Journal: Theranostics

Article Title: Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice

doi: 10.7150/thno.30030

Figure Lengend Snippet: Exosome uptake by murine K7M2 osteosarcoma cells. Exosomes derived from human umbilical cord-derived mesenchymal stromal cells were labeled with Rhodamine B (Exo-RhB) were added to K7M2 mouse osteosarcoma cells and imaged by confocal microscopy. K7M2 murine osteosarcoma cells (10 4 /cm 2 ) were seeded in 8-well chambered coverglass and incubated for 24 h at 37 ºC and 5% CO 2 . Next, K7M2 cells were exposed to 25 μg of Exo-RhB in a 50:1 ratio (Exo:RhB) and incubated for an additional 24 h. Rhodamine B lipid alone (RhB-Control) or Dulbecco's phosphate buffered saline (Negative Control) were used as controls. Next, the cells fixed and their DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) prior to confocal microscopy. (A) Exosome uptake shown by confocal images. (B) Integrated density of Exo-RhB and RhB-control shows that significantly more RhB fluorescence is found within K7M2 cells after exposure to Exo-RhB compared to RhB-Control. (C) A stack of confocal images generated a 3D visualization of exosome location within the cytoplasm of K7M2 cells. The red rectangle in panel A indicates the cells imaged in panel C. Graph shows average ± one standard deviation, n = 3. Two-tailed t-test, ** p < 0.01.

Article Snippet: The cancer cell lines such as 143B (ATCC ® CRL-8303) human osteosarcoma and K7M2 (ATCC ® CRL-2836) murine osteosarcoma were gifted by Dr. Deryl Troyer (Department of Anatomy and Physiology, Kansas State University).

Techniques: Derivative Assay, Labeling, Confocal Microscopy, Incubation, Control, Saline, Negative Control, Staining, Fluorescence, Generated, Standard Deviation, Two Tailed Test

Journal: Theranostics

Article Title: Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice

doi: 10.7150/thno.30030

Figure Lengend Snippet: Exosome uptake quantified by flow cytometry. Murine osteosarcoma K7M2 cells were incubated with unlabeled mesenchymal stromal cell-derived exosomes (Control) or Rhodamine B (RhB)- labeled exosomes (Exo-RhB) and the time-dependent uptake was measured by flow cytometry (10000 events per histogram). (A) Flow cytometry histograms indicate RhB-positive events (P2) for unlabeled exosomes (Control), or after 6 h, 12 h, 24 h, and 48 h of incubation with Exo-RhB. (B) Integrated fluorescence intensity (RhB intensity) of Exo-RhB within K7M2 cells. Note that RhB intensity with K7M2 cells increases over the first 24 h. Data shown is median area under the curve ± 1 standard deviation.

Article Snippet: The cancer cell lines such as 143B (ATCC ® CRL-8303) human osteosarcoma and K7M2 (ATCC ® CRL-2836) murine osteosarcoma were gifted by Dr. Deryl Troyer (Department of Anatomy and Physiology, Kansas State University).

Techniques: Flow Cytometry, Incubation, Derivative Assay, Control, Labeling, Fluorescence, Standard Deviation

Journal: Theranostics

Article Title: Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice

doi: 10.7150/thno.30030

Figure Lengend Snippet: Effect of exosomes on osteosarcoma cell proliferation. A , B and C . K7M2 mouse osteosarcoma cells were exposed to 10 ng, 20 ng or 30 ng of unlabeled (naive)-exosomes or gadolinium-labeled exosomes (Exo-GdL) and their proliferation was assayed using a colorimetric assay for measuring cell metabolic activity based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), to insoluble formazan. Dulbecco's modified eagle medium (DMEM) without exosomes was added as the Control. Note that naïve exosomes produced a dose-dependent decrease in K7M2 cell proliferation, but Exo-GdL did not. D , E and F . 143B human osteosarcoma cells were exposed to 10 ng, 20 ng or 30 ng of naïve-exosomes, Exo-GdL, or exosomes labeled with both GdL and Rhodamine B (Exo-GdL-RhB) and assayed using MTT. Note that, similar to the response of K7M2 cells ( A-C ), naive exosomes produced a dose-dependent decrease in 143B cell proliferation, but Exo-GdL did not. In contrast, Exo-GdL-RhB produced a dose-dependent increase in 143B cell proliferation. To compare the effect of exosomes isolated from different human mesenchymal stromal cell (MSC) lines, line HUC-257 was used in C and exosomes from line HUC-293 was used in F . Note that the exosomes produced from different MSC lines had similar effects on osteosarcoma cell proliferation. Data are averages of technical triplicates from five independent cell lines (n=5) ± one standard deviation. Statistical analysis by ANOVA followed by post hoc planned comparisons. * p < 0.05).

Article Snippet: The cancer cell lines such as 143B (ATCC ® CRL-8303) human osteosarcoma and K7M2 (ATCC ® CRL-2836) murine osteosarcoma were gifted by Dr. Deryl Troyer (Department of Anatomy and Physiology, Kansas State University).

Techniques: Labeling, Colorimetric Assay, Activity Assay, Modification, Control, Produced, Isolation, Standard Deviation

Journal: Theranostics

Article Title: Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice

doi: 10.7150/thno.30030

Figure Lengend Snippet: Effect of exosomes on murine K7M2 cell apoptosis. K7M2 cells were exposed to exosomes derived from human umbilical cord mesenchymal stromal cells (Naive-Exo) or exsomes labeled with gadolinium (Exo-GdL) at three different concentrations (30 ng/cm 2 = 1X, 300 ng/cm 2 = 10X, or 3000 ng/cm 2 = 100X). Exosomes were suspended in 1 mL of Dulbecco's modified eagle medium (DMEM) supplemented with 10% pooled human platelet lysate (HPL) depleted of exosomes. 1 mL of the same culture media was used as negative control (Negative Control) and DMEM with 500 µM of hydrogen peroxide (H 2 O 2 ) was used as a positive control (Positive Control). (A) Representative dot plots of PI vs. Annexin-V stained cells. Cells in quadrant 2 (Q2) correspond to dead cells. Viable cells are found in Q1 and Q3, and cells in early apoptosis are found in Q4. (B) Percent distribution of live (Viable, gray bars), early apoptosis (black bars) or dead cells (hashed bar) after exposure to different treatments for 24 h. Note that the positive control (peroxide treatment) tended to increase cell death and early apoptosis in K7M2 cells, as expected. In contrast, neither K7M2 cell death nor early apoptosis were not affected by exposure to either naive-Exo or GdL-Exo, even up to 100X concentration.

Article Snippet: The cancer cell lines such as 143B (ATCC ® CRL-8303) human osteosarcoma and K7M2 (ATCC ® CRL-2836) murine osteosarcoma were gifted by Dr. Deryl Troyer (Department of Anatomy and Physiology, Kansas State University).

Techniques: Derivative Assay, Labeling, Modification, Negative Control, Positive Control, Staining, Concentration Assay

Journal: Theranostics

Article Title: Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice

doi: 10.7150/thno.30030

Figure Lengend Snippet: Exosomes effect apoptosis of osteosarcoma cells. Murine K7M2 osteosarcoma cells were exposed to 30 ng/cm 2 (1X) of unlabeled human mesenchymal stromal cell derived exosomes (Naive Exo) or 3000 ng/cm 2 (100X) or 30,000 ng/cm 2 (1000X) gadolinium-labeled exosomes (Exo-GdL) suspended in Dulbecco's modified eagle medium (DMEM) supplemented with 10% pooled human platelet lysate depleted of exosomes (dpHPL). K7M2 cells were incubated for 24 h and observed under confocal microscopy (the calibration bar is 20 μm). DMEM with 10% dpHPL media was used as negative control and DMEM with 10% dpHPL with 500 µM of H 2 O 2 was the positive control. K7M2 cells were stained with Annexin-V FITC (green) and propidium iodide (PI, red). Double-negative (no staining) were healthy cells, Annexin V-positive stained cells were in early apoptosis (green), cells Annexin V-positive and PI-positive were dead or necrotic cells (green/red). Note that in the Naive Exo panels, one cell in the lower right part of the panel is undergoing cytokinesis and is staining with Annexin V and PI.

Article Snippet: The cancer cell lines such as 143B (ATCC ® CRL-8303) human osteosarcoma and K7M2 (ATCC ® CRL-2836) murine osteosarcoma were gifted by Dr. Deryl Troyer (Department of Anatomy and Physiology, Kansas State University).

Techniques: Derivative Assay, Labeling, Modification, Incubation, Confocal Microscopy, Negative Control, Positive Control, Staining

Journal: Theranostics

Article Title: Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice

doi: 10.7150/thno.30030

Figure Lengend Snippet: Distribution of gadolinium-labeled exosomes in tumor-bearing mice using magnetic resonance imaging (MRI). (A) Representative MR images of mouse K7M2 osteosarcoma ectopic tumor-bearing mouse demonstrating the enhancement of positive contrast at tumor site after 30 min and 90 min post injection of gadolinium-labeled exosomes (Exo-GdL). The images were taken at echo time (TE) of 1.6 ms, repetition time (TR) of 600 ms, and flip angle (FA) of 80º using a 14.1 T MRI. (B) 24 h after Exo-GdL injection, the mice were sacrificed, the organs and tumor were disrupted with concentrated HNO 3 and gadolinium concentration was measured using inductively coupled plasma mass spectroscopy (ICP-MS). Note that the liver had approximately 37% of the gadolinium and the tumor had approximately 16%. Graph data is the average ± one standard deviation, n=3.

Article Snippet: The cancer cell lines such as 143B (ATCC ® CRL-8303) human osteosarcoma and K7M2 (ATCC ® CRL-2836) murine osteosarcoma were gifted by Dr. Deryl Troyer (Department of Anatomy and Physiology, Kansas State University).

Techniques: Labeling, Magnetic Resonance Imaging, Injection, Concentration Assay, Clinical Proteomics, Mass Spectrometry, Standard Deviation

Journal: Theranostics

Article Title: Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice

doi: 10.7150/thno.30030

Figure Lengend Snippet: Near infrared dye (DiR) fluorescence within mouse K7M2 osteosarcoma tumors. (A) Fluorescent bioimaging of mouse osteosarcoma tumors after sacrifice, 48 h after intravenous injection of either by DiR-labeled exosomes (Exo-DiR) or the synthetic control nanoparticle labeled with DiR (PEGNP-DiR). Note that the tumor size was consistent between the two groups. (B) Quantitative signal intensity per gram tumor demonstrates the tumor accumulation of exosomes compared with the control nanoparticles. Data is shown as averages ± one standard deviation, n = 3.

Article Snippet: The cancer cell lines such as 143B (ATCC ® CRL-8303) human osteosarcoma and K7M2 (ATCC ® CRL-2836) murine osteosarcoma were gifted by Dr. Deryl Troyer (Department of Anatomy and Physiology, Kansas State University).

Techniques: Fluorescence, Injection, Labeling, Control, Standard Deviation

Journal: bioRxiv

Article Title: Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma

doi: 10.1101/2024.03.02.582994

Figure Lengend Snippet: Trabectedin improves oHSV antitumor efficacy with reproducibility in immunocompetent osteosarcoma mouse models. The best response for each treated tumor through 28 days, the average tumor burden, spider plots, and individual body weights are shown for treated osteosarcoma models A) K7M2 and B) F420 in immunocompetent BALB/c and B6-albino mice, respectively. Body weight plots include mice that were excluded from the tumor burden analyses due to early non-tumor-related endpoints. PBS and oHSV (1.0 x 10 pfu) were given intratumorally (i.Tu.) on Days 0, 2, 4. Trabectedin (0.15 mg/kg) was given intravenously (I.V.) on Days 0 & 7. Statistical analyses of the disease control rates (CR + PR + SD) were performed using a pairwise Fisher’s exact test with p values adjusted using the Benjamini-Hochberg procedure; *p < 0.05, **p < 0.01, ***p < 0.001. Summarized data with error bars depict mean ± SEM.

Article Snippet: The A673 human Ewing sarcoma cell line (Cat# CCL-81), Vero green monkey kidney cell line (Cat# CRL-1598), and K7M2 mouse osteosarcoma cell line (Cat# CRL-2836) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Control

Journal: bioRxiv

Article Title: Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma

doi: 10.1101/2024.03.02.582994

Figure Lengend Snippet: Trabectedin reduces immune suppression and enhances effector cell activation to synergize with oHSV. A) scRNAseq UMAP plot of the treated K7M2 tumor microenvironments. Datasets from all treatment groups were merged with no need for batch correction. B) Bar plot displaying the normalized enrichment score (NES) of the significant KEGG pathways from gene set enrichment analysis in NK cells from the tumor microenvironment. C) FACS of tumor infiltrating immune cells showed decreased immunosuppressive cells and increased activated T cells post-treatment (arrow indicates control for comparison, *p < 0.05, **p < 0.01, ***p < 0.001). D) Violin plots showing increased cytotoxic expression in all NK and T cells of combination-treated K7M2 (arrow indicates control for comparison, percent fold change = 100*2log2FC). PBS and oHSV (1.0 x 10 pfu) were given i.Tu. on Days 0 & 2. Trabectedin (0.15 mg/kg) was given I.V. on Day 0. scRNAseq samples were collected on Day 3. Flow cytometry samples were collected on Day 7.

Article Snippet: The A673 human Ewing sarcoma cell line (Cat# CCL-81), Vero green monkey kidney cell line (Cat# CRL-1598), and K7M2 mouse osteosarcoma cell line (Cat# CRL-2836) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Activation Assay, Control, Comparison, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma

doi: 10.1101/2024.03.02.582994

Figure Lengend Snippet: Combination efficacy in immunocompetent osteosarcoma models results from augmentation of antitumor NK and T cell responses. The best response for each treated tumor through 28 days, the average tumor burden, and spider plots tracking individual tumor volumes over the full study period are displayed for A) K7M2 tumor-bearing nude mice (lack T and B cells), B) K7M2-bearing BALB/c mice treated with anti-asialo NK cell depletion (given I.V. on Days 0, 7, 14, 21), C) K7M2-bearing BALB/c mice treated with antibody-mediated CD4 and CD8 T cell depletion (given I.V. on Days 0, 4, 8, 12, 16, 20, 24, 28). PBS and oHSV (1.0 x 10 pfu) were given i.Tu. on Days 0, 2, & 4. Trabectedin (0.15 mg/kg) was given I.V. on Days 0 & 7. Statistical analyses of the disease control rates (CR + PR + SD) were performed using a pairwise Fisher’s exact test with p values adjusted using the Benjamini-Hochberg procedure; *p < 0.05, **p < 0.01, ***p < 0.001. Summarized data with error bars depict mean ± SEM.

Article Snippet: The A673 human Ewing sarcoma cell line (Cat# CCL-81), Vero green monkey kidney cell line (Cat# CRL-1598), and K7M2 mouse osteosarcoma cell line (Cat# CRL-2836) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Avicularin Reduces the Expression of Mediators of Inflammation and Oxidative Stress in Bradykinin-Treated MG-63 Human Osteoblastic Osteosarcoma Cells

doi: 10.12659/MSM.921957

Figure Lengend Snippet: The effects of avicularin on the viability of MG-63 human osteoblastic osteosarcoma cells. MG-63 cells were treated with increasing concentrations of avicularin (10 μM, 30 μM, 100 μM, and 300 μM) for 48 h. Cell viability was measured using the MTT assay. Data are presented as the mean±standard deviation (SD) from three independent experiments.

Article Snippet: MG-63 human osteoblastic osteosarcoma cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) (Cat. No. CRL-1427).

Techniques: MTT Assay, Standard Deviation

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Avicularin Reduces the Expression of Mediators of Inflammation and Oxidative Stress in Bradykinin-Treated MG-63 Human Osteoblastic Osteosarcoma Cells

doi: 10.12659/MSM.921957

Figure Lengend Snippet: The effects of avicularin on the expression of inflammatory factors in bradykinin-treated MG-63 human osteoblastic osteosarcoma cells. MG-63 cells were treated with increasing concentrations of avicularin (10 μM, 30 μM, 100 μM, and 300 μM) for 48 h, then treated with 1 μM bradykinin for 24 h. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of tumor necrosis factor-α (TNF-α) ( A ), interleukin-1β (IL-1β) ( B ), and IL-6 ( C ) in MG-63 cells from the control groups and bradykinin-treated groups. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of TNF-a ( D ), IL-1β ( E ), and IL-6 ( F ) mRNA expression in MG-63 cells from the control groups and bradykinin-treated groups. Data were presented as the mean±SD from three independent experiments. ** p<0.01 vs. the control group; # , ## p<0.05, p<0.01 vs. the bradykinin group.

Article Snippet: MG-63 human osteoblastic osteosarcoma cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) (Cat. No. CRL-1427).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Avicularin Reduces the Expression of Mediators of Inflammation and Oxidative Stress in Bradykinin-Treated MG-63 Human Osteoblastic Osteosarcoma Cells

doi: 10.12659/MSM.921957

Figure Lengend Snippet: The effects of avicularin on the levels of MDA, SOD, and catalase in bradykinin-treated MG-63 human osteoblastic osteosarcoma cells. Increasing concentrations of avicularin treated MG-63 cells for 48 h, then we used 1 μM bradykinin to treat the cells for 24 h. ( A ) The malondialdehyde (MDA) content was determined by MDA assay kit. ( B ) Superoxide dismutase (SOD) activity was determined by the SOD assay kit. ( C ) Catalase activity was measured using a catalase assay kit. Data are presented as the mean±standard deviation (SD) from three independent experiments. ** p<0.01 vs. the control group; # , ## p<0.05, p<0.01 vs. the bradykinin group.

Article Snippet: MG-63 human osteoblastic osteosarcoma cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) (Cat. No. CRL-1427).

Techniques: Multiple Displacement Amplification, Activity Assay, Standard Deviation, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Avicularin Reduces the Expression of Mediators of Inflammation and Oxidative Stress in Bradykinin-Treated MG-63 Human Osteoblastic Osteosarcoma Cells

doi: 10.12659/MSM.921957

Figure Lengend Snippet: The effects of avicularin on p38MAPK/NF-κB pathway in bradykinin-treated MG-63 human osteoblastic osteosarcoma cells. MG-63 cells were treated with increasing concentrations of avicularin for 48 h, and 1 μM of bradykinin was used to treat the cells for 24 h. ( A ) Western blot detected the expression of p-p38 and p-p65 at the protein level. ( B ) The ratio of p-p38/p38. ( C ) The ratio of p-p65/p65. Quantitative real-time polymerase chain reaction (qRT-PCR) measured the mRNA expression level of p38 ( D ) and p65 ( E ) at the mRNA level. Data are presented as the mean±SD from three independent experiments. ** p<0.01 vs. the control group; # , ## p<0.05, p<0.01 vs. the bradykinin group.

Article Snippet: MG-63 human osteoblastic osteosarcoma cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) (Cat. No. CRL-1427).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Avicularin Reduces the Expression of Mediators of Inflammation and Oxidative Stress in Bradykinin-Treated MG-63 Human Osteoblastic Osteosarcoma Cells

doi: 10.12659/MSM.921957

Figure Lengend Snippet: The effects of avicularin on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in bradykinin-treated MG-63 human osteoblastic osteosarcoma cells. MG-63 cells were treated with increasing concentrations of avicularin for 48 h, and 1 μM of bradykinin was used to treat the cells for 24 h. ( A ) Western blot assay was performed to measure the protein expression of iNOS and COX-2. ( B ) The ratio of iNOS/GAPDH is presented. ( C ) The ratio of COX-2 GAPDH is presented. Quantitative real-time polymerase chain reaction (qRT-PCR) detected the expression level of iNOS ( D ) and COX-2 ( E ) at the mRNA level. Data are presented as the mean±standard deviation (SD) from three independent experiments. ** p<0.01 vs. the control group; # , ## p<0.05, p<0.01 vs. the bradykinin group.

Article Snippet: MG-63 human osteoblastic osteosarcoma cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) (Cat. No. CRL-1427).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Control