Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Rational design and construction of the Anti-MICB-CAR. ( A ) Schematic of the Anti-MICB chimeric antigen receptor (Anti-MICB-CAR). ( B ) The plasmid structure of the Anti-MICB chimeric antigen receptor lentiviral vector. ( C ) The expression of Anti-MICB-CAR in NK cells was analyzed via flow cytometry. ( D ) The secretion of IL-15 by Anti-MICB-CAR-NK cells was detected by ELISA. NC: supernatant of 1 × 10 6 NK cells, Anti-MICB–CAR–NK: supernatant of 1 × 10 6 Anti-MICB-CAR-NK cells. Supernatants of Anti-MICB-CAR-NK cells were significantly elevated up to 48.88 pg/mL compared to the control NK cell supernatants. p = 0.0024. ( E ) The supernatant of 1 × 10 6 Anti-MICB-CAR-NK cells was filtered and incubated with PANC-1 for 1 h. MICB Protein hFc and IgG1 Fc FITC antibody staining were performed and free Anti-MICB-scFv was detected by flow cytometry. Statistical analysis was performed using a two-tailed Student’s t -test; ** p < 0.01.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Plasmid Preparation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Incubation, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Anti-MICB-CAR-NK cells demonstrate potent in vitro activity against tumors with high expression of MICB. ( A ) Western blot detection of MICB expression in PANC-1, A549, HepG2, BxPC-3, and AsPC-1 human tumor cell lines. ( B ) The qPCR results showed in the MICB gene expression compared to the five tumor cell lines. ( C ) The Effect-to-Target Ratio (E/T Ratio) of Anti-MICB-CAR-NK cells on tumor cell PANC-1. ( D ) The mortality rate of five types of tumor cells treated with Anti-MICB-CAR-NK cells after 24 h with CCK-8 Assay, PANC-1(71.37%), A549(31.78%), HepG2(53.83%), BxPC-3(62.26%), and AsPC-1(39.86%), E/T Ratio 1:1. ( E ) Anti-MICB-CAR-NK treatment of AsPC-1 and PANC–1 tumor cells with differential MICB expression for 24 h by flow cytometry, E/T Ratio 1:1. The results showed that the viability of AsPC-1 was 68.9% and the viability of PANC-1 was 54.6%. ( F – H ) PANC-1, HepG2, and A549 tumor cells treated with NK cells, Anti-MICB-CAR-NK cells, and Anti-MICB-CAR-NK supernatant + NK cells for 24 h, E/T Ratio 1:1; CCK8 detects mortality rate. Anti-MICB-CAR-NK cells significantly enhanced the anti-tumor ability compared to NK cells. PANC-1: 68.37%, p = 0.0005. HepG2: 57.3%, p = 0.0027. A549: 33.18%, p = 0.0342. Supernatant of Anti-MICB-CAR-NK cells co-treated tumor cells with NK cells also promoted tumor cell killing compared to NK. PANC-1: 57.66%, p = 0.0006. HepG2: 48.21%, p = 0.0037. A549: 31.55%, p = 0.0446. ( I ) NC-NK cells, non-transduced NK(NT-NK), NT-NK supernatant + NK cells, Anti-MICB-CAR-NK supernatant + NK cells, Anti-MICB-CAR-NK cells treated with PANC-1 tumor cells for 24 h, E/T Ratio 1:1. The viability of each group was detected by flow cytometry and the results showed that NK cells: 89.4%, non-transduced NK(NT-NK): 86.6%, NT-NK supernatant + NK cells: 88.7%, Anti-MICB-CAR-NK supernatant + NK cells: 62.6%, and Anti-MICB-CAR-NK cells: 55.5%. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, *** p < 0.001.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: In Vitro, Activity Assay, Expressing, Western Blot, Gene Expression, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: The anti-tumor mechanism induced by Anti-MICB-CAR-NK cells. ( A – D ) The release of perforin, granzyme B, TNF-α, and IFN-γ from PANC-1, HepG2, and A549 cells. NK cells and Anti-MICB-CAR-NK cells were treated with tumor cells for 24 h, with an E/T ratio of 1:1. Control represents complete medium cultivation. The supernatant was collected and detected by ELISA. Compared to the NK group, the Anti-MICB-CAR-NK group exhibited significantly higher levels of TNF-α expression (PANC-1, p = 0.0072; AsPC-1, p = 0.035; HepG2, p = 0.0076; A549, p = 0.0066) and IFN-γ expression (PANC-1, p = 0.0072; AsPC-1, p = 0.0162; HepG2, p = 0.002; A549, p = 0.0006) following treatment with PANC-1, AsPC-1, HepG2, and A549 tumor cells. Similarly, Granzyme B expression levels were significantly elevated in the Anti-MICB-CAR-NK group (PANC-1, p = 0.0007; HepG2, p = 0.0023; A549, p = 0.0004), as were Perforin expression levels (PANC-1, p = 0.0021; HepG2, p = 0.042; A549, p = 0.0022) after treatment with PANC-1, HepG2, and A549 tumor cells. Additionally, the Anti-MICB-CAR-NK group demonstrated significantly increased IL-15 expression following treatment with PANC-1 and A549 tumor cells (PANC-1, p = 0.0012; A549, p = 0.0025) compared to the NK group. ( E , F ) Elisa and flow cytometry were used to detect the levels of IL-15 and Anti-MICB-scFv in the tumor cells’ supernatants treated with Anti-MICB-CAR-NK cells for 24 h. ( G ) Study on MICB expression in PANC-1 treated with Anti-MICB-CAR-NK cells for 24 h using Western blot technique. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Anti-MICB-CAR-NK cells exhibit tumor regression in the PANC-1 Xenograft Model. ( A ) Flow chart of Anti-MICB-CAR-NK cells treatment for PANC-1 Transplanted Tumors. On the 0th day, PANC-1 cells were inoculated and on the 13th day, PBS, NK cells, and Anti-MICB-CAR-NK cells were injected into mice via tail vein at a dose of 5 × 10 6 cells per mouse. Record tumor growth observed every other day. ( B ) Growth curve of mouse transplanted tumor. ( C , D ) Resected tumors from each group were imaged and Weighed at the end of the experiment. NC: 834.31 ± 197.26 (mg), NK: 606.69 ± 141.37 (mg), Anti-MICB-CAR-NK: 447.32 ± 136.13 (mg). ( E ) Anti-tumor rate of Anti-MICB-CAR-NK cells against PANC-1 transplanted tumors. The tumor inhibition rate of NK compared to NC was 27.28%, p = 0.07. The tumor inhibition rate of Anti-MICB-CAR-NK compared to NC was 46.38%, p = 0.007. ( F ) Mouse serum was collected at the end of the experiment and the amounts of IL-15, IFN-γ, and TNF-α were measured by ELISA. The IL-15, IFN—γ, and TNF—α in the Anti-MICB-CAR-NK group were significantly increased compared to the NK group, with a p -value of 0.0013 for IL-15, 0.013 for IFN—γ, and 0.003 for TNF—α. ( G , H ) Mouse tumor tissues were analyzed by immunohistochemistry for MICB expression and NK cell infiltration within the tumor tissue. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, ** p < 0.01.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Injection, Inhibition, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Expressing, Two Tailed Test

Journal:

Article Title: ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor

doi: 10.1073/pnas.161290898

Figure Lengend Snippet: Ligation of flagellin receptor activates the MAP kinase cascade, which is required for mucin induction. (A) HM3 cells grown in six-well plates were lysed with SDS sample buffer 5 min after the addition of agonist antibody, α-ASGM1. Equal amounts of lysate were run on a 12% SDS gel, transferred to nitrocellulose, immunoblotted with a phospho-specific Erk 1/2 antibody, and visualized by chemiluminescence. Blots were stripped and reprobed with Erk 1/2 antibody to show equal loading. P-Erk Cntrl, positive control from vendor. (B) Effect of MEK 1/2 inhibitor PD98059 (37 μM, 30 min) and dominant negative mutant MEK K97R on MUC 2 promoter activity. (c) Inhibition of Erk phosphorylation by the calcium chelator, BAPTA/AM (30 μM, 45 min). Fold increase expressed as the ratio of phosphorylated Erk to baseline Erk 1/2 as determined by densitometry.

Article Snippet: Phosphospecific anti-Erk 1/2 (mouse monoclonal) was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Ligation, SDS-Gel, Positive Control, Dominant Negative Mutation, Activity Assay, Inhibition

Journal:

Article Title: ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor

doi: 10.1073/pnas.161290898

Figure Lengend Snippet: Cartoon depicting events in flagellin-triggered host cell signaling. Flagellin binds to asialoGM1, a membrane glycolipid, which causes the extracellular release of ATP, which then binds to a nucleotide receptor. Downstream events include G-protein activation, the cleavage of PIP2 by PLC, the formation of IP3, Ca2+ mobilization, the phosphorylation of MEK 1/2 and Erk 1/2 [via an unknown calcium binding protein (CBP)], and mucin (MUC 2) transcription. Flagellin-induced signaling bifurcates after Ca2+ mobilization, with ≈50% of the response giving rise to downstream events that are Erk dependent whereas the remaining 50% is Erk independent.

Article Snippet: Phosphospecific anti-Erk 1/2 (mouse monoclonal) was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Binding Assay

Journal: PLoS ONE

Article Title: Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans

doi: 10.1371/journal.pone.0182926

Figure Lengend Snippet: PC3 cells were treated with 2.5 μM [Alexa594]—(Cys 0 ) -DRS-B2 (red) for either 5 min. (1A-C) or 1 hour (2A-D). After fixation, the membrane was visualized by the anti-CD147 antibody labeled with an anti-mouse antibody A488 (green) and nuclei by DAPI (blue). The confocal images were obtained using an Olympus IX81 inverted microscope (60x oil immersion objective NA 1.25) with a spinning disk confocal system DSU (Olympus), coupled to a CCD camera R2 Orca (Hamamatsu Corporation, Japan). Image processing was performed using the ImageJ software, and the program Zoom in Images and Stack. z is the distance from the base to the top of the cell in 0.5 μm steps. The boxes are zoom lenses, the zoom factor = 2.

Article Snippet: Purified mouse anti-human EMMPRIN/CD147 antibodies was from BD Pharmingen (Bedford, UK)

Techniques: Labeling, Inverted Microscopy, Software

Journal: PLoS ONE

Article Title: Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans

doi: 10.1371/journal.pone.0182926

Figure Lengend Snippet: U87MG cells were treated with 2.5 μM of [Alexa594]—(Cys 0 ) -DRS-B2 (red) for either 5 min. (1A-D) or 1 hour (2A-C). After fixation, the membrane was visualized using anti CD147 antibody labeled with an anti-mouse antibody A488 (green) and nuclei by DAPI (blue). The confocal images were acquired using Olympus IX81 inverted microscope (60x oil immersion objective NA 1.25) with a spinning disk confocal system DSU (Olympus), coupled to a CCD camera R2 Orca (Hamamatsu Corporation, Japan). Image processing was performed using the program Zoom in Images and Stack. z is the distance from the base to the top of the cell in 0.5 μm steps. The boxes are zoom lenses, the zoom factor = 2.

Article Snippet: Purified mouse anti-human EMMPRIN/CD147 antibodies was from BD Pharmingen (Bedford, UK)

Techniques: Labeling, Inverted Microscopy

Journal: BMC Cancer

Article Title: Pentoxifylline sensitizes human cervical tumor cells to cisplatin-induced apoptosis by suppressing NF-kappa B and decreased cell senescence

doi: 10.1186/1471-2407-11-483

Figure Lengend Snippet: Determination of phosphorylated ERK 1/2, p38, and p65 in HeLa, SiHa and HaCaT cell treatment with pentoxifylline or cisplatin either alone or in combination . 24 hours later the cells were harvested and the phosphorylated ERK1/2, p38 and p65 proteins were determined by flow cytometry. A total of 20,000 events were registered in each test. The results represent the mean ± SD of 3 independent experiments carried out in triplicate. (*) P < 0.001 vs untreated control cells. (♦) = P < 0.001 vs CIS.

Article Snippet: In normal untreated and treated cell cultures, we determinated the Alexa Fluor ® 647mouse anti-human Bcl-2 and Alexa Fluor ® 647 mouse anti human Bcl-XL proteins (Santa Cruz CA) and phosphorylated ERK1/2 (pT202/pY204) PE-Cy™7 mouse anti-human, Alexa Fluor ® 488 mouse anti-human anti-phospho (P)-p38 (pT180/pY182) and Alexa Fluor ® 647 mouse anti-human NF-κB p65 (pS529) BD Biosciences by flow cytometry.

Techniques: Flow Cytometry