Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) Strategy for identification of the overlapped genes that were upregulated in both GBM-associated microglia and bone marrow–derived macrophage (BMDMs) compared with normal monocytes. The analysis was based on RNA-Seq data ( GSE86573 ) from GBM-associated microglia/BMDMs isolated from the GL261 tumor model and RCAS tumor model and normal microglia and monocytes. ( B ) The expression pattern of genes in CD45 – GBM cells and tumor-associated immune cells (e.g., BMDMs, microglia, neutrophils, CD4 + T cells, and CD8 + T cells) isolated from human GBM tumors based on the Brain TIME dataset . ( C ) Coimmunofluorescence staining for GPNMB (red) and F4/80 (macrophage marker, green) or CX3CR1 (microglia marker, green) in CT2A tumors implanted in C57BL/6 mice. Scale bar: 50 μm. ( D – G ) Flow cytometry analysis of GPNMB expression in CD11b + CD45 hi CD68 + macrophages ( D and E ) isolated from bone marrow and CD11b + CD45 lo CX3CR1 + microglia ( F and G ) isolated from brain tissues of tumor-free C57BL/6 mice and CT2A and QPP7 tumor-bearing C57BL/6 mice. n = 3–6 independent samples. One-way ANOVA test. ( H – K ) Flow cytometry analysis of GPNMB expression in CD11b + CD45 hi CD11c + DCs, CD11b + CD45 lo CX3CR1 + microglia (Total MG), CD11b + CD45 lo CX3CR1 + CD206 + microglia (CD206 + MG), CD11b + CD45 lo CX3CR1 + CD206 – microglia (CD206 – MG), CD11b + CD45 hi CD68 + macrophages (Total MΦ), CD11b + CD45 hi CD68 + CD206 + macrophages (CD206 + MΦ), CD11b + CD45 hi CD68 + CD206 – macrophages (CD206 – MΦ), CD11b + CD68 + Ly6G lo Ly6C hi monocytic immature myeloid cells (M-IMCs), and CD11b + CD68 + Ly6G hi Ly6C lo polymorphonuclear immature myeloid cells (PMN-IMCs) isolated from QPP7 tumors ( H and I ) and CT2A tumors ( J and K ) implanted in C57BL/6 mice. n = 3 independent samples. One-way ANOVA test. ( L ) Immunoblots for GPNMB in lysates of Raw264.7 macrophages and SIM-A9 microglia treated with the conditioned media (CM) of QPP7 GSCs for 24 hours. ( M ) Immunoblots for GPNMB in lysates of THP-1 macrophage and HMC3 microglia treated with the CM of GSC272 cells for 24 hours. ** P < 0.01, *** P < 0.001.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: Derivative Assay, RNA Sequencing, Isolation, Expressing, Staining, Marker, Flow Cytometry, Western Blot

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) GSEA analysis of single-cell RNA-Seq (scRNA-Seq) data from human GBM tumors (EGAS00001004871) shows top enriched WikiPathways signatures in tumors with TAMs expressing high GPNMB compared to low GPNMB . ( B and C ) Extracellular acidification rate (ECAR) of GSC272 cells ( B ) and CT2A cells ( C ) treated with GPNMB recombinant protein (100 ng/mL) for 24 hours. ECAR was obtained from the Seahorse experiments, and glucose was added at the indicated time point. n = 3 independent samples. ( D and E ) ECAR of GSC272 cells treated with the conditioned media (CM) of THP-1 macrophages ( D ) and HMC3 microglia ( E ) expressing shRNA control (shC) or GPNMB shRNA (sh GPNMB ) for 24 hours. ECAR was obtained from the Seahorse experiments, and glucose was added at the indicated time point. n = 6 independent samples. ( F – H ) Quantification of relative L-lactate levels in GSC272 ( F ), GSC2 ( G ), and CT2A cells ( H ) treated with GPNMB recombinant protein (100 ng/mL) for 24 hours. n = 3 independent samples. Student’s t test. ( I and J ) Quantification of relative L-lactate levels in GSC272 cells treated with the CM of THP-1 macrophages ( I ) and HMC3 microglia ( J ) expressing shC or sh GPNMB for 24 hours. n = 3 independent samples. One-way ANOVA test. ( K and L ) Quantification of relative L-lactate levels in GSC2 cells treated with the CM of THP-1 macrophages ( K ) and HMC3 microglia ( L ) expressing shC or sh GPNMB for 24 hours. n = 3 independent samples. One-way ANOVA test. ( M and N ) Quantification of relative L-lactate levels in CT2A cells treated with the CM of Raw264.7 macrophages ( M ) and SIM-A9 microglia ( N ) expressing shC or sh Gpnmb for 24 hours. n = 3 independent samples. One-way ANOVA test. * P < 0.05, ** P < 0.01.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: RNA Sequencing, Expressing, Recombinant, shRNA, Control

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) Strategy to cluster GBM samples into GPNMB -low and GPNMB -high subgroups based on 4 published single-cell RNA-Seq (scRNA-Seq) datasets ( GSE131928 , GSE148842 , GSE89567 , and EGAS00001004422). ( B ) GSC signature score in GPNMB -low ( n = 19) and GPNMB -high ( n = 20) subgroups based on the above 4 scRNA-Seq datasets. Student’s t test. ( C and D ) Immunoblots for CD133 and SOX2 in lysates of GSC272 ( C ) and GSC2 ( D ) cells treated with GPNMB recombinant protein at the different indicated concentrations for 24 hours. ( E and F ) Immunoblots for CD133 and SOX2 in lysates of GSC272 cells treated with the conditioned media (CM) of THP-1 macrophages ( E ) and HMC3 microglia ( F ) expressing shRNA control (shC) or GPNMB shRNA (sh GPNMB ) for 24 hours. ( G and H ) Immunoblots for CD133 and SOX2 in lysates of GSC2 cells treated with the CM of THP-1 macrophages ( G ) and HMC3 microglia ( H ) expressing shC and sh GPNMB for 24 hours. ( I – L ) Representative images and quantification of tumorspheres of GSC272 cells treated with the CM of THP-1 macrophages ( I and J ) or HMC3 microglia ( K and L ) expressing shC and sh GPNMB for 2 weeks. Scale bar: 200 μm. n = 5 independent samples. One-way ANOVA test. ( M – P ) Representative images and quantification of tumorspheres of GSC2 cells treated with the CM of THP-1 macrophages ( M and N ) or HMC3 microglia ( O and P ) expressing shC and sh GPNMB for 2 weeks. Scale bar: 200 μm. n = 5 independent samples. One-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: RNA Sequencing, Western Blot, Recombinant, Expressing, shRNA, Control

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A and B ) Representative images ( A ) and quantification ( B ) of human phospho-kinases in GSC2 cells treated with or without GPNMB recombinant protein (100 ng/mL) for 1 hour. Affected kinases are indicated. ( C and D ) Immunoblots for P-RSK2, RSK2, P-PYK2, and PYK2 in lysates of GSC272 cells treated with GPNMB recombinant protein at the indicated concentrations and time points. ( E ) Extracellular acidification rate (ECAR) of GSC272 cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PYK2 inhibitor PF-271 (15 nM) or RSK1/2 inhibitor SL0101 (100 μM) for 24 hours. n = 6 independent samples. ( F ) Relative L-lactate levels in GSC272 cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM) or SL0101 (100 μM) for 24 hours. n = 3 independent samples. One-way ANOVA test. ( G ) ECAR of CT2A cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM) or SL0101 (100 μM) for 24 hours. n = 6 independent samples. ( H ) Relative L-lactate levels in CT2A cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM) or SL0101 (100 μM) for 24 hours. n = 3 independent samples. One-way ANOVA test. ( I and J ) Immunoblots for CD133 and SOX2 in lysates of GSC272 cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM, I ) or SL0101 (100 μM, J ) for 24 hours. ( K – N ) Tumorsphere formation assays of GSC272 cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM, K and L) or SL0101 (100 μM, M and N ) for 2 weeks. Scale bar: 200 μm. n = 3–5 independent samples. One-way ANOVA test. ** P < 0.01, *** P < 0.001, n.s., not significant ( P > 0.05).

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: Recombinant, Western Blot

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) T-distributed stochastic neighbor embedding (t-SNE) dimensional reduction of cancer cells and immune cells from GBM tumor samples based on single-cell RNA-Seq (scRNA-Seq) dataset ( GSE182109 ). ( B ) Pattern representing single-cell gene expression (left) and quantification (right) of GPNMB in TAMs and CD44 in GSCs/GBM cells based on scRNA-Seq dataset ( GSE182109 ). ( C ) Relationship between GPNMB expression in TAMs and CD44 expression in GSCs/GBM cells based on scRNA-Seq dataset ( GSE182109 ). P and r values are shown. Pearson’s test. ( D ) Relationship between GPNMB expression in IMCs and CD44 expression in GSCs/GBM cells based on scRNA-Seq dataset ( GSE182109 ). P and r values are shown. Pearson’s test. ( E ) Immunoblots for CD44 in lysates of GSC272 cells expressing shRNA control (shC) or CD44 shRNA (sh CD44 ). ( F ) Immunoblots for P-RSK2, RSK2, P-PYK2, and PYK2 in lysates of GSC272 cells expressing shC and sh CD44 treated with or without GPNMB recombinant protein (100 ng/mL) for 2 hours. ( G ) Extracellular acidification rate (ECAR) of GSC272 cells expressing shC and sh CD44 treated with or without GPNMB recombinant protein (100 ng/mL) for 24 hours. ECAR was obtained from the Seahorse experiments, and glucose was added at the indicated time point. n = 3 independent samples. ( H ) Immunoblots for CD133 and SOX2 in lysates of GSC272 cells expressing shC and sh CD44 treated with or without GPNMB recombinant protein (100 ng/mL) for 24 hours. ( I and J ) Representative images ( I ) and quantification ( J ) of tumorspheres of GSC272 cells expressing shC and sh CD44 treated with or without GPNMB recombinant protein (100 ng/mL) for 2 weeks. Scale bar: 200 μm. n = 4 independent samples. One-way ANOVA test. ** P < 0.01, *** P < 0.001, n.s., not significant ( P > 0.05).

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: RNA Sequencing, Gene Expression, Expressing, Western Blot, shRNA, Control, Recombinant

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A and B ) Survival curves of nude mice coimplanted with GSC272 cells (1 × 10 5 cells/mouse) and THP-1 macrophages (1 × 10 5 cells/mouse, A ) or HMC3 microglia (1 × 10 5 cells/mouse, B ) expressing shRNA control (shC) or GPNMB shRNA (sh GPNMB ). n = 9–10 mice per group. Median survival days are shown. Log-rank test. ( C and D ) Survival curves of C57BL/6 mice coimplanted with CT2A cells (1 × 10 4 cells/mouse) and Raw264.7 macrophages (1 × 10 4 cells/mouse, C ) or SIM-A9 microglia (1 × 10 4 cells/mouse, D ) expressing shC or sh Gpnmb . n = 7 mice per group. Median survival days are shown. Log-rank test. ( E – H ) Representative images and quantification of immunofluorescence staining of SOX2 in tumors from the brains of nude mice intracranially coimplanted with GSC272 cells and THP-1 macrophages ( E and F ) or HMC3 microglia ( G and H ) expressing shC or sh GPNMB . Scale bar: 50 μm. n = 3 independent samples. One-way ANOVA test. ( I – K ) Representative images ( I ) and quantification ( J and K ) of immunofluorescence staining of Ki67 ( I and J ) and CC3 ( I and K ) in tumors from the brains of nude mice intracranially coimplanted with GSC272 cells and THP-1 macrophages expressing shC or sh GPNMB . Scale bar: 50 μm. n = 3 independent samples. One-way ANOVA test. ( L – N ) Representative images ( L ) and quantification ( M and N ) of immunofluorescence staining of Ki67 ( L and M ) and CC3 ( L and N ) in tumors from the brains of nude mice intracranially coimplanted with GSC272 cells and HMC3 microglia expressing shC or sh GPNMB . Scale bar: 50 μm. n = 3 independent samples. One-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: Expressing, shRNA, Control, Immunofluorescence, Staining

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) GPNMB expression levels in different types of immune cells from GBM tumors based on single-cell RNA-Seq (scRNA-Seq) dataset (EGAS00001004871). ( B ) Pattern representing single-cell gene expression of GPNMB in macrophages, microglia, M-IMCs, and PNM-IMCs from GBM tumors based on scRNA-Seq dataset (EGAS00001004871). ( C ) Coimmunofluorescence staining for GPNMB (red) and F4/80 (macrophage marker, green) or CX3CR1 (microglia marker, green) in human GBM tumor samples. Scale bar: 50 μm. ( D ) Correlation between GPNMB expression and macrophage/microglia signature scores in IDH1-WT TCGA samples from patients with GBM. P and r values are shown. Pearson’s test. ( E ) The expression of GPNMB in macrophage-high ( n = 185) and macrophage-low ( n = 187) as well as microglia-high ( n = 185) and microglia-low ( n = 187) groups of IDH1-WT TCGA samples from patients with GBM. Student’s t test. ( F and G ) Representative images ( F ) and correlation quantification analysis ( G ) between GPNMB and SOX2 expression in human GBM tumor samples ( n = 30) based on immunofluorescence staining. Scale bar: 50 μm. P and r values are shown. Pearson’s test. ( H ) Relationship between histological Ki67 score in tumors and GPNMB expression level in the plasma of patients with GBM ( n = 36). P and r values are shown. Pearson’s test. (I and J) Survival curves of patients with GBM with high and low GPNMB expression in IDH1-WT TCGA (I) and Gravendeel (J) GBM datasets from GlioVis ( https://gliovis.bioinfo.cnio.es/ ). Log-rank test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: Expressing, RNA Sequencing, Gene Expression, Staining, Marker, Immunofluorescence, Clinical Proteomics

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: Generation of CRISPR-engineered CD38 KO /CD38-CAR human primary NK cells using Cas9/RNP and AAV. (A) Schemata of steps for CRISPR/RNP knockout of the CD38 gene and directed insertion of a CD38-CAR encoding DNA delivered by AAV6 vector with homology arms for CD38 targeting site. (B) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry for binding of CD38 antigen, 7 days after stimulation. Constructs contain a 41BB signaling domain, a CD8α transmembrane domain/hinge, a CD3ζ stimulatory domain, and reversed orderings of light and heavy chain orientations. (C) Relative percentage and intensity of CD38-CAR expression (n = 10; mean ± standard deviation [SD]). (D) Fold expansion of WT and CD38-CAR NK cells over 12 days after activation with irradiated, modified mbIL21-K562 cells and IL-2 show no significant change from WT human NK cells (n = 10; mean ± SD). P values were calculated using a 2-way analysis of variance (ANOVA); ∗ P = .0332; ∗∗ P = .0021; ∗∗∗ P = .0002; ∗∗∗∗ P < .0001. (E) Cytotoxicity observed for V3 and V4 CD38 KO /CD38-CAR NK cells against high CD38-expressing MM (H929), BL (Raji), and AML (MV-11) (n = 5; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: CRISPR, Knock-Out, Plasmid Preparation, Expressing, Flow Cytometry, Binding Assay, Construct, Standard Deviation, Activation Assay, Irradiation, Modification

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: CD38-CAR NK cells exhibit enhanced cytotoxic function and cytokine secretion. CD38-CAR NK cells were tested against CD38-expressing AML, MM, and T-cell malignancies collected from patients at baseline. (A) NK and CD38-CAR NK killing of AML-1 (n = 3; mean ± SD). (B) NK and CD38-CAR NK killing of samples from patients with MM (MM-1 and MM-2; n = 1; mean ± SD). (C) NK and CD38-CAR NK killing of T-cell malignancies (hepatosplenic T-cell lymphoma and T-PLL [T-PLL-1 and T-PLL-2]; n = 1; mean ± SD). All cytotoxicity P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (D) Bio-Plex Pro Human Cytokine assay was performed on the supernatant of WT and CD38-CAR NK cells cocultured with CD38 + malignancies (n = 9; mean ± SD). P values were calculated using a paired Student t test; ∗ P = .05; ∗∗ P = .01; ∗∗∗ P = .001. GM-CSF, IFN-γ, MCP-1, MIP-1α, TNF-α.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Cytokine Assay

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: ATRA upregulates CD38 expression on tumor cells and can enhance antitumor activity. (A) CD38 cell surface expression as measured by flow cytometry across the hematologic malignancies MM, AML, BL, and T-ALL after treatment with 10 nM of ATRA for 48 hours. (B) Mean fluorescence intensity (MFI) of CD38 expression on cell lines with and without ATRA treatment. (C) Cytotoxicity assays performed by coculturing WT and CD38-CAR NK cells against AML, MM, BL, and T-cell malignancies with and without 48-hour, 10-nM ATRA pretreatments. MM1S (n = 4), H929 (n = 4), AML-10 (n = 4), MV4-11 (n = 4), Raji (n = 4), Daudi (n = 4), and primary cells from patients with T-ALL (n = 3; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P <.01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Activity Assay, Flow Cytometry, Fluorescence

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: Mass cytometry analysis shows combination ATRA and CD38-CAR NK-cell treatment decreases live AML population. Mass cytometry analysis was performed 24 hours after the coculture of a primary AML cell line (AML-1) with WT or CD38-CAR NK cells with and without 48-hour, 10-nM ATRA pretreatment. Eight distinct conditions were analyzed: WT NK cells alone (A), CD38-CAR NK cells alone (B), AML cells alone (C), AML plus WT NK cells (D), AML plus CD38-CAR NK cells (E), AML cells with ATRA (F), AML plus ATRA plus WT NK cells (G), and AML plus ATRA plus CD38-CAR NK cells (H). (I) Heat map of surface marker appearing on the live AML cells after treatment analyzed by mass cytometry. Original values are ln(x)-transformed. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage using ClustVis.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Mass Cytometry, Marker, Transformation Assay

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: CD38 KO /CD38-CAR T cells generated by Cas9/RNP and AAV6 show antitumor activity. (A) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry. (B) Relative percentage of CD38-CAR expression (n = 5; mean ± SD). (C) Ten-day fold expansion of generated CD38-CAR T cells with matched donors (n = 4; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P = .0332; ∗∗ P = .0021; ∗∗∗ P = .0002; ∗∗∗∗ P < .0001. (D) Cytotoxicity of CD38 KO /CD38-CAR T cells toward H929, Raji, and MV4-11 cell lines (n = 4; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Generated, Activity Assay, Expressing, Flow Cytometry

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: AAVS1KO/CD38-CAR NK cells avoid fratricide. (A) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry for the CD38-CAR inserted into different loci. All CAR NK cells were generated with matched donors. (B) Fold expansion of WT and CD38-CAR NK cells over 12 days (n = 6; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (C) Relative percentage of CD38-CAR expression (n = 6; mean ± SD). (D) Reverse-transcription qPCR was performed using CD38 primer probes to detect transcription of the CD38 gene in the WT and AAVS1KO/CD38-CAR NK cells. (E) CD38 expression measured by flow cytometry using a polyclonal anti-CD38 antibody.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Flow Cytometry, Generated, Reverse Transcription

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: AAVS1KO/CD38-CAR NK cells display enhanced cytotoxicity and metabolism. CD38-CAR NK cells were tested against CD38-expressing AML, MM, and BL. (A) CD38-CAR NK-cell killing of CD38-expressing cell lines (n = 3; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (B) Oxygen consumption rate (OCR) for CD38-CAR NK cells. (C) Glycolytic capacity, measured by the extracellular acidification rate (ECAR), observed in CD38-CAR NK cells. (D) Spare respiratory capacity, a measure of the cell's ability to produce adenosine triphosphate (ATP) in response to stress, measured in CAR NK cells. (E) CAR NK-cell maximal respiration rates compared with WT.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing