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14 pages, 2945 KiB

Open AccessArticle

MLN4924, a Protein Neddylation Inhibitor, Suppresses the Growth of Human Chondrosarcoma through Inhibiting Cell Proliferation and Inducing Endoplasmic Reticulum Stress-Related Apoptosis

by Meng-Huang Wu, Ching-Yu Lee, Tsung-Jen Huang, Kuo-Yuan Huang, Chih-Hsin Tang, Shing-Hwa Liu, Kuan-Lin Kuo, Feng-Che Kuan, Wei-Chou Lin and Chung-Sheng Shi

Int. J. Mol. Sci. 2019, 20(1), 72; https://doi.org/10.3390/ijms20010072 - 24 Dec 2018

Cited by 29 | Viewed by 5461

Abstract

Chondrosarcoma, a heterogeneous malignant bone tumor, commonly produces cartilage matrix, which generally has no response to conventional therapies. Studies have reported that MLN4924, a NEDD8-activating enzyme inhibitor, achieves antitumor effects against numerous malignancies. In this study, the suppressive effects of MLN4924 on human [...] Read more.

Chondrosarcoma, a heterogeneous malignant bone tumor, commonly produces cartilage matrix, which generally has no response to conventional therapies. Studies have reported that MLN4924, a NEDD8-activating enzyme inhibitor, achieves antitumor effects against numerous malignancies. In this study, the suppressive effects of MLN4924 on human chondrosarcoma cell lines were investigated using in vitro and in vivo assays, which involved measuring cell viability, cytotoxicity, apoptosis, proliferation, cell cycles, molecule-associated cell cycles, apoptosis, endoplasmic reticulum (ER) stress, and tumor growth in a xenograft mouse model. Our results demonstrated that MLN4924 significantly suppressed cell viability, exhibited cytotoxicity, and stimulated apoptosis through the activation of caspase-3 and caspase-7 in chondrosarcoma cell lines. Furthermore, MLN4924 significantly inhibited cell proliferation by diminishing the phosphorylation of histone H3 to cause G2/M cell cycle arrest. In addition, MLN4924 activated ER stress–related apoptosis by upregulating the phosphorylation of c-Jun N-terminal kinase (JNK), enhancing the expression of GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP, an inducer of endoplasmic ER stress–related apoptosis) and activating the cleavage of caspase-4. Moreover, MLN4924 considerably inhibited the growth of chondrosarcoma tumors in a xenograft mouse model. Finally, MLN4924-mediated antichondrosarcoma properties can be accompanied by the stimulation of ER stress–related apoptosis, implying that targeting neddylation by MLN4924 is a novel therapeutic strategy for treating chondrosarcoma. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Graphical abstract

Graphical abstract
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MLN4924 reduced cellular viability and induced cytotoxicity in human chondrosarcoma cells. (a) NAE-1 expression in human chondrosarcoma cell lines (jj012, sw-1353), and normal chondrocyte cell line (C28/I2). (b) The jj012, sw-1353, and C28/I2 cells were treated with various concentrations of MLN4924 for 24 h. A WST-1 assay was performed to assess cell viability. (c) The jj012 and sw-1353 cells were treated with various concentrations of MLN4924 for 24 and 48 h in a WST-1 assay. (d) The jj012 and sw-1353 cells were treated with dimethyl sulfoxide ([DMSO], as a control treatment) or MLN4924 (500 and 750 nM) for 48 h, and cytotoxicity was determined through an LDH assay. * p < 0.05. Full article ">Figure 2
MLN4924 inhibited cell proliferation and caused G2/M cell cycle arrest in two human chondrosarcoma cells. (a) The jj012 and sw-1353 cells were exposed to mock (untreated) treatment or MLN4924 treatment (750 nM) for 48 h. After incubation, the status of DNA synthesis in terms of representing cell proliferation was determined using a BrdU incorporation assay. (b) Starved jj012 and sw-1353 cells were treated with or without various concentrations of MLN4924 for 24 h. After treatment, cells were subjected to propidium iodide (PI) staining to determine DNA content. (c) jj012 and sw-1353 cells were treated with or without various concentrations of MLN4924 (250, 500, and 750 nM) for 48 h. After treatment, the expression levels of cell cycle regulatory proteins, including histone-H3 and phospho-histone-H3 (Ser10), in total cell lysates were analyzed using Western blot analysis. The results are representative of at least three independent experiments. * p < 0.05. Full article ">Figure 3
MLN4924 induced apoptosis through caspase-3/7 activation in human chondrosarcoma cell lines. (a) The jj012 and sw-1353 cells were treated with 750 nM MLN4924 and DMSO (for the nontreated control group) for 48 h. The activation of caspase-3/7 on apoptotic cells was analyzed using fluorescence-activated cell-sorting flow cytometry. (b) After they were harvested, total cell lysates were analyzed by conducting a Western blot analysis that used specifically cleaved caspase-3/-7, casepase-8 (pro-form), Bcl-2, and Bcl-XL antibodies. Similar results were obtained in at least three independent experiments. Full article ">Figure 4
MLN4924 activated ER stress-related apoptosis in human chondrosarcoma cells. The jj012 and sw-1353 cells were treated with various concentrations of MLN4924 (250, 500, and 750 nM) and DMSO (for the control group) for 48 h. After they were harvested, cell lysates were analyzed by a Western blot analysis that used specific antibodies to molecules related to ER stress-induced apoptosis, including (a) c-Jun N-terminal kinase (JNK) and phospho-JNK, (b) GRP78, CHOP, and caspase-4. The results are representative of at least three independent experiments. Full article ">Figure 5
MLN4924 significantly inhibited the growth of the chondrosarcoma xenograft in vivo. Mice with jj012 or sw-1353 cells were grouped for treatment with DMSO or MLN4924 (10 mg/kg/day) through intraperitoneal injection for 5 weeks. (a,c) Representative images of excised chondrosarcoma tumors from each group of cells. (b,d) Tumor volumes were recorded to assess tumor growth. On the last day of treatment, tumors from MLN4924-treated and DMSO control mice were compared. * p < 0.05 versus DMSO control mice. The weights and volumes of tumors are represented herein as mean ± standard error of the mean; * p < 0.05. Full article ">Figure 5 Cont.
MLN4924 significantly inhibited the growth of the chondrosarcoma xenograft in vivo. Mice with jj012 or sw-1353 cells were grouped for treatment with DMSO or MLN4924 (10 mg/kg/day) through intraperitoneal injection for 5 weeks. (a,c) Representative images of excised chondrosarcoma tumors from each group of cells. (b,d) Tumor volumes were recorded to assess tumor growth. On the last day of treatment, tumors from MLN4924-treated and DMSO control mice were compared. * p < 0.05 versus DMSO control mice. The weights and volumes of tumors are represented herein as mean ± standard error of the mean; * p < 0.05. Full article ">

23 pages, 2767 KiB

Open AccessArticle

Defining a Characteristic Gene Expression Set Responsible for Cancer Stem Cell-Like Features in a Sub-Population of Ewing Sarcoma Cells CADO-ES1

by Marc Hotfilder, Nikhil Mallela, Jochen Seggewiß, Uta Dirksen and Eberhard Korsching

Int. J. Mol. Sci. 2018, 19(12), 3908; https://doi.org/10.3390/ijms19123908 - 6 Dec 2018

Cited by 12 | Viewed by 4801

Abstract

One of the still open questions in Ewing sarcoma, a rare bone tumor with weak therapeutic options, is to identify the tumor-driving cell (sub) population and to understand the specifics in the biological network of these cells. This basic scientific insight might foster [...] Read more.

One of the still open questions in Ewing sarcoma, a rare bone tumor with weak therapeutic options, is to identify the tumor-driving cell (sub) population and to understand the specifics in the biological network of these cells. This basic scientific insight might foster the development of more specific therapeutic target patterns. The experimental approach is based on a side population (SP) of Ewing cells, based on the model cell line CADO-ES1. The SP is established by flow cytometry and defined by the idea that tumor stem-like cells can be identified by the time-course in clearing a given artificial dye. The SP was characterized by a higher colony forming activity, by a higher differentiation potential, by higher resistance to cytotoxic drugs, and by morphology. Several SP and non-SP cell fractions and bone marrow-derived mesenchymal stem cell reference were analyzed by short read sequencing of the full transcriptome. The double-differential analysis leads to an altered expression structure of SP cells centered around the AP-1 and APC/c complex. The SP cells share only a limited proportion of the full mesenchymal stem cell stemness set of genes. This is in line with the expectation that tumor stem-like cells share only a limited subset of stemness features which are relevant for tumor survival. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Figure 1

Figure 1
Stemness features of the side population. In image (A) respective panel (D the left drawing), the colony-forming potential of SP compared to non-SP cells is illustrated. The image shows representative colonies. Panel (B) shows OIL-RED-O-stained cells after adipogenic differentiation. More dark red cells indicate more adipogeneic differentiation. The slightly smaller SP cells are seen in Panel (C) (May-Grünwald-Giemsa staining). In panel (D), the two rightmost diagrams show the higher resistance of SP cells compared to non-SP cells (reference no-sort) after treatment with cytotoxic drugs for 72 h. Doxorubicine was effective at 0.1 µM while Etoposide is shown at 1 µM. A star indicates a significant difference using the t test and an alpha error of 5%. ‘n’ denotes the number of biological replicates behind a condition. Horizontal brackets with a star indicate a significant difference between bars (alpha < 0.05). Full article ">Figure 2
Workflow of the data analysis. This graph explains how the different gene sets are created and in which type of analysis they are utilized. The starting point is the top left. Full article ">Figure 3
Heatmap of the counts for 42 up-regulated genes from SET-2. The counts are quantile normalized (preprocessCore) and rlog transformed (DESeq2) prior to plotting. The color bar on the right is defining the fold change values (FC) concerning gene average. Green colors denote levels of down-regulation, while red colors indicate levels of up-regulation. The order of SP and non-SP experiments reflects their paired nature (1,2,3,1,2,3). The hierarchical cluster tree on the left is based on the Euclidean measure. Full article ">Figure 4
Intersecting number of genes between the SET-1 DEGs and the member of the pathway involved in the Activation of APC/c and CDC20-mediated degradation of mitotic proteins. The nine intersecting members include PLK1, CDK1, MAD2L1, BUB1B, UBE2C, CCNA2, CDC20, NEK2, and CCNB1. Full article ">Figure 5
Subset of the PPI relevance network with the genes from SET-2. The gene products are represented by circles and their interactions are represented by edges. The size of the circles indicate the degree of connectivity to other partners. The larger the circle, the greater the degree. Red circles represent the products of up-regulated DEGs and green circles represent the products of down-regulated DEGs. The intensity of the colors corresponds to the log2 fold changes. The higher the fold change, the higher the color intensity. The blue color around the circles represents the p-value. The lower the p-value, the higher the color intensity. The PPI is underlining the relevance of AP-1 and the histone cluster. Full article ">Figure 6
Highest scoring subnetworks of SET-1. (A) shows the first highest scoring subnetwork with six nodes (Module 1, see <a href="#app1-ijms-19-03908" class="html-app">supplementary Figure S3</a>). (B) shows the second highest scoring subnetwork with 36 nodes (Module 2). Full article ">

16 pages, 3239 KiB

Open AccessArticle

Low HIF-1α and low EGFR mRNA Expression Significantly Associate with Poor Survival in Soft Tissue Sarcoma Patients; the Proteins React Differently

by Swetlana Rot, Helge Taubert, Matthias Bache, Thomas Greither, Peter Würl, Hans-Jürgen Holzhausen, Alexander W. Eckert, Dirk Vordermark and Matthias Kappler

Int. J. Mol. Sci. 2018, 19(12), 3842; https://doi.org/10.3390/ijms19123842 - 3 Dec 2018

Cited by 8 | Viewed by 3883

Abstract

In various tumors, the hypoxia inducible factor-1α (HIF-1α) and the epidermal growth factor-receptor (EGFR) have an impact on survival. Nevertheless, the prognostic impact of both markers for soft tissue sarcoma (STS) is not well studied. We examined 114 frozen [...] Read more.

In various tumors, the hypoxia inducible factor-1α (HIF-1α) and the epidermal growth factor-receptor (EGFR) have an impact on survival. Nevertheless, the prognostic impact of both markers for soft tissue sarcoma (STS) is not well studied. We examined 114 frozen tumor samples from adult soft tissue sarcoma patients and 19 frozen normal tissue samples. The mRNA levels of HIF-1α, EGFR, and the reference gene hypoxanthine phosphoribosyltransferase (HPRT) were quantified using a multiplex qPCR technique. In addition, levels of EGFR or HIF-1α protein were determined from 74 corresponding protein samples using ELISA techniques. Our analysis showed that a low level of HIF-1α or EGFR mRNA (respectively, relative risk (RR) = 2.8; p = 0.001 and RR = 1.9; p = 0.04; multivariate Cox´s regression analysis) is significantly associated with a poor prognosis in STS patients. The combination of both mRNAs in a multivariate Cox’s regression analysis resulted in an increased risk of early tumor-specific death of patients (RR = 3.1, p = 0.003) when both mRNA levels in the tumors were low. The EGFR protein level had no association with the survival of the patient’s cohort studied, and a higher level of HIF-1α protein associated only with a trend to significance (multivariate Cox’s regression analysis) to a poor prognosis in STS patients (RR = 1.9, p = 0.09). However, patients with low levels of HIF-1α protein and a high content of EGFR protein in the tumor had a three-fold better survival compared to patients without such constellation regarding the protein level of HIF-1α and EGFR. In a bivariate two-sided Spearman’s rank correlation, a significant correlation between the expression of HIF-1α mRNA and expression of EGFR mRNA (p < 0.001) or EGFR protein (p = 0.001) was found, additionally, EGFR mRNA correlated with EGFR protein level (p < 0.001). Our results show that low levels of HIF-1α mRNA or EGFR mRNA are negative independent prognostic markers for STS patients, especially after combination of both parameters. The protein levels showed a different effect on the prognosis. In addition, our analysis suggests a possible association between HIF-1α and EGFR expression in STS. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Figure 1
Multivariate Cox’s hazard regression model for HIF-1α mRNA expression and tumor-specific survival in STS patients. The expression of HIF-1α mRNA for 114 STS patients was associated with survival. The model was adjusted for tumor stage, tumor localization, tumor entity, and the type of tumor resection. The high and low cut-off values for HIF-1α were >4.7 and ≤4.7 copies of HIF-1α mRNA relative to the number of copies of hypoxanthine phosphoribosyltransferase (HPRT) mRNA, respectively (relative risk (RR) = 2.8, p = 0.001). Full article ">Figure 2
Multivariate Cox’s hazard regression model for EGFR mRNA expression and tumor-specific survival in STS patients.Expression of EGFR for 114 STS patients was associated with survival. The model was adjusted for tumor stage, tumor localization, tumor entity, and the type of tumor resection. The high and low cut-off values for EGFR were >1.5 and ≤1.5 copies of EGFR mRNA relative to the number of copies of HPRT mRNA, respectively (RR = 1.9, p = 0.04). Full article ">Figure 3
Multivariate Cox’s hazard regression model for the combination of HIF-1α and EGFR mRNA expression and tumor-specific survival in STS patients. The expression of HIF-1α mRNA and EGFR mRNA for 114 STS patients was associated with survival. The model was adjusted for tumor stage, tumor localization, tumor entity, and the type of tumor resection. If both markers had a low expression in the tumor, the prognosis of the patients was worse (RR = 3.1, p = 0.003) compared to high expression of both markers in the tumor. Full article ">Figure 4
Multivariate Cox’s hazard regression model for HIF-1α protein expression and tumor-specific survival in STS patients. The expression of HIF-1α protein for 74 STS patients was associated with survival. The model was adjusted for tumor stage, tumor localization, tumor entity, and the type of tumor resection. The high and low cut-off values for HIF-1α were >55 and ≤55 pg HIF-1α per µg protein, respectively (RR = 1.9, p = 0.09). Full article ">Figure 5
Multivariate Cox’s hazard regression model for EGFR protein expression and tumor-specific survival in STS patients. Expression of EGFR protein for 74 STS patients was associated with survival. The model was adjusted for tumor stage, tumor localization, tumor entity, and the type of tumor resection. The high and low cut-off values for EGFR were >0.76 and ≤0.76 ng EGFR protein per µg protein, respectively (RR = 1.5, p = 0.30). Full article ">Figure 6
Multivariate Cox’s hazard regression model for the combination of HIF-1α and EGFR protein expression and tumor-specific survival in STS patients. The expression of HIF-1α protein and EGFR protein for 74 STS patients was associated with survival. The model was adjusted for tumor stage, tumor localization, tumor entity, and the type of tumor resection. A low HIF-1α protein and a high EGFR protein level in the tumor resulted in a significantly better prognosis in STS patients compared to that of groups II and III. Full article ">Figure 7
Comparative data on to the impact of EGFR (7A) and HIF-1α (7B) mRNA expression on the overall survival of breast (a), ovarian (b), lung (c), and gastric (d) cancer patients. Plots were drawn by the Kaplan-Meier Plotter algorithm (Available online: <a href="http://kmplot.com/analysis" target="_blank">http://kmplot.com/analysis</a>) (accessed on 22 October 2018.) usingdata of the EGFR probe 1565483-at and of the HIF-1α probe 200989-at. Abbreviations: HR—hazard ratio. Full article ">

16 pages, 3094 KiB

Open AccessArticle

Functionalized Keratin as Nanotechnology-Based Drug Delivery System for the Pharmacological Treatment of Osteosarcoma

by Elisa Martella, Claudia Ferroni, Andrea Guerrini, Marco Ballestri, Marta Columbaro, Spartaco Santi, Giovanna Sotgiu, Massimo Serra, Davide Maria Donati, Enrico Lucarelli, Greta Varchi and Serena Duchi

Int. J. Mol. Sci. 2018, 19(11), 3670; https://doi.org/10.3390/ijms19113670 - 20 Nov 2018

Cited by 37 | Viewed by 7057

Abstract

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin [...] Read more.

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin nanoparticles as a novel drug delivery system to treat osteosarcoma. The nanoparticles are prepared from high molecular weight and hydrosoluble keratin, suitably functionalized with the photosensitizer Chlorin-e6 (Ce6) and then loaded with the chemotherapeutic drug Paclitaxel (PTX). This multi-modal PTX-Ce6@Ker nanoformulation is prepared by both drug-induced aggregation and desolvation methods, and a comprehensive physicochemical characterization is performed. PTX-Ce6@Ker efficacy is tested on osteosarcoma tumor cell lines, including chemo-resistant cells, using 2D and 3D model systems. The single and combined contributions of PTX and Ce6 is evaluated, and results show that PTX retains its activity while being vehiculated through keratin. Moreover, PTX and Ce6 act in an additive manner, demonstrating that the combination of the cytostatic blockage of PTX and the oxidative damage of ROS upon light irradiation have a far superior effect compared to singularly administered PTX or Ce6. Our findings provide the proof of principle for the development of a novel, nanotechnology-based drug delivery system for the treatment of osteosarcoma. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Graphical abstract
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Synthesis and characterization on PTX-Ce6@ker nanoparticles. (A) Schematic representation of PTX-Ce6@ker synthesis through the (i) drug-induced aggregation (ag), and (ii) desolvation (ds) methods; (B,C) Trasmission Electron Microscopy (TEM) micrograph of PTX-Ce6@kerag; (D) PTX-Ce6@kerag colloidal stability in Phosphate buffered solution (PBS) at 37 °C. Results are expressed as means of three independent experiments; (E) release profiles of PTX-Ce6@kerag and PTX-Ce6@kerds performed at 37 °C under stirring for 24 h. All results are expressed as the mean ± SD (from at least three independent experiments performed in triplicate). Full article ">Figure 2
Sensitivity of osteosarcoma (OS) cell lines to free Paclitaxel (PTX), and PTX loaded onto keratin nanoparticles. The graphs show the WST-1 assay performed on OS cell lines exposed to PTX (black circles), PTX@kerag (blue squares), PTX@kerds (red triangles), PTX-Ce6@kerag (pink squares), or PTX-Ce6@kerds (green triangles). All results are expressed as the mean ± SD (from at least three independent experiments performed in triplicate). Full article ">Figure 3
PTX-Ce6@kerag localization and the effect of PTX on MG63 cells. (A–C) Representative confocal images of MG63 treated for 24 h with PTX-Ce6@kerag and immunostained with EEA1, Lamp-1, and lysosomes, respectively (green channel in all pictures). The nuclei staining is shown in blue, the Ce6 signal in red, and colocalization in white; (D) Phalloidin (green channel) and β-tubulin (red channel) stainings were evaluated on MG63 at the end of the 24 h of treatment with Ce6@ker or PTX-Ce6@kerag. Images are representative of at least three independent experiments. Scale bar: 20 µm. Full article ">Figure 4
Impact of medium dosage of PTX-Ce6@Kerag on OS cells’ viability in a 2D system, and the additive effect of PTX and photodynamic therapy (PDT) on OS cell viability. OS cell lines were treated for 24 h with Ce6@ker, PTX@kerag, or PTX-Ce6@kerag at medium concentration. The graphs show the Alamar blue assay performed immediately after keratin nanoparticle treatments (−PDT), and 24 h after irradiation of the same samples (+PDT). Data, normalized to untreated cells (Ctrl) at the first time-point, are expressed as the mean ± SD (N = 2 biological replicates; N = 3 technical replicates) and analyzed using the one-way ANOVA test, and Tukey’s multiple comparison test as a post-test. Results were considered to be statistically significant at p values < 0.05 (*** p values < 0.001). Full article ">Figure 5
Impact of keratin nanoformulation on chemoresistant SaOS-2/DX580 cells. (A,B) SaOS-2 and SaOS-2/DX580 were treated for 24 h with Ce6 or PTX-Ce6@ker at a [Ce6] concentration of 3.35 µM. (A) Representative confocal microscopy images of cells treated with Ce6 or PTX-Ce6@kerag. Scale bar: 25 µm. (B) the graphs show the Ce6 fluorescence after internalization of the photosensitizer by itself (blue line) or loaded into keratin nanoparticles (red line) quantified by flow cytometry analysis (Control, black line). (C) the graphs show the Alamar blue assay on SaoOS-2/DX580 after 24 h treatment with PTX, PTX@kerag, or PTX-Ce6@kerag at an equivalent concentration of [PTX] of 13.4 µM (High) and 24 h after irradiation (+PDT). All data are normalized to untreated cells (Ctrl) and expressed as the mean ± SD (from at least two independent experiments performed in triplicate) and analyzed using a one-way ANOVA test, and Tukey’s multiple comparison test as a post-test. Results were considered to be statistically significant at p values < 0.05 (*** p values < 0.001). Full article ">Figure 6
Impact of keratin nanoformulation on 3D OS tumor model. (A) Schematic representation of cell spheroid formation and treatment with PTX-Ce6@kerag at a [Ce6] concentration of 6.7 µM; (B, C) the graphs show the Cell Titer Glo assay performed 1 day (B) or 6 days (C) after keratin nanoparticle treatment and −/+ irradiation (−/+PDT). The results are normalized to Ctrl at day 1 (no treated cells) and expressed as mean ± SD (N = 6 technical replicates and N = 2 individual replicates experiments) using the one-way ANOVA test, and Tukey’s multiple comparison test as a post-test. Results were considered to be statistically significant at p values < 0.05. * p-values < 0.05, ** p-values < 0.01 and *** p-values < 0.001; (D) transmission electron microscopy of treated MG63 spheroids with PTX@kerag and PTX-Ce6@kerag one day after irradiation (+PDT). The black squares highlight the mitocondrial alterations after nanoparticle treatment, compared to Ctrl spheroids. The white square in PTX@kerag highlights the necrosis area, and the red star in PTX-Ce6@kerag -PDT highlights the vesicle containing semidigested keratin nanoparticles. Scale bar: 2 µm. Full article ">

15 pages, 4301 KiB

Open AccessArticle

Preclinical Evaluation of Vemurafenib as Therapy for BRAF^V600E Mutated Sarcomas

by Sarina Gouravan, Leonardo A. Meza-Zepeda, Ola Myklebost, Eva W. Stratford and Else Munthe

Int. J. Mol. Sci. 2018, 19(4), 969; https://doi.org/10.3390/ijms19040969 - 23 Mar 2018

Cited by 13 | Viewed by 4904

Abstract

The BRAF^V600E mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas and we here evaluate the therapeutic potential in sarcoma cell lines. Methods: Four sarcoma cell lines harboring the BRAFV600E mutation, representing liposarcomas (SA-4 and SW872), Ewing sarcoma [...] Read more.

The BRAF^V600E mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas and we here evaluate the therapeutic potential in sarcoma cell lines. Methods: Four sarcoma cell lines harboring the BRAFV600E mutation, representing liposarcomas (SA-4 and SW872), Ewing sarcoma (A673) and atypical synovial sarcoma (SW982), were treated with vemurafenib and the effects on cell growth, apoptosis, cell cycle progression and cell signaling were determined. Results: Vemurafenib induced a strong cytostatic effect in SA-4 cells, mainly due to cell cycle arrest, whereas only moderate levels of apoptosis were observed. However, a high dose was required compared to BRAF^V600E mutated melanoma cells, and removal of vemurafenib demonstrated that the continuous presence of drug was required for sustained growth inhibition. A limited growth inhibition was observed in the other three cell lines. Protein analyses demonstrated reduced phosphorylation of ERK during treatment with vemurafenib in all the four sarcoma cell lines confirming that the MAPK pathway is active in these cell lines, and that the pathway can be inhibited by vemurafenib, but also that these cells can proliferate despite this. Conclusions: These findings indicate that vemurafenib alone would not be an efficient therapy against BRAF^V600E mutated sarcomas. However, further investigations of combination with other drugs are warranted. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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10 pages, 2155 KiB

Open AccessArticle

SK-216, a Novel Inhibitor of Plasminogen Activator Inhibitor-1, Suppresses Lung Metastasis of Human Osteosarcoma

by Minori Tsuge, Mitsuhiko Osaki, Ryo Sasaki, Mio Hirahata and Futoshi Okada

Int. J. Mol. Sci. 2018, 19(3), 736; https://doi.org/10.3390/ijms19030736 - 5 Mar 2018

Cited by 8 | Viewed by 4283

Abstract

Lung metastasis constitutes the leading cause of the death in patients with osteosarcoma. We have previously reported that plasminogen activator inhibitor-1 (PAI-1) regulates the invasion and lung metastasis of osteosarcoma cells in a mouse model and as well as in clinical samples. In [...] Read more.

Lung metastasis constitutes the leading cause of the death in patients with osteosarcoma. We have previously reported that plasminogen activator inhibitor-1 (PAI-1) regulates the invasion and lung metastasis of osteosarcoma cells in a mouse model and as well as in clinical samples. In the present study, we examined the anti-metastatic effect of SK-216, a small compound PAI-1 inhibitor, in human 143B osteosarcoma cells. An in vitro study showed that SK-216 treatment suppressed invasion activity by inhibiting PAI-1 expression in 143B cells, but had no influence on their proliferation or migration. 143B cells treated with SK-216 exhibited reduced matrix metalloproteinase-13 (MMP-13) secretion in a dose-dependent manner. Moreover, intraperitoneal injection of SK-216 into mouse models resulted in downregulation of PAI-1 expression levels in the primary tumors and showed suppression of lung metastases without influencing the proliferative activity of the tumor cells in the primary lesions. These results indicate that SK-216, a PAI-1 inhibitor, may serve as a novel drug to prevent lung metastasis in human osteosarcoma. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Figure 1

Figure 1
The PAI-1 inhibitor SK-216 suppresses invasion with no influence on proliferation or migration of 143B cells. (a) Western blot analyses of PAI-1 expression in SK-216 treated 143B cells. PAI-1 expression was quantified using Image Studio Lite (LI-COR) and normalized to β-actin. Expression is shown relative to that in non-treated cells (0 μM); (b) Matrigel assay of the invasion of SK-216-treated cells. The ratio of the number of pores containing invading cells to the total number of all pores is shown. Bar graphs show means ± SD ** p < 0.01; (c) Proliferation assay indicating absorbance (450 nm) measured at 0, 24, 48, or 72 h after SK-216 treatment for 143B cells is shown (n = 5 wells per group); (d) Scratch assay of the migration of 143B cells after 48 h treatment of SK-216. The migrated areas were analyzed at about 30 h after being scratched. Bar graphs show means ± SD. Full article ">Figure 2
Intraperitoneal injection of SK-216 suppresses lung metastasis of 143B cells in a mouse model. (a) 143B-Luc cells were inoculated into the right knee of a model mouse. Lung metastases at 5 weeks after inoculation are reflected in bioluminescence; (b) the areas of metastatic lesion on the lung in the model mice were plotted. The black bar indicates the mean value. Representative hematoxylin and eosin (H & E) staining of the lung at five weeks after inoculation are shown. Scale bars, 500 μm; (c) Primary tumors at five weeks after cell-inoculation are reflected in bioluminescence. Total Flux (photons/seconds) measured in the obtained IVIS images of mice. The black bar indicates the mean value; (d) the rates of tumor cells that were positive for Ki-67 in primary tumors were calculated by counting 10 visual fields at high magnification. Representative staining of Ki-67 from control and SK-216 treated mice are shown. Scale bars, 50 μm. Full article ">Figure 3
SK-216 suppresses PAI-1 expression of osteosarcoma cells in primary tumors. (a) Western blot analysis of PAI-1 expression (arrow) in primary tumors at 2 weeks after cell inoculation; (b) Western blot analysis of PAI-1 expression in 143B cells, which were transfected with PAI-1 siRNA or Control siRNA. Open circles and closed circles indicate upper and lower bands recognized by the anti-PAI-1 antibody used in this study, respectively; (c) PAI-1 expression in primary tumors was quantified using Image Studio Lite (LI-COR) and normalized to β-actin (±SD, n = 3 per group. * p < 0.05). Full article ">Figure 4
SK-216 attenuates MMP-13 secretion from 143B cells. MMP-13 levels in the conditioned media of SK-216-treated 143B cells were determined using the Sensolyte MMP-13 assay kit. The reaction was initiated by adding 50 μL of the substrate solution. The fluorescence intensity of the reaction (Fl) was determined by calculation of the ratio of λ emission (Em) = 485 nm/λ excitation (Ex) = 520 nm (±SD, n = 3 per group. ** p < 0.01). Full article ">

16 pages, 2374 KiB

Open AccessArticle

Integrated Molecular Characterization of Gastrointestinal Stromal Tumors (GIST) Harboring the Rare D842V Mutation in PDGFRA Gene

by Valentina Indio, Annalisa Astolfi, Giuseppe Tarantino, Milena Urbini, Janice Patterson, Margherita Nannini, Maristella Saponara, Lidia Gatto, Donatella Santini, Italo F. Do Valle, Gastone Castellani, Daniel Remondini, Michelangelo Fiorentino, Margaret Von Mehren, Giovanni Brandi, Guido Biasco, Michael C. Heinrich and Maria Abbondanza Pantaleo

Int. J. Mol. Sci. 2018, 19(3), 732; https://doi.org/10.3390/ijms19030732 - 4 Mar 2018

Cited by 29 | Viewed by 6675

Abstract

Gastrointestinal stromal tumors (GIST) carrying the D842V activating mutation in the platelet-derived growth factor receptor alpha (PDGFRA) gene are a very rare subgroup of GIST (about 10%) known to be resistant to conventional tyrosine kinase inhibitors (TKIs) and to show an [...] Read more.

Gastrointestinal stromal tumors (GIST) carrying the D842V activating mutation in the platelet-derived growth factor receptor alpha (PDGFRA) gene are a very rare subgroup of GIST (about 10%) known to be resistant to conventional tyrosine kinase inhibitors (TKIs) and to show an indolent behavior. In this study, we performed an integrated molecular characterization of D842V mutant GIST by whole-transcriptome and whole-exome sequencing coupled with protein–ligand interaction modelling to identify the molecular signature and any additional recurrent genomic event related to their clinical course. We found a very specific gene expression profile of D842V mutant tumors showing the activation of G-protein-coupled receptor (GPCR) signaling and a relative downregulation of cell cycle processes. Beyond D842V, no recurrently mutated genes were found in our cohort. Nevertheless, many private, clinically relevant alterations were found in each tumor (TP53, IDH1, FBXW7, SDH-complex). Molecular modeling of PDGFRA D842V suggests that the mutant protein binds imatinib with lower affinity with respect to wild-type structure, showing higher stability during the interaction with other type I TKIs (like crenolanib). D842V mutant GIST do not show any actionable recurrent molecular events of therapeutic significance, therefore this study supports the rationale of novel TKIs development that are currently being evaluated in clinical studies for the treatment of D842V mutant GIST. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Transcriptome profile of D842V mutant versus KIT mutant GIST. (A) Principal component analysis performed on the whole set of expressed genes (n = 15,134) shows a very uniform profile of D842V mutant tumors (red dots) that is distinctly separated from KIT mutant GIST (blue dots); (B) Heatmap of 494 overexpressed and 144 downregulated genes in D842V GIST; hierarchical clustering was performed to groups genes adopting Manhattan distance and Ward method; (C) Graphical representation of genes and pathways emerging as enriched (red) or depleted (green) in D842V mutant GIST. Genes and the corresponding pathways are also reported in <a href="#app1-ijms-19-00732" class="html-app">Supplementary Table S2</a>. Full article ">Figure 2
Mutational burden of D842V mutant GIST. The histogram bars indicate the number of somatic mutations per magabases (Mb) of coding region. Full article ">Figure 3
Graphic representation of clonal evolution of metastatic tumors T07–11 and T04–05. The full filled squares indicate the somatic mutations in the corresponding gene (blue and pink respectively for patient P06 and P04). Full article ">Figure 4
Percentage of samples with copy number gains (red) and losses (blue) for each chromosome. Full article ">Figure 5
(A) Schematic representation of the platelet-derived growth factor receptor alpha (PDGFRA) receptor; (B) Structure alignment performed using the jCE algorithm, between the crystallized structure of the c-Kit–Imatinib complex (PDB: 1T46) in purple and the structure of PDGFRA (PDB: 5K5X) in gold. Highlighted in green is the Activation loop; (C) Polar interactions of the 842 residue with wild-type Aspartic Acid; (D) Polar interactions of the 842 residue carrying the mutated Valine amino acid; (E) Representation of the best docked pose of crenolanib in the PDGFRA ATP binding site. Full article ">

21 pages, 4550 KiB

Open AccessArticle

Comparison of Tumor- and Bone Marrow-Derived Mesenchymal Stromal/Stem Cells from Patients with High-Grade Osteosarcoma

by Louis-Romée Le Nail, Meadhbh Brennan, Philippe Rosset, Frédéric Deschaseaux, Philippe Piloquet, Olivier Pichon, Cédric Le Caignec, Vincent Crenn, Pierre Layrolle, Olivier Hérault, Gonzague De Pinieux and Valérie Trichet

Int. J. Mol. Sci. 2018, 19(3), 707; https://doi.org/10.3390/ijms19030707 - 1 Mar 2018

Cited by 20 | Viewed by 6432

Abstract

Osteosarcoma (OS) is suspected to originate from dysfunctional mesenchymal stromal/stem cells (MSC). We sought to identify OS-derived cells (OSDC) with potential cancer stem cell (CSC) properties by comparing OSDC to MSC derived from bone marrow of patients. This study included in vitro characterization [...] Read more.

Osteosarcoma (OS) is suspected to originate from dysfunctional mesenchymal stromal/stem cells (MSC). We sought to identify OS-derived cells (OSDC) with potential cancer stem cell (CSC) properties by comparing OSDC to MSC derived from bone marrow of patients. This study included in vitro characterization with sphere forming assays, differentiation assays, cytogenetic analysis, and in vivo investigations of their tumorigenicity and tumor supportive capacities. Primary cell lines were isolated from nine high-grade OS samples. All primary cell lines demonstrated stromal cell characteristics. Compared to MSC, OSDC presented a higher ability to form sphere clones, indicating a potential CSC phenotype, and were more efficient at differentiation towards osteoblasts. None of the OSDC displayed the complex chromosome rearrangements typical of high grade OS and none of them induced tumors in immunodeficient mice. However, two OSDC demonstrated focused genomic abnormalities. Three out of seven, and six out of seven OSDC showed a supportive role on local tumor development, and on metastatic progression to the lungs, respectively, when co-injected with OS cells in nude mice. The observation of OS-associated stromal cells with rare genetic abnormalities and with the capacity to sustain tumor progression may have implications for future tumor treatments. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Figure 1
Microscopic observation of primary cell lines derived from bone marrow (MSC) or OS samples (OSDC) on treated culture dishes. Representative images are shown for MSC and OSDC at indicated passages (p). (A) Optical microscopy observation of adherent cells in standard culture conditions; (B) Optical microscopy observation of X-gal staining (upper panel, senescent cells have blue cytoplasmic color) and quantification of senescent cells (middle and bottom panels). Quantification was performed on 50 nuclei identified by two associated nucleoli using ImageJ. Non-senescent cells and senescent cells are shown with dark or light blue points, respectively; (C) Optical fluorescent microscopy immunodetection of alpha-smooth muscle actin (ASMA) (green), with nuclei colored in blue. Full article ">Figure 2
Histograms of relative fluorescent unites (RFU) for cluster of differentiation (CD) antigens on primary cell lines derived from OS samples (OSDC) or bone marrow (MSC) obtained from patients 6–9. RFU identified as CT were acquired with the control isotype (IgG-PE). MSC from patient 8 and 9 were not available. Colors are used to simplify reading. Full article ">Figure 3
Osteosarcoma derived cells (OSDC) in anchorage-independent, osteogenic or adipogenic culture conditions. (A) Optical microscopy observation of sphere clones in anchorage-independent culture conditions; (B) Calcium deposition observed with alizarin red staining following 21 days of cell culture with (+OM) or without (-OM) osteogenic induction media. Images of stained-culture wells are shown for OSDC or MSC plated in triplicate for each culture condition. Bound alizarin red was solubilized and optical density (OD) was measured for each well. The histogram represents mean values with standard deviation of each triplicate; (C) Representative images of MSC and OSDC from patient 7 following 14 days of cell culture with adipogenic induction medium are shown. Adipocytes containing small Nile Red-positive lipid droplets are easily observed (red vesicles) whereas undifferentiated cells were not labeled with Nile Red. Nuclei were counterstained (blue color) with 4′,6-diamidino-2-phenylindole (DAPI). Full article ">Figure 4
Few chromosomal rearrangements on OSDC-3 and 6. (A) Karyotype analysis on OSDC-3 and 6. Abnormal chromosomes are indicated (arrows). Only abnormal chromosomes that were observed in more than 10 metaphases are indicated by arrow; (B) Analysis of array comparative genomic hybridization on OSDC-3. Full article ">Figure 5
Complex chromosomal rearrangements on tumor samples of patient 3. Analysis of array comparative genomic hybridization on DNA from patient-3, of (A) the OS biopsy before poly-chemotherapy and (B) of pulmonary metastasis 18 months after the initial treatment. Black arrows show the 17(q22q25.3) hyperploidy that is found in the biopsy sample but not in the metastasis sample. Full article ">Figure 6
Co-injection of OS-inducing cells (MNNG-HOS cells (HOS)) with OSDC-1 in athymic mouse. (A) Mean tumor volumes are presented for three groups of mice (n = 8) injected with 1 million MNNG-HOS cells either alone, or with 0.5 million of MSC or OSDC-1. Significant differences between the MNNG-HOS and the MNNG-HOS + MSC/OSDC-1 groups are indicated. Differences were significant when p <0.05. Individual tumor volumes are represented as dots and mean tumor volumes in <a href="#app1-ijms-19-00707" class="html-app">supplementary Figure S3</a>; (B) Images of in vivo bioluminescence detection are shown for excised lung lobes. Luciferase-activity was detected at day 28 after cell injections for each group. Counts per second (cps) of selected areas with value above background were added together as cps total. Full article ">

15 pages, 3622 KiB

Open AccessArticle

Proteomic Differences in Feline Fibrosarcomas Grown Using Doxorubicin-Sensitive and -Resistant Cell Lines in the Chick Embryo Model

by Katarzyna Zabielska-Koczywąs, Katarzyna Michalak, Anna Wojtalewicz, Mateusz Winiarczyk, Łukasz Adaszek, Stanisław Winiarczyk and Roman Lechowski

Int. J. Mol. Sci. 2018, 19(2), 576; https://doi.org/10.3390/ijms19020576 - 14 Feb 2018

Cited by 6 | Viewed by 4413

Abstract

Proteomic analyses are rapid and powerful tools that are used to increase the understanding of cancer pathogenesis, discover cancer biomarkers and predictive markers, and select and monitor novel targets for cancer therapy. Feline injection-site sarcomas (FISS) are aggressive skin tumours with high recurrence [...] Read more.

Proteomic analyses are rapid and powerful tools that are used to increase the understanding of cancer pathogenesis, discover cancer biomarkers and predictive markers, and select and monitor novel targets for cancer therapy. Feline injection-site sarcomas (FISS) are aggressive skin tumours with high recurrence rates, despite treatment with surgery, radiotherapy, and chemotherapy. Doxorubicin is a drug of choice for soft tissue sarcomas, including FISS. However, multidrug resistance is one of the major causes of chemotherapy failure. The main aim of the present study was to identify proteins that differentiate doxorubicin-resistant from doxorubicin-sensitive FISS using two-dimensional gel electrophoresis (2DE), followed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using the three-dimensional (3D) preclinical in ovo model, which resembles features of spontaneous fibrosarcomas, three significantly (p ≤ 0.05) differentially expressed proteins were identified in tumours grown from doxorubicin-resistant fibrosarcoma cell lines (FFS1 and FFS3) in comparison to the doxorubicin-sensitive one (FFS5): Annexin A5 (ANXA5), Annexin A3 (ANXA3), and meiosis-specific nuclear structural protein 1 (MNS1). Moreover, nine other proteins were significantly differentially expressed in tumours grown from the high doxorubicin-resistant cell line (FFS1) in comparison to sensitive one (FFS5). This study may be the first proteomic fingerprinting of FISS reported, identifying potential candidates for specific predictive biomarkers and research targets for doxorubicin-resistant FISS. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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9336 KiB

Open AccessArticle

Primary Culture of Undifferentiated Pleomorphic Sarcoma: Molecular Characterization and Response to Anticancer Agents

by Alessandro De Vita, Federica Recine, Laura Mercatali, Giacomo Miserocchi, Chiara Spadazzi, Chiara Liverani, Alberto Bongiovanni, Federica Pieri, Roberto Casadei, Nada Riva, Valentina Fausti, Dino Amadori and Toni Ibrahim

Int. J. Mol. Sci. 2017, 18(12), 2662; https://doi.org/10.3390/ijms18122662 - 8 Dec 2017

Cited by 29 | Viewed by 5343

Abstract

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal neoplasm with no specific line of differentiation. Eribulin, a novel synthetic microtubule inhibitor, has shown anticancer activity in several tumors, including soft tissue sarcomas (STS). We investigated the molecular biology of UPS, and the mechanisms [...] Read more.

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal neoplasm with no specific line of differentiation. Eribulin, a novel synthetic microtubule inhibitor, has shown anticancer activity in several tumors, including soft tissue sarcomas (STS). We investigated the molecular biology of UPS, and the mechanisms of action of this innovative microtubule-depolymerizing drug. A primary culture from a patient with UPS was established and characterized in terms of gene expression. The activity of eribulin was also compared with that of other drugs currently used for STS treatment, including trabectedin. Finally, Western blot analysis was performed to better elucidate the activity of eribulin. Our results showed an upregulation of epithelial mesenchymal transition-related genes, and a downregulation of epithelial markers. Furthermore, genes involved in chemoresistance were upregulated. Pharmacological analysis confirmed limited sensitivity to chemotherapy. Interestingly, eribulin exhibited a similar activity to that of standard treatments. Molecular analysis revealed the expression of cell cycle arrest-related and pro-apoptotic-related proteins. These findings are suggestive of aggressive behavior in UPS. Furthermore, the identification of chemoresistance-related genes could facilitate the development of innovative drugs to improve patient outcome. Overall, the results from the present study furnish a rationale for elucidating the role of eribulin for the treatment of UPS. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Chemical structure of eribulin. Full article ">Figure 2
(a) Coronal post-contrast MRI image showing the presence of a large solid mass in the vastus lateralis of the right quadriceps femoris muscle (longitudinal diameter 20 cm). Absence of focal abnormalities in the remaining muscle–tendon structures of the thigh bilaterally; (b) Axial MRI image of the lesion (transverse diameter 53 mm × 88 mm) located in the middle–distal third of the quadriceps femoris muscle; (c) H&E staining of the surgical specimen showing the tumor-infiltrated muscle (20×). Margins were negative (R0 resection); (d) H&E staining of cytospun healthy cells from the patient-derived UPS primary culture (20×); (e) H&E staining of the surgical specimen showing undifferentiated pleomorphic sarcoma cells (light blue stroma) (20×); (f) H&E staining of cytospun UPS primary culture showing undifferentiated pleomorphic sarcoma cells (light blue stroma) (20×); (g) Immunohistochemical expression of CD34 in the tumor specimen showing undifferentiated pleomorphic sarcoma cells (brown cytoplasmic staining); (h) Immunohistochemical expression of CD34 in the cytospun UPS primary culture showing undifferentiated pleomorphic sarcoma cells (brown cytoplasmic staining) and cell nuclei stained with hematoxylin (blue spots). Full article ">Figure 3
(a) Relative expression of EMT-related genes in the UPS primary culture (K) and matched healthy tissue (H); (b) Relative expression of chemoresistance-related genes and CD109 gene in the UPS primary culture and matched healthy tissue. Differences between groups were assessed by a two-tailed Student’s t-test, and accepted as significant * p < 0.05. Full article ">Figure 4
(a) Cytotoxicity assay in UPS primary culture exposed to the following drugs: epirubicin(EPI) plus ifosfamide (IFO), doxorubicin (DOXO), eribulin (ERI), trabectedin (TRABE), and dacarbazine (DACA). Differences between groups were assessed by a two-tailed Student’s t-test, and accepted as significant at p < 0.05; (b) Images of the primary culture after treatment (2× and 10× magnification, scale bar 2000 µm and 400 µm, respectively); (c) Morphological features observed in the primary culture after ERI treatment (20× and 40× magnification, scale bar 200 µm and 100 µm, respectively); (d) Western blot analysis of apoptosis-related proteins (p21, BAX, Bcl-xL; caspase-3 and caspase-9). Vinculin was used as loading control. Fold changes of band density were normalized to the band of the CTR group. Full article ">Figure 5
Potential mechanism of action of the cytotoxic effect of eribulin in our patient-derived UPS primary culture. Full article ">

1707 KiB

Open AccessArticle

CMG2 Expression Is an Independent Prognostic Factor for Soft Tissue Sarcoma Patients

by Thomas Greither, Alice Wedler, Swetlana Rot, Jacqueline Keßler, Astrid Kehlen, Hans-Jürgen Holzhausen, Matthias Bache, Peter Würl, Helge Taubert and Matthias Kappler

Int. J. Mol. Sci. 2017, 18(12), 2648; https://doi.org/10.3390/ijms18122648 - 7 Dec 2017

Cited by 11 | Viewed by 8284

Abstract

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with [...] Read more.

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (r_s = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients’ disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Box-Plot overview on CMG2 mRNA expression in the different soft tissue sarcoma histological subtypes (a) and tumor stages (b). Numbers in bracket depict the cases (n) per category. Median CMG2 mRNA expression was slightly, but not significantly higher in leimosarcoma and NOS (a), and in stage 1 or stage 4 tumors (b). Abbreviations: LS—liposarcoma; FS—fibrosarcoma; RMS—rhabdomyosarcoma; LMS—leiomyosarcoma; NS—neuronal sarcoma; Syn—synovial sarcoma; NOS—not other specified. Full article ">Figure 2
Bivariate correlation analyses of CMG2 mRNA expression and CD26 mRNA (a), uPA mRNA (b), PAI-1 mRNA (c) and CMG2 protein (d) expression. Full article ">Figure 3
Survival analyses for CMG2 mRNA expression and protein expression in soft tissue sarcoma patients. Kaplan-Meier survival analyses revealed a significant worsened prognosis for patients with a low CMG2 mRNA expression in their tumors (a), but not for protein expression (c). Multivariate Cox’s regression analyses exhibited the detrimental effect of low CMG2 mRNA (b) and CMG2 protein (d) expression on patients’ disease-specific survival. Full article ">Figure 4
Comparative data on to the impact of CMG2 mRNA expression on the overall survival of breast (a), ovarian (b), lung (c), and gastric (d) cancer patients. Plots were drawn by the Kaplan-Meier Plotter algorithm (Available online: <a href="http://kmplot.com/analysis" target="_blank">http://kmplot.com/analysis</a>) facilitating microarray data of the CMG2/ANTXR2 probe 225524_at. Abbreviations: HR—hazard ratio. Full article ">

878 KiB

Open AccessArticle

Clinical Usefulness of the Platelet-to Lymphocyte Ratio in Patients with Angiosarcoma of the Face and Scalp

by Gen Suzuki, Hideya Yamazaki, Norihiro Aibe, Koji Masui, Naomi Sasaki, Daisuke Shimizu, Takuya Kimoto, Jun Asai, Makoto Wada, Satoshi Komori, Norito Katoh and Kei Yamada

Int. J. Mol. Sci. 2017, 18(11), 2402; https://doi.org/10.3390/ijms18112402 - 13 Nov 2017

Cited by 8 | Viewed by 4027

Abstract

Angiosarcoma of the face and scalp (ASFS) is an extremely aggressive tumor that frequently metastasizes, often leading to death. The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) are inflammatory markers that predict outcome of various cancers. We aimed to examine [...] Read more.

Angiosarcoma of the face and scalp (ASFS) is an extremely aggressive tumor that frequently metastasizes, often leading to death. The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) are inflammatory markers that predict outcome of various cancers. We aimed to examine the relationship between pretreatment inflammatory markers and ASFS outcome. We included 17 patients with ASFS and a control group of 56 age- and gender-matched healthy individuals. Total white blood counts, neutrophil, lymphocyte, monocyte, and platelet counts were recorded; NLR, PLR, and LMR were calculated. Kaplan–Meier curves were used to calculate overall survival (OS) and distant metastasis-free survival (DMFS). Optimal cut-off values for each inflammatory marker were calculated using receiver operating curve analysis. Median follow-up was 22 months (range, 6–75). There was a statistically significant difference in absolute neutrophil counts and NLR between patient and control groups. Two-year OS and DMFS rates were 41% and 35%, respectively. In patients with tumors < 10 cm, PLR was highly correlated with DMFS, with the 2-year DMFS for those with a high PLR being 50% compared with 100% for those with a low PLR (p = 0.06). This study suggests that PLR is superior to NLR and LMR, and is a clinically useful marker in patients with ASFS with small tumors. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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18 pages, 317 KiB

Open AccessReview

Therapeutic Targets for Bone and Soft-Tissue Sarcomas

by Shinji Miwa, Norio Yamamoto, Katsuhiro Hayashi, Akihiko Takeuchi, Kentaro Igarashi and Hiroyuki Tsuchiya

Int. J. Mol. Sci. 2019, 20(1), 170; https://doi.org/10.3390/ijms20010170 - 4 Jan 2019

Cited by 50 | Viewed by 6111

Abstract

Due to the rarity and heterogeneity of bone and soft-tissue sarcomas, investigation into molecular targets and new treatments has been particularly challenging. Although intensive chemotherapy and establishment of surgical procedures have improved the outcomes of patients with sarcoma, the curative rate of recurrent [...] Read more.

Due to the rarity and heterogeneity of bone and soft-tissue sarcomas, investigation into molecular targets and new treatments has been particularly challenging. Although intensive chemotherapy and establishment of surgical procedures have improved the outcomes of patients with sarcoma, the curative rate of recurrent and metastatic sarcomas is still not satisfactory. Recent basic science research has revealed some of the mechanisms of progression and metastasis of malignancies including proliferation, apoptosis, angiogenesis, tumor microenvironment, migration, invasion, and regulation of antitumor immune systems. Based on these basic studies, new anticancer drugs, including pazopanib, trabectedin, eribulin, and immune checkpoint inhibitors have been developed and the efficacies and safety of the new drugs have been assessed by clinical trials. This review summarizes new molecular therapeutic targets and advances in the treatment for bone and soft tissue sarcomas. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

19 pages, 626 KiB

Open AccessReview

Translocation-Related Sarcomas

by Kenji Nakano and Shunji Takahashi

Int. J. Mol. Sci. 2018, 19(12), 3784; https://doi.org/10.3390/ijms19123784 - 28 Nov 2018

Cited by 33 | Viewed by 7661

Abstract

Chromosomal translocations are observed in approximately 20% of soft tissue sarcomas (STS). With the advances in pathological examination technology, the identification of translocations has enabled precise diagnoses and classifications of STS, and it has been suggested that the presence of and differences in [...] Read more.

Chromosomal translocations are observed in approximately 20% of soft tissue sarcomas (STS). With the advances in pathological examination technology, the identification of translocations has enabled precise diagnoses and classifications of STS, and it has been suggested that the presence of and differences in translocations could be prognostic factors in some translocation-related sarcomas. Most of the translocations in STS were not regarded as targets of molecular therapies until recently. However, trabectedin, an alkylating agent, has shown clinical benefits against translocation-related sarcoma based on a modulation of the transcription of the tumor’s oncogenic fusion proteins. Many molecular-targeted drugs that are specific to translocations (e.g., anaplastic lymphoma kinase and tropomyosin kinase related fusion proteins) have emerged. The progress in gene technologies has allowed researchers to identify and even induce new translocations and fusion proteins, which might become targets of molecular-targeted therapies. In this review, we discuss the clinical significance of translocation-related sarcomas, including their diagnoses and targeted therapies. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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14 pages, 11053 KiB

Open AccessReview

GFRA1: A Novel Molecular Target for the Prevention of Osteosarcoma Chemoresistance

by Mihwa Kim and Dae Joon Kim

Int. J. Mol. Sci. 2018, 19(4), 1078; https://doi.org/10.3390/ijms19041078 - 4 Apr 2018

Cited by 35 | Viewed by 8665

Abstract

The glycosylphosphatidylinositol-linked GDNF (glial cell derived neurotrophic factor) receptor alpha (GFRA), a coreceptor that recognizes the GDNF family of ligands, has a crucial role in the development and maintenance of the nervous system. Of the four identified GFRA isoforms, GFRA1 specifically recognizes GDNF [...] Read more.

The glycosylphosphatidylinositol-linked GDNF (glial cell derived neurotrophic factor) receptor alpha (GFRA), a coreceptor that recognizes the GDNF family of ligands, has a crucial role in the development and maintenance of the nervous system. Of the four identified GFRA isoforms, GFRA1 specifically recognizes GDNF and is involved in the regulation of proliferation, differentiation, and migration of neuronal cells. GFRA1 has also been implicated in cancer cell progression and metastasis. Recent findings show that GFRA1 can contribute to the development of chemoresistance in osteosarcoma. GFRA1 expression was induced following treatment of osteosarcoma cells with the popular anticancer drug, cisplatin and induction of GFRA1 expression significantly suppressed apoptosis mediated by cisplatin in osteosarcoma cells. GFRA1 expression promotes autophagy by activating the SRC-AMPK signaling axis following cisplatin treatment, resulting in enhanced osteosarcoma cell survival. GFRA1-induced autophagy promoted tumor growth in mouse xenograft models, suggesting a novel function of GFRA1 in osteosarcoma chemoresistance. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Figure 1
The interaction of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) with their receptors. Four homodimeric GFLs (GDNF, NRTN, ARTN, and PSPN) recognize their corresponding GDNF family receptor α (GFRα/GFRA1-4). GDNF specifically binds to GFRA1, NRTN to GFRA2, ARTN to GFRA3, and PSPN to GFRA4. Dotted arrow indicates cross-interaction of GFLs with GFRA which has been observed. Binding of GFL with GFRA activates protein tyrosine kinase RET. Neural cell adhesion molecule (NCAM) has been identified as an alternative signaling receptor for GFLs. RET also has been known as the representative receptor tyrosine kinases (RTKs) for these neurotrophic factor receptors. Activation of RET or NCAM via the GFL/GFRA complex is involved in various physiological functions, such as neuronal cell growth, differentiation, migration, and osteosarcoma chemoresistance via autophagy. Gray-colored area of the membrane indicates lipid raft. NRTN, neurturin; ARTN, artemin; PSPN, persephin. Full article ">Figure 2
Two hypothetical modes of RET interaction/stimulation by GFL/GFRA. The extracellular domain of RET tyrosine kinase binds with GFL/GFRA which is linked to the plasma membrane via a glycosyl phosphatidylinositol (GPI) anchor. The interaction of RET with GFL/GFRA leads to the phosphorylation of tyrosine residues located in intracellular domain of RET and activates downstream signaling pathways. (A) GFL dimer binds to the GPI-anchored GFRA in lipid rafts and recruits RET; (B) GFRA without ligand forms a complex with RET that can serve as a receptor for GFL dimer. Full article ">Figure 3
Schematic presentation of regulatory mechanisms of GFL-GFRA1 signaling. (A) RET is recruited to the lipid microdomain (or lipid rafts) by GPI-anchored GFRA1 (left side, cis-signaling) and by soluble GFRA1 (right side, sGFRA1, trans-signaling) released from plasma membrane. During cis-signaling, GFRA1 is anchored to lipid rafts by GPI, allowing GFLs to bind to GFRA1 to transduce their signals to co-receptor RET which can then activate downstream targets like Src kinase, PLCγ, or PI3K/AKT. Trans-signaling occurs when the GDNF/sGFRA1 complex binds to RET on another peripheral neuron which does not express GFRA1. GDNF/sGFRA1/RET can then transduce their signal to downstream targets such as PI3K/AKT and PLCγ/Cdk5, resulting in neuronal survival and neurite outgrowth; (B) In the absence of GFLs, short-range signaling by homophilic interactions between p140NCAM can be disrupted by the presence of GFRA, resulting in the inhibition of cell adhesion, or extracellular signaling crosstalk (left side). In the presence GFLs, the association of membrane-bound GFRA with p140NCAM results in the activation of the Fyn-FAK-MAPK signaling pathway (right side); (C) Ligand-induced cell adhesion; (D) Recycling of GDNF/GFRA1/RET complex by SorLA. SorLA/GFRA1-mediated GDNF uptake occurs by RET-independent endocytosis. SorLA/GFRA1 complex directs GDNF to lysosome. SorLA/GFRA1-mediated RET endocytosis is independent of GDNF. Full article ">Figure 3 Cont.
Schematic presentation of regulatory mechanisms of GFL-GFRA1 signaling. (A) RET is recruited to the lipid microdomain (or lipid rafts) by GPI-anchored GFRA1 (left side, cis-signaling) and by soluble GFRA1 (right side, sGFRA1, trans-signaling) released from plasma membrane. During cis-signaling, GFRA1 is anchored to lipid rafts by GPI, allowing GFLs to bind to GFRA1 to transduce their signals to co-receptor RET which can then activate downstream targets like Src kinase, PLCγ, or PI3K/AKT. Trans-signaling occurs when the GDNF/sGFRA1 complex binds to RET on another peripheral neuron which does not express GFRA1. GDNF/sGFRA1/RET can then transduce their signal to downstream targets such as PI3K/AKT and PLCγ/Cdk5, resulting in neuronal survival and neurite outgrowth; (B) In the absence of GFLs, short-range signaling by homophilic interactions between p140NCAM can be disrupted by the presence of GFRA, resulting in the inhibition of cell adhesion, or extracellular signaling crosstalk (left side). In the presence GFLs, the association of membrane-bound GFRA with p140NCAM results in the activation of the Fyn-FAK-MAPK signaling pathway (right side); (C) Ligand-induced cell adhesion; (D) Recycling of GDNF/GFRA1/RET complex by SorLA. SorLA/GFRA1-mediated GDNF uptake occurs by RET-independent endocytosis. SorLA/GFRA1 complex directs GDNF to lysosome. SorLA/GFRA1-mediated RET endocytosis is independent of GDNF. Full article ">Figure 4
Schematic diagram for autophagy mediated by GFRA1 in cisplatin-resistant osteosarcoma. Cisplatin induces GFRA1 through NFκB, and GFRA1 activates autophagy by SRC/AMPK signaling. Finally, GFRA1-mediated autophagy inhibits cisplatin-induced apoptosis, resulting in cell proliferation and migration. Full article ">

21 pages, 281 KiB

Open AccessReview

Current Molecular Targeted Therapies for Bone and Soft Tissue Sarcomas

by Kenji Nakano and Shunji Takahashi

Int. J. Mol. Sci. 2018, 19(3), 739; https://doi.org/10.3390/ijms19030739 - 5 Mar 2018

Cited by 40 | Viewed by 6919

Abstract

Systemic treatment options for bone and soft tissue sarcomas remained unchanged until the 2000s. These cancers presented challenges in new drug development partly because of their rarity and heterogeneity. Many new molecular targeting drugs have been tried in the 2010s, and some were [...] Read more.

Systemic treatment options for bone and soft tissue sarcomas remained unchanged until the 2000s. These cancers presented challenges in new drug development partly because of their rarity and heterogeneity. Many new molecular targeting drugs have been tried in the 2010s, and some were approved for bone and soft tissue sarcoma. As one of the first molecular targeted drugs approved for solid malignant tumors, imatinib’s approval as a treatment for gastrointestinal stromal tumors (GISTs) has been a great achievement. Following imatinib, other tyrosine kinase inhibitors (TKIs) have been approved for GISTs such as sunitinib and regorafenib, and pazopanib was approved for non-GIST soft tissue sarcomas. Olaratumab, the monoclonal antibody that targets platelet-derived growth factor receptor (PDGFR)-α, was shown to extend the overall survival of soft tissue sarcoma patients and was approved in 2016 in the U.S. as a breakthrough therapy. For bone tumors, new drugs are limited to denosumab, a receptor activator of nuclear factor κB ligand (RANKL) inhibitor, for treating giant cell tumors of bone. In this review, we explain and summarize the current molecular targeting therapies approved and in development for bone and soft tissue sarcomas. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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15 pages, 795 KiB

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Sarcoma Spheroids and Organoids—Promising Tools in the Era of Personalized Medicine

by Gianluca Colella, Flavio Fazioli, Michele Gallo, Annarosaria De Chiara, Gaetano Apice, Carlo Ruosi, Amelia Cimmino and Filomena De Nigris

Int. J. Mol. Sci. 2018, 19(2), 615; https://doi.org/10.3390/ijms19020615 - 21 Feb 2018

Cited by 53 | Viewed by 10943

Abstract

Cancer treatment is rapidly evolving toward personalized medicine, which takes into account the individual molecular and genetic variability of tumors. Sophisticated new in vitro disease models, such as three-dimensional cell cultures, may provide a tool for genetic, epigenetic, biomedical, and pharmacological research, and [...] Read more.

Cancer treatment is rapidly evolving toward personalized medicine, which takes into account the individual molecular and genetic variability of tumors. Sophisticated new in vitro disease models, such as three-dimensional cell cultures, may provide a tool for genetic, epigenetic, biomedical, and pharmacological research, and help determine the most promising individual treatment. Sarcomas, malignant neoplasms originating from mesenchymal cells, may have a multitude of genomic aberrations that give rise to more than 70 different histopathological subtypes. Their low incidence and high level of histopathological heterogeneity have greatly limited progress in their treatment, and trials of clinical sarcoma are less frequent than trials of other carcinomas. The main advantage of 3D cultures from tumor cells or biopsy is that they provide patient-specific models of solid tumors, and they overcome some limitations of traditional 2D monolayer cultures by reflecting cell heterogeneity, native histologic architectures, and cell–extracellular matrix interactions. Recent advances promise that these models can help bridge the gap between preclinical and clinical research by providing a relevant in vitro model of human cancer useful for drug testing and studying metastatic and dormancy mechanisms. However, additional improvements of 3D models are expected in the future, specifically the inclusion of tumor vasculature and the immune system, to enhance their full ability to capture the biological features of native tumors in high-throughput screening. Here, we summarize recent advances and future perspectives of spheroid and organoid in vitro models of rare sarcomas that can be used to investigate individual molecular biology and predict clinical responses. We also highlight how spheroid and organoid culture models could facilitate the personalization of sarcoma treatment, provide specific clinical scenarios, and discuss the relative strengths and limitations of these models. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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26 pages, 3155 KiB

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Chondrosarcoma: A Rare Misfortune in Aging Human Cartilage? The Role of Stem and Progenitor Cells in Proliferation, Malignant Degeneration and Therapeutic Resistance

by Karen A. Boehme, Sabine B. Schleicher, Frank Traub and Bernd Rolauffs

Int. J. Mol. Sci. 2018, 19(1), 311; https://doi.org/10.3390/ijms19010311 - 21 Jan 2018

Cited by 53 | Viewed by 10355

Abstract

Unlike other malignant bone tumors including osteosarcomas and Ewing sarcomas with a peak incidence in adolescents and young adults, conventional and dedifferentiated chondrosarcomas mainly affect people in the 4th to 7th decade of life. To date, the cell type of chondrosarcoma origin is [...] Read more.

Unlike other malignant bone tumors including osteosarcomas and Ewing sarcomas with a peak incidence in adolescents and young adults, conventional and dedifferentiated chondrosarcomas mainly affect people in the 4th to 7th decade of life. To date, the cell type of chondrosarcoma origin is not clearly defined. However, it seems that mesenchymal stem and progenitor cells (MSPC) in the bone marrow facing a pro-proliferative as well as predominantly chondrogenic differentiation milieu, as is implicated in early stage osteoarthritis (OA) at that age, are the source of chondrosarcoma genesis. But how can MSPC become malignant? Indeed, only one person in 1,000,000 will develop a chondrosarcoma, whereas the incidence of OA is a thousandfold higher. This means a rare coincidence of factors allowing escape from senescence and apoptosis together with induction of angiogenesis and migration is needed to generate a chondrosarcoma. At early stages, chondrosarcomas are still assumed to be an intermediate type of tumor which rarely metastasizes. Unfortunately, advanced stages show a pronounced resistance both against chemo- and radiation-therapy and frequently metastasize. In this review, we elucidate signaling pathways involved in the genesis and therapeutic resistance of chondrosarcomas with a focus on MSPC compared to signaling in articular cartilage (AC). Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Cold Atmospheric Plasma in the Treatment of Osteosarcoma

by Denis Gümbel, Sander Bekeschus, Nadine Gelbrich, Matthias Napp, Axel Ekkernkamp, Axel Kramer and Matthias B. Stope

Int. J. Mol. Sci. 2017, 18(9), 2004; https://doi.org/10.3390/ijms18092004 - 19 Sep 2017

Cited by 45 | Viewed by 8577

Abstract

Human osteosarcoma (OS) is the most common primary malignant bone tumor occurring most commonly in adolescents and young adults. Major improvements in disease-free survival have been achieved by implementing a combination therapy consisting of radical surgical resection of the tumor and systemic multi-agent [...] Read more.

Human osteosarcoma (OS) is the most common primary malignant bone tumor occurring most commonly in adolescents and young adults. Major improvements in disease-free survival have been achieved by implementing a combination therapy consisting of radical surgical resection of the tumor and systemic multi-agent chemotherapy. However, long-term survival remains poor, so novel targeted therapies to improve outcomes for patients with osteosarcoma remains an area of active research. This includes immunotherapy, photodynamic therapy, or treatment with nanoparticles. Cold atmospheric plasma (CAP), a highly reactive (partially) ionized physical state, has been shown to inherit a significant anticancer capacity, leading to a new field in medicine called “plasma oncology.” The current article summarizes the potential of CAP in the treatment of human OS and reviews the underlying molecular mode of action. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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