD characteristics were examined. In addition, patients were prospectively monitored for up to 12 months after enrollment. We found that, in both patients with UC and those with CD, the gut microbiome was significantly more similar to the oral microbiome than in HCs, suggesting that ectopic gut colonization by oral bacteria is increased in patients with IBD. Incipient periodontitis did not further enhance gut colonization by oral bacteria. The presence of incipient periodontitis did not significantly affect the clinical outcomes of patients with UC and CD. However, the short CD activity index increased in patients with CD with incipient periodontitis but declined or was unchanged during the study period in patients without periodontitis. Thus, early periodontitis may associate with worse clinically symptoms in some patients with CD.

DUOX2 variants associate with preclinical disturbances in microbiota-immune homeostasis and increased inflammatory bowel disease risk.

A primordial gut-epithelial innate defense response is the release of hydrogen peroxide by dual NADPH oxidase (DUOX). In inflammatory bowel disease (IBD), a condition characterized by an imbalanced gut microbiota-immune homeostasis, DUOX2 isoenzyme is the highest induced gene. Performing multiomic analyses using 2872 human participants of a wellness program, we detected a substantial burden of rare protein-altering DUOX2 gene variants of unknown physiologic significance. We identified a significant association between these rare loss-of-function variants and increased plasma levels of interleukin-17C, which is induced also in mucosal biopsies of patients with IBD. DUOX2-deficient mice replicated increased IL-17C induction in the intestine, with outlier high Il17c expression linked to the mucosal expansion of specific Proteobacteria pathobionts. Integrated microbiota/host gene expression analyses in patients with IBD corroborated IL-17C as a marker for epithelial activation by gram-negative bacteria. Finally, the impact of DUOX2 variants on IL-17C induction provided a rationale for variant stratification in case control studies that substantiated DUOX2 as an IBD risk gene. Thus, our study identifies an association of deleterious DUOX2 variants with a preclinical hallmark of disturbed microbiota-immune homeostasis that appears to precede the manifestation of IBD.

Interleukin-22-mediated host glycosylation prevents Clostridioides difficile infection by modulating the metabolic activity of the gut microbiota.

The involvement of host immunity in the gut microbiota-mediated colonization resistance to Clostridioides difficile infection (CDI) is incompletely understood. Here, we show that interleukin (IL)-22, induced by colonization of the gut microbiota, is crucial for the prevention of CDI in human microbiota-associated (HMA) mice. IL-22 signaling in HMA mice regulated host glycosylation, which enabled the growth of succinate-consuming bacteria Phascolarctobacterium spp. within the gut microbiome. Phascolarctobacterium reduced the availability of luminal succinate, a crucial metabolite for the growth of C. difficile, and therefore prevented the growth of C. difficile. IL-22-mediated host N-glycosylation is likely impaired in patients with ulcerative colitis (UC) and renders UC-HMA mice more susceptible to CDI. Transplantation of healthy human-derived microbiota or Phascolarctobacterium reduced luminal succinate levels and restored colonization resistance in UC-HMA mice. IL-22-mediated host glycosylation thus fosters the growth of commensal bacteria that compete with C. difficile for the nutritional niche.

Dietary L-serine confers a competitive fitness advantage to Enterobacteriaceae in the inflamed gut.

Metabolic reprogramming is associated with the adaptation of host cells to the disease environment, such as inflammation and cancer. However, little is known about microbial metabolic reprogramming or the role it plays in regulating the fitness of commensal and pathogenic bacteria in the gut. Here, we report that intestinal inflammation reprograms the metabolic pathways of Enterobacteriaceae, such as Escherichia coli LF82, in the gut to adapt to the inflammatory environment. We found that E. coli LF82 shifts its metabolism to catabolize L-serine in the inflamed gut in order to maximize its growth potential. However, L-serine catabolism has a minimal effect on its fitness in the healthy gut. In fact, the absence of genes involved in L-serine utilization reduces the competitive fitness of E. coli LF82 and Citrobacter rodentium only during inflammation. The concentration of luminal L-serine is largely dependent on dietary intake. Accordingly, withholding amino acids from the diet markedly reduces their availability in the gut lumen. Hence, inflammation-induced blooms of E. coli LF82 are significantly blunted when amino acids-particularly L-serine-are removed from the diet. Thus, the ability to catabolize L-serine increases bacterial fitness and provides Enterobacteriaceae with a growth advantage against competitors in the inflamed gut.

IL-10 produced by macrophages regulates epithelial integrity in the small intestine.

Macrophages (Mϕs) are known to be major producers of the anti-inflammatory cytokine interleukin-10 (IL-10) in the intestine, thus playing an important role in maintaining gastrointestinal homeostasis. Mϕs that reside in the small intestine (SI) have been previously shown to be regulated by dietary antigens, while colonic Mϕs are regulated by the microbiota. However, the role which resident Mϕs play in SI homeostasis has not yet been fully elucidated. Here, we show that SI Mϕs regulate the integrity of the epithelial barrier via secretion of IL-10. We used an animal model of non-steroidal anti-inflammatory drug (NSAID)-induced SI epithelial injury to show that IL-10 is mainly produced by MHCII+ CD64+ Ly6Clow Mϕs early in injury and that it is involved in the restoration of the epithelial barrier. We found that a lack of IL-10, particularly its secretion by Mϕs, compromised the recovery of SI epithelial barrier. IL-10 production by MHCII+ CD64+ Ly6Clow Mϕs in the SI is not regulated by the gut microbiota, hence depletion of the microbiota did not influence epithelial regeneration in the SI. Collectively, these results highlight the critical role IL-10-producing Mϕs play in recovery from intestinal epithelial injury induced by NSAID.

Flagellin-mediated activation of IL-33-ST2 signaling by a pathobiont promotes intestinal fibrosis.

Intestinal fibrosis is a severe complication in patients with Crohn's disease (CD). Unfortunately, the trigger leading to the development of intestinal fibrosis in the context of CD remains elusive. Here, we show that colonization by a CD-associated pathobiont adherent-invasive Escherichia coli (AIEC) promotes the development of intestinal fibrosis. Exogenously inoculated AIEC strain LF82 and commensal E. coli HS were gradually eradicated from the intestine in healthy mice. In Salmonella- or dextran sodium sulfate-induced colitis models, AIEC exploited inflammation and stably colonize the gut. Consequently, persistent colonization by AIEC LF82 led to substantial fibrosis. In contrast, commensal E. coli HS was unable to derive a growth advantage from inflammation, thereby failing to colonize the inflamed intestine or promote intestinal fibrosis. AIEC colonization potentiated the expression of the IL-33 receptor ST2 in the intestinal epithelium, which is crucial for the development of intestinal fibrosis. The induction of ST2 by AIEC LF82 was mediated by flagellin, as the ΔfliC mutant failed to induce ST2. These observations provide novel insights into pathobiont-driven intestinal fibrosis and can lead to the development of novel therapeutic approaches for the treatment of intestinal fibrosis in the context of CD that target AIEC and/or its downstream IL-33-ST2 signaling.

Host-microbial Cross-talk in Inflammatory Bowel Disease.

A vast community of commensal microorganisms, commonly referred to as the gut microbiota, colonizes the gastrointestinal tract (GI). The involvement of the gut microbiota in the maintenance of the gut ecosystem is two-fold: it educates host immune cells and protects the host from pathogens. However, when healthy microbial composition and function are disrupted (dysbiosis), the dysbiotic gut microbiota can trigger the initiation and development of various GI diseases, including inflammatory bowel disease (IBD). IBD, primarily includes ulcerative colitis (UC) and Crohn's disease (CD), is a major global public health problem affecting over 1 million patients in the United States alone. Accumulating evidence suggests that various environmental and genetic factors contribute to the pathogenesis of IBD. In particular, the gut microbiota is a key factor associated with the triggering and presentation of disease. Gut dysbiosis in patients with IBD is defined as a reduction of beneficial commensal bacteria and an enrichment of potentially harmful commensal bacteria (pathobionts). However, as of now it is largely unknown whether gut dysbiosis is a cause or a consequence of IBD. Recent technological advances have made it possible to address this question and investigate the functional impact of dysbiotic microbiota on IBD. In this review, we will discuss the recent advances in the field, focusing on host-microbial cross-talk in IBD.

Regulation of virulence: the rise and fall of gastrointestinal pathogens.

Colonization resistance by the commensal microbiota is a key defense against infectious pathogens in the gastrointestinal tract. The microbiota directly competes with incoming pathogens by occupying the colonization niche, depleting nutrients in the gut lumen as well as indirectly inhibiting the growth of pathogens through activation of host immunity. Enteric pathogens have evolved strategies to cope with microbiota-mediated colonization resistance. Pathogens utilize a wide array of virulence factors to outcompete their commensal rivals in the gut. However, since the expression of virulence factors is costly to maintain and reduces bacterial fitness, pathogens need to regulate their virulence properly in order to maximize their fitness. To this end, most pathogens use environmental cues to regulate their virulence gene expression. Thus, a dynamic regulation of virulence factor expression is a key invasion strategy utilized by enteric pathogens. On the other hand, host immunity selectively targets virulent pathogens in order to counter infection in the gut. The host immune system is generally tolerant of harmless microorganisms, such as the commensal microbiota. Moreover, the host relies on its commensal microbiota to contribute, in concert with its immune system, to the elimination of pathogens. Collectively, regulation of virulence determines the fate of enteric pathogens, from the establishment of infection to the eventual elimination. Here, we will review the dynamics of virulence and its role in infection.

Pathogenic role of the gut microbiota in gastrointestinal diseases.

The gastrointestinal (GI) tract is colonized by a dense community of commensal microorganisms referred to as the gut microbiota. The gut microbiota and the host have co-evolved, and they engage in a myriad of immunogenic and metabolic interactions. The gut microbiota contributes to the maintenance of host health. However, when healthy microbial structure is perturbed, a condition termed dysbiosis, the altered gut microbiota can trigger the development of various GI diseases including inflammatory bowel disease, colon cancer, celiac disease, and irritable bowel syndrome. There is a growing body of evidence suggesting that multiple intrinsic and extrinsic factors, such as genetic variations, diet, stress, and medication, can dramatically affect the balance of the gut microbiota. Therefore, these factors regulate the development and progression of GI diseases by inducing dysbiosis. Herein, we will review the recent advances in the field, focusing on the mechanisms through which intrinsic and extrinsic factors induce dysbiosis and the role a dysbiotic microbiota plays in the pathogenesis of GI diseases.

Combination of Hedgehog inhibitors and standard anticancer agents synergistically prevent osteosarcoma growth.

High-dose chemotherapy and surgical intervention have improved long-term prognosis for non-metastatic osteosarcoma to 50-80%. However, metastatic osteosarcoma exhibits resistance to standard chemotherapy. We and others have investigated the function of Hedgehog pathway in osteosarcoma. To apply our previous findings in clinical settings, we examined the effects of Hedgehog inhibitors including arsenic trioxide (ATO) and vismodegib combined with standard anticancer agents. We performed WST-1 assays using ATO, cisplatin (CDDP), ifosfamide (IFO), doxorubicin (DOX), and vismodegib. Combination-index (CI) was used to examine synergism using CalcuSyn software. Xenograft models were used to examine the synergism in vivo. WST-1 assays showed that 143B and Saos2 cell proliferation was inhibited by ATO combined with CDDP, IFO, DOX, and vismodegib. Combination of ATO and CDDP, IFO, DOX or vismodegib was synergistic when the two compounds were used on proliferating 143B and Saos2 human osteosarcoma cells. An osteosarcoma xenograft model showed that treatment with ATO and CDDP, IFO, or vismodegib significantly prevented osteosarcoma growth in vivo compared with vehicle treatment. Our findings indicate that combination of Hedgehog pathway inhibitors and standard FDA-approved anticancer agents with established safety for human use may be an attractive therapeutic method for treating osteosarcoma.