Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: A QPCR results showed that CB1 was downregulated in colorectal cancer cells. B QPCR results showed that EGFR was upregulated in colorectal cancer cells. C, D Western blot analysis showed that CB1 was downregulated and EGFR was upregulated in colorectal cancer cells. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Western Blot

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: A Western blot assay tested the protein level of CB1 after ACEA and AM251 treatment. B Western blot analysis showed that CB1 activation significantly decreased the expression of EGFR while its inhibition increased the expression of EGFR. C, D Colony forming assay showed that CB1 activation suppressed colony formation of colorectal cancer cells, while its inhibition promoted colony formation. E, F Wound healing test showed that CB1 activation prevented cell migration while CB1 inhibition enhanced cell migration of colorectal cancer cells. G, H Transwell invasion assay demonstrated that CB1 activation blocked cell invasion while CB1 inhibition enhanced cell invasion of colorectal cancer cells. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Western Blot, Activation Assay, Expressing, Inhibition, Migration, Transwell Invasion Assay

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Cells were transfected with EGFR plasmids and simultaneously treated with ACEA. A QPCR analysis showed that EGFR plasmids transfection increased the expression of EGFR in SW480 and SW620 cells. B , C Colony-forming assay demonstrated that EGFR overexpression blocked the proliferation suppression induced by CB1 activation. D , E Wound healing test showed that EGFR overexpression counteracted the migration inhibition induced by CB1 activation. F , G Transwell invasion assay showed that EGFR overexpression prevented the decrease in cell invasion caused by CB1 activation. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, Expressing, Over Expression, Activation Assay, Migration, Inhibition, Transwell Invasion Assay

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Colorectal cancer cells were transfected with EGFR plasmids and simultaneously treated with ACEA. Then PMA induced THP-1 cells were co-cultured with the culture medium of colorectal cancer cells. A – F QPCR results demonstrated that EGFR overexpression blocked CB1 activation caused increase in the expression of IL-6 and TNF-α, and decrease in IL-10, CCL22, Arg-1 and CD206. G – J ELISA assay showed that EGFR overexpression blocked CB1 activation caused increase in the release of IL-6 and TNF-α, and decrease in the release of IL-10 and CCL22. K , L Western blot showed that EGFR overexpression prevented the decrease in the expression of Arg-1 and CD206. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, Cell Culture, Over Expression, Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Colorectal cells were transfected with plasmids bearing shRNA against EGFR and simultaneously treated with AM251. Culture medium for these colorectal cells were taken to incubate PMA-induced THP-1 cells. A QPCR analysis showed that sh-EGFR effectively suppressed the expression of EGFR. B , C Colony formation test demonstrated that EGFR knockdown reversed the enhancement in colony formation induced by AM251. D – I QPCR results demonstrated that EGFR knockdown reversed the CB1 inhibition caused decrease in the expression of IL-6 and TNF-α, and increase in IL-10, CCL22, Arg-1 and CD206. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, shRNA, Expressing, Knockdown, Inhibition

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: SW480 cells were subcutaneously injected into nude mice and the tumors were treated with control or ACEA (1.5 mg/kg/d). A CB1 activation suppressed tumor growth. Representative tumors ( B ) and tumor weight ( C ) from different experimental groups. D QPCR analysis showed that CB1 activation increased the expression of IL-6 and TNF-α, and decreased the expression of IL-10, CCL-22, Arg-1 and CD206. E Western blot analysis showed that the expression of EGFR, Arg-1 and CD206 were decreased in the tumors of ACEA-treated mice. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Injection, Control, Activation Assay, Expressing, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Tumor-associated antigen expression (normalized MFI, mean fluorescence intensity) in PDAC cell lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Expressing, Fluorescence

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Structure and in vitro activity of PDAC-directed T cell-engaging bispecific antibodies ( A ) IgG-[L]-scFv structure of BsAbs. CH, constant heavy chain; CL, constant light chain; scFv, single chain variable fragment; VH, variable heavy chain; VL, variabl light chain. ( B ) Flow cytometric analysis displaying fluorescent intensities of IgG-[L]-scFv BsAbs bound to human PDAC cells. IgG-[L]-scFv BsAbs evaluated were specific for human EGFR, HER2, MSLN, c-MET, and B7-H3. ( C ) Antibody-dependent T cell-mediated cytotoxicity (ADTC) as a function of increasing doses of BsAbs against PDAC cell lines. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro, Activity Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: In vitro sensitivities (EC50, pM) to target antigen-specific bispecific antibodies in PDAC cell lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: EGFR and HER2 T-BsAb heterodimerization and in vitro kinetics ( A ) Schematic of BsAb heterodimerization by controlled Fab Arm Exchange. Parental BsAbs bore 2 Fabs specific to EGFR or HER2 and 2 scFvs specific for CD3 (‘2 + 2’). Reduction of inter-H-chain disulfide bonds and subsequent heterodimerization (driven by F405L and K409R amino acid substitutions) yielded EGFRxHER2 BsAbs bearing 1 EGFR Fab, 1 HER2 Fab, and 2 CD3 scFv (‘1 + 1 + 2’). ( B ) SPR analysis of T-BsAbs binding to EGFR (left panel), HER2 (middle panel), and EGFR x HER2 (right panel) proteins. Representative normalized sensorgrams at 20nM were shown. ( C ) IHC staining of two cell-derived PDAC xenograft tumors SW1990 (top) and BxPC-3 (bottom). OCT-embedded tumor sections were stained with EGFRxEGFR, HER2xHER2, or EGFRxHER2 BsAbs. Control slides were either unstained (SW1990) or incubated with a control antibody (BxPC-3). ( D ) Antibody-dependent T cell-mediated cytotoxicity assays (ADTC) against SW1990 (left) and BxPC-3 (right). Cytotoxicity measured by Chromium-51 release. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1. EC50s (SW1990; BxPC-3): EGFRxEGFR: 165.8fM; 31.5fM, HER2xHER2: 18.5pM; 7.14pM, EGFRxHER2: 699.6fM; 128.8fM. CD33xCD33 BsAb was included as a negative control

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro, Binding Assay, Immunohistochemistry, Derivative Assay, Staining, Control, Incubation, Negative Control

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Avidities of EGFR and HER2 T-BsAbs

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Protein Binding

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Heterodimeric EGFR and HER2 T-BsAbs impede PDAC growth ( A ) In vivo antitumor effect of BsAbs in the presence of human T cells against SW1990 cell line xenografts; 3 × 10 6 cells of SW1990 were subcutaneously implanted into mice. T-BsAbs were administered twice per week and 2 × 10 7 of luciferase-transduced T cells (Luc(+) T cells) were administered once per week for 2 weeks to treat the tumors. ( B ) In vivo anti-tumor effects of 10 µg EGFR and HER2 T-BsAbs. ( C ) Relative body weight of mice during treatment and ( D ) overall survival were plotted. ( E ) Bioluminescence imaging (BLI) of Luc(+) T cells. Quantitation of bioluminescence intensity in the lesions of tumors. Representative images (right) were taken on day 7

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vivo, Luciferase, Imaging, Quantitation Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: T-BsAbs drive T cell infiltration, activation, and function in PDAC ( A ) Treatment of SW1990 tumor-bearing mice with EGFR and HER2 T-BsAbs; 3 × 10 6 cells of SW1990 were subcutaneously implanted into mice. Once tumor reached ∼500mm 3 , mice received a single infusion of 2 × 10 7 T cells and were treated with 10µg T-BsAb, administered twice per week. Tumors were harvested for analysis on day 10 after initiation of treatment. Mice that exhibited 50% or more reduction in tumor volume by day 10 were excluded from this and subsequent analyses. ( B ) Flow cytometric analysis of the in vivo effect of T-BsAb treatments on T cell infiltration into harvested SW1990 PDAC tumors. Infiltrating T cells were identified by detection of human CD45 + and human CD4 + or CD8 + . ( C ) Frequency of T cell activation indicated by detection of IFN-γ + TNF-α + T cells and expression of CD69 among CD4 + or CD8 + T cells. ( D ) Frequency of CD11b + intratumoral myeloid cells and prevalence of PDL1 expression

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Activation Assay, In Vivo, Expressing

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: ADTC sensitivities (EC50, pM) of SW1990 lines to EGFR and HER2 T-BsAbs

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques:

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: FACS binding (EC50, pM) of EGFR and HER2 T-BsAbs to SW1990 lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Binding Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: EGFRxHER2 T-BsAbs efficacy is lost in EGFR and HER2-single-positive PDAC ( A ) Mean fluorescent intensities determined by flow cytometry and ( B ) antibody-dependent T cell-mediated cytotoxicity (ADTC) against SW1990-EGFR-KO (left) and SW1990-HER2-KO (right) cell lines. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1. ( C ) Tumor growth of subcutaneous SW1990 EGFR-KO (left) and HER2-KO (right) tumors. One infusion of 2 × 10 7 T cells was administered. Two doses of T-BsAbs (10 µg each) were given by i.v. injection

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Flow Cytometry, Injection