Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 2. Expression of CD70 on solid tumors and confirmation of target expression and CAR T cell transduction efficiency. (A) Gene expression of CD70 in various solid tumors compared to normal matched tissues was analyzed using data from The University of Alabama at Birmingham Cancer data analysis (UALCAN) and The Cancer Genome Atlas program (TCGA). B-C) Top: confirmation of CD70 (target) expression on cancer cells post transduction – (B) glioblastoma (GBM) and (C) osteosarcoma (OS) models - and Bottom: transduction of CAR construct in (B) C57BL/6 mice - derived T cells (CAR T Kr158B ) and (C) in Balb/c mice - derived T cells (CAR T K7M2 ) as indicated by GFP reporter. On average, (D) the mean CD70 expression was found to be 73% for GBM and 99% for OS models. (E) The mean transduction efficiency was 66% for CAR T Kr158B and 60% for CAR T K7M2 . The names of the cell lines and tumor models, GBM for Kr158B and OS for K7M2, will be used interchangeably in the study. Number of biological replicates (N) is indicated on the graphs.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Expressing, Transduction, Gene Expression, Construct, Derivative Assay

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 3. CAR T cell exhibit directed motion towards the target tumor. A) Representative confocal snapshots showed CD70-specific CAR T cells (green) navigating through supported COL1-LLS RhB microgels (red) and infiltrating the target GBM tumor (white), with blue arrows indicating the paths of CAR T cell migration. (B, D) Mean velocity of CAR T cells co-cultured with CD70-positive tumors and the wild-type (WT) control were quantified for GBM (B) and OS (D) tumor models. The number of tracks (n) and biological replicates (N) were indicated on the plots, and statistical significance was determined using an unpaired two-tailed Student’s t-test with p values indicated on the plots. (C, E) Tumor-infiltrating CAR T cells were quantified as a percentage of total CAR T cells on average from 0 to 72 h for GBM (C) and OS (E) tumors, displayed as box plots showing 25th and 75th percentiles, median, and mean, with whiskers representing the minimum to maximum observations. An unpaired two-tailed Student’s t-test was performed for statistical analysis, and p values were indicated on the plots. (F) Top panel: maximum intensity z projection showed snapshots of CAR T – GBM tumor interaction at 0, 24, and 72 h. Middle panel: the segmentation of CAR T with colors indicating individual CAR T cells at each frame. Bottom panel: maximum intensity projection of segmented CAR T cell velocity tracks over time with color-coded velocity gradient, revealing accumulation of CAR T cells inside the tumor. The segmentation employed a deep learning-based method, as discussed in the methods section, and the cells were tracked using a Linear Assignment Problem (LAP) tracker with maximum frame-to-frame linking and allowable track segment gap closing of 150 μm. (G) Evidence of chemotaxis and upregulation in migratory pathways for CAR T cells co-cultured with their target tumors was demonstrated for GBM (top panel) and OS (bottom panel) tumor models. Notably, evidence of immune-mediated cytotoxic function was shown through IFN γ detection. Error bars represent standard deviation.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Migration, Cell Culture, Control, Two Tailed Test, Chemotaxis Assay, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 4. CAR T expansion, activation, and killing. (A) Representative confocal time-lapse images of the FITC channel (green) illustrating immune activation, expansion, and killing of the target tumors. CAR T cell clustering and rapid expansion were observed in almost all conditions with efficient anti-tumor activity. White arrows indicate CAR T cell clusters. (B, C) The number of CAR T cell clusters rapidly increased within the first 24 h and remained steady for over 72 h in (B) GBM and (C) OS models. (D, E) Cluster size in all co-cultures with target tumors steadily increased over time, but not in WT controls. (F, G) Expansion of CAR T cell clusters revealed an inverse correlation with tumor size. n = 3 for all CD70 pos samples and n = 2 for all WT samples. (H, I) Endpoint flow cytometry data measuring CAR T cell expansion after 96 h of co-culture with cancer cells in the 2D assay for both tumor models. (J, K) A comparison in IFN γ secretion (pg/mL) between 2D and 3D cultures for (J) GBM and (K) OS. Error bars indicate SD and the number of biological replicates is n = 2. (L) Top row: confocal 3D snapshots of GBM tumors after co-cultured with CAR T cells (E:T = 1:4) for 24 h, showing highly tortuous tumor margins in CD70 pos tumors compared to WT counterparts. Bottom row: cross-section view (at z depth: 70 μm) of the sample on the top row revealing infiltrating CAR T cells within the tumor mass. (M) Measurement of tumor tortuosity factors revealed more than 3-fold change for the CD70 pos tumors within the first 48 h of co-culture. The tumor tortuosity factor was calculated as the ratio between the perimeter of the tumor outline and the perimeter of a circle with the same pixel area. Data was obtained from GBM samples ( n = 3) at an initial E:T ratio of 1:2 and from osteosarcoma (K7M2) samples ( n = 3) at an initial E:T ratio of 1:1. Error bars represent standard deviation and biological samples ( n = 3) for each group were performed unless indicated otherwise.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Activation Assay, Activity Assay, Cytometry, Co-Culture Assay, Two-Dimensional Assay, Comparison, Cell Culture, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 5. Quantification of CAR T–tumor interactions . Top rows: confocal timelapse images at 0, 24, 48, and 72 h showing dynamic immune-cancer interactions and antitumor activities of CAR T cells in (A) GBM and (B) OS tumor models. The bottom row of each group shows a digital reconstruction of the confocal data with distinct segmentation of tumor and immune cell populations. The initial CAR T to tumor cell ratio or effector-to-tar get (E:T) ratio is indicated on the graph. (C, D) Quantification of CAR T–tumor interactions revealing an inverse correlation between tumor size and infiltrating CAR T cells for (C) GBM and D) OS tumors. The percentage of tumor size (left axis) and CAR T cells infiltrating the tumor (right axis) were normalized to the original tumor size at time 0. (E, F) Killing rates over time were calculated as derivatives from C and D, respectively. (G, H) The measured CAR T to cancer cell area ratios (E:T) as a function of time. The E:T ratios dynamically changed over time, while for WT tumors, the ratios remained constant. (I) RNA sequencing data from single CAR T cells isolated from a 3D co-culture with target CD70 pos versus WT tumors. All genes shown in the results have a statistical significance of p ≤0.05. Error bars represent standard deviation and biological samples n = 3 for all CD70 pos samples and n = 2 for all WT samples.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: RNA Sequencing, Isolation, Co-Culture Assay, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 7. Sensitivity of anti-tumor activity to various CAR T: cancer cell (E:T) ratios. (A,B) confocal time-lapse images of CAR T – tumor co-culture in iVITA at different E:T ratios. (C,D) show the digital image reconstruction of confocal data quantifying bulk tumor mass, migrating single cancer cells, immune cells, and immune cell clusters, and killing activity over time. (A,C) GBM CD70 pos tumors and (B,D) OS CD70 pos tumors were co-cultured with their respective CAR T cells at different concentrations corresponding to E:T of 1:4, 1:2, and 4:1 for the GBM model and E:T of 1:4, 1:1, and 4:1 for the OS model. (E–H) CAR T expansion as a function of initial E:T seeding ratios. (E,F) CAR T expansion on average in both models from 0 to 72 h. For each seeding E:T, CAR T expansion at each time point was normalized to the CAR T at time 0, and the average was calculated for all frames. G,H) the number of CAR T clusters counted every 1.5 h for 72 h for each group. The box plots display 25th and 75th percentiles, a line at the median a plus sign at the mean, from the minimum to the maximum observation. (I,J) Quantification of tumor size over time at the initial E:T = 1:4, 1:2, and 4:1 for I) GBM model and at E:T = 1:4, 1:1, and 4:1 for (J) OS model. (K,L) The E:T ratio dynamically changed over time. The CAR T expansion and tumor-killing were presented by the exponential increase of E:T ratios. (M,N) tumor killing rates were calculated as derivatives from (I) and (J), respectively. Error bars represent standard deviation. Statistical analysis was performed using Ordinary One-Way ANOVA. ( n = 3 unless indicated otherwise, ∗∗= p < 0.01, and ∗∗∗∗= p < 0.0 0 01).

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Activity Assay, Co-Culture Assay, Cell Culture, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Validation of CD70 as a GB target for CAR-T cell therapy (A) Expression of CD70 and selected markers from an in-house glioma patient single-cell RNA seq dataset. (B) CD70 expression in GB samples from Heidelberg University Hospital, analyzed by bulk RNAseq. Each dot represents a patient. A Mann-Whitney test was performed to assess statistical significance. (C) CD70 expression in 20 matched pairs from (B). A two-tailed paired t test was used to assess significance. (D) IHC staining of three Heidelberg University Hospital GB patients from (B) with high CD70 expression based on RNAseq. Scale bars, 200 μm. Patient-1 and Patient-3: primary GB; Patient-2: recurrent GB. (E) Measurement of CD70 protein levels on GB cells by flow cytometry. (F) Measurement of CD70 gene expression levels in glioma cell lines by RT-qPCR. N = 3 technical replicates per cell line. (G) Correlation between CD70 gene expression and protein levels from (E) and (F). (H) Measurement of CD70 on the surface of generated primary GB models by flow cytometry (blue histograms). An isotype control antibody (red histograms) was used. For (E) and (H), data were gated on single live cells. Data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Biomarker Discovery, Expressing, Single Cell, RNA Sequencing, RNA sequencing, MANN-WHITNEY, Two Tailed Test, Immunohistochemistry, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Generated, Control

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Generation and phenotyping of anti-CD70 CAR-Tcells (A) CAR construct design. (B) Transduction efficiency of primary human T cells by flow cytometry. A non-transduced (NT) sample from each donor was used to determine gating. Data gated on single live CD3 + cells. (C) CD8a/CD4 expression on transduced T cells, determined by flow cytometry. A one-way ANOVA followed by a Dunnett’s multiple comparisons test was performed to assess significance. (D) Expression of exhaustion receptors on transduced T cells, determined by flow cytometry. (E) Quantification of PD1 + /LAG3 + /TIM3 + T cells from (D). (F) Expression of immune memory-associated markers on transduced T cells by flow cytometry. For (C), (D), (E), and (F), N = 3 biological replicates per group. Isotype controls were used to determine gating and displayed data are gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Data presented as mean (SD). A one-way ANOVA followed by a post-hoc Šídák’s multiple comparisons test was performed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Construct, Transduction, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vitro assessment of the CD70-directed CAR-T cell anti-GB cytotoxicity (A) Measurement of secreted TNF-α and IFN-γ in the SN of GB/CAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. For comparison between MCS and CD70 (upper bar plots), an unpaired two-tailed t test was used. For comparisons among constructs (bottom), a one-way ANOVA followed by a Holm-Šídák multiple comparisons test was used. (B) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. A two-tailed Student’s t test was performed using the values of the last measured time point to determine statistical significance. For (A) and (B), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test, Construct, Co-Culture Assay

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Assessment of anti-CD70 CAR-T cell killing in naturally CD70 expressing cell lines (A) CD70 expression levels (blue histogram) on primary GB cell line MMKI by flow cytometry. Data gated on single live cells. An isotype control (red histogram) was used. (B) Quantification of activation marker CD137 after O/N co-culture of MMKI cells with CD70-targeting CAR-T cells by flow cytometry. N = 4 biological replicates per group. Data gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Isotype control antibodies were used for gating. A one-way ANOVA followed by a Tukey’s multiple comparisons test was applied for significance. (C) Measurement of CD70 (blue histograms) on the surface of generated genetic knockout models by flow cytometry. An isotype control (red histogram) was used. Data gated on single live EGFP + cells. (D) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. Statistical significance was assessed as in B). For (B) and (D), data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Expressing, Flow Cytometry, Control, Activation Assay, Marker, Co-Culture Assay, Generated, Knock-Out

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Evaluation of CD70-directed CAR-T cell effector function in cerebral organoids (A) Confocal microscopy of cerebral organoids, infiltrated by generated GB models. (B) Quantification of CD70 signal in organoids from (A). Each dot represents an organoid. A Welch’s t test was used to assess significance. (C) Immunofluorescence analysis of endogenous CD70 expression in cerebral organoids. (D) Confocal microscopy of cerebral organoids previously invaded by GB cells and subsequently treated with CAR-T cells for 3 d. (E) Quantification of Granzyme-B signal from (D). A two-tailed t test was used to determine significance. (F) Measurement of secreted Granzyme-B and IFN-γ levels in the SN of co-cultures from (D) by ELISA. N = 3 biological replicates per group. (G) CAR construct direct comparisons from (F). A one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons was used. For (A), (C), and (D), scale bars, 200 μm. For (D) and (E), N ≥ 3 organoids per group. For (E) and (F), a two-tailed t test was used to assess significance. For (B), (E), and (F), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Confocal Microscopy, Generated, Immunofluorescence, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Construct

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vivo evaluation of the anti-CD70 CAR-T cell killing efficacy (A) In vivo pipeline. (B) Measurement of tumor cell signal in mice orthotopically implanted with P3/CD70_NLuc/EGFP cells and subsequently treated ICT with anti-CD70 CAR-T cells by BLI. (C) Quantification of BLI signals from B). N = 8 animals per group. (D) Overall survival of treated mice from B). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0083. (E) Analysis of CAR-T cell persistence and CD70 expression in recurrent tumors of treated mice from (A) by IF. Representative images from N = 3 animals per group. Scale bars, 100 μm.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vivo, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based CAR constructs lead to regression of syngeneic GB tumors in vivo (A) Schematic representation of the syngeneic model in vivo experiment. (B) Evaluation of mCAR expression on transduced murine T cells on treatment day by flow cytometry. A non-transduced sample was used to determine gating. Data gated on single live mCD3 + cells. (C) Weekly tumor volume evaluation in C57BL/6J mice orthotopically implanted with GL261/CD70 cells and subsequently treated ICT with anti-mCD70 CAR-T cells by MRI. A one-way ANOVA with a Dunnett’s multiple comparisons test was used to assess significance. (D) Indicative T2-weighed MRI sections (coronal plane) from N = 3 mice of each treatment group from (C), before and after treatment with anti-mCD70 CAR-T cells. (E) Comparison of tumor volumes among treatment groups from (C). For day-14, a one-way ANOVA followed by a Tukey’s multiple comparisons test was used. For day-21, a Welch’s ANOVA followed by a Dunnett’s T3 multiple comparisons test was used. For day-28, a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used. Data presented as mean (SD). (F) Overall survival of treated mice from (C). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0166. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Construct, In Vivo, Expressing, Flow Cytometry, Comparison

Journal: Cancer & Metabolism

Article Title: HIF prolyl hydroxylase PHD3 regulates translational machinery and glucose metabolism in clear cell renal cell carcinoma

doi: 10.1186/s40170-017-0167-y

Figure Lengend Snippet: Significantly regulated proteins in response to PHD3 silencing according to PECA analysis under hypoxia

Article Snippet: Western blot analyses with the following antibodies were performed: PHD3 (NB100-139, Novus Biologicals), CD70 (CD27 Ligand) (MAB2738, R&D Systems), Integrin β1 (610468, BD Transduction Laboratories), LDHA (#3582, Cell Signaling Technologies), MDH2 (ab181873, Abcam), STAT1 (#9176, Cell Signaling Technologies), Fibronectin (F3648, Sigma), alpha-actinin 4 (ALX-210-356-C050, Enzo Life Sciences), GLUT1 (ab14683, Abcam), glyceraldehyde phosphate dehydrogenase (GAPDH) (5G4-6C5, HyTest), S6 ribosomal protein (#2217, Cell Signaling Technologies), pS6 ribosomal protein S235/236 (#2211, Cell Signaling Technologies), pS6 ribosomal protein S240/244 (#3564, Cell Signaling Technologies), p70 S6 kinase (#2708, Cell Signaling Technologies), p-p70 S6 kinase T389 (#9234, Cell Signaling Technologies), α-tubulin (sc-23948, Santa Cruz Biotechnology), β-actin (AC-74, Sigma-Aldrich), anti-mouse-HRP (DAKO), and anti-rabbit HRP (DAKO).

Techniques:

Journal: bioRxiv

Article Title: Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell interactions

doi: 10.1101/2023.02.17.529033

Figure Lengend Snippet: CD70 expression correlates with survival in patients with solid tumors. A) (top) CD70 gene expression for different solid tumors compared with normal matched tissues. Sources: The University of Alabama at Birmingham Cancer data analysis (UALCAN) and The Cancer Genome Atlas program (TCGA). (Bottom panels) survival rates of patients diagnosed with renal carcinoma and lung cancer with low and high CD70 expression. Source: The Human Protein Atlas program. B-C) Confirmation of CD70 (target) expression on cancer cells – B) glioblastoma (GBM) and C) osteosarcoma (OS) models - and (bottom) transduction of chimeric antigen receptor (CAR) construct in B) C57BL/6 mice - derived T cells (CAR T Kr158B ) and C) in Balb/c mice - derived T cells (CAR T K7M2 ) as indicated by GFP reporter. On average, D) percentage of CD70 expression is 73% for glioblastoma and 99% for osteosarcoma models. E) Mean transduction of 66 % for CAR T Kr158B and 60% for CAR T K7M2 . The name of the cell lines and tumor models will be used interchangeably – GBM for Kr158B and OS for K7M2.

Article Snippet: 293T cell line (ATCC, CRL-3216) was used for lentiviral plasmid production encoding for mouse full length CD70 surface antigen (GenBank: U78091.1).

Techniques: Expressing, Gene Expression, Transduction, Construct, Derivative Assay

Journal: bioRxiv

Article Title: Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell interactions

doi: 10.1101/2023.02.17.529033

Figure Lengend Snippet: CAR T cell directed motion towards the target tumor. A) Representative confocal snapshots of CAR T cells infiltrating a GBM solid tumor in 3D. The panel shows CD70-specific CAR T cells (green) navigating through the supported COL1-LLS RhB microgels (red) and infiltrating the target GBM tumor (white). Blue arrows indicate paths of CAR T migration. B, D) mean velocity of CAR T cells cocultured with CD70 pos tumors and the WT control for B) GBM and D) OS tumor models. The number of tracks (n) is indicated on the plots. The number of biological replicates is N=2. An unpaired two-tailed Student’s t-test was performed. Statistical significance with p values was indicated on the plots. C, E) Quantifying tumor-infiltrating CAR T cells on average from 0–72h as a percent of total CAR T cells for C) GBM and E) OS tumors. The box plots display 25 th and 75 th percentiles, a line at the median, and a plus sign at the mean, from the minimum to the maximum observation. Unpaired two-tailed Student’s t-test was performed (n=234, N=3, statistical significance with p values were indicated on the plots). F) Top panel: Maximum intensity z projection (top panel) showing snapshots of CAR T – GBM tumor interaction at 0, 24, and 72h. Middle panel: the segmentation of CAR T with colors indicated by individual CAR T cells at each frame. The segmentation employed a deep learning-based method (as discussed in the method section). Bottom panel: maximum intensity projection of the segmented CAR T cell velocity tracks overtime. The velocity gradient was color-coded, showing accumulation of CAR T inside the tumor. The segmented cells were tracked using Linear Assignment Problem (LAP) tracker at the maximum frame-to-frame linking and allowable track segment gap closing of 150 μm (~ 3 cell diameter). G) Evidence of chemotaxis and upregulation in migratory pathways for CAR T cells co-cultured with their target tumors for GBM (top panel) and OS (bottom panel). Noticeably, evidence of immune-mediated cytotoxic function is demonstrated via IFNγ detection.

Article Snippet: 293T cell line (ATCC, CRL-3216) was used for lentiviral plasmid production encoding for mouse full length CD70 surface antigen (GenBank: U78091.1).

Techniques: Migration, Control, Two Tailed Test, Chemotaxis Assay, Cell Culture

Journal: bioRxiv

Article Title: Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell interactions

doi: 10.1101/2023.02.17.529033

Figure Lengend Snippet: CAR T expansion, activation, and killing. A) Representative confocal timelapse images of the FITC channel (green) that show immune activation, expansion, and killing of the target tumors. The patterns of CAR T clustering and rapid expansion were observed in almost all conditions with efficient anti-tumor activity. White arrows locate CAR T cell clusters. B, C) The number of CAR T clusters rapidly increased during the first 24h and steadily maintained for more than 72h in B) GBM and C) OS models. D, E) Cluster size in all cocultures with target tumors steadily increased over time but not in the WT controls. F, G) Expansion of CAR T clusters revealed an inverse correlation with tumor size. (n=3 for all CD70 pos samples and n=2 for all WT samples). H, I) Endpoint flow cytometry data measuring CAR T expansion after 96h of CAR T – cancer cells coculture in the 2D assay for both tumor models. J, K) A comparison in IFNγ secretion (pg/mL) for 2D vs 3D for J) GBM and K) OS. The number of biological replicates is N=2. L) Top row: confocal 3D snapshots of GBM tumors after co-cultured with CAR T cells for 24h showing highly tortuous tumor margin of CD70 pos tumor as compared to the WT counterpart. Bottom row: cross-section view (z depth: 70 μm) of the sample on the top row exposing infiltrating CAR T cells within the tumor mass. M) Measurement of tumor tortuosity factors revealed more than 3-fold change for the CD70 pos tumors within the first 48h of coculture. The tumor tortuosity factor was calculated as a ratio between the perimeter of the tumor outline to the perimeter of a circle of the same pixel area. Data was obtained from GBM samples (n=3) at an initial E:T ratio of 1:2, and from osteosarcoma (K7M2) samples (n=3) at an initial E:T ratio of 1:1. Biological samples for each group (n= 3), and technical repetitions (n= 3) were performed.

Article Snippet: 293T cell line (ATCC, CRL-3216) was used for lentiviral plasmid production encoding for mouse full length CD70 surface antigen (GenBank: U78091.1).

Techniques: Activation Assay, Activity Assay, Flow Cytometry, Two-Dimensional Assay, Comparison, Cell Culture

Journal: bioRxiv

Article Title: Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell interactions

doi: 10.1101/2023.02.17.529033

Figure Lengend Snippet: Sensitivity of anti-tumor activity to various CAR T: cancer cell (E:T) ratios. A,B) confocal time-lapse images of CAR T – tumor co-culture in iVITA at different E:T ratios. C,D) show the digital image reconstruction of confocal data quantifying bulk tumor mass, migrating single cancer cells, immune cells, and immune cell clusters, and killing activity over time. A,C) GBM CD70 pos tumors and B,D) OS CD70 pos tumors were co-cultured with their respective CAR T cells at different concentrations corresponding to E:T of 1:4, 1:2, and 4:1 for the GBM model and E:T of 1:4, 1:1, and 4:1 for the OS model. At 72h and E:T= 4:1, almost 100% tumor elimination was observed in both models. E-H) CAR T expansion as a function of initial E:T seeding ratios. E,F) CAR T expansion on average in both models from 0–72h. For each seeding E:T, CAR T expansion at each time point was normalized to the CAR T at time 0, and the average was calculated for all frames. G,H) the number of CAR T clusters counted every 1.5h for 72h for each group. The box plots display 25th and 75th percentiles, a line at the median a plus sign at the mean, from the minimum to the maximum observation. I,J) Quantification of tumor size over time at the initial E:T= 1:4, 1:2, and 4:1 for I) GBM model and at E:T= 1:4, 1:1, and 4:1 for J) OS model. K,L) The E:T ratio dynamically changed over time. The CAR T expansion and tumor-killing were presented by the exponential increase of E:T ratios. M,N) tumor killing rates were calculated as derivatives from I) and J), respectively. Statistical analysis was performed using Ordinary One-Way ANOVA. (n=3 unless indicated otherwise, ** = p < 0.01, and **** = p < 0.0001).

Article Snippet: 293T cell line (ATCC, CRL-3216) was used for lentiviral plasmid production encoding for mouse full length CD70 surface antigen (GenBank: U78091.1).

Techniques: Activity Assay, Co-Culture Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: Single-cell and bulk transcriptomics reveal a CD8 + T-cell gene signature predicting prognosis in diffuse large B-cell lymphoma

doi: 10.3389/fimmu.2025.1685541

Figure Lengend Snippet: Validation cohort for predicting the risk signature of DLBCL survival based on the discovery cohort. (A) Expression of CD69 on infiltrating CD8 + T cells in DLBCL (400X). CD69 (red), CD8 (green), DAPI (blue). (B) Kaplan–Meier curves of OS in DLBCL patients with CD69 + /CD8 + and CD69 + /CD8 + . Cases were classified as CD69 + /CD8 + when ≥10% of infiltrating CD8 + T cells expressed CD69. (C) Expression of CD70 on infiltrating CD8 + T cells in DLBCL (400X). CD70 (green), CD8 (red), DAPI (blue). (D) Kaplan–Meier curves of OS in DLBCL patients with CD70 + /CD8 + and CD70 + /CD8 + . Cases were classified as CD70 + /CD8 + when ≥10% of infiltrating CD8 + T cells expressed CD70. (E) The Kaplan-Meier OS curve of the validation cohort (this work. (n=66)) patients between low risk group (n=34) and high risk group (n=32). This work samples were stratified by risk score. (F) Univariate Cox regression analysis of Risk Score: high risk, IPI: low-mid, IPI: mid-high and IPI: high in this work. (G) The Kaplan-Meier OS curve of the validation cohort ( GSE181063 (n=773)) patients between low risk group (n=387) and high risk group (n=386). GSE181063 samples were stratified by risk score. (H) Univariate Cox regression analysis of Risk Score: high risk, IPI: low-mid, IPI: mid-high and IPI: high in GSE181063 . (I) The Kaplan-Meier OS curve of the validation cohort ( GSE117556 (n=469)) patients between low risk group (n=235) and high risk group (n=234). GSE117556 samples were stratified by risk score. (J) Univariate Cox regression analysis of Risk Score: high risk, IPI: low-mid, IPI: mid-high and IPI: high in GSE117556 . Log-rank tests were used to derive p-values for comparisons between two groups.

Article Snippet: The primary antibodies are as follows: CD69 Polyclonal antibody (Proteintech, 10803-1-AP), CD70 Monoclonal antibody (Proteintech, 67749-1-Ig), CD8a Monoclonal antibody (Proteintech, 66868-1-Ig), Anti-CD8 alpha antibody (Abcam, ab93278).

Techniques: Biomarker Discovery, Expressing

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Expression of CD70 and B7-H3 on human tumor tissues. (A) Representative images of IHC staining of CD70 and B7-H3 on human tumor tissue microarrays were shown. (Scale bar, 20 μm) (B) IHC result of CD70 and B7-H3 staining in normal tissues including brain, esophagus, stomach, intestine, pancreas, appendix. The representative images were shown. (Scale bar, 50 μm) (C) Differential expression profile analysis of B7-H3 and CD70 in tumor and normal tissues based on the TCGA database.

Article Snippet: Tumor cell surface expression of CD70 and B7-H3 were detected using CD70 scFv.mFc and B7-H3 scFv.hFc chimeric proteins followed by Cy3-conjuncted goat anti-mouse Fc (Proteintech) for CD70 scFv.hFc and Alexa Fluor 594-conjugated goat anti-human Fc (Jackson ImmunoResearch) for B7-H3 scFv.hFc.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Expression of CD70 and B7-H3 in human solid tumor cell lines. (A, B) Representative images showed the immunofluorescence staining of B7-H3 and CD70 together with DAPI in NCI-H460, A375, MDA-MB-435 and 786-O tumor cells. (Scale bar: 20 μm) (C, D) Flow cytometry result indicated high expression of CD70 and B7-H3 on the four solid tumor cell lines.

Article Snippet: Tumor cell surface expression of CD70 and B7-H3 were detected using CD70 scFv.mFc and B7-H3 scFv.hFc chimeric proteins followed by Cy3-conjuncted goat anti-mouse Fc (Proteintech) for CD70 scFv.hFc and Alexa Fluor 594-conjugated goat anti-human Fc (Jackson ImmunoResearch) for B7-H3 scFv.hFc.

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Construct of CAR (A) Schematic diagrams showing the composition of the four CARs used in this study: CD70 CAR 1 , CD70 CAR 2 , B7-H3 CAR and TanCAR. (B) The lentiviral backbone plasmid encodes the TanCAR. (C) Cartoon depicted of TanCAR targeting respective tumor antigens.

Article Snippet: Tumor cell surface expression of CD70 and B7-H3 were detected using CD70 scFv.mFc and B7-H3 scFv.hFc chimeric proteins followed by Cy3-conjuncted goat anti-mouse Fc (Proteintech) for CD70 scFv.hFc and Alexa Fluor 594-conjugated goat anti-human Fc (Jackson ImmunoResearch) for B7-H3 scFv.hFc.

Techniques: Construct, Plasmid Preparation

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Functional analysis of CD70 CAR 1 and CD70 CAR 2 . (A) To assess the affinity of two CD70 binding fragments, immunofluorescence was performed using human trCD27.mFc and CD70 scFv.hFc chimeric protein as the primary antibody. Images showed the immunofluorescence staining of CD70 by NCI-H460 and A375 tumor cells. (Scale bar: 20 μm) (B) 4-hour 51 Cr cytotoxicity assays indicated a higher tumor killing of CD70 CAR 2 -T cells against target cells. (C) Microscopy images were captured 8 hours after A375 or H460 cells cocultured with CD70 CAR 1 , CD70 CAR 2 and NT T cells at a ratio of 2 effector cell to 1 target cells. (Scale bar: 50 μm) (D) ELISA results showed the IFN-γ and IL-2 secretion levels by CD70 CAR 1 , CD70 CAR2 and NT T cells encountering A375 or H460 cells.

Article Snippet: Tumor cell surface expression of CD70 and B7-H3 were detected using CD70 scFv.mFc and B7-H3 scFv.hFc chimeric proteins followed by Cy3-conjuncted goat anti-mouse Fc (Proteintech) for CD70 scFv.hFc and Alexa Fluor 594-conjugated goat anti-human Fc (Jackson ImmunoResearch) for B7-H3 scFv.hFc.

Techniques: Functional Assay, Binding Assay, Immunofluorescence, Staining, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Activity of TanCAR-T cells against tumor cells expressing CD70 and/or B7-H3. (A) Four-hour 51 Cr cytotoxicity assays of TanCAR-T cells against tumor cells expressing CD70 and/or B7-H3, compared with unispecific CAR and NT T cells. (B) Analysis of IFNγ and IL2 secretion level from supernatants of co-cultures of TanCAR, B7-H3 CAR, CD70 CAR 2 and NT T cells with multiple tumor cells, as detected by ELISA. Shown are pooled data from 3 independent experiments done in triplicates.

Article Snippet: Tumor cell surface expression of CD70 and B7-H3 were detected using CD70 scFv.mFc and B7-H3 scFv.hFc chimeric proteins followed by Cy3-conjuncted goat anti-mouse Fc (Proteintech) for CD70 scFv.hFc and Alexa Fluor 594-conjugated goat anti-human Fc (Jackson ImmunoResearch) for B7-H3 scFv.hFc.

Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Antitumor response of TanCAR-T cells against CD70 and B7-H3 positive tumors in vivo . (A) The treatment scheme showed the timing of subcutaneous injection of tumor cells, vein-tail injection of CAR-T cells T cells and in vivo optical imaging. (B) Antitumor response of TanCAR-T cells in human subcutaneous xenograft models. NCI-H460.ffLuc or A375.ffLuc tumor bearing (confirmed by imaging 6 days after tumor implantation, 8/group) mice were adoptively transferred through tail vein injection with NT, CD70 CAR 2 , B7-H3 CAR or high/low doses (5×10 6 or 1×10 6 /mouse) of TanCAR T cells on 7 days and 10 days post tumor inoculation. (C) Tumor growth was measured weekly by using Living Image software, and mean values per treated group were shown. (D) Kaplan-Meier survival curve were performed 75 days after T cells injection. Mice treated with TanCAR-T cells had a significantly longer survival probability in comparison with mice with NT, CD70 CAR 2 or B7-H3 CAR-T cells.

Article Snippet: Tumor cell surface expression of CD70 and B7-H3 were detected using CD70 scFv.mFc and B7-H3 scFv.hFc chimeric proteins followed by Cy3-conjuncted goat anti-mouse Fc (Proteintech) for CD70 scFv.hFc and Alexa Fluor 594-conjugated goat anti-human Fc (Jackson ImmunoResearch) for B7-H3 scFv.hFc.

Techniques: In Vivo, Injection, Optical Imaging, Imaging, Tumor Implantation, Software

Journal: PLoS ONE

Article Title: Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method

doi: 10.1371/journal.pone.0134727

Figure Lengend Snippet: HCV29, KK47, and YTS1 cells were cultured and stained with six antibodies directed to identified proteins (MAGEA4, THY1, IGF2BP1, VIM, CTNNB1, FN1) labeled with Cy3 as described in M&M. Images are shown of merge images of Cy3-conjugated antibodies and DAPI staining of nuclei (objective magnification 60×). Scale bars: 70 μm.

Article Snippet: The primary antibodies were rabbit anti-insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) (#AP10466b, Abgent; San Diego, CA, USA), rabbit anti-melanoma-associated antigen 4 (MAGEA4) (#AP6166a, Abgent), rabbit anti-Thy-1 membrane glycoprotein (THY1) (#AP2050a, Abgent), 14-3-3 protein sigma (SFN) (#sc-365539, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-fibronectin (FN1) (#F3648, Sigma-Aldrich), mouse anti-vimentin (VIM) (#V5255, Sigma-Aldrich), CD70 antigen (CD70) (#sc-7681, Santa Cruz Biotechnology), and mouse anti-β-catenin (CTNNB1) (#610153, BD Biosciences; San Jose, CA, USA).

Techniques: Cell Culture, Staining, Labeling