Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 2 The expression of selected hypoxia-related markers (IDH1, IDH2, HIF1a, HIF1b, HIF2a, EGFR, PTEN, VEGFA, VEGFC and STAT3) in primary glioma cells GBMLe3, GBMLe4 and GBMDo2 (A) and in cryopreserved samples corresponding to the tumor used for particular primary glioma culture derivation (B) at mRNA level. The expression of mRNA was determined by RT-PCR. Data are expressed as fold increase ± SD of averages from two independent experi ments. Beta-2-microglobulin was used as a housekeeping gene. * p < 0.05 GBM26 vs. GBM43; # p < 0.05 GBM26 vs. GBM59

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 3 Comparison of tumor growth and drug accumulation in Foxn1-nu mice after implantation of glioma cell lines followed by TMZ treatment. Tumor size of implanted (n = 4) (A) U87MG IDH1wt, U87MG STAT3 KO with and without TMZ (0.9 mg/kg) treatment. Evaluation of accumulation of (B) TMZ and (C) its metabolites AIC inside the brain, tumor and plasma in tumor bearing mice with implanted U87MG IDH1wt, resp. U87MG STAT3 KO. The administration of drug (TMZ– 0.9 mg/kg) begins two weeks after implantation (from day 15. to day 28. daily). Organs were collected 15 min after last TMZ application. Confidence interval values of tumor size are shown as mean ± SD. The data of drug accumulation are expressed as ng per mg of tissue. Measurements were performed in two independent experiments

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Comparison, Clinical Proteomics

Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 4 The expression of selected markers related with hypoxia (IDH1, IDH2, HIF1a, HIF1b, HIF2a, EGFR, PTEN, VEGFA, VEGFC and STAT3) in glioma U87MG and U87MG STAT3 KO glioma cell lines (A) and glioma samples collected from Foxn1-nu mice with implanted U87MG and U87MG STAT3 KO glioma cells on mRNA level (B). Tumors were collected 28 days after glioma cell implantation and processed as described in Materials and methods section

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Expressing

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 1. GRB2 complexes with AGO2 under non-stimulated conditions. Schematic diagram of, (a) AGO2 and, (b) GRB2 domain structures. Domains are named and colour coded and attributed amino acid sequence number. Red arrows indicate positions of PXXP motifs investigated in this work. (c) Western blot of AGO2 co-immunoprecipitated with GRB2 in serum starved HEK293T, A498 and PC3 cells. A longer exposure was used to capture AGO2 bands than for GRB2 and GAPDH. All images are taken from the same western blot. (d) Fluorescence and fluorescence resonance energy transfer signals of RFP-tagged GRB2 and GFP-tagged AGO2. HEK293T cells overexpressing fluorescent proteins were serum-starved before imaging. N = 3. Scale bars are 10 μm.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Sequencing, Western Blot, Immunoprecipitation, Fluorescence, Förster Resonance Energy Transfer, Imaging

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 2. Binding of GRB2 to AGO2 is mediated by GRB2 NSH3 and a PXXP motif in AGO2 PAZ domain. (a) Isothermal titration calorimetry (ITC) of a peptide spanning the proline-rich motif 323PHLP326 in AGO2 PAZ domain. (KD = 4.27 ± 1.17 µM). (b, c) ITC of MBP-tagged AGO2 PAZ domain titrated into GRB2. (b) PAZ WT (KD = 585 ± 61 nM). (c) No binding observed for mutation of PXXP (MBP-PAZ 4A). N = 2. (d) Fluorescence resonance energy transfer (FRET) between wild type (WT) and 323AAAA326 (4A) mutant GFP-tagged AGO2 and RFP-tagged GRB2 in HEK293T cells under conditions of serum starvation. White arrows indicate intracellular puncta which show increased FRET when WT AGO2 is expressed. N = 2. Scale bars are 10 μm. (e) Fluorescence lifetime imaging microscopy of RFP-tagged GRB2 proteins and GFP-AGO2 overexpressed in serum-starved HEK293T cells. The formation of a protein complex results in a reduction in fluorescent lifetime represented by a shift to the left of the population of fluorophores (measured in number of pixels). Lifetime population distribution shown by red line on graphs. x = Lifetime (ns), y = number of pixels. Solid black line corresponds to average fluorescent lifetime for GFP, 2.1 ns. Scale bars 25 μm. (f) Expanded region of interest (ROI) further exemplifying left-shift for AGO2/NSH3-SH2 interaction.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Mutagenesis, Fluorescence, Förster Resonance Energy Transfer, Imaging, Microscopy

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 3. Impact of GRB2-AGO2 complex on interaction with DICER1 and miRNA. (a) Western blot of AGO2 and DICER1 pulldown by GST-GRB2 in HEK293T cells. HEK293T cells were serum-starved before lysis. Bands captured with both a long and short exposure are shown for DICER1, whereas only the image captured with a short exposure is shown for AGO2. GST proteins were detected by ponceau stain. All images are taken from the same western blot. N = 3. (b–d) MST of AGO2 binding to DICER1 C-terminal region, upon pre-incubation of AGO2 with increasing concentrations of GRB2. The difference in binding affinity was negligible. (e) MST of GRB2 with DICER1 C-terminal region. No binding is observed within a physiologically relevant range hence the two do not interact directly. (f) Expanded ribbon model of molecular docking of GRB2 (green; PDB: 1GRI77) to AGO2 PAZ domain (cyan; red and blue indicate positive and negative charges respectively; PDB: 6RA478). The 323PHLP326 sequence is shown (yellow). GRB2 W36 (magenta) interacts with AGO2 P249 (red). Other residues in GRB2 which may contribute towards the interaction are shown in orange. Also shown is space-filling representation of AGO2 PAZ domain with PRM shown (below); and ribbon model of PAZ domain rotated by 90° to highlight juxtaposition of GRB2 binding site PRM and docking site for miRNA (right). Figures generated using PyMOL.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Western Blot, Lysis, Staining, Binding Assay, Incubation, Sequencing, Generated

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 4. GRB2 regulates miRNA expression in HEK293T cells. (a) Western blot of GRB2 expression in wild type (293 T) and depleted (G1) HEK293T clones 1 (G1.1) and 2 (G1.2). While G1.1 is a complete knockout, G1.2 contains a deletion and large insertion in the N-terminal SH3 domain. GRB2 was blotted with an antibody which recognised the C-terminal SH3 domain. Both long and short exposures were used to capture the GRB2 bands, whereas the GAPDH image was captured using a short exposure only. All images are taken from the same western blot. N = 3. (b) Heat plot highlighting miRNAs which show significant log2(fold changes) in expression (p < 0.05) between wild type HEK293T and G1 cells, measured by small RNA sequencing. Cells were deprived of growth factor. miRNAs demonstrated positive (red) and negative (blue) expression changes. N = 2. (c, d) RT-qPCR analysis of fold-change in mean expression of precursor miRNA transcripts (precursor and primary, pre-mir-, hashed bars) and mature miRNA (miR-, plain bars) derived from serum-starved G1 or wild type HEK293T cells. Two groups of miRNAs were observed: (c) miRNAs which diminished at both the level of the precursor and mature transcripts and, (d) miRNAs which were enhanced as mature transcripts but not as precursors. Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 4. ns = not significant.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Expressing, Western Blot, Clone Assay, Knock-Out, RNA Sequencing, Quantitative RT-PCR, Derivative Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 5. The GRB2-let-7 axis regulates oncogene expression. (a) RT-qPCR measurement of fold change in mean expression of let-7 g-5p miRNA and five target mRNAs in serum-starved GRB2 knockout cells (G1) compared to wild type HEK293T (293 T). Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 3. (b) Western blot and (c) quantification of mean protein expression of let-7 targets in growth-factor-deprived G1 and HEK293T cells. The higher molecular band detected by the GRB2 antibody in G1 corresponds to an NSH3-mutated GRB2 polypeptide. For blot 1, a longer exposure was used to capture the DICER1 and GRB2 bands than was used for LIN28B and α-Tubulin. For blot 2, HMGA2 bands were captured using a longer exposure than that required for GRB2 and GAPDH. (d) Quantification of the area covered by migration of HEK293T cells expressing GFP-tagged wild type AGO2 (WT) or an AGO2 mutant which is incapable of binding GRB2 (4A), under conditions of reduced growth factor. Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 3.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Two Tailed Test, Western Blot, Migration, Mutagenesis, Binding Assay

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 1. Increased expression of RIPK2 promotes cachexia in the TgRC mice. (A) The method of generation of RIPK2-tgflox/+ (TgR) mice based on Rosa26 locus (CAG-STOP-RIPK2-EGFP-Rosa26) using the CRISPR/Cas9 system. (B) The generation of TgR/Rosa26-CreERT2 (TgRC) mice using tamoxifen (TAM) injection. (C) The genotype analysis of RIPK2 protein expression in tail tissue of the TgR and TgRC mice. (D) Mouse body weight post tamoxifen (TAM) administration in TgR and TgRC mice (n = 10). (E) Fat mass, lean mass, and weight loss post tamoxifen (TAM) administration in TgR and TgRC mice (n = 10). Mice body composition was analyzed by EchoMRI. (F) Representative images of H&E staining showing the histopathological changes of muscle and WAT tissue in TgR and TgRC mice (n = 5). (G-M) Indicated mice were maintained in metabolic cages. Experimental mice administrated with TAM at the first light cycle. Values are hourly means. The energy balance (G), food intake (H), total energy expenditure (TEE) (I), oxygen consumption (VO2) (J), carbon dioxide production (VCO2) (K), respiratory exchange ratio (RER) (L) and locomotor activity (M) of TgR and TgRC mice post TAM injection were determined (n = 10). (N) The serum albumin, BUN, ALP and GGT contents in the TgR and TgRC mice (n = 10). (O) Kaplan-meier (KM) survival curves of mice. Data were shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns., no significance.

Article Snippet: The RIPK2 gene knockout kit (CRISPR) was obtained from OriGene Technologies, Wuxi, China.

Techniques: Expressing, CRISPR, Injection, Staining, Activity Assay

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 2. Methylnissolin protects against overexpressed RIPK2-associated cachexia in the TgRC mice. (A) A protocol of methylnissolin treatment on TgRC mice with TAM injection. (B) Mouse body weight analysis in TgRC and TgRC mice with increasing dosage of methylnissolin (10 mg/kg, 20 mg/kg and 40 mg/kg) in response to TAM injection (n = 10). (C, D) Fat mass (C) and lean mass (D) in TgRC and TgRC mice (n = 10). (E) Representative images of H&E staining showing the histopathological changes of muscle and WAT tissue in the indicated mice group (n = 5). (F-L) Indicated mice were maintained in metabolic cages. Values are hourly means. The energy balance (F), food intake (G), total energy expenditure (TEE) (H), oxygen consumption (VO2) (I), carbon dioxide production (VCO2) (J), respiratory exchange ratio (RER) (K) and locomotor activity (L) of TgRC + DMSO and TgRC + methylnissolin (40 mg/kg) mice were determined (n = 10). (M) The serum albumin, BUN, ALP and GGT contents in the indicated mice (n = 10). (N) Kaplan-meier (KM) survival curves of mice. Data were shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns., no significance.

Article Snippet: The RIPK2 gene knockout kit (CRISPR) was obtained from OriGene Technologies, Wuxi, China.

Techniques: Injection, Staining, Activity Assay

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 3. Overexpression of RIPK2 impairs lipid homeostasis and blocks lipid biosynthesis. (A) Heatmap showing the serum TG contents alterations in the TgR and TgRC mice in response to fed or fasting conditions (n = 5). (B) Analysis of small (LC-TGs) and large (LC-TGs) levels in fed or fasted TgR and TgRC mice (n = 5). (C) Analysis of small (LC-TGs) and large (LC-TGs) levels in fed or fasted TgR and TgRC mice (n = 5). (D) Heatmap showing the serum TG contents alterations in the fed or fasted TgR and TgRC mice (n = 5). (E) Heatmap showing the serum TG contents in the stomach tissue of Balb/c mice with/without MKN-45-RIPK2 WT or MKN-45-RIPK2 KO tumors (n = 10). (F, G) Lipid biosynthesis analysis in TgR and TgRC mice with TAM administration (F) and in Balb/c mice with/without MKN-45- RIPK2 WT or MKN-45-RIPK2 KO tumors (G) (n = 10). Data were shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns., no significance.

Article Snippet: The RIPK2 gene knockout kit (CRISPR) was obtained from OriGene Technologies, Wuxi, China.

Techniques: Over Expression

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 5. Increased expression of RIPK2 inhibits the ASK signaling pathway. (A) The RNA-seq analysis showing the gene expression profiles in the stomach tissue after TAM administration TgR and TgRC mice (n = 5). (B) KEGG analysis of DEGs by the DAVID database. (C) GSEA enrichment bars for lipid metabolism and ASK signaling pathway. (D) RNA-seq analysis indicating the genes expression alterations of ASK, PPARα, PPARβ, PPARδ, PPARγ, PPARGC1a and PPARGC1b in TgR and TgRC mice (n = 5). (E, F) Analysis of mRNA and protein expression of PPARα, ACLY, CD36, FASN and ACSL1 determined by qPCR (E) and western blotting assay (F) in TgR and TgRC mice (n = 5). (G, H) Analysis of mRNA and protein expression of p-ASK1, PPARα, ACLY, CD36, FASN and ACSL1 determined by qPCR (G) and western blotting assay (H) in TgR and TgRC mice (n = 5). Data were shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns., no significance.

Article Snippet: The RIPK2 gene knockout kit (CRISPR) was obtained from OriGene Technologies, Wuxi, China.

Techniques: Expressing, RNA Sequencing, Gene Expression, Western Blot

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 7. The molecular mechanism diagram by which RIPK2 induces cachexia, and protective effect of methylnissolin on gastric cancer and its associated cachexia progression.

Article Snippet: The RIPK2 gene knockout kit (CRISPR) was obtained from OriGene Technologies, Wuxi, China.

Techniques:

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: PD-L1 expression in GBM and PD-1 recruitment to the CARIS with GBM. A, Constitutive (UPN01) and inducible (UPN06) surface expression of PD-L1 in primary GBM cells after 24–48 hours of IFN-γ exposure. Representative results from two samples shown. UPN, unique patient number. B, PD-L1 median fluorescent intensity (MFI) on primary GBM ( n = 14) before and at 48 hours of IFN-γ (10 ng/mL) exposure; ***, P < 0.001, Wilcoxon signed-rank test. C, Fold-change in PD-L1 MFI in GBM cells from baseline, at 24, 48, and 72 hours. Each color represents a single patient donor, with some measured at multiple time points. Data are shown as individual values with the mean ± SD. D, PD-L1 expression in WT LN229-GBM cells and LN229 with PD-L1 deletion (LN229-PD-L1 KO) at baseline and at 24 hours of exposure to IFN-γ (10 ng/mL) or CAR28ζ T cells. E, MFI of PD-1 in the immune synapse between CARζ cells and WT LN229-GBM cells at 15, 30, and 60 minutes (****, P < 0.0001; ns, P > 0.5, Kruskal–Wallis and Wilcox pairwise); ≥20,000 events were captured, and 300–1,000 CAR + conjugates were examined for PD-1 recruitment to CARIS. The white box represents the IQR with horizontal lines at 25%, 50%, and 75%. F, Representative image capture showing the CARIS with tumor cell (WT LN229-GBM) and other parameters evaluated. Gating strategy is shown in the Supplementary material. G, Spearman correlation between PD-1 intensity and CAR intensity in the immune synapse at 15, 30, and 60 minutes. H, Spearman correlation between PD-1 intensity and actin intensity in the CARIS at 15, 30, and 60 minutes. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Design and functional screening of PD-1 TR and CPR28 molecules. A, Schematic representation of the bicistronic vectors encoding for the HER2-CAR with truncated PD-1 (PD-1 TR ) or CPR28. The CPR28 dimer included a membrane proximal extracellular cysteine residue (C141) required for CD28 homodimerization, as indicated. B, Flow cytometry analysis showing coexpression of HER2-CAR and PD-1 CPR on T cells. CAR28ζ and NT T cells from the same donor were used as controls for assessment of CPR expression determined by surface PD-1 detection. C, Long-term cytolytic function of CAR28ζ/PD-1 TR , compared with CAR28ζ cells, against U373-GBM cells at effector to target ratios of 1:5 and 1:10 assessed using a cell-impedance based assay (xCELLigence). D, Comparison of the cytolytic ability of patient-derived CAR28ζ cells ( n = 3) coexpressing CPR28 dimer or CPR28 monomer against autologous HER2 + GBM cells at an effector to target ratio of 1:10 by assessment of tumor cell viability in an xCELLigence assay. In C and D , error bars represent the mean ± SD at each time point. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. E, IL-2 and ( F ) IFN-γ release by CAR28ζ, CAR28ζ/PD-1 TR , and CAR28ζ/CPR28 cells (100,000 T cells/well) upon stimulation with Fc-conjugated HER2 (0–2 μg/mL) and PD-L1 (0–5 μg/mL) proteins. Median values from a representative donor shown. G, CAR28ζ/CPR28 cell–induced lysis of Raji cells modified to express HER2 or HER2 and PD-L1 across different T-cell to tumor cell ratios but not the WT Raji cells (HER2 − /PD-L1 − ) or those expressing PD-L1 alone in a 51 Cr-release assay. T cells with CPR28 alone had no cytotoxic effect against HER2 + or PD-L1 + Raji cells, like NT T cells from the same donor. A, Created in BioRender. Navai, S. (2024) BioRender.com/l86h941 .

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Membrane, Residue, Flow Cytometry, Expressing, Comparison, Derivative Assay, Lysis, Modification, Release Assay

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Effect of decoupling signal 2 from the CAR on T-cell function. A, Illustration depicting the bipartite T-cell activation through signal 1 delivery from CAR engaging the HER2 antigen and signal 2 from binding of CPR with PD-L1 (or PD-L2). B, Percent increase in IFN-γ production by CAR28ζ/CPR28 and CARζ/CPR28 compared with CAR28ζ cells (50,000 T cells/well) at 24 hours of coculture with autologous GBM cells ( n = 5 patients). Effector (100,000 T cells) to target ratio of 1:1. **, P < 0.01; ****, P < 0.0001, one-way ANOVA with the Tukey multiple comparisons test. C, Cytolytic function of CAR28ζ/CPR28 and CARζ/CPR28 cells assessed using 4-hour 51 Cr-release assay at baseline and at 7 days of persistent T-cell stimulation through the CAR. NT T cells had poor viability after prolonged stimulation without added homeostatic cytokines and were not evaluable. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. D, Percent of total T cells expressing HER2-CAR on cell surface, and percent of CAR + T cells detected with PD-1 (surrogate marker for CPR), respectively, amongst different CPR/CART groups ( n = 4 patients) using flow cytometry. E, Median fluorescent intensity (MFI) of HER2-CAR detected in transduced T cells shown in D . In D and E , only statistically significant differences are shown, *, P < 0.05; **, P < 0.01; ****, P < 0.0001, one-way ANOVA with the Tukey multiple comparisons test. F, Multiplex analysis for proinflammatory cytokines (IL-2, MIP-1α, TNF-α, and GM-CSF) in autologous T-cell and GBM coculture ( n = 4 patients) supernatants at 24 hours. UPN, unique patient number. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. G, Assessment of long-term cytolytic function of CPR/CART against autologous GBM cells ( n = 3 patients) using cell impedance–based xCELLigence assay. Statistical differences (denoted by the color key) shown are in comparison to control treatment CAR28ζ cells overtime. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. In G, black arrow indicates addition of T cells. A, Created in BioRender. Navai, S. (2019) BioRender.com/p40z816 .

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Cell Function Assay, Activation Assay, Binding Assay, Release Assay, Cell Stimulation, Expressing, Marker, Flow Cytometry, Multiplex Assay, Comparison, Control

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Phenotypic and functional profile of CART receiving bipartite activation signals through CPR41BB costimulation. A, In vivo functional screening of CARζ and CAR28ζ cells coexpressing CPR28 or CPR41BB against orthotopic xenografts of HER2 + PD-L1 + U373-GBM in SCID mice ( n = 5 per group). B, Fold change in tumor burden after treatment relative to the tumor volume before intratumoral injection of T cells (day 0), quantified by serial BLI. *, P < 0.05; **, P < 0.01, two-way ANOVA with the Tukey multiple comparisons test. Data are shown as the mean ± SD. C, CD8 + : CD4 + ratio in CAR-expressing T cells among CPR/CART from patients with GBM ( n = 4) as assessed by flow cytometry. Box plots show minimum to maximum with individual values. ns, P > 0.5, one-way ANOVA with the Tukey multiple comparisons test. D, Pie graph demonstrating immunophenotype distribution of CARζ/CPR41BB cells ( n = 4 patients) at baseline. Percentages shown represent the mean value. Immunophenotype is defined as follows: naïve, CCR7 + /CD45RA + ; central memory, CCR7 + /CD45RA − ; effector memory, CCR7 − /CD45RA − ; and terminal effector, CCR7 − /CD45RA + . E, The percentage CAR + CD8 + central memory (CCR7 + /CD45RA − ) T cells did not significantly differ amongst different CPR/CART groups ( n = 4 patients). The mean ± SEM is shown. ns, P > 0.5, one-way ANOVA with the Dunnett multiple comparisons test. F, Pie graph demonstrating immunophenotype distribution of CARζ/CPR41BB cells ( n = 4 patients) after 7 days of continued stimulation by repeat cocultures with autologous GBM cells. Percentages shown represent the mean value. On day 7 of repeated stimulation, CARζ/CPR41BB cells ( n = 4 patients) demonstrated ( G ) a significantly higher proportion of CAR + /CD8 + cells with the central memory phenotype compared with other CPR/CART groups and CAR28ζ, and ( H ) a significantly lower proportion of CAR + /CD8 + cells with the effector memory phenotype compared with CAR28ζ/CPR28 cells. The mean ± SEM is shown. *, P < 0.05; **, P < 0.01, one-way ANOVA with the Dunnett multiple comparisons test. I, Log-change in the median fluorescent intensity (MFI) of T-cell surface PD-1, LAG3, and TIM3 at 7 days of repeat coculture with autologous GBM cells ( n = 4 patients) from baseline. Box plots show minimum to maximum with individual values. *, P < 0.05; **, P < 0.01, two-way ANOVA with the Tukey multiple comparisons test. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Activation Assay, In Vivo, Injection, Expressing, Flow Cytometry

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Dynamics of CARζ/CPR41BB T-cell activation and CARIS formation in comparison with CAR41BBζ cells. A, CARζ/CPR41BB cells ( n = 3 donors) demonstrated significantly lower IL-2 and IFN-γ release compared with CAR41BBζ cells at 24 hours of coculture with LN229-GBM cells, with CAR28ζ cells consistently showing the higher Th1 cytokine production. Data are shown as the mean ± SD. **, P < 0.000; ****, P < 0.0001; two-way ANOVA with the Tukey multiple comparisons test. B, Western blot analysis for CAR-phosphoCD3ζ (pCD3) in T cells in a resting state (maintained in culture with IL-7/IL-15). The pCD3 to CD3 ratio is normalized to CARζ in each donor. *, P < 0.05; one-way ANOVA with the Tukey multiple comparisons test. C, Representative image capture showing the CARIS with tumor cell (LN229-GBM) and different CARζ/CPR41BB immune synapse parameters evaluated. Gating strategy is shown in the Supplementary Material. Spearman correlation ( D ) between the intensity of CAR and CPR at the CARIS and ( E ) between the intensity of actin and CPR at the CARIS, both assessed by imaging flow cytometry at 15, 30, and 60 minutes. F, CARζ/CPR41BB and CAR41BBζ cells show significantly higher percent (%) of F-actin at the CARIS compared with CARζ cells. ns, P > 0.5; **, P < 0.01; ***, P < 0.0001, two-way ANOVA with the Tukey post hoc test. Data are shown as the mean with 95% confidence interval (CI). G, CARζ/CPR41BB cells show significantly higher CPR intensity in the CARIS with WT LN229 GBM cells at 15, 30, and 60 minutes compared with conjugates with LN229-PD-L1 KO (Kruskal–Wallis and Wilcox pairwise). CPR intensity increased over time in both conditions. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Activation Assay, Comparison, Western Blot, Imaging, Flow Cytometry

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Metabolomic parameters of CARζ/CPR41BB cells in comparison with CAR41BBζ cells. A, OCR measurements of resting (cultured in media containing in IL-7 and IL-15) CARζ/CPR41BB and CAR41BBζ cells ( n = 3 donors; 200,000 T cells per well) under basal metabolic conditions and after the addition of mitochondrial inhibitors. B, Comparison of maximal respiration between CARζ/CPR41BB cells and CAR41BBζ cells at baseline (day 0). P = 0.059, Student two-tailed t test. C, Basal OCR, maximal respiration, and SRC between CAR41BBζ and CARζ/CPR41BB cells after 7 days of stimulation with Fc-conjugated HER2 and PD-L1 proteins. ns, P > 0.05, Student two-tailed t test. D, The ECAR in resting CARζ/CPR41BB and CAR41BBζ cells. E, The ECAR of CARζ/CPR41BB and CAR41BBζ at baseline and at 7 days of continued stimulation with plate-bound HER2 and PD-L1 proteins. ns, P > 0.05; **, P < 0.01, Student two-tailed t test. F, Basal OCR to ECAR ratio in CARζ/CPR41BB compared with CAR41BBζ cells at rest and at 7 days of continued stimulation with HER2 and PD-L1 proteins. ns, P > 0.05; *, P < 0.05, Student two-tailed t test. G, The OCR in CD8 + CARζ/CPR41BB and CAR41BBζ cells ( n = 3 donors; 150,000 T cells per well) following 48-hour coculture with LN229-GBM cells (effector to tumor ratio 1:2). H, The SRC of CD8 + CARζ/CPR41BB and CAR41BBζ cells after 48 hours of coculture with LN229 GBM cells. ns, P > 0.05, Student two-tailed t test. The OCR and ECAR measurements are shown as the mean ± SEM. Data shown denote the IQR in the violin plot and the mean ± SEM in the bar graphs. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Comparison, Cell Culture, Two Tailed Test