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: JCI Insight

Article Title: CD34 hi subset of synovial fibroblasts contributes to fibrotic phenotype of human knee osteoarthritis

doi: 10.1172/jci.insight.183690

Figure Lengend Snippet: ( A ) Dot plots of representative DEGs from fibrotic group synovium in 11 synovial cell subsets . ( B ) Expression levels of CD70 in 2 OA synovium groups, determined by knee OA synovium RNA-Seq. All data are expressed as dot plots and mean ± SEM. Mann-Whitney U test. * P < 0.05. ( C ) Expression levels of CD70 in 4 OA synovium subgroups, determined by knee OA synovium RNA-Seq. All data are expressed as dot plots and mean ± SEM. Kruskal-Wallis test. * P < 0.05. ( D ) UMAP of 2 CD34 hi fibroblast clusters identified by scRNA-Seq. ( E ) Percentage of fibroblast subsets in the 4 synovium samples. Infla, inflammatory group; Fibro, fibrotic group. ( F ) Dot plots of stable state marker genes and CD70 in CD34 hi CD70 lo fibroblasts and CD34 hi CD70 hi fibroblasts. ( G ) Feature plots of stable state marker genes and CD70 in CD34 hi CD70 lo fibroblasts and CD34 hi CD70 hi fibroblasts. ( H ) Immunofluorescence images of Podoplanin (PDPN), CD34, and CD70 in human knee OA synovium tissue around vessels. Arrowheads indicate PDPN + CD34 + CD70 + cells. Scale bar: 100 μm.

Article Snippet: In the coculture experiments, after 2 days of culture, synovial and cartilage tissues were transferred to coculture plates, and each well was treated with anti–human CD70 antibody (NOVUS Bio, NBP3-11587) or isotype control (R&D Systems, MAB002) at a concentration of 5 μg/mL.

Techniques: Expressing, RNA Sequencing, MANN-WHITNEY, Marker, Immunofluorescence

Journal: JCI Insight

Article Title: CD34 hi subset of synovial fibroblasts contributes to fibrotic phenotype of human knee osteoarthritis

doi: 10.1172/jci.insight.183690

Figure Lengend Snippet: ( A ) T cell proliferation assay cocultured with CD70 transcript variant 1 or 2 or with GFP-transfected fibroblasts cell line (MRC-5). ( B ) The percentage of CD45 + cells and CD4 + T cells in live cells cocultured with CD70 transcript variant 1 or 2 or with GFP-transfected MRC-5 cells. ( C ) Graphical schema of OA synovial and cartilage tissues coculture experiments. ( D ) Time-course mRNA levels of IL1B , IL6 , and TNF in cocultured synovial tissues and COL2A1 , ACAN , and SOX9 mRNA levels in cocultured cartilage tissues with anti-CD70 antibody or isotype control. n = 6 biologically independent experiments. ( E ) The percentage of CD45 + cells and T cell subsets in cocultured synovial tissues with anti-CD70 antibody or isotype control. n = 10 biologically independent experiments. ( F ) The percentage of IL-1B + DCs, macrophages (Mφ), and T cells in cocultured synovial tissues with anti-CD70 antibody or isotype control. n = 6 biologically independent experiments. Data are expressed as dot plots and mean ± SEM. Mann-Whitney U test. * P < 0.05, ** P < 0.01.

Article Snippet: In the coculture experiments, after 2 days of culture, synovial and cartilage tissues were transferred to coculture plates, and each well was treated with anti–human CD70 antibody (NOVUS Bio, NBP3-11587) or isotype control (R&D Systems, MAB002) at a concentration of 5 μg/mL.

Techniques: Proliferation Assay, Variant Assay, Transfection, Control, MANN-WHITNEY

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: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

Techniques: Expressing, Immunohistochemistry, Staining, Quantitative Proteomics

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: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

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: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

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: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

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: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

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: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

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

Journal: Journal for Immunotherapy of Cancer

Article Title: Characterization of the human T cell response to in vitro CD27 costimulation with varlilumab

doi: 10.1186/s40425-015-0080-2

Figure Lengend Snippet: Cytokine induction, kinetics and TCR dependence of anti-CD27 costimulation of T cells by varlilumab or natural ligand (CD70). T cells were stimulated concurrently for 72 h with anti-CD3 (OKT3) and anti-CD27 (varlilumab; varli) antibodies. a Boxed whisker plot showing direct comparison of varlilumab stimulation of T cells with that of 4-1BB, OX40 and GITR-specific Abs [ n = 6]. Statistical comparisons of IFNγ release by the different TNFR stimulations was performed using Welch Two Sample t -test; significance is shown as * p ≤ 0.05; ** p ≤ 0.01. b Cell culture supernatants were tested for Th1/Th2 cytokines at the time points indicated by standard ELISA; representative data of 3 donor samples is shown; c T cells were stimulated for 72 h with OKT3 and irradiated 293-CD70 or 293-mock cell lines and assessed for IFNγ and IL-13. Additionally, CD70-dependent IFNγ release was assessed in the presence of a blocking Ab (BU.69) or control Ab (mIgG, left panel; n = 3) and IL-13 (right panel; n = 4); d T cells were first pre-activated for 46 h with anti-CD3 and further stimulated with varlilumab or hIgG or in combination with anti-CD3 for 24 h and tested for IFNγ ( n = 4); NS, not significant ( p = 0.09). e NFκB signaling by varlilumab-costimulated T cells for 72 h showing differential release of IFNγ in the presence of known pathway-specific inhibitors (top to bottom)- NFκB (Celastrol), ERK1/2 (PD98059), PKR (2-aminopurine), p38 MAPK (SB203580), IκBα (BAY11-7082), and JAK2 (AG490); bars represent mean ± SD ( n = 4)

Article Snippet: A vector containing the cDNA for human CD70 (accession # NM_001252.2) was purchased from Origene (Rockville, MD).

Techniques: Whisker Assay, Comparison, Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation, Blocking Assay, Control