Bavachin Induces Ferroptosis through the STAT3/P53/SLC7A11 Axis in Osteosarcoma Cells.

Ferroptosis is a new form of regulated cell death, which is mediated by intracellular iron. Although it is reported that bavachin has antitumour effects on several tumour cells and prompts the reactive oxygen species (ROS) generation, it is unclear whether ferroptosis can be induced by bavachin in osteosarcoma (OS) cells. In this study, we found that bavachin inhibits the viability of MG63 and HOS OS cell lines along with an increase in the ferrous iron level, ROS accumulation, malondialdehyde overexpression, and glutathione depletion. Moreover, iron chelators (deferoxamine), antioxidants (Vit E), and ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) reverse bavachin-induced cell death. Bavachin also altered the mitochondrial morphology of OS cells, leading to smaller mitochondria, higher density of the mitochondrial membrane, and reduced mitochondrial cristae. Further investigation showed that bavachin upregulated the expression of transferrin receptor, divalent metal transporter-1, and P53, along with downregulating the expression of ferritin light chain, ferritin heavy chain, p-STAT3 (705), SLC7A11, and glutathione peroxidase-4 in OS cells. More importantly, STAT3 overexpression, SLC7A11 overexpression, and pretreatment with pifithrin-α (P53 inhibitor) rescued OS cell ferroptosis induced by bavachin. The results show that bavachin induces ferroptosis via the STAT3/P53/SLC7A11 axis in OS cells.

Scaffold-based 3D cellular models mimicking the heterogeneity of osteosarcoma stem cell niche.

The failure of the osteosarcoma conventional therapies leads to the growing need for novel therapeutic strategies. The lack of specificity for the Cancer Stem Cells (CSCs) population has been recently identified as the main limitation in the current therapies. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing and screening as well as in the study of cell and molecular biology, are affected by a poor in vitro-in vivo translation ability. To overcome these limitations, this work provides two tumour engineering approaches as new tools to address osteosarcoma and improve therapy outcomes. In detail, two different hydroxyapatite-based bone-mimicking scaffolds were used to recapitulate aspects of the in vivo tumour microenvironment, focusing on CSCs niche. The biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched-CSCs were deeply analysed in these complex cell culture models. The results highlight the fundamental role of the tumour microenvironment proving the mimicry of osteosarcoma stem cell niche by the use of CSCs together with the biomimetic scaffolds, compared to conventional 2D culture systems. These advanced 3D cell culture in vitro tumour models could improve the predictivity of preclinical studies and strongly enhance the clinical translation.

The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma.

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

Co-delivery of doxorubicin and paclitaxel by reduction/pH dual responsive nanocarriers for osteosarcoma therapy.

Nanoparticle-based drug delivery system offers a promising platform for combination cancer therapy. However, the inefficient drug release in cells reduces the therapeutic efficacy of cancer nanomedicines. Herein, a PEGylated poly(α-lipoic acid) copolymer (mPEG-PαLA) was prepared and used as a reduction/pH dual responsive nanocarrier to simultaneously deliver paclitaxel (PTX) and doxorubicin (DOX) for osteosarcoma therapy. The amphiphilic mPEG-PαLA could efficiently encapsulate both PTX and DOX during its self-assembly into micelles in aqueous solution to generate PTX and DOX co-loaded nanoparticles (NP-PTX-DOX). The as-prepared NP-PTX-DOX showed enhanced PTX and DOX release in response to reductive and acidic stimuli. Moreover, the dual-drug loaded nanoparticles were efficiently internalized by K7 osteosarcoma cells and released drugs intracellularly, as confirmed by flow cytometry analysis and confocal laser scanning microscopy. Consequently, NP-PTX-DOX exhibited synergistic therapeutic effects and induced enhanced cell apoptosis in K7 cells. Furthermore, NP-PTX-DOX presented improved biodistribution and higher tumor growth inhibition efficacy compared to the control groups in a murine osteosarcoma model. Altogether, the results of this work indicate that the proposed strategy is promising for osteosarcoma therapy using mPEG-PαLA copolymer as a dual-responsive nanocarrier to co-deliver anticancer drugs.

Myoepithelioma of bone: ultrastructural, immunohistochemical and molecular study of three cases.

Primary intraosseous myoepithelial tumors are rare neoplasms with only a handful of cases described in the medical literature. To date, intraosseous variant of benign myoepithelioma, due to its rarity, has not been studied ultrastructurally, and only one case of a malignant intraosseous myoepithelioma has been described. Three cases were retrieved from the files at the Massachusetts General Hospital (MGH). A diagnosis of benign myoepithelioma was made in case 1 and malignant epithelioma in cases 2 and 3. Ultrastructurally, intermediate filaments (without dense bodies) were found in each case with an abundance in case 1 and lesser amounts in cases 2 and 3. Surprisingly, cell junctions were not identified in case 1. However, they were found occasionally as intermediate junctions in case 2 and were easily identified as desmosome like junctions in case 3. The nucleus was irregular in the neoplastic cells of benign myoepithelioma which contrasted with cases 2 and 3 where the nuclei were oval yet had visible nucleoli. Herein, we add three new cases, including two new cases of malignant myoepithelioma. We also provide the first ultrastructural description of benign myoepithelioma of bone.

Roles of microRNA-22 in Suppressing Proliferation and Promoting Sensitivity of Osteosarcoma Cells via Metadherin-mediated Autophagy.

OBJECTIVE: To analyze the effect of microRNA-22 on autophagy and proliferation and to investigate the underlying molecular mechanism of osteosarcoma cell chemotherapy sensitivity. METHODS: MG-63 cells were divided into four groups, including a control group, a negative control (NC) group, a cisplatin group, and a cisplatin + miR-22 group. Proliferation of MG-63 cells that had been treated with cisplatin and transfected with miR-22 mimics was determined using MTT assay and colony formation assay. We assessed the degree of autophagy using flow cytometry through cellular staining of the autophagy lysosomal marker monodansylcadaverine (MDC). The effect of microRNA-22 on autophagy was observed along with the expression levels of Beclin1, LC3, metadherin (MTDH) and ATG5 by western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Luciferase reporter assay revealed the targeted binding site between miR-22 and the 3'-untranslated region (3'-UTR) of MTDH mRNA. Western blot and qRT-PCR were used to explore the level of MTDH in the control group, the NC group, the cisplatin group, and the miR-22 group for 6, 12, and 24 h. RESULTS: In the in vitro study, the MTT results indicated that the MG-63 cells with overexpression of miR-22 exhibited a significant decline in the proliferation capacity compared with the control group (0.513 ± 0.001, P < 0.0005). Similar to the MTT results, MG-63 cells that were transfected with miR-22 mimic (101.0 ± 10.58) formed fewer colonies compared with the cisplatin group (129.7 ± 4.163). MDC staining revealed that miR-22-overexpressing osteosarcoma (OS) cells treated with cisplatin showed a significant decrease in the expression of autophagy (7.747 ± 0.117, P < 0.0001). Our data revealed that miR-22 could regulate not only autophagy but also proliferation in the chemosensitivity of osteosarcoma cells. We found that miR-22 sensitized osteosarcoma cells to cisplatin treatment by regulating autophagy-related genes. In addition, Luciferase Reporter Assay revealed that miR-22 negatively regulated autophagy through direct targeting of MTDH. We performed western blot analysis to detect the MTDH expression level. The results revealed that the overexpression of miR-22 obviously decreased the expression of MTDH (1.081 ± 0.023, P < 0.001). CONCLUSION: Inhibition of miR-22 ameliorated the anticancer drug-induced cell proliferation decrease in osteosarcoma cells. MTDH was identified as the miR-22 target in OS cells and MTDH-triggered autophagy played a key function in the miR-22-associated chemotherapy sensitivity.

Paeoniflorin induces G2/M cell cycle arrest and caspase-dependent apoptosis through the upregulation of Bcl-2 X-associated protein and downregulation of B-cell lymphoma 2 in human osteosarcoma cells.

Paeoniflorin (PF), extracted from the peony root, has been proved to possess antineoplastic activity in different cancer cell lines. However, it remains unclear whether PF has an antineoplastic effect against osteosarcoma cells. The present study investigated the effects and the specific mechanism of PF on various human osteosarcoma cell lines. Using the multiple methods to detect the activity of PF on HOS and Saos­2 human osteosarcoma cell lines, including an MTS assay, flow cytometry, transmission electron microscopy and western blotting, it was demonstrated that PF induces inhibition of proliferation, G2/M phase cell cycle arrest and apoptosis in the osteosarcoma cell lines in vitro, and activation of cleaved­caspase­3 and cleaved­poly (ADPribose) polymerase in a dose­dependent manner. Furthermore, the pro­apoptotic factors Bcl­2 X­associated protein and BH3 interacting domain death agonist were uregulated, while the anti­apoptotic factors B­cell lymphoma 2 (Bcl­2) and Bcl­2­extra large were downregulated. In conclusion, these results demonstrated that PF has a promising therapeutic potential in for osteosarcoma.

The tumor microenvironment promotes cancer progression and cell migration.

The tumor microenvironment contributes to cancer progression, in part through interactions between tumor and normal stromal cells. This study analyzed morphological and molecular changes induced in co-cultured human fibroblasts (HFs) and the MG-63 osteosarcoma cell line. Co-cultured cell monolayers were morphologically analyzed using high resolution scanning electron microscopy (HR-SEM), and trans-well assays were performed to assess cell migration and invasion. Proteins involved in inflammatory responses, cancer cell invasion, and angiogenesis were assessed using western blotting. HR-SEM showed progressive spatial orientation loss by fibroblasts in contact with MG-63s, while MG-63s proliferated rapidly and invaded HF space. Trans-well assays showed enhanced MG-63 migration in the presence of HFs. IL-6 expression was increased in co-cultured HFs, possibly stimulated by TNF-α. HFs do not normally express YKL-40 but did so in co-culture. Band densitometric analyses showed that increasing YKL-40 expression was followed by VEGF overexpression, especially in MG-63s. Finally, our results confirmed fibroblasts as the main matrix metalloproteinase source in this tumor microenvironment. Our study sheds new light on how tumor-stroma interactions promote tumor development and progression, and may support identification of novel anti-cancer therapeutics.

The oncolytic peptide LTX-315 triggers necrotic cell death.

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.

High-performance thin-layer chromatography for the evaluation of voacamine intracellular concentration related to its cytotoxic effect.

Previous investigations demonstrated that pretreatment with non-cytotoxic concentrations of voacamine had a chemosensitizing effect on cultured multidrug resistant osteosarcoma cells exposed to doxorubicin; whereas when used alone at high concentrations voacamine induced apoptosis-independent cell death on both sensitive and resistant cells. To gain insight into the mechanism of action of voacamine at the subcellular level, we developed an analytical high-performance thin-layer chromatography technique to assess the intracellular content of voacamine that could be correlated with the induction of cell death and consequent morphological and ultrastructural changes. The results of the quantitative analysis not only did allow us to measure both the amount of unmodified voacamine molecules (determined by the method) and the amount of molecules which reacted with cellular components (undetectable), but also to confirm the findings of our previous studies and support the validity of this method.

Diffusion-weighted imaging and dynamic contrast-enhanced MRI of experimental breast cancer bone metastases--a correlation study with histology.

PURPOSE: To validate imaging parameters from diffusion-weighted imaging and dynamic contrast-enhanced MRI with immunohistology and to non-invasively assess microstructure of experimental breast cancer bone metastases. MATERIALS AND METHODS: Animals bearing breast cancer bone metastases were imaged in a clinical 1.5 T MRI scanner. HASTE sequences were performed to calculate apparent diffusion coefficients. Saturation recovery turbo FLASH sequences were conducted while infusing 0.1 mmol/l Gd-DTPA for dynamic contrast-enhanced MRI to quantify parameters amplitude A and exchange rate constant kep. After imaging, bone metastases were analyzed immunohistologically. RESULTS: We found correlations of the apparent diffusion coefficients from diffusion-weighted imaging with tumor cellularity as assessed with cell nuclei staining. Histological vessel maturity was correlated negatively with parameters A and kep from dynamic contrast-enhanced MRI. Tumor size correlated inversely with cell density and vessel permeability as well as positively with mean vessel calibers. Parameters from the rim of bone metastases differed significantly from values of the center. CONCLUSION: In vivo diffusion-weighted imaging and dynamic contrast-enhanced MRI in experimental bone metastases provide information about tumor cellularity and vascularity and correlate well with immunohistology.

Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.

In vitro ultrastructural changes of MCF-7 for metastasise bone cancer and induction of apoptosis via mitochondrial cytochrome C released by CaCO3/Dox nanocrystals.

Bones are the most frequent site for breast cancer cells to settle and spread (metastasise); bone metastasis is considered to have a substantial impact on the quality of patients with common cancers. However, majority of breast cancers develop insensitivity to conventional chemotherapy which provides only palliation and can induce systemic side effects. In this study we evaluated the effect of free Dox and CaCO3/Dox nanocrystal on MCF-7 breast cancer using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), neural red, and lactate dehydrogenase colorimetric assays while DNA fragmentation and BrdU genotoxicity were also examined. Apoptogenic protein Bax, cytochrome C, and caspase-3 protein were analysed. Morphological changes of MCF-7 were determined using contrast light microscope and scanning and transmission electron microscope (SEM and TEM). The findings of the analysis revealed higher toxicity of CaCO3/Dox nanocrystal and effective cells killing compared to free Dox, morphological changes such as formation of apoptotic bodies, membrane blebbing, and absent of microvilli as indicated by the SEM analysis while TEM revealed the presence of chromatin condensation, chromosomal DNA fragmentation, cell shrinkage, and nuclear fragmentation. Results of TUNEL assay verified that most of the cells undergoes apoptosis by internucleosomal fragmentation of genomic DNA whereas the extent of apoptotic cells was calculated using the apoptotic index (AI). Therefore, the biobased calcium carbonate nanocrystals such as Dox carriers may serve as an alternative to conventional delivery system.

Myofibroblastic differentiation of stromal cells in giant cell tumor of bone: an immunohistochemical and ultrastructural study.

The nature of the mononuclear stromal cells (MSCs) in giant cell tumor of bone (GCTB) has not been thoroughly investigated. The purpose of this study was to evaluate the degree and significance of myofibroblastic differentiation in 18 cases of GCTB by immunohistochemistry (IH) and/or electron microscopy (EM). All immunostained cases were found positive for smooth muscle actin (SMA) and/or muscle specific actin (MSA), most in 1-33% of the MSCs. Ultrastructurally, most MSCs were fibroblasts, and a significant number of cells displayed myofibroblastic differentiation. Myofibroblasts are an important component of MSCs in GCTB. The myofibroblastic population may be responsible in part for the production of matrix metalloproteinases (MMPs), which probably play a role in bone destruction, tumor aggression, and recurrence.

Modeling Ewing sarcoma tumors in vitro with 3D scaffolds.

The pronounced biological influence of the tumor microenvironment on cancer progression and metastasis has gained increased recognition over the past decade, yet most preclinical antineoplastic drug testing is still reliant on conventional 2D cell culture systems. Although monolayer cultures recapitulate some of the phenotypic traits observed clinically, they are limited in their ability to model the full range of microenvironmental cues, such as ones elicited by 3D cell-cell and cell-extracellular matrix interactions. To address these shortcomings, we established an ex vivo 3D Ewing sarcoma model that closely mimics the morphology, growth kinetics, and protein expression profile of human tumors. We observed that Ewing sarcoma cells cultured in porous 3D electrospun poly(ε-caprolactone) scaffolds not only were more resistant to traditional cytotoxic drugs than were cells in 2D monolayer culture but also exhibited remarkable differences in the expression pattern of the insulin-like growth factor-1 receptor/mammalian target of rapamycin pathway. This 3D model of the bone microenvironment may have broad applicability for mechanistic studies of bone sarcomas and exhibits the potential to augment preclinical evaluation of antineoplastic drug candidates for these malignancies.

Selective effects of hydroxyapatite nanoparticles on osteosarcoma cells and osteoblasts.

The effects of hydroxyapatite nanoparticles (HA-NPs) on two kinds of cells, human MG63 cells and the normal osteoblasts were investigated. According to the MTT assay and fluorescent staining assay, it was proved that HA-NPs could inhibit the growth of MG63 cells but slightly support proliferation of the osteoblasts. Meanwhile, transmission electron microscopy (TEM) was employed to observe the ultrastructural alterations of both cells. The TEM results showed that HA-NPs had entered the two kinds of cells. Typical apoptosis was observed in the MG63 cells, especially in the group of 250 µg/mL with 5 days culture. While no apoptosis could be found in the normal osteoblasts at any concentration group of HA-NPs. Our results suggested that the HA-NPs had selective effects to different kinds of cells: supporting proliferation to the normal bone cells while causing apoptosis to the osteosarcoma cells.

Establishment and characterization of a novel chordoma cell line: CH22.

Chordoma is a rare primary malignant bone tumor and there exist only a few established human chordoma cell lines. The scarcity of robust chordoma cell lines has limited the ability to study this tumor. In this report, we describe the establishment of a novel chordoma cell line and characterize its in vitro and in vivo behaviors. The tumor tissue was isolated from a patient with recurrent chordoma of the sacrum. After 6 months in culture, the chordoma cell line, referred here as CH22, was established. Microscopic analysis of two-dimensional culture confirmed that the CH22 cells exhibited a typical vacuolated cytoplasm similar to the well-established chordoma cell line U-CH1. Electron microscopy showed cohesive cells with numerous surface filopodia, pockets of glycogen and aggregates of intermediate tonofilaments in cytoplasm. Three-dimensional culture revealed that the CH22 cells could grow and form clusters by day 8. The MTT assays demonstrated that, compared with sensitive osteosarcoma cell lines, CH22 cells were relatively resistant to conventional chemotherapeutic drugs. Western blotting and immunofluorescence analysis confirmed that the CH22 cells expressed brachyury, vimentin, and cytokeratin. Finally, histological analysis of CH22 xenograft tumor tissues demonstrated the appearance of physaliphorous cells and positive staining of brachyury, cytokeratin, and S100. By CT and MRI, imaging xenografts showed the typical appearances seen in human chordomas. These findings suggest that the established novel human chordoma cell line CH22 and its tumorigenecity in SCID nude mice may serve as an important model for studying chordoma cell biology and the development of new therapeutic modalities.

Ultrastructual morphology of juvenile psammomatoid ossifying fibroma.

We investigated the ultrastructural morphology of the hard tissue in a fibro-osseous lesion. Bone samples were obtained from a juvenile psammomatoid ossifying fibroma and were observed under a scanning electron microscope. The lesion had a spongy lamellar structure containing bony tissue. On histological examination, resorbing preexisting lamellar bone associated with a large number of cathepsin K-positive osteoclasts was confirmed. Scanning electron microscopy revealed the bony material to have a cribriform structure and to indicate resorption related to osteoclasts throughout the tumor. These characteristic findings revealed the presence of active bony reconstruction and destruction in this lesion. The spongy calcified structure observed by scanning electron microscopy expressed the characteristic ground glass appearance in computed tomography of this patient. This remarkable activation of osteoclasts may deeply relate to characteristic calcified structure in this lesion. Observation of hard tissue structure under a scanning electron microscope may shed light on the pathology of fibro-osseous lesions in the head and neck.

Zoledronic acid inhibits vasculogenic mimicry in murine osteosarcoma cell line in vitro.

BACKGROUND: To study the effects of zoledronic acid (ZA) on the vasculogenic mimicry of osteosarcoma cells in vitro. METHODS: A Three-dimensional culture of LM8 osteosarcoma cells on a type I collagen matrix was used to investigate whether osteosarcoma cells can develop vasculogenic mimicry, and to determine the effects of ZA on this process. In addition, the cellular ultrastructural changes were observed using scanning electron microscopy and laser confocal microscopy. The effects of ZA on the translocation of RhoA protein from the cytosol to the membrane in LM8 cells were measured via immunoblotting. RESULTS: ZA inhibited the development of vasculogenic mimicry by the LM8 osteosarcoma cells, decreased microvilli formation on the cell surface, and disrupted the F-actin cytoskeleton. ZA prevented translocation of RhoA protein from the cytosol to the membrane in LM8 cells. CONCLUSIONS: ZA can impair RhoA membrane localization in LM8 cells, causing obvious changes in the ultrastructure of osteosarcoma cells and induce cell apoptosis, which may be one of the underlying mechanisms by which the agent inhibits the development of vasculogenic mimicry by the LM8 cells.

Primary kaposiform hemangioendothelioma of a long bone: two cases in unusual locations with long-term follow up.

Kaposiform hemangioendothelioma (KHE) is a rare vascular neoplasm of low malignant potential that mainly affects infants and adolescents. The tumor almost exclusively occurs in somatic soft tissue or the retroperitoneum. We report herein two cases of primary KHE occurring in a long bone without cutaneous changes with long-term follow up in young patients. The patients were a 9-year-old girl and 5-year-old boy presenting with lytic lesions of the femur and humerus, respectively, without cutaneous lesions. Histologically, the neoplasms were comprised of nodules of spindle- to oval-shaped cells growing in an infiltrative fashion. The neoplastic cells formed poorly canalized or slit-like blood vessels alternating with solid spindle areas. Immunohistochemical studies showed that the tumor cells expressed CD31, CD34 and Fli1, but not HHV8, LNA-1 or GLUT1. D2-40 stained the neoplastic spindle cells and lymphatic channels adjacent to vascular lobules. The girl remains well with 15 years and 6 months follow up after a second complete excision. The boy has no signs of recurrence or metastasis nearly 5 years after local complete excision. To our best knowledge, this is the first report in the English literature of primary long bone occurrences of KHE without cutaneous changes with long-term follow up.