Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 1. The human anti-CD99 dAbd C7 inhibits tumor growth and increases macrophages infiltration. A, Tumor volume after treatment with the anti-CD99 dAbd C7 in xenografts derived from injection of 6647 cells in nude mice. Control, n ¼ 10; dAbd C7, n ¼ 6 (left) or PDX-EW#2 tumors developed in NSG mice; control, n ¼ 4; dAbd C7, n ¼ 5 (right). Dots represent single tumor volumes, mean SD are displayed. Mann–Whitney U test, , P < 0.05. B, Representative H&E images of 6647 (left) or PDX-EW#2 (right) xenografts treated or not with dAbd C7 (scale bar, 50 mm). The tumor regions with nuclear condensation and the total area of the samples were identified by manually marking with Image J software. Histogram represents the percentage of tumor area with nuclear condensation (vs. total tumor area). Data are expressed as the median and range (minimum–maximum). Mann–Whitney U test, , P < 0.05. C, Representative images of double staining IHC for F4/80 murine macrophages (brown) and CD99 EWS cells (red) in 6647 (left) or PDX-EW#2 (right) xenografts after treatment with dAbd C7 (scale bar, 50 mm). The enlarged box shows a mouse macrophage with engulfed EWS cells; the histograms represent the percentage of F4/80-positive cells calculated after evaluation of at least ten fields. Data are expressed as the median and range (minimum–maximum. Mann–Whitney U test; , P < 0.001.

Article Snippet: For immunoprecipitation, 500 mg of cell lysates (as described above) obtained from 5 106 EWS cells (6647, TC-71 and TC-CD99-shRNA#2) were incubated for 16 hours with Protein G-Plus agarose beads (#sc-2003, Santa Cruz Biotechnology) in the presence of 2 mg anti-CD99 12E7 mAb (sc-53148, Santa Cruz, mouse monoclonal, RRID: AB_629249), anti-CD47 (#ab218810, rabbit monoclonal Abcam), or control IgG antibody (MOPC-21, #M7894, Sigma-Aldrich, RRID: AB_1163632).

Techniques: Derivative Assay, Injection, Control, MANN-WHITNEY, Software, Double Staining

Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 2. Macrophage-mediated phagocytosis and cytotoxicity of EWS cells after anti-CD99 antibodies treatment. A, Phagocytic index of M0-like macrophages cocultured for 6 hours with TC-EGFP cells being exposed to anti-CD99 antibodies (12E7 mAb; 0662 mAb; dAbd C7 or to irrelevant MOPC21 antibody used as isotype control) for 30 minutes. Phagocytic index indicated the number of EWS cells phagocytosed per 100 macrophages. Dots represent single fields and data are expressed as the median and range (minimum–maximum) of at least three independent experiments. One-way ANOVA: , P < 0.05; , P < 0.01; , P < 0.001. B, Representative images of M0-like macrophages phagocytosing EWS cells after treatment with anti-CD99 antibodies. Arrows point to phagocytosed tumor cells (scale bar, 50 mm). C, EWS cell survival in the presence or not of M0-like macrophages (Mj) detected by Trypan blue vital counting. Data are expressed as percentage compared with untreated TC-71 cells. Bars represent the mean SD of at least three independent experiments. Mann–Whitney U test: , P < 0.05; , P < 0.01.

Article Snippet: For immunoprecipitation, 500 mg of cell lysates (as described above) obtained from 5 106 EWS cells (6647, TC-71 and TC-CD99-shRNA#2) were incubated for 16 hours with Protein G-Plus agarose beads (#sc-2003, Santa Cruz Biotechnology) in the presence of 2 mg anti-CD99 12E7 mAb (sc-53148, Santa Cruz, mouse monoclonal, RRID: AB_629249), anti-CD47 (#ab218810, rabbit monoclonal Abcam), or control IgG antibody (MOPC-21, #M7894, Sigma-Aldrich, RRID: AB_1163632).

Techniques: Control, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 3. Modulation of ‘eat-me’ and ‘don’t eat-me’ molecules on EWS cell surface after CD99 engagement. A, Cytofluorimetric profile of PS and CALR expression in EWS cells treated with anti-CD99 antibodies (expressed as percentage of positive cells). B, MFI of CD47 expression in EWS cells treated with anti-CD99 antibodies. All cells are 100% positive to CD47 even after treatments. Bars indicated the mean SD of at least three independent experiments. One-way ANOVA: , P < 0.05; , P < 0.01; , P < 0.001. Gating strategy and representative profiles of each type of experiment (A–B), are reported in Supplementary Figs. S4 and S5. C, Confocal microscopy images of CD47 in TC-71 cells treated with 0662 mAb (10 mg/mL), indicating intracellular localization of CD47 (arrows). Enlarged views of the boxed regions are also displayed (scale bar, 50 mm, representative images of two independent experiments are shown). D, Western blot analysis of CD47 on total cell lysates from 6647 and TC-71 cells treated with anti-CD99 antibodies (0662 mAb; dAbd C7) in the presence or absence of the proteasome inhibitor MG132. Data are representative of at least three independent experiments. Beta-tubulin was included as loading control. E, Representative images of IHC staining of CD47 in 6647 (left) or PDX-EW#2 (right) xenografts after treatment with dAbd C7 (scale bar, 50 mm). Histograms represent the percentage of CD47 positive cells calculated after evaluation of at least ten fields. Data are presented as the median and range (minimum–maximum). Mann–Whitney U test: , P < 0.05; , P < 0.001.

Article Snippet: For immunoprecipitation, 500 mg of cell lysates (as described above) obtained from 5 106 EWS cells (6647, TC-71 and TC-CD99-shRNA#2) were incubated for 16 hours with Protein G-Plus agarose beads (#sc-2003, Santa Cruz Biotechnology) in the presence of 2 mg anti-CD99 12E7 mAb (sc-53148, Santa Cruz, mouse monoclonal, RRID: AB_629249), anti-CD47 (#ab218810, rabbit monoclonal Abcam), or control IgG antibody (MOPC-21, #M7894, Sigma-Aldrich, RRID: AB_1163632).

Techniques: Expressing, Confocal Microscopy, Western Blot, Control, Immunohistochemistry, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 4. The presence of CD99 on EWS cell surface influences CD47 status. A,ExpressionofCD47inCD99-silenced cells. Bars indicate MFI. Mean SD of at least three independent experiments, Kruskal–Wallis test: , P < 0.05. B, Cytofluorimetric profile of CD47 expression in TC-CD99-shRNA#2 EWS cells treated with anti-CD99 antibodies. Bars indicate the mean SD of at least three independent experiments. Gating strategy and representative profiles of each type of experiment (A and B), are reported in Supplementary Fig. S7. C, Immunoprecipitation of CD99 with CD47 in 6647, TC-71, and TC-CD99- shRNA#2 EWS cells. Blots are repre- sentative of at least three independent experiments.

Article Snippet: For immunoprecipitation, 500 mg of cell lysates (as described above) obtained from 5 106 EWS cells (6647, TC-71 and TC-CD99-shRNA#2) were incubated for 16 hours with Protein G-Plus agarose beads (#sc-2003, Santa Cruz Biotechnology) in the presence of 2 mg anti-CD99 12E7 mAb (sc-53148, Santa Cruz, mouse monoclonal, RRID: AB_629249), anti-CD47 (#ab218810, rabbit monoclonal Abcam), or control IgG antibody (MOPC-21, #M7894, Sigma-Aldrich, RRID: AB_1163632).

Techniques: Expressing, shRNA, Immunoprecipitation

Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 6. Engagement of CD99 on ex vivo undifferentiated M0-like macrophages induces M1-like polarization and increases their phagocytosis capabilities against EWS cells. A, Phagocytic index of M0-like macrophages exposed to anti-CD99 antibodies (0662 mAb; dAbd C7) or to irrelevant MOPC21 antibody (used as isotype control) for 3 hours and then cocultured with TC-EGFP cells for an additional 3 hours as indicated in the Materials and Methods. The phagocytic index indicates the number of EWS cells phagocytosed per 100 macrophages. Dots represent single fields, and data are expressed as the median and range (minimum–maximum) of at least three independent experiments. Kruskal–Wallis test: , P < 0.05; , P < 0.01. B, TC-EGFP cell survival in the presence of M0-like macrophages exposed to anti-CD99 antibodies. Trypan blue vital cell count was used. Data are expressed as percentages compared with untreated EWS cells. Bars indicate mean SD from at least three independent experiments. Kruskal–Wallis test: , P < 0.05; , P < 0.01. C, Relative expression of CD80, CD86, CD163, and CD206 was analyzed by qPCR in M0-like macrophages treated with anti-CD99 0662 mAb or dAbd C7 for 1 to 6hours. Data are expressed as the mean SD. Two-wayANOVA test: , P < 0.05; , P < 0.01; , P < 0.001. D, Multiplex cytokine assay analysis of IL1b, IL6, TNFa, and IL4 release in M0-like macrophages treated with anti-CD99 0662 mAb or dAbd C7 as indicated in the Materials and Methods. Data are expressed as mean SD of the fold increase versus control M0-like ¼ 1. Two-way ANOVA test: , P < 0.05; , P < 0.01; , P < 0.001. E, Western blot analysis of intracellular signaling molecules in M0-like macrophages treated as indicated. GAPDH was used as the loading control. Representative blots from three independent experiments are shown.

Article Snippet: For immunoprecipitation, 500 mg of cell lysates (as described above) obtained from 5 106 EWS cells (6647, TC-71 and TC-CD99-shRNA#2) were incubated for 16 hours with Protein G-Plus agarose beads (#sc-2003, Santa Cruz Biotechnology) in the presence of 2 mg anti-CD99 12E7 mAb (sc-53148, Santa Cruz, mouse monoclonal, RRID: AB_629249), anti-CD47 (#ab218810, rabbit monoclonal Abcam), or control IgG antibody (MOPC-21, #M7894, Sigma-Aldrich, RRID: AB_1163632).

Techniques: Ex Vivo, Control, Cell Counting, Expressing, Multiplex Assay, Cytokine Assay, Western Blot

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME is a 153–amino acid transmembrane glycoprotein. A, Western blots of TC-71 lysates transfected with EV or ROME-HA for 24 hours blotted with the commercially available anti-HA antibody, ICD-6 (monoclonal antibody against the predicted ICD of ROME), and ECD-5 (monoclonal antibody against the predicted ECD of ROME) are shown. B, ROME was knocked out via CRISPR/Cas9 in TC-71 cells. Successful knockout was confirmed by immunoprecipitation and Western blotting with a ROME monoclonal antibody (ICD-6). C, Coomassie blue staining was used to verify the purity of recombinant ROME-FL and ECD proteins and the synthetic ICD peptide. Western blotting with ROME ICD-6 and ECD-5 monoclonal antibodies was performed to confirm protein identities. D, SPR sensorgrams demonstrating the binding of the anti-ROME monoclonal antibody ICD-6 to the ROME ICD and FL proteins and the binding of the anti-ROME monoclonal antibody ECD-5 to the ROME ECD and FL proteins. The antibodies were injected in duplicate at 200, 100, 50, 25, 12.5, and 6.25 nmol/L. The red lines are the actual data and the black lines indicate the curve fit. E, Three-dimensional structure of ROME predicted by AlphaFold. F, A diagram of the ROME amino acid sequence with three predicted domains is shown. Circles correspond to phosphorylation sites and triangles correspond to glycosylation sites. The black color designates sites predicted in silico and the blue color designates sites confirmed by MS. G, Homology between human, mouse, and zebrafish ROME proteins is shown. The Drosophila inaF motif (from the INAF-D protein) is shown for comparison. The black line box indicates the transmembrane domain. Asterisks (*) indicate identical amino acids (aa) and colons (:) indicate amino acids with similarity among all three species. H, Phylogenetic tree showing the high evolutionary conservation of the ROME protein in vertebrates. I, Compared with the eGFP control, ROME-GFP localizes to the plasma membrane of TC-71 cells (quantification shown in the graph to the right). J, ROME-HA was expressed in TC-71 cells and detected primarily in the membrane fraction, as well as the soluble nuclear fraction. α-Tubulin was used as a cytoplasmic fraction control, CD99 was used as a membrane fraction control, and lamin A/C was used as a soluble nuclear fraction control. K, Lysates from TC-71 cells transfected with EV or ROME-FLAG were treated with a deglycosylase mixture. Western blotting with anti-FLAG antibody was performed to detect ROME-FLAG.

Article Snippet: For the tail vein experiments, 1 million TC-71 cells (WT and ROME KO or stably expressing ROME cDNA versus an EV control cell line) were injected into the tail vein of 6-week-old female SCID beige mice (Charles River Laboratories, RRID: IMSR_CRL:250).

Techniques: Western Blot, Transfection, CRISPR, Knock-Out, Immunoprecipitation, Staining, Recombinant, Bioprocessing, Binding Assay, Injection, Sequencing, Phospho-proteomics, Glycoproteomics, In Silico, Comparison, Control, Clinical Proteomics, Membrane

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: Genome-wide CRISPR/Cas9 transcriptional activation screen reveals novel prometastatic genes in Ewing sarcoma. A, Schematic overview of the in vivo CRISPR/Cas9 genome-wide transcriptional activation screening experiment (TSS, transcription start site). B, Four Ewing sarcoma cell lines were injected into 48 hpf zebrafish embryos at three different cell densities ( n = 12 for each condition) and the number of fish with metastasis to the tail was scored every day for 5 days. C, Read counts per million for each gRNA detected from the genome-wide CRISPR/Cas9 transcriptional activation screening experiment. The eight genes chosen for further study are indicated in bold. D, Eight candidate genes from the screen were stably overexpressed in TC-71 cells and tested in zebrafish xenograft assays (*, P < 0.05; **, P < 0.01; χ 2 test). The red line indicates the baseline intravasation of TC-71 cells transfected with EV. The means and SEMs are shown. The dots represent individual experiments and the total number of fish in all the experiments combined is provided above each condition. E, TC-71 cells stably overexpressing eight different genes were tested via a scratch assay. The dots indicate independent experiments. The means and SEMs are shown (*, P < 0.05, Student t test). The red line indicates the average percent wound closure of TC-71 cells transfected with EV. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/pw6o2c1 .]

Article Snippet: For the tail vein experiments, 1 million TC-71 cells (WT and ROME KO or stably expressing ROME cDNA versus an EV control cell line) were injected into the tail vein of 6-week-old female SCID beige mice (Charles River Laboratories, RRID: IMSR_CRL:250).

Techniques: Genome Wide, CRISPR, Activation Assay, In Vivo, Injection, Stable Transfection, Transfection, Wound Healing Assay

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME drives intravasation through vimentin. A, Timeline of zebrafish xenograft experiments. B, Zebrafish xenograft experiments with three different ROME-overexpressing Ewing sarcoma cell lines. Statistical significance was determined via the Fisher exact test (two-sided). C, Zebrafish xenograft experiments comparing WT and ROME-KO TC-71 cells. Statistical significance was determined via the Fisher exact test (one-sided). D, ROME and vimentin proteins co-immunoprecipitate in three different Ewing sarcoma cell lines. For TC-32 ROME blot, line denotes higher exposure on left to visualize input and lower exposure on right. E, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and vimentin protein. Vimentin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 2,500, 800, 280, 90, 30, and 10 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. F, ROME expression reduces vimentin phosphorylation at serine 56 (S56) in a tet-inducible ROME-HA expression system (DOX = doxycycline 250 ng/mL). Densitometry analysis of Western blot bands is shown in the bottom right corner ( n = 4 independent experiments). G, Western blotting was used to confirm vimentin knockdown by siRNA in STA-ET-7.2 cells with EV or ROME overexpression. H, Vimentin knockdown selectively reduced ROME-driven intravasation in ROME-overexpressing STA-ET-7.2 cells compared with an EV control [ n = 102 (EV + siNT), 116 (ROME + siNT), 88 (EV + siVimentin), and 119 (ROME + siVimentin)]. I, ROME mRNA expression positively correlates with vimentin mRNA expression in analysis of 13,313 patient samples. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/fp8c676 .]

Article Snippet: For the tail vein experiments, 1 million TC-71 cells (WT and ROME KO or stably expressing ROME cDNA versus an EV control cell line) were injected into the tail vein of 6-week-old female SCID beige mice (Charles River Laboratories, RRID: IMSR_CRL:250).

Techniques: Binding Assay, Recombinant, Injection, Expressing, Phospho-proteomics, Western Blot, Knockdown, Over Expression, Control

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME expression drives metastasis development in vivo . A, Timeline of tail vein metastasis experiments. B, In a tail vein metastasis model, mice injected with ROME-overexpressing TC-71 cells ( n = 11) had a higher total mass of metastatic nodules than did controls ( n = 10). C, In a tail vein metastasis model, mice injected with ROME-KO TC-71 cells ( n = 11) had a lower total mass of metastases than controls injected with WT TC71 cells ( n = 11). Statistical significance was determined via an unpaired t test (one-tailed) for B and C . D, Diagram of the orthotopic Ewing sarcoma leg amputation experimental timeline. E, Representative gross images of lungs from mice injected with TC-71 cells overexpressing ROME or with EV (left) with quantification (middle). Representative images of H&E staining are shown on the right confirming metastatic nodules in the lungs of SCID mice. Statistical significance was calculated by the Fisher exact test (one-sided). F, TC-71 ROME-KO cells displayed significantly reduced primary tumor recurrence [ n = 10 per group, log-rank (Mantel–Cox) test]. G, ROME is expressed throughout many different tumor types (CNS, central nervous system; HEPAC, hepatocellular carcinoma and cholangioma; KICH, kidney chromophobe and other renal carcinoma; KIPCC, papillary cell renal carcinoma; OV, ovarian cystadenocarcinoma; PAAD, pancreatic ductal adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; SCC/BLCA, squamous cell or bladder carcinoma; TGCT SEM, testicular germ cell tumor, seminoma). H, Kaplan–Meier curves of patient survival stratified by high (red line) and low (black line) ROME expression (from the Kaplan–Meier plotter database). [ A and D, Created in BioRender. Lab, S. (2026) https://BioRender.com/atgnfw0 .]

Article Snippet: For the tail vein experiments, 1 million TC-71 cells (WT and ROME KO or stably expressing ROME cDNA versus an EV control cell line) were injected into the tail vein of 6-week-old female SCID beige mice (Charles River Laboratories, RRID: IMSR_CRL:250).

Techniques: Expressing, In Vivo, Injection, One-tailed Test, Staining

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME protein negatively regulates the Wnt pathway and calcium signaling in human cancer cells. A and B, Compared with EV control cells, TC-71 ( A ) and TC-32 ( B ) cells stably overexpressing ROME were less responsive to recombinant human Wnt3a protein as measured by TOPFlash β-catenin–responsive luciferase reporter. C, TC-71 cells with ROME knockdown by siRNA were more responsive to Wnt3a. D, Endogenous Wnt pathway activity in the colorectal cancer cell line HCT116 was significantly decreased by ROME expression (Western blots confirming ROME-HA expression on the right). Statistical significance was calculated by an unpaired t test (two-tailed) for A–D . E, ROME co-immunoprecipitates with β-catenin in β-catenin pulldown experiments. F, β-Catenin co-immunoprecipitates with ROME in ROME pulldown experiments. G, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and β-catenin protein. β-Catenin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 1,250, 416.7, 138.9, 46.3, 15.4, and 5.12 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. H, ROME expression decreases β-catenin and TCF4 interaction (measured by co-IP). I–K, The Ca 2+ response was decreased in A4573, STA-ET-7.2, and TC-71 cells overexpressing ROME compared with that in EV-transfected cells when the cells were stimulated with ATP or FBS after overnight serum starvation. L, The Ca 2+ response was greater in the TC-71 cells with ROME KO than in the WT cells. Statistical significance was calculated by an unpaired t test (two-tailed) for I–L .

Article Snippet: For the tail vein experiments, 1 million TC-71 cells (WT and ROME KO or stably expressing ROME cDNA versus an EV control cell line) were injected into the tail vein of 6-week-old female SCID beige mice (Charles River Laboratories, RRID: IMSR_CRL:250).

Techniques: Control, Stable Transfection, Recombinant, Luciferase, Knockdown, Activity Assay, Expressing, Western Blot, Two Tailed Test, Binding Assay, Injection, Co-Immunoprecipitation Assay, Transfection

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME expression increases cell motility and chemotaxis in vitro . A, TC-71 cells stably overexpressing ROME demonstrated increased wound closure compared with that of a control cell line, and TC-71 cells with ROME KO by CRISPR/Cas9 demonstrated decreased wound closure compared with that of WT cells in scratch assays. At least two independent experiments were performed, and average wound closure with SEM was quantified. Statistical significance was calculated by an unpaired t test (two-tailed). B–E, Compared with EV control cells, A4573, TC-71, and Hs746.T cells stably expressing ROME presented increased chemotaxis in Boyden chamber assays. F, CHLA-10 cells with ROME KO via CRISPR/Cas9 showed decreased chemotaxis compared with that of WT cells. G–I, ROME KO (TC-71) or knockdown (769-P and Hs746.T) decreased the chemotaxis of cancer cells. J and K, Rescue of ROME expression by an exogenous vector restored chemotaxis in both CHLA-10 and TC-71 cells. At least two independent experiments with three biological replicates were performed for each Boyden chamber assay. Statistical significance was calculated by an unpaired t test (two-tailed).

Article Snippet: For the tail vein experiments, 1 million TC-71 cells (WT and ROME KO or stably expressing ROME cDNA versus an EV control cell line) were injected into the tail vein of 6-week-old female SCID beige mice (Charles River Laboratories, RRID: IMSR_CRL:250).

Techniques: Expressing, Chemotaxis Assay, In Vitro, Stable Transfection, Control, CRISPR, Two Tailed Test, Knockdown, Plasmid Preparation, Boyden Chamber Assay

Journal: Oncotarget

Article Title: Inhibition of porcupine prolongs metastasis free survival in a mouse xenograft model of Ewing sarcoma

doi: 10.18632/oncotarget.19432

Figure Lengend Snippet: A. The indicated cells were treated with or without 1 μM WNT974 for 48 hours, and relative cell number was quantified using the Cell Counting Kit-8. Data shown are the mean relative cell number, with untreated cells defined as 100%, from experiments performed in triplicate. Error bars represent standard error of the mean. Experiments were repeated 3 times and representative results are presented. B. The indicated cells were grown under nonadherent conditions for 7 days in the presence or absence of 1 μM WNT974 and spheres were counted manually. Data shown are the mean relative number of spheres per well, from triplicate wells, with untreated cells defined as 1.0. Error bars represent standard error of the mean. Experiments were repeated 3 times and representative results are presented. C. Representative TC71 sarcospheres are shown (40x magnification). D. Migration of the indicated cell lines in a Boyden chamber assay was quantified in the presence or absence of 1 μM WNT974. Data shown are the relative number of cells migrating through the barrier, performed in triplicate, with untreated cells defined as 1.0. Error bars represent standard error of the mean. Experiments were repeated 3 times and representative results are presented. E. The indicated cells were grown for 48 hours in the presence or absence of 1 μM WNT974 and then fixed and stained with the Cytopainter F-Actin Staining Kit. Long cytoplasmic extensions were counted and data shown are the relative number of extensions per cell, quantified from triplicate wells and normalized to a value of 1.0 for control cells. Error bars represent standard error of the mean. Experiments were repeated 3 times and representative results are presented. F. Representative images of SK-ES-1 cells stained with the Cytopainter F-Actin Staining Kit after growing in vehicle (left image) or 1 μM WNT974 (right image) for 48 hours (100x magnification).

Article Snippet: NOD/SCID/IL-2Rγ-null (NSG) mice bred by Johns Hopkins University Research Animal Resources were implanted with 3 mm fragments of either a TC71 cell line xenograft or of the patient derived xenograft (PDX) EWS1 or EWS4 in the pre-tibial space as previously described [ ].

Techniques: Cell Counting, Migration, Boyden Chamber Assay, Staining, Control

Journal: Oncotarget

Article Title: Inhibition of porcupine prolongs metastasis free survival in a mouse xenograft model of Ewing sarcoma

doi: 10.18632/oncotarget.19432

Figure Lengend Snippet: A. The indicated cell lines were treated with or without 1 μM WNT974 for 48 hours, and mRNA was isolated, reverse transcribed, and analyzed using an EMT gene-focused PCR array. Expression of the indicated genes in treated cells was quantified relative to control cells by the ΔΔCt method after normalization based on beta-2 microglobulin expression. A heatmap of the results is presented. The deepest blue represents expression level of 0.1 in treated cells compared with control, and the deepest red represents an expression level of 10.0 in treated cells compared with control. Mice bearing the EWS4 PDX B. or a xenograft derived from the TC71 cell line C. were treated with or without WNT974 at 5mg/kg/dose twice daily for 3 days. Tumors were harvested and expression of the indicated genes evaluated by RT-PCR, using beta-2-microglobulin expression for normalization. Fold expression refers to expression relative to tumors harvested from untreated mice. Data shown are the results of triplicate assays, and the error bar shows standard error of the mean. With the exception of TWIST1, each difference is statistically significant, with p < 0.01.

Article Snippet: NOD/SCID/IL-2Rγ-null (NSG) mice bred by Johns Hopkins University Research Animal Resources were implanted with 3 mm fragments of either a TC71 cell line xenograft or of the patient derived xenograft (PDX) EWS1 or EWS4 in the pre-tibial space as previously described [ ].

Techniques: Isolation, Reverse Transcription, Expressing, Control, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Inhibition of porcupine prolongs metastasis free survival in a mouse xenograft model of Ewing sarcoma

doi: 10.18632/oncotarget.19432

Figure Lengend Snippet: Mice were implanted with fragments of a TC71 xenograft A. and D. , the EWS1 PDX B. and E. or the EWS4 PDX C. and F. and then treated with or without WNT974 at 5mg/kg/dose twice daily. Tumor growth was quantified twice weekly A. - C. After amputation, mice were followed until death D. - F. Tumor growth data show the mean leg circumference, with error bars showing standard deviation. Differences were evaluated for statistical significance by 2-way ANOVA but none of the growth curves showed a statistically significant difference. Kaplan-Meier survival curves were tested for significance by the log-rank test. Histological and immunofluorescent evaluation of the primary tumor G. and I. and of metastases isolated from the lung at necropsy H. and J. demonstrate a small round blue cell tumor G. and H. of human origin (based on immunofluorescent staining with anti-human mitochondrion antibodies; I. and J. , consistent with Ewing sarcoma. Panels G. and H. show a primary tumor (red arrow) and a pulmonary metastasis (green arrow) stained with haematoxylin and eosin at 4x G. and 20x H. magnification. Panels I. and J. show a primary tumor (red arrow) and 2 pulmonary metastases (yellow arrows) stained with Alexa Fluor 488 -labeled anti-human mitochondrion antibody (green) and counterstained with DAPI (blue) at 4x I. and 20x J. magnification.

Article Snippet: NOD/SCID/IL-2Rγ-null (NSG) mice bred by Johns Hopkins University Research Animal Resources were implanted with 3 mm fragments of either a TC71 cell line xenograft or of the patient derived xenograft (PDX) EWS1 or EWS4 in the pre-tibial space as previously described [ ].

Techniques: Standard Deviation, Isolation, Staining, Labeling

Journal: Oncotarget

Article Title: Inhibition of porcupine prolongs metastasis free survival in a mouse xenograft model of Ewing sarcoma

doi: 10.18632/oncotarget.19432

Figure Lengend Snippet: Ten thousand TC71 cells A. or cells from a single cell suspension made from the EWS4 PDX B. were injected into the tail vein of mice treated with or without (control) WNT974 at 5mg/kg/dose twice daily. Mice were followed until death from metastatic disease.

Article Snippet: NOD/SCID/IL-2Rγ-null (NSG) mice bred by Johns Hopkins University Research Animal Resources were implanted with 3 mm fragments of either a TC71 cell line xenograft or of the patient derived xenograft (PDX) EWS1 or EWS4 in the pre-tibial space as previously described [ ].

Techniques: Suspension, Injection, Control

Journal: Oncotarget

Article Title: Inhibition of porcupine prolongs metastasis free survival in a mouse xenograft model of Ewing sarcoma

doi: 10.18632/oncotarget.19432

Figure Lengend Snippet: Fragments of a TC71 xenograft were implanted in the pretibial space of NSG mice which were then treated with or without WNT974 at 5mg/kg/dose twice daily. Mice were imaged weekly MRI using the Bruker 9.4T horizontal bore spectrometer, and images were evaluated by an investigator blinded to the status of the mice for the presence or absence of lung metastases. Differences in time to the development of first lung metastasis were evaluated by log-rank test A. or Student’s t test B. , and both methods confirmed a statistically significant difference.

Article Snippet: NOD/SCID/IL-2Rγ-null (NSG) mice bred by Johns Hopkins University Research Animal Resources were implanted with 3 mm fragments of either a TC71 cell line xenograft or of the patient derived xenograft (PDX) EWS1 or EWS4 in the pre-tibial space as previously described [ ].

Techniques: