panc02 13  (ATCC)

Journal: Journal for Immunotherapy of Cancer

Article Title: Loss of Annexin A1 in macrophages restrains efferocytosis and remodels immune microenvironment in pancreatic cancer by activating the cGAS/STING pathway

doi: 10.1136/jitc-2024-009318

Figure Lengend Snippet: Myeloid-specific Anxa1 deficiency causes accumulation of apoptotic cells in tumors. ( A ) Flow cytometry analysis of apoptotic level of tumors from Anxa1 ΔMɸ and Anxa1 WT mice. Annexin-V-APC and PI were used to detect apoptotic cells and dead cells. ( B ) Immunoblot analysis of c-Casp3 expression in tumors from Anxa1 ΔMɸ and Anxa1 WT mice. ( C ) Quantification of host-derived cfDNA in the plasma collected from Anxa1 ΔMɸ and Anxa1 WT Panc02-tumor-bearing mice. Each group contained four mice. ( D ) Representative images of tumors from Anxa1 ΔMɸ and Anxa1 WT mice (left). Quantification of tumor volume and weight are shown (right). Each group contained five mice. ( E ) Immunoblot analysis of Bcl2 and Bax expression in Panc02 tumor cells that were untreated, irradiated with 25 mJ/cm 2 UV-C (UV), or treated with 100 µM BIP-V5 (Bax inhibitor) and irradiated with 25 mJ/cm 2 UV-C (BIP-V5+UV). ( F ) Flow cytometry analysis of apoptotic level of Panc02 tumor cells that from these groups. ( G ) Flow cytometry analysis of apoptotic level of tumors from these groups. ( H ) The growth of Panc02 tumor cells that were treated with control solvent or BIP-V5 (200 ug/day) in Anxa1 ΔMɸ and Anxa1 WT mice. Each group contained five mice. ( I ) Immunoblot analysis of ANXA1 in BMDM cells that transfected with lentiviruses control (BMDM-ctrl) and carrying plasmids overexpressing ANXA1 (BMDM-ANXA1). ( J ) Representative images of tumors from the PDAC model that macrophages were reconstituted by BMDM-ctrl and BMDM-ANXA1 (left). Quantification of tumor volume and weight are shown (right). Each group contained five mice. ( K ) Immunoblot analysis of c-Casp3 expression in tumors from BMDM-ctrl and BMDM-ANXA1 mice. The data presented are the means±SD. ANXA1, Annexin A1; BIP-V5, Bax inhibitory peptide V5; BMDM, bone marrow-derived macrophage; c-Casp3, cleaved Caspase-3; cfDNA, cell-free DNA; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: The viability of Panc02 cells were determined using the Cell Counting Kit-8 (Dojindo, Japan) and measured at 450 nM with the BioTek Gen5 system (BioTek, Winooski, Vermont, USA).

Techniques: Flow Cytometry, Western Blot, Expressing, Derivative Assay, Clinical Proteomics, Irradiation, Control, Solvent, Transfection

Journal: Journal for Immunotherapy of Cancer

Article Title: Loss of Annexin A1 in macrophages restrains efferocytosis and remodels immune microenvironment in pancreatic cancer by activating the cGAS/STING pathway

doi: 10.1136/jitc-2024-009318

Figure Lengend Snippet: Macrophage Anxa1 deficiency activates cGAS-STING pathway to induce type I interferon in tumors. ( A ) GO enrichment analysis of upregulated genes in tumors from Anxa1 ΔMɸ versus Anxa1 WT mice. ( B ) GO enrichment analysis of upregulated proteins in tumors from Anxa1 ΔMɸ versus Anxa1 WT mice. ( C ) qPCR analysis of mRNA expression of Ifnb1, Usp18, Oas3 and Ifit1 in tumors from Anxa1 ΔMɸ and Anxa1 WT mice. Results represent the averages of three independent experiments. ( D ) Measurement of IFN-β protein in tumor homogenates. Tumors were collected 3 weeks after tumor cells implanted. ( E, G ) Immunoblot analysis of IFN-β, cGAS, STING, P65, p-P65TBK1, p-TBK1, IRF3 and p-IRF3 in tumor tissues from Anxa1 ΔMɸ and Anxa1 WT mice. ( F ) Shown are the image of the subcutaneous tumor of Anxa1 ΔMɸ mice treated with IgG or anti-IFNAR1 (left). Quantification of tumor volume and weight are shown (right). Each group contained five mice. ( H ) qPCR analysis of mRNA expression of Ifnb1, Usp18, Oas3 and Ifit1 in tumors of Anxa1 ΔMɸ mice treated with control solvent or H151. Results represent the averages of three independent experiments. ( I ) Measurement of IFN-β protein in tumor homogenates from Anxa1 ΔMɸ mice treated with control solvent or H151. ( J ) Shown are the image of the subcutaneous tumor of Anxa1 ΔMɸ mice treated with control solvent or H151 (left). Quantification of tumor volume and weight are shown (right). Each group contained five mice. ( K ) qPCR analysis of mRNA expression of Ifnb1, Usp18, Oas3 and Ifit1 in tumors of Anxa1 ΔMɸ mice treated with control solvent or RU521. Results represent the averages of three independent experiments. ( L ) Measurement of IFN-β protein in tumor homogenates from Anxa1 ΔMɸ mice treated with control solvent or RU521. ( M ) Shown are the image of the subcutaneous tumors of Anxa1 ΔMɸ mice treated with control solvent or RU521 (left). Quantification of tumor volume and weight are shown (right). Each group contained five mice. ( N ) Shown are the image of the subcutaneous tumor size of Anxa1 ΔMɸ mice treated with IgG or anti-CSF1R. Each group contained five mice. Quantification of tumor volume and weight that from Anxa1 ΔMɸ mice treated with IgG or anti-CSF1R are shown. ( O ) qPCR analysis of mRNA expression of Ifnb1, Usp18, Oas3 and Ifit1 in tumors from Anxa1 ΔMɸ mice treated with IgG or anti-CSF1R. Results represent the averages of three independent experiments. ( P ) Measurement of IFN-β protein in tumor homogenates from Anxa1 ΔMɸ mice treated with IgG or anti-CSF1R. ( Q ) Immunoblot analysis of c-Casp3 and STING in tumor tissues from Anxa1 ΔMɸ that treated IgG and anti-CSF1R mice. ( R ) Immunoblot analysis of ANXA1 in THP1 cells that transfected with lentiviruses control (THP1-ctrl) and carrying plasmids knockdown ANXA1 (THP1-shANXA1). ( S ) Representative images of tumors from the humanized mouse model of PDAC that ANXA1 was knocked down in macrophages (left). Quantification of tumor volume and weight are shown (right). Each group contained five mice. ( T ) Immunoblot analysis of c-Casp3, STING and IFN-β expression in tumors from THP1-ctrl and THP1-shANXA1 mice. ( U ) Immunoblot analysis of ANXA1 in THP1 cells that transfected with lentiviruses control (THP1-ctrl) and carrying plasmids overexpressing ANXA1 (THP1-ANXA1). ( V ) Representative images of tumors from the humanized mouse model of PDAC that ANXA1 was overexpressed in macrophages (left). Quantification of tumor volume and weight are shown (right). Each group contained five mice. ( W ) Immunoblot analysis of c-Casp3, STING and IFN-β expression in tumors from THP1-ctrl and THP1-ANXA1 mice. The Panc02 cells line was used in the mouse PDAC tumor model involved in ( A–Q ). The data presented are the means±SD. ANXA1, Annexin A1; c-Casp3, cleaved Caspase-3; CSF1R, colony-stimulating factor 1 receptor; GO, gene ontology; IFN, interferon; IFNAR1, IFN-alpha/beta receptor 1; mRNA, messenger RNA; PDAC, pancreatic ductal adenocarcinoma; qPCR, quantitative PCR; THP1, human monocytic cell line.

Article Snippet: The viability of Panc02 cells were determined using the Cell Counting Kit-8 (Dojindo, Japan) and measured at 450 nM with the BioTek Gen5 system (BioTek, Winooski, Vermont, USA).

Techniques: Expressing, Western Blot, Control, Solvent, Transfection, Knockdown, Real-time Polymerase Chain Reaction

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Activated Stellate Cell Paracrine HGF Exacerbated Pancreatic Cancer Cell Ferroptosis Resistance

doi: 10.1155/2022/2985249

Figure Lengend Snippet: The activated PSCs promote pancreatic cancer cell ferroptosis resistance. (a) A proposed model illustrating the coculture system of Panc02 cells. (b) Panc02 cells were treated with different concentrations of Erastin (0, 0.5, and 1 μ M) or RSL3 (0, 0.05, and 0.1 μ M) for 72 hours under normal or coculture and Fer-1 conditions. Cell viability was measured by CCK-8 kits. (c) qRT-PCR analysis of the mRNA expression levels of ferroptosis indicators (NRF2, SLC7A11, GPX4, FTH1, and NCOA4) in Panc02 cells under normal and coculture conditions. ACTB mRNA expression was detected as a loading control. (d) Western blot analysis of the protein expression levels of ferroptosis indicators (SLC7A11 and GPX4) in Panc02 treated with DMSO, coculture, Erastin (2 μ M), and coculture+Erastin (2 μ M). β -tubulin expression was detected as a loading control. (e–h) Panc02 cells were treated with DMSO, coculture, Erastin (2 μ M)/RSL3 (0.1 μ M), and coculture+Erastin (2 μ M)/RSL3 (0.1 μ M). Iron (Fe 2+ ) level of Panc02 was evaluated by flow cytometry (e). The relative GSH (f) and MDA (g) concentrations of Panc02 were analyzed. Lipid ROS level of Panc02 was evaluated by flow cytometry (h). Fer-1 represents ferrostatin-1. Co-Panc02 represents Panc02 cells which were cocultured with activated PSCs. Experiments were repeated three times, and data were expressed as the mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The logarithmic growth phase Panc02 cells were exposed to different conditions for 72 h, and then, the cell viability was detected with the Cell Counting Kit-8 (CCK-8, Dojindo, #CK04) reagent.

Techniques: CCK-8 Assay, Quantitative RT-PCR, Expressing, Control, Western Blot, Flow Cytometry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Activated Stellate Cell Paracrine HGF Exacerbated Pancreatic Cancer Cell Ferroptosis Resistance

doi: 10.1155/2022/2985249

Figure Lengend Snippet: HGF secreted by activated PSCs mediated ferroptosis resistance in pancreatic cancer cells. (a) qRT-PCR analysis of mRNA expression levels of multiple secreted factors (CXCL-16, TNF- α , CCL7, HGF, TGF- β , PDGF, CCL2, VEGF, SDF1-A, IGF-1, IL-6, b-FGF, and CCL5) in PSCs under normal or coculture conditions. (b) ELISA-based analysis of the HGF concentration in PSCs under normal and coculture conditions. (c) Panc02 cells were treated with HGF (5 ng/ml) and different concentrations of Erastin (0, 0.2, 0.4, 0.6, 0.8, and 1 μ M) or RSL3 (0, 0.02, 0.04, 0.06, 0.08, and 0.1 μ M) for 72 hours. Cell viability was measured by CCK-8 kits. (d, e) qRT-PCR and western blot analysis of the mRNA and protein expression levels of ferroptosis indicators (SLC7A11 and GPX4) in Panc02 cells treated with DMSO, HGF (5 ng/ml), Erastin (2 μ M), or HGF (5 ng/ml)+Erastin (2 μ M). ACTB mRNA expression was detected as a loading control for qRT-PCR. β -tubulin expression was detected as a loading control for western blot. (f–h) Panc02 cells were treated with DMSO, HGF (5 ng/ml), Erastin (2 μ M)/RSL3 (0.1 μ M), or HGF (5 ng/ml)+Erastin (2 μ M)/RSL3 (0.1 μ M). The relative levels of GSH (f) and MDA (g) of Panc02 were assayed by test kits, and the relative levels of Panc02 lipid ROS were detected by flow cytometry (h). Experiments were repeated three times, and data were expressed as the mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The logarithmic growth phase Panc02 cells were exposed to different conditions for 72 h, and then, the cell viability was detected with the Cell Counting Kit-8 (CCK-8, Dojindo, #CK04) reagent.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, CCK-8 Assay, Western Blot, Control, Flow Cytometry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Activated Stellate Cell Paracrine HGF Exacerbated Pancreatic Cancer Cell Ferroptosis Resistance

doi: 10.1155/2022/2985249

Figure Lengend Snippet: Inhibition of c-MET sensitizes pancreatic cancer cells to ferroptosis with the presence of activated PSCs. (a, b) Panc02 cells treated with DMSO, c-MET-IN-1, Erastin, Erastin+IN-1, RSL3, and RSL3+IN-1 cultured under normal or coculture conditions. Representative images showing the induction of cell death in Panc02 cells (a) and cell viability was measured by CCK-8 kits (b). (c–f) Panc02 cells were cocultured with activated PSCs and then treated with DMSO, IN-1 (0.05 μ M), Erastin (2 μ M), Erastin (2 μ M)+c-MET-IN-1 (0.05 μ M), RSL3 (0.1 μ M), and RSL3 (0.1 μ M)+c-MET-IN-1 (0.05 μ M). Western blot analysis of the protein expression levels of c-MET, SLC7A11, and GPX4 in Co-Panc02 (c). β -tubulin expression was detected as a loading control. The relative GSH (d) and MDA (e) concentrations of Co-Panc02 were analyzed; lipid ROS level of Co-Panc02 was evaluated by flow cytometry (f). (g) qRT-PCR and western blot analysis of shRNA-mediated knockdown of c-MET mRNA and protein levels in Panc02 cells. ACTB mRNA expression was detected as a loading control for qRT-PCR. β -tubulin expression was detected as a loading control for western blot. (h) Co-Panc02 cells (Ctrl shRNA, c-MET shRNA1, and c-MET shRNA2) were treated with different concentrations of Erastin (0, 0.5, and 1 μ M) or RSL3 (0, 0.05, and 0.1 μ M) for 72 hours. Cell viability was measured by CCK-8 kits. (i) qRT-PCR analysis of SLC7A11 and GPX4 mRNA expression in Panc02 (Ctrl shRNA) and c-MET knockdown Panc02 (c-MET shRNA1, c-MET shRNA2, and c-MET shRNA3). (j–l) Co-Panc02 cells (Ctrl shRNA, c-MET shRNA1, and c-MET shRNA2) were treated with Erastin (2 μ M) or RSL3 (0.1 μ M). Western blot analysis of the protein expression levels of c-MET, SLC7A11, and GPX4 in Co-Panc02. β -tubulin expression was detected as a loading control (j). The relative GSH and MDA concentrations of Co-Panc02 were analyzed (k); lipid ROS level of Co-Panc02 was evaluated by flow cytometry (l). IN-1 represents c-MET-IN-1. Experiments were repeated three times, and data were expressed as the mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The logarithmic growth phase Panc02 cells were exposed to different conditions for 72 h, and then, the cell viability was detected with the Cell Counting Kit-8 (CCK-8, Dojindo, #CK04) reagent.

Techniques: Inhibition, Cell Culture, CCK-8 Assay, Western Blot, Expressing, Control, Flow Cytometry, Quantitative RT-PCR, shRNA, Knockdown

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Activated Stellate Cell Paracrine HGF Exacerbated Pancreatic Cancer Cell Ferroptosis Resistance

doi: 10.1155/2022/2985249

Figure Lengend Snippet: Activated PSCs promoted ferroptosis resistance in pancreatic cancer in vivo . (a–g) C57BL/6 mice were injected subcutaneously with Panc02 (Panc02 groups, 2 × 10 5 cells/mouse) or Panc02+PSCs (Co-Panc02 groups, 1.5 × 10 5 Panc02 cells + 0.5 × 10 5 PSCs). They were divided into Panc02 groups (treated with DMSO), Co-Panc02 groups (DMSO), Panc02+H (HGF, 50 ng/i.h., every two days), Panc02+E (Piperazine Erastin, 30 mg/kg/i.h., every two days), Co-Panc02+E (Piperazine Erastin, 30 mg/kg/i.h., every two days), and Panc02+H+E (HGF, 50 ng/i.h., Piperazine Erastin, 30 mg/kg/i.h., every two days). (a) Representative photographs of isolated tumor tissues in each treatment group at day 14. (b) Tumor volume was detected every two days. (c) The body weight of mice in each treatment group was measured every two days. (d, e)D-E. The GSH (d) and MDA (e) levels in isolated tumors were assayed at day 14 after different treatments. (f) Western blot analysis of protein expression levels of ferroptosis-related indicators (SLC7A11 and GPX4) of isolated tumor tissues in each treatment group. (g) Immunohistochemistry analysis of the expression of c-MET in isolated tumor tissues. (h) The expression of c-MET in PDAC patients and normal pancreatic tissues (T: tumor; N: normal) and the correlation between the expression of c-MET and the survival of PDAC patients were analyzed with the GEPIA database. (i) Analysis of the TCGA and GEPIA databases for the correlation c-MET mRNA expression level with the ferroptosis-related indicators (NRF2, SLC7A11, and GPX4). The TCGA database result was presented by heat map: n = 183. PE: Piperazine Erastin. Experiments were repeated three times, and the data were expressed as the mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The logarithmic growth phase Panc02 cells were exposed to different conditions for 72 h, and then, the cell viability was detected with the Cell Counting Kit-8 (CCK-8, Dojindo, #CK04) reagent.

Techniques: In Vivo, Injection, Isolation, Western Blot, Expressing, Immunohistochemistry