tumor cell lines a673  (ATCC)

Journal: Advanced healthcare materials

Article Title: Detection of Extracellular Vesicles with Colocalized Surface Markers via a Capture–Release–Capture Strategy for Treatment Monitoring in Ewing Sarcoma

doi: 10.1002/adhm.202505917

Figure Lengend Snippet: Validation of Paired Surface Markers, CD99 and B7-H3, using ES TMA and A673 ES Cells. (A) Representative H&E staining shows ES tissue morphology, while IHC staining demonstrates strong membrane expression of CD99 and B7-H3 on ES TMA slides. Scale bars = 100 μm. (B) Heatmap and pie charts (CD99, n = 57; B7-H3, n = 47) summarizing IHC staining intensities as strong (3+), moderate (2+), or weak (1+); pie charts exclude detached TMA samples. (C) Representative IF images of A673 ES cells showing colocalized membrane expression of CD99 and B7-H3, with nuclei counterstained by DAPI. Blue: DAPI; green: CD99; red: B7-H3; Schematic illustration depicts antibody binding on the ES cell surface. Scale bar = 10 μm. ES, Ewing Sarcoma; TMA, tissue microarray; H&E, hematoxylin and eosin; IHC, immunohistochemistry; ICC, immunocytochemistry; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The A673 cell line (RRID: CVCL_0080) was purchased from American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Staining, Immunohistochemistry, Membrane, Expressing, Binding Assay, Microarray, Immunocytochemistry

Journal: Advanced healthcare materials

Article Title: Detection of Extracellular Vesicles with Colocalized Surface Markers via a Capture–Release–Capture Strategy for Treatment Monitoring in Ewing Sarcoma

doi: 10.1002/adhm.202505917

Figure Lengend Snippet: Characterization of A673-Derived ES EVs Immobilized via DTB- and Click Chemistry-Mediated Enrichment. (A) Schematic of independent enrichment of A673 ES EVs using DTB-grafted anti-CD99 with SA–Dynabeads and TCO-grafted anti-B7-H3 with EV Click MagBeads, followed by immunogold staining for CD63 or B7-H3 to confirm EV identity and marker colocalization. (B) SEM images showing immobilization of CD99 + and B7-H3 + ES EVs on SA–Dynabeads and EV Click MagBeads, respectively. Scale bars = 1 μm. (C) TEM validation of EV marker expression and colocalization: CD63 (canonical EV marker) was detected on both CD99 + and B7-H3 + EVs using 12 nm nanogold-conjugated antibodies, while B7-H3 was additionally confirmed on CD99 + EVs, establishing the presence of CD99 + /B7-H3 + ES EVs. Scale bars = 100 nm. ES, Ewing Sarcoma; EV, Extracellular Vesicle; DTB, desthiobiotin; SA, streptavidin; TCO, trans-cyclooctene; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

Article Snippet: The A673 cell line (RRID: CVCL_0080) was purchased from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Staining, Marker, Biomarker Discovery, Expressing, Electron Microscopy, Transmission Assay

Journal: Advanced healthcare materials

Article Title: Detection of Extracellular Vesicles with Colocalized Surface Markers via a Capture–Release–Capture Strategy for Treatment Monitoring in Ewing Sarcoma

doi: 10.1002/adhm.202505917

Figure Lengend Snippet: Validation of the ES EV CaReCa Assay Using Synthetic Plasma Samples. (A) Schematic workflow of the two-step ES EV CaReCa assay applied to synthetic plasma generated by spiking defined quantities of A673 ES EVs into EV-depleted HD plasma, followed by ACTB mRNA quantification via RT-dPCR. (B) Quantification of ACTB mRNA from enriched ES EVs demonstrates a strong linear correlation (R 2 = 0.964) between ACTB mRNA copy number and input quantity of A673 ES EVs, confirming the assay’s quantitative performance. All data points are presented as mean ± SD ( n = 3). ES, Ewing Sarcoma; EV, Extracellular Vesicle; CaReCa, Capture–Release–Capture; HD, Healthy Donor; ACTB , β -actin; RT-dPCR, Reverse Transcription Digital Droplet PCR; DTB, Desthiobiotin.

Article Snippet: The A673 cell line (RRID: CVCL_0080) was purchased from American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Clinical Proteomics, Generated, Reverse Transcription

Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) EwS TMA sections stained for CD99 (tumor marker) with the low (left) and high (right) pSMAD2 S465/S467 signal. ( B ) Nuclear pSMAD2 S465/467 and cytoplasmic/membranous CD99 IF average intensity per segmented cell. Gates define positive versus negative populations (conservative threshold of pSMAD2 or CD99 true positives). Red box: CD99+ (likely tumor cell) population ( C ) % CD99+ cells that were also pSMAD2+ for each sample in the TMA ( n = 27, table S2); red line: median. ( D ) H&E and IF from EwS TMA sections stained for CD99, pSMAD2 S465/467, and TNC (ECM protein). Right: Zoomed insets showing the CD99 hi /pSMAD low /TNC low region versus CD99 hi /pSMAD2 hi /TNC hi region. ( E ) H&E of the tumor formed 3 weeks post–renal subcapsular injection of A673 cells in NSG mice. Right: IF for pSMAD2 S465/467 and ECM marker TNC (human-specific antibody).

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Staining, Marker, Injection

Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) Real-time measurements of confluence (Incucyte) for TC71, A673, CHLA10, and PDX305 cells treated with vehicle controls, TGFβ1 (10 ng/ml), vactosertib (1 μM), or TGFβ1 + vactosertib. n = 2; error bars: SEM. P values: unpaired t tests of confluence at 48 hours relative to 0 hours within each line (nonsignificant comparisons not shown). hr, hours. ( B ) Measurement of the cell area at 48 versus 0 hours of Incucyte imaging in parental cell lines or BlastR control versus dominant-negative TGFBR2 transduced CHLA10 cells. n = 2. P values: unpaired t test within each line. ( C ) Cell viability measurements by CellTiter-Glo after 72 hours of culture with vehicle control, TGFβ1 (10 ng/ml), or vactosertib (1 μM) in parental cell lines or BlastR control versus dominant-negative TGFBR2 transduced CHLA10 cells. n = 3; P values: unpaired t test within each line. ( D ) Soft agar colony-forming assays of A673 (day 14) and CHLA10 (day 14) cells treated with vehicle control, TGFβ1 (10 ng/ml), or vactosertib (1 μM). ( E ) Number of colonies formed in soft agar for TC71 (day 14), A673 (day 14), CHLA10 (day 14), and PDX305 cells (day 35), relative to the number of colonies in the vehicle control well. n = 2; P values: unpaired t tests within each line. ( F ) Invasive morphology of spheroids cultured for 4 days in 3D rat tail collagen I–rich gels treated with vehicle controls, TGFβ1 (10 ng/ml), or vactosertib (10 μM). Stained with phalloidin to mark F-actin. Yellow arrows: invasive strands. Representative of n = 2. For all panels: n.s., P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001. hr, hours.

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Imaging, Control, Dominant Negative Mutation, Cell Culture, Staining

Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) % GFP+ after transduction with SBE-GFP SMAD reporter −/+ TGFβ1 (48 hours, n = 2). ( B ) TGFB1 and TGFB2 expression in CAF-like (top 30% expression CAF-like gene signature) versus non-CAF-like cells by scSeq (nine EwS lines). ( C ) ELISAs of TGFβ1 in conditioned media (3 days of culture). n = 3. Dashed line indicates the TGFβ1 level detected in media-only controls. ( D ) ELISAs of secreted total TGFβ2 in conditioned media (3 days of culture). n = 3. TGFβ2 not detected in media-only controls. ( E ) Genes significantly ( P adj < 0.05) up-regulated by TGFβ1 or TGFβ2 in A673, CHLA10, and PDX305 (bulk RNA-seq, n = 3). ( F ) Crystal violet–stained cells 24 hours after seeding on Matrigel-coated transwells to measure invasion. Each cell marked with an asterisk. ( G ) Quantification of Matrigel-coated transwell invasion 24 hours postseeding ( n = 3 to 4; each dot is a biological replicate). P values: unpaired t tests within each line. ( H and I ) ELISAs of secreted total (active and latent) (H) TGFβ2 and (I) TGFβ1 after 3 days of culture of CHLA10 dominant-negative TGFBR2 or empty vector transduced cells. n = 3. P values: unpaired t tests. ( J ) Expression of TNC , TGFB1 , and TGFB2 in PDX305 cells treated with TGFβ1 (10 ng/ml) −/+ vactosertib, TGFβ2 (10 ng/ml) −/+ vactosertib, or vactosertib alone (1 μM). n = 2; P values: unpaired t tests of treatment versus vehicle (nonsignificant comparisons not shown). ( K ) Expression of TGFβ-induced genes in CHLA10 cells treated with TGFβ ligand–blocking antibodies (10 μg/ml, 4 days). n = 2; P values: unpaired t tests. ( L ) IF of TNC in CHLA10 cells treated with TGβ ligand–blocking antibodies versus IgG control (10 μg/ml, 4 days). P values: unpaired t tests. For all panels: n.s., P > 0.05; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Transduction, Expressing, RNA Sequencing, Staining, Dominant Negative Mutation, Plasmid Preparation, Blocking Assay, Control