Journal: Molecular Cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: TREM2 is a potential marker of colorectal cancer (CRC). (A) Kaplan-Meier analysis representing the association of TREM2 expression with overall survival in CRC patients from The Cancer Genome Atlas cohort; ≥ 50% quantile of TREM2 expression is defined as the high-expressing group, and others are considered in the low-expressing group. (B) Immunohistochemistry staining representing TREM2 (brown) expression in CRC tissues. (C) Bar plots summarizing the immunohistochemistry semi-quantitative analysis; score 1, ≤ 25%; 2, 26–50%; 3, 51–75%; 4, > 75%. Scale bar: 100 μm; magnification: 200× (n = 4). (D) Relative TREM2 mRNA levels in CRC patients (n = 4). ( E ) CRC tissues or normal tissues were double stained with anti-CD206 (marked macrophages) (green) and anti-TREM2 (red) antibodies, and then observed by fluorescent microscopy. DAPI, blue (n = 10). Scale bars: 20 μm. Data are expressed as means ± SD; * P < 0.05, ** P < 0.01, by unpaired Student’s t test ( C - D ). TREM2, triggering-receptor-expressed on myeloid cells 2; CD, cluster of differentiation; DAPI, 4′,6-diamidino-2-phenylindole

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bi-specific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Marker, Expressing, Immunohistochemistry, Staining, Microscopy

Journal: Molecular Cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Construction and characterization of the bi-specific scFv antibody (BsAb). (A) Schematic representation of PD-1-scFv, TREM2-scFv, and BsAb. (B) SDS-PAGE analysis of PD-1 scFv, TREM2 scFv, and BsAb derived from the supernatants of CHO cells using nickel column purification and concentration. (C) Flow cytometric histograms demonstrating His-tag detection of PD-1 scFv, TREM2 scFv, and BsAb binding to 293T stably expressing PD-1 (293T-PD-1). The 293T-PD-1 cells were treated with PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL and MFI of His was detected by flow cytometry. Bar plots summarizing the data are shown on the right. (D) Flow cytometric histograms demonstrating His-tag detection of PD-1 scFv, TREM2 scFv, and BsAb binding to TREM2 on the RAW 264.7 cells. PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL and MFI of His was detected by flow cytometry. Bar plots summarizing the data are shown on the right. (E-F) Relative viability of MC38 cells, mouse primary hepatocytes, and mouse primary intestinal cells treated with PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL for 24 h analyzed using the CCK-8 kit. Data are expressed as means ± SD; * P < 0.05, ** P < 0.01 (n = 3), by unpaired Student’s t test ( C - D ). PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; RAW 264.7, Mouse Mononuclear Macrophages Cells; 293T, human embryonic kidneys; CCK, cell counting kit; SD, standard deviation

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bi-specific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: SDS Page, Derivative Assay, Nickel Column, Purification, Concentration Assay, Binding Assay, Stable Transfection, Expressing, Flow Cytometry, CCK-8 Assay, Polyacrylamide Gel Electrophoresis, Cell Counting, Standard Deviation

Journal: Molecular Cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: The bi-specific scFv antibody (BsAb) delays tumor growth and modulates intratumoral cytokines and the proportions of infiltrating immune cells, MDSCs, and M2 TAMs. (A) Schematic representation of the animal experiment. Six-week-old C57BL/6J mice were subcutaneously injected with 2 × 10 6 MC38 cells expressing CEA. On day 7, mice received intravenous injection of PD-1 scFv, TREM2 scFv, or the BsAb at 200 µg/mouse daily; the PBS-treated group served as the control. (B) Bioluminescence images of mice treated with PD-1 scFv, TREM2 scFv, and BsAb at days 7, 14, and 21 (n = 4). (C) Bioluminescence signals of individual groups at day 7, 14, and 21 are shown on the Y-axis. The PBS group is indicated as black, the PD-1 scFv group is in orange, the TREM2 scFv group is in blue, and the BsAb group is in red. (D-E) Tumor volume and body weight of mice subjected to the indicated treatments (n = 4). (F-H) MC38-CEA tumors excised from individual groups of sacrificed mice on day 23. Tumor volume and weight of mice subjected to the indicated treatments (n = 4). (I) Effect of different treatments on the survival of tumor-bearing mice. (J-L) Effect of different treatment modalities on the infiltration of CD8-positive T cells ( J ), MDSCs ( K ), and M2 TAMs (L) . Proportions of tumor-infiltrating CD8-positive T cells, MDSCs, and M2 TAMs were detected by flow cytometry on day 23. All cells gated on CD45, CD45 + CD11b Gr1 + were marked as MDSCs, whereas cells gated at CD45 + CD11b F4/80 CD206 were marked as M2 TAMs. Representative plots are shown on the right (n = 4). Data are expressed as means ± SD; * P < 0.05; ** P < 0.01, by unpaired Student’s t test and one-way ANOVA. scFv, single-chain fragment variable; CEA, carcinoembryonic antigen; PD-1, programmed death-1; TREM2, triggering-receptor-expressed on myeloid cells 2; PBS, phosphate buffered saline; MDSCs, myeloid-derived suppressor cells; TAMs, tumor associated macrophages; CD, cluster of differentiation

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bi-specific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Injection, Expressing, Control, Flow Cytometry, Saline, Derivative Assay

Journal: Molecular Cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Construction and characterization of bi-specific scFv antibody (BsAb)- secreting CAR-T cells. ( A ) Schematic representation of Mock, PD-1 scFv, TREM2 scFv, and BsAb-secreting CAR constructs targeting CEA. CART-CEA was constructed as a control. ( B ) Efficient transduction of all constructs in mouse primary T cells as observed by fluorescence microscopy. Scale bar: 50 μm. ( C, D ) Flow cytometric assay analysis ( C ) and bar plot summary ( D ) indicating the transduction efficiency of all constructs in mouse primary T cells. ( E ) Western blot detecting the His-tag for CAR protein in mouse primary T cells. ( F ) Cytotoxicity of all constructed CAR-T cells against MC38 cells. All groups of CAR-T cells were co-cultured with MC38 cells at 1:1, 4:1, 8:1, and 16:1, ratios. MC38 cell lysis was detected by an LDH cytotoxicity kit. Data are expressed as means ± SD; * P < 0.05; ** P < 0.01, by one-way ANOVA. CAR-T, chimeric antigen receptor-modified-T; PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; CEA, carcinoembryonic antigen; LDH, lactate dehydrogenase

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bi-specific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Construct, Control, Transduction, Fluorescence, Microscopy, Flow Cytometry, Western Blot, Cell Culture, Lysis, Modification

Journal: Molecular Cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Anti-tumor effects of armed CAR-T cells in tumor-bearing mice. ( A ) Time-course diagram of the animal experiment. Six-week-old C57BL/6J mice were subcutaneously injected with 2 × 10 6 MC38 cells expressing CEA. On day 7, mice from all groups were individually injected with 1 × 10 7 constructed CAR-T cells through the tail vein. Tumor imaging was performed once a week. ( B ) Bioluminescence images of mice treated with CART-CEA, CART-CEA.sPD-1scFv, CART-CEA.sTREM2 scFv, and CART-CEA.sBsAb at days 7, 14, and 21; PBS treatment served as the control (n = 4). ( C ) Bioluminescence signals of mice in each group at days 7, 14, and 21 are shown on the Y-axis. The PBS group is shown in black, the CART-CEA group in purple, the CART-CEA.sPD-1 scFv group in orange, the CART-CEA.sTREM2 scFv group in blue, and the CART-CEA.sBsAb group in red. ( D ) Comparison of tumor volume over time in each group of tumor-bearing mice. ( E ) Weight development of mice during treatment in each group. ( F ) Effect of different treatments on the survival of tumor-bearing mice. Data are expressed as means ± SD; * P < 0.05; ** P < 0.01, by unpaired Student’s t test. CAR-T, chimeric antigen receptor-modified-T; CEA, carcinoembryonic antigen; PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; PBS, phosphate-buffered saline

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bi-specific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Injection, Expressing, Construct, Imaging, Control, Comparison, Modification, Saline

Journal: BMC Plant Biology

Article Title: The elicitation of a systemic resistance by Pseudomonas putida BTP1 in tomato involves the stimulation of two lipoxygenase isoforms

doi: 10.1186/1471-2229-11-29

Figure Lengend Snippet: Characterisation of TomLOXF . A . The detection of his-tagged proteins was realized on Western blot with an anti-His-tag antibody (1 and 2) and we evaluated the purity of the protein through SDS-PAGE and Coomassie blue staining (3). 1: Page Ruler Plus Prestained Protein Ladder (Fermentas), 1: total proteins extracted from TomLOXF-expressing E. coli , 2 and 3: partially-purified protein extracted from TomLOXF-expressing clone by nickel affinity chromatography. B . LOX activity was evaluated on partially-purified recombinant TomLOXF with linoleic (C18:2) and linolenic (C18:3) acids as substrate. Reaction was performed at pH 6.0, room temperature. C . Linolenic and linoleic acids were both incubated with extracts of E. coli expressing TomLOXF in oxygenated buffer. Produced hydroperoxides were separated by HPLC, and the profile of compounds absorbing at 234 nm was compared with the profile of pure 13-HPOT, 13-HPOD, 9-HPOT and 9-HPOD.

Article Snippet: His-tagged proteins were purified by nickel column affinity chromatography (Äkta, Amersham Pharmacia Biotech) according the method described in Boutaud and Brash [ ].

Techniques: Western Blot, SDS Page, Staining, Expressing, Purification, Affinity Chromatography, Activity Assay, Recombinant, Incubation, Produced