Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 1. CXCR4+ DTC cells exhibit enhanced self-renewal activity, tumorigenic potential and IR resistance compared to CXCR4- cells. (a) MTT assay, (b) sphere-forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR cl, 22onogenic survival assay (black bar 50 mm), and (f) Western blot (left) and RT-PCR (right) analyses of DTC cells after sorting with apc-conjugated CXCR4 antibody. *p < .05. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: Activity Assay, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 2. Silencing of CXCR4 via shRNA infection suppresses self-renewal activity, tumorigenic potential and IR resistance in DTC cells. (a) MTT assay, (b) sphere-forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR clonogenic survival assay (black bar 50 mm), (f) Western blot (left) and RT-PCR (right), and (g) immunofluorescence (white bar 100 μm) analyses of DTC cells infected with shControl (shCTL), shCXCR4 a or D (upper) and quantification of the results (lower). *p < .05, **p < .01, ***p < .001. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: shRNA, Infection, Activity Assay, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 5. CXCR4 overexpressing U-CH1 cells exhibited higher self-renewal activity, tumorigenic potential and IR resistance than control cells. (a) MTT assay, (b) sphere- forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR clonogenic survival assay (black bar 50 mm), (f) Western blot (left) and RT-PCR (right), and (g) immunofluorescence (white bar 100 μm) analyses of DTC cells of transduced with control (pQCXIP) and CXCR4 carrying retroviruses in U-CH1 cells (upper) and quantification of the results (lower). *p < .05, **p < .01. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: Activity Assay, Control, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Transduction

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 6. Suppression of CXCR4 reduced tumorigenic potential and enhanced IR sensitivity of DTC cell in an in vivo xenograft model. (a) A representative image of tumor-bearing mice (left) and measurement of in vivo tumor growth rate (right) in shControl (shCTL) and shCXCR4-infected DTC cells subcutaneously transplanted in mice. (b) Combination therapy of IR and CXCR4 inhibitor (AMD3100, 5 mg/kg) in xenograft mouse model. (c) Immunohistochemistry (white bar 50 μm) of xenograft tissues probed with Sox2 antibody (left) and quantitation of the results (right) *p < .05, ***p < .001.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: In Vivo, Infection, Immunohistochemistry, Quantitation Assay

Journal: Cell Reports

Article Title: Aryl Hydrocarbon Receptor Contributes to the Transcriptional Program of IL-10-Producing Regulatory B Cells

doi: 10.1016/j.celrep.2019.10.018

Figure Lengend Snippet:

Article Snippet: Negative CD43- Isolation Kit , Miltenyi Biotec , Cat# 130-049-801.

Techniques: Western Blot, Recombinant, Methylation, Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, cDNA Synthesis, SYBR Green Assay, DNA Library Preparation, Purification, Bicinchoninic Acid Protein Assay, Microarray, Ex Vivo, Generated, Software, Red Blood Cell Lysis, Staining, Lysis

Journal: Genes to Cells

Article Title: Functional Role of COP1 Gene in Hepatocellular Carcinoma Lipid Metabolism and Stemness

doi: 10.1111/gtc.70108

Figure Lengend Snippet: COP1 knockdown reduces motility, and stemness in HCC cells. (a‐b) Migration (a) or invasion (b) assay showing reduced motility in Huh7 and HepG2 cells treated with COP1‐siRNA, as quantified by the relative migration or invaded area. Statistical significance: *, p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. (c) Sphere formation assay images and quantitative data from Huh7 and HepG2 cells, demonstrating a decrease in both the number and size of spheres in COP1‐siRNA‐treated cells. (d) Sphere formation assay images and quantitative data from PLC/PRF/5 CD133+ cells. Compared to the control grpup, both the sphere number and size decreased upon COP1‐siRNA treatment. Scale bar, 100 μm Statistical significance: *, p < 0.05; *** p < 0.001 vs. control.

Article Snippet: The cells were treated with FcR Blocking Reagent and CD133 MicroBeads (130‐100‐857, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions and incubated for 15 min at 4°C in the dark.

Techniques: Knockdown, Migration, Control, Tube Formation Assay

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A, Schematic experiment setting. B, Increase in total number of cells of known (KIF-P1 and -P2, SYT-T1, -T2, -P1, -P2) and newly identified (KIF-sc1 and -sc2) TCRs upon antigen-specific stimulation and CD137-enrichment with dominance of KIF-P1 and -P2 harboring high precursor frequency. The two newly identified KIF2C-TCRs were selected based on fold change of TCR frequency and highest absolute frequency in the stimulated sample. C, Assessment of antigen-specific IFN-γ-secretion for the two newly identified TCRs KIF-sc1 and -sc2 in comparison to the known TCR KIF-P2. Cytokine secretion was measured by IFN-γ-ELISA upon 24h of co-culture of TCR-tg T cells from one representative donor with Mel15-LCLs transgenic for the mutated KIF2C P13L minigene (mut mg) and the wildtype KIF2C minigene (wt mg) as well as pulsed for 2h at 37°C with the mutated and wildtype peptide (mut pep and wt pep). An irrelevant peptide (irr peptide), target cells (LCL only) or T cells alone (T cell only) served as negative controls. D, Frequency of KIF-sc1 and -sc2 in relation to the previously identified TCR-sequences identified by deep sequencing of the TCR-β-chain in intestinal (MInt) and lung metastases (MLung) as well as corresponding non-malignant draining lymph nodes (MInt-LN1, MInt-LN2 and MLung-LN) of patient Mel15. Non-td: non-transduced.

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Transgenic Assay, Sequencing

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A-C, Mel15 LCLs were pulsed with titrated peptide concentrations (2h, 37°C) and co- incubated with TCR-tg T cells with subsequent ELISA-based assessment of IFN-γ-secretion within 24h of co-culture (A). The cellular activation level was determined after 24h by FACS staining of the extracellular level of CD137 (B) and PD-1 (C) expression (reflected by geometric mean of all CD3 + CD8 + /TCRmu + cells). The mean for ELISA data is depicted for technical triplicates of one donor; triplicates from the same donor have been pooled prior to EC FACS- staining. E:T = 1:1 (15.000 tg T cells:15.000 tumor cells). D-G, EC FACS staining at different timepoints after co-culture setup displays temporal dynamics of T cell activation marker CD137 (D, E) and inhibitory receptor LAG-3 (F, G) for TCR-tg T cells upon co-culture with JJN3-B27 peptide-pulsed target cells. A weak (0.01 µM for peptide pulsing; D, F) versus a strong (1 µM for peptide pulsing; E, G) stimulus were compared. E:T = 1:1 (10.000 tg T cells:10.000 tumor cells). H, Annexin-V/PI-staining was employed for detection of activation induced cell death (AICD) after 20h of co-culture upon strong stimulation with 1µM mut-peptide pulsed Mel15 LCLs (early apoptotic = AnnexinV + PI - , late apoptotic = AnnexinV + PI + ). E:T = 1:1 (30.000 tg T cells:30.000 tumor cells). I, Representative FACS plot of a healthy donor of CTV-analysis for all TCRmu + cells depicted after 4 days of co-culture with 1µM mut-peptide pulsed Mel15 LCLs (colors were chosen according to Figure B-E; representative wt mg-control depicted in grey). E:T = 1:1 (30.000 tg T cells:30.000 tumor cells). For all co-cultures in D-I technical triplicates per donor were pooled prior to staining; the mean and SD for biological replicates from three different human donors are shown.

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Activation Assay, Staining, Expressing, Marker, Control

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A, Schematic experiment setting of xenograft neoTCR-tumor rejection experiment with newly transduced T cells. B, Tumor growth kinetics are displayed as tumor area (in cm 2 ) for mut mg-U698M-tumor- bearing NSG-mice comparing neoTCR-tg T cells to the irrelevant TCR 2.5D6 until day 20. Mean values and SDs for each group of mice display rejection dynamics (n=6). Parts of this dataset were already published before . In this renewed version, tumor rejection kinetics of KIF-sc1 and -sc2 analyzed in the same experiment are included. C, Kaplan-Meier-survival curve is displayed until day 20 for tumor-bearing mice injected with different neoTCR-tg T cells. D, schematic experiment setting of xenograft neoTCR-TIL-rechallenge experiment. E-G, Ex vivo restimulation of T cells derived from TIL products on day 21 after tumor explant (TIL-P) compared to newly transduced (NEW) TCR-tg T cells from the same human donor stained for CD137 (EC; E), IFN-γ (IC; F) and GzmB (IC; G); expression was analyzed using geometric mean of all CD3 + CD8 + /TCRmu + cells after 18h of co-culture. Mut-mg and wt-mg U698M cells were used as target cells in E:T = 1:1 (50.000 tg T cells:50.000 tumor cells). Mean and SD are shown for three experimental replicates. Statistical significance is calculated with one-way ANOVA and Tukey’s multiple comparison test (*p≤0.05, ****p≤0.0001). H, Tumor growth kinetics are displayed as tumor area (in cm 2 ) for NSG-mice continuing the experiment until day 17 after second injection of in total 5x10 6 neoTCR-tg T cells (transduction rate of 55% equalized for all groups). For the TIL- P-groups, TIL-P from two mice per TCR (highest numbers of expanded neoTCR-tg T cells) were pooled. Mean values and SEMs for each group of mice display rejection dynamics (n=5 for experimental groups, n=3 for 2.5D6; due to achievement of humane endpoint criteria one 2.5D6- control mouse was sacrificed on day 13 and excluded from this graph). Statistical significance is calculated for the tumor area on day 17 with one-way ANOVA and Tukey’s multiple comparison test (***p≤0.001, ****p≤0.0001). I , Kaplan-Meier-survival curve is displayed for tumor-bearing mice injected with different TCR-tg T cells (n=5 for experimental groups, n=4 for 2.5D6 control). Survival of mice receiving TIL-P-KIF-P2 compared to TIL-P-KIF-sc1 was significantly prolonged (p=0.0019, Mantel-Cox test).

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Injection, Ex Vivo, Derivative Assay, Staining, Expressing, Co-Culture Assay, Comparison, Transduction, Control