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: In vitro cell cultures We used the following human patient-derived EWS cell lines: TC-71 (DSMZ, Catalog no. ACC-516, RRID: CVCL_2213) and 6647 (RRID: CVCL_H722) both kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA); H-825 kindly provided by Prof. A. Llombart-Bosch (University of Valencia, Spain); and A673 cells (RRID: CVCL_0080) kindly provided by H. Kovar (St. Anna Kinderkrebsforschung).

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: In vitro cell cultures We used the following human patient-derived EWS cell lines: TC-71 (DSMZ, Catalog no. ACC-516, RRID: CVCL_2213) and 6647 (RRID: CVCL_H722) both kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA); H-825 kindly provided by Prof. A. Llombart-Bosch (University of Valencia, Spain); and A673 cells (RRID: CVCL_0080) kindly provided by H. Kovar (St. Anna Kinderkrebsforschung).

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: In vitro cell cultures We used the following human patient-derived EWS cell lines: TC-71 (DSMZ, Catalog no. ACC-516, RRID: CVCL_2213) and 6647 (RRID: CVCL_H722) both kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA); H-825 kindly provided by Prof. A. Llombart-Bosch (University of Valencia, Spain); and A673 cells (RRID: CVCL_0080) kindly provided by H. Kovar (St. Anna Kinderkrebsforschung).

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: In vitro cell cultures We used the following human patient-derived EWS cell lines: TC-71 (DSMZ, Catalog no. ACC-516, RRID: CVCL_2213) and 6647 (RRID: CVCL_H722) both kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA); H-825 kindly provided by Prof. A. Llombart-Bosch (University of Valencia, Spain); and A673 cells (RRID: CVCL_0080) kindly provided by H. Kovar (St. Anna Kinderkrebsforschung).

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 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