Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: IFI16 was overexpressed in human PAAD and correlated with poor survival of the patients. (A) Expression data of inflammasome-related proteins were retrieved from the human database GEPIA. A comparison of expression between normal pancreatic tissues and PAAD tissues was done. Genes with a significantly different expression between normal and tumor tissues are shown in red. (B) The data on overall survival and disease-free survival of PAAD patients were retrieved. Only NLRP1, among the genes with significantly different expression, showed a negative correlation with patients’ overall survival. Overall survival (C) but not disease-free survival (D) was adversely correlated with the expression of IFI16 in PAAD. We further extracted expression data of IFI16 from the human GEO database, including GDS4336 (E) and GDS4103 (F) . IFI16 was significantly overexpressed in the tumor tissues of PAAD compared with that in the non-tumor adjacent normal pancreas. (G) Pearson correlation between the expression of IFI16 and IL-1β was analyzed, which showed a positive correlation in human PAAD. * p < 0.05.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Expressing, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: Overexpression of IFI16 promoted tumor growth in an experimental model of PAAD. (A) The IFI16-overexpressing PAAD cell line was established by transfecting the CRISPR activation plasmid of IFI16 into the Panc-2 cells. Cells were selected with a culture medium containing 1 μg/ml of puromycin, until a stable IFI16-overexpressing clone was established. Overexpression of IFI16 was validated with immunoblotting. (B) Panc-2 of a stable clone expressing the luciferase reporter was established. Panc-2 cells were transfected with the PGL3 vector expressing firefly luciferase and selected by neomycin (50 μg/ml). Approximately 20 μg Matrigel matrix, containing 1 × 10 8 Panc-2 cells, was orthotopically injected into the pancreas of mice. The luciferase signal intensity was measured by intraperitoneal injection of luciferin (30 mg/kg) and quantification under a live animal imager once per week. Overexpression of IFI16 significantly accelerated the orthotopic growth of the pancreatic tumor. (C) At the end of the study, the mice were sacrificed, and the pancreas along with the spleen was dissected out. The tumor weight was measured, and tumor size was calibrated by the diameters of the tumor. Overexpression of IFI16 potently increased the tumor size and weight in the experimental PAAD model. The black arrow shows an obvious surface tumor nodule found in the pancreas. (D) The dissected tumor was then digested in 0.8 mg/ml of collagenase IV for 30 min at 37°C with gentle shaking. The immune cells were enriched with Ficoll methods. The profile of immune cells in the tumor microenvironment was measured with flow cytometry. Overexpression of IFI16 significantly increased the population of TAMs but not dendritic cells, T helper cells, or cytotoxic T cells. (E) The F4/80 + CD11b + TAMs were collected using a cell sorter, and expressions of HIF-1α, CCL2, and PECAM1 were measured with qRT-PCR. TAMs from IFI16-overexpressing tumors exhibited a significantly high expression of TAM markers, including HIF-1α, CCL2, and PECAM1. * p < 0.05.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Over Expression, CRISPR, Activation Assay, Plasmid Preparation, Western Blot, Stable Transfection, Expressing, Luciferase, Transfection, Injection, Gentle, Flow Cytometry, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: Depletion of TAMs attenuated IFI16-induced tumor growth of PAAD. (A) Flowchart of TAM depletion treatment. To minimize early recruitment of TAMs, mice received a single injection of liposome PBS (as sham control) or liposome clodronate, 3 days before orthotopic implantation of tumor cells, and received subsequent injection twice per week after implantation at a dose of 15 mg/kg. (B) The tumor was dissected out, and TAMs were enriched with Ficoll methods. The enriched cells were stained with an antibody against F4/80 and subjected to flow cytometric analysis. Treatment with liposome clodronate could potently remove TAMs from mice with orthotopic PAAD tumors. (C) Panc-2 of a stable clone expressing the luciferase reporter was established. Panc-2 cells were transfected with the PGL3 vector expressing firefly luciferase and selected by neomycin (50 μg/ml). Approximately 20 μl Matrigel matrix, containing 1 × 10 8 Panc-2 cells, was orthotopically injected into the pancreas of mice. The luciferase signal intensity was measured by intraperitoneal injection of luciferin (30 mg/kg) and quantification under a live animal imager once per week. Depletion of TAMs attenuated the tumor growth of PAAD induced by IFI16 overexpression. (D) At the end of the study, the mice were sacrificed, and the pancreas along with the spleen was dissected out. The tumor weight was measured, and tumor size was calibrated by the diameters of the tumor. Depletion of TAMs abolished the IFI16 overexpression-induced increase in tumor size and weight in the experimental PAAD model. The black arrow shows an obvious surface tumor nodule found on the pancreas. * p < 0.05 and *** p < 0.001.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Injection, Control, Staining, Stable Transfection, Expressing, Luciferase, Transfection, Plasmid Preparation, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: Conditional medium from IFI16-overexpressing PAAD cells increased the TAM population. (A) The culture medium of wild-type and IFI16-overexpressing PAAD cells was collected. BMDMs were cultured with 30% of the aforementioned conditional medium for 7 days. Cells were then collected and stained with antibodies against CD11b and F4/80 and subjected to flow cytometric analysis. BMDMs cultured with IFI16-overexpressing PAAD cells showed a higher level of CD11b + F4/80 + cells. Total RNA was extracted, and the expressions of HIF-1α, CCL2, and PECAM1 were analyzed with qRT-PCR. BMDMs cultured with conditional medium from IFI16-overexpressing PAAD cells showed significantly higher expressions of HIF-1α, CCL2, and PECAM1. (B) The Panc-2 cells with or without IFI16 overexpression were cultured on the apical side of the 0.8-μM-pore size Transwell, while BMDMs were seeded on the receiving chamber with the non-attached surface, supplemented with 10 ng/ml of M-CSF. This co-culture was maintained for 7 days to allow all possible non-contact interaction between the Panc-2 cells and BMDMs. BMDMs were then collected for analysis of the TAM population with flow cytometry and qPCR. Co-culture of Panc-2 cells with IFI16 overexpression significantly increased the population of TAMs from the BMDM culture compared to the co-culture of vector-expressing Panc-2 cells. (C) Added to the culture medium was 10 μM of BrdU 4 h prior to sample collection by trypsinization. BrdU-incorporated CD11b + F4/80 + cells were stained with anti-BrdU antibody and detected with a flow cytometer. BMDMs cultured with the conditional medium from IFI16-overexpressing PAAD cells showed significantly higher incorporation of BrdU into the DNA. (D) Approximately 2 × 10 5 BMDMs cultured with the conditional medium from Panc-2 cells were seeded at the apical side of the Transwell insert with serum-free medium. A culture medium supplemented with chemotaxis MCP-1 (10 ng/ml) was added into the receiving chambers and cultured for 3 h. Cells remaining at the apical side of inserts were scraped away, and cells at the basal side of the membrane were collected by trypsinization. The cells were then collected and stained with 50 μM calcein AM and quantified with a fluorescence microplate reader. BMDMs cultured with the conditional medium from IFI16-overexpressing PAAD cells showed significantly higher motility. (E) Intraperitoneally injected into the mice was 10 mg/kg of BrdU 24 h prior to sample collection by enriching TAMs from the dissected tumor by Ficoll methods. BrdU-incorporated CD11b + F4/80 + cells were stained with an anti-BrdU antibody and detected with a flow cytometer. TAMs from tumors with IFI16 overexpression showed significantly high incorporation into the DNA. (F) BMDMs were isolated from the femurs and cultured into macrophages in a medium containing 10 μg/ml of M-CSF for 7 days. Cells were then stained with 100 μM PKH26PCL for labeling. Labeled cells were then intraperitoneally injected into the mice bearing PAAD tumors with or without IFI16 overexpression and allowed circulation for 24 h. The tumor was then dissected out, and the number of PKH26PCL-labeled cells infiltrated into the tumor was measured with a flow cytometer. IFI16-overexpressing tumors showed more cell infiltration into the tumor microenvironment. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Cell Culture, Staining, Quantitative RT-PCR, Over Expression, Pore Size, Co-Culture Assay, Flow Cytometry, Plasmid Preparation, Expressing, Chemotaxis Assay, Membrane, Fluorescence, Injection, Isolation, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: IL-1β is responsible for the IFI16-induced TAM profile changes in PAAD. (A) Protein expression of Panc-2 cells at intracellular and extracellular levels was measured with immunoblotting. IFI16 overexpression induced further activation of the inflammasome, as evidenced by the cleavage of intracellular pro-caspase-1 and pro-IL-1β, as well as the extracellular expression of cleaved IL-1β and caspase-1 in the culture medium. (B) IL-1β levels were quantified with ELISA. IFI16 overexpression significantly increased IL-1β production and secretion in Panc-2 cells in the presence or absence of poly dA:dT. Supplementation with an IL-1β-neutralizing antibody mitigated the function of secreted IL-1β in the conditional medium from Panc-2 cells. This method was adopted from a published protocol, with minor modifications. For the blocking experiments, the BMDMs were preincubated with 10 μg/ml of the IL-1β-neutralizing antibody, for 15 min before the functional studies. (C) Cells were then collected and stained with antibodies against CD11b and F4/80 and subjected to flow cytometry analysis. Neutralization of IL-1β significantly blocked the increase in TAM population cultured in the conditioned medium from IFI16-overexpressing Panc-2 cells. (D) Added to the culture medium was 10 μM of BrdU 4 h prior to sample collection by trypsinization. BrdU-incorporated CD11b + F4/80 + cells were stained with anti-BrdU antibody and detected using a flow cytometer. Neutralization of IL-1β significantly blocked the increase in BrdU incorporation into the DNA of BMDMs cultured in conditioned medium from IFI16-overexpressing Panc-2 cells. (E) Approximately 2 × 10 5 BMDMs cultured with the conditioned medium from Panc-2 cells were seeded at the apical side of Transwell inserts with serum-free medium. Culture medium supplemented with chemotaxis MCP-1 (10 ng/ml) was added to the receiving chambers, followed by culture for 3 h. Cells remaining on the apical side of the inserts were scraped away, and cells at the basal side of the membrane were collected by trypsinization. The cells were then collected and stained with 50 μM calcein AM and quantified using a fluorescence microplate reader. Neutralization of IL-1β significantly blocked the increased motility of the BMDMs cultured in the conditioned medium from IFI16-overexpressing Panc-2 cells. * p < 0.05.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Expressing, Western Blot, Over Expression, Activation Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Functional Assay, Staining, Flow Cytometry, Neutralization, Cell Culture, BrdU Incorporation Assay, Chemotaxis Assay, Membrane, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: Suppression of IFI16 improves gemcitabine sensitivity in PAAD tumors. (A) Flowchart of gemcitabine treatment. Approximately 20 μl Matrigel matrix, containing 1 × 10 8 Panc-2 cells, was orthotopically injected into the pancreas of mice. Mice were then orally administered gemcitabine at a dose of 100 mg/kg or vehicle every day. (B) The IFI16-knockdown PAAD cell line was established by transfecting the shRNA plasmid of IFI16 into the Panc-2 cells. Cells were selected with a culture medium containing 1 μg/ml of puromycin, until a stable IFI16-knockdown clone was established. Knockdown of IFI16 was validated with immunoblotting, which showed that RNA interference using shRNA against IFI16 can significantly attenuate gemcitabine-induced IFI16 upregulation. (C) The luciferase signal intensity was measured by intraperitoneal injection of luciferin (30 mg/kg) and quantification under a live animal imager once per week. Knockdown of IFI16 could significantly improve the suppression of tumor growth by gemcitabine. (D) At the end of the study, the mice were sacrificed, and the pancreas along with the spleen was dissected out. The tumor weight was measured, and tumor size was calibrated by the diameters of the tumor. Knockdown of IFI16 in gemcitabine-treated mice further reduced the size and weight of the tumor in the experimental PAAD model. The black arrow shows an obvious surface tumor nodule found on the pancreas. (E) The tumor was dissected out, and TAMs were enriched by Ficoll methods. The enriched cells were stained with an antibody against F4/80 and subjected to flow cytometry analysis. Knockdown of IFI16 could attenuate the infiltration of TAMs induced by gemcitabine in the tumor microenvironment of PAAD. (F) The major M1/M2/TAM markers were quantified using qPCR. The ratio of expression of IFN-γ and TGF-β was calculated to represent the M1/M2 ratio in the tumor microenvironment. Gemcitabine had no significant effect on the polarization of macrophages toward either phenotype but potently activated the expression of TAM markers HIF-1α, CCL2, and PECAM1. (G) Expression of IFI16 was measured by immunoblotting in different PAAD cell lines. Expression of IFI16 was highest in BxPC3 cells, then SW1990 cells, and then Panc-2 cells, while Panc-1 cells expressed the lowest level of IFI16. (H) Expression of IFI16 was forcefully activated and knocked down in SW1990 cells. (I) BMDMs were co-cultured with SW1990 cells with or without IFI16 knockdown in the presence or absence of 100 nM gemcitabine for 7 days. The activation of BMDMs was then examined by flow cytometry. Knockdown of IFI16 reduced co-cultured BMDM activation in the presence or absence of gemcitabine. (J) BMDMs were co-cultured with SW1990 cells with or without IFI16 overexpression in the presence or absence of 100 nM gemcitabine for 7 days. The activation of BMDMs was then examined by flow cytometry. Overexpression of IFI16 further increased co-cultured BMDM activation in the presence or absence of gemcitabine. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Injection, Knockdown, shRNA, Plasmid Preparation, Western Blot, Luciferase, Staining, Flow Cytometry, Expressing, Cell Culture, Activation Assay, Over Expression

Journal: PLoS ONE

Article Title: IFI16 Protein Mediates the Anti-inflammatory Actions of the Type-I Interferons through Suppression of Activation of Caspase-1 by Inflammasomes

doi: 10.1371/journal.pone.0027040

Figure Lengend Snippet: ( A ) Puromycin-resistant THP-1 cells either infected with control lentivirus (lanes 1 an2) or the virus expressing the shIFI16 RNA (lanes 3–8; cell populations from three different wells) were either left untreated (lanes 1, 3, 5, 7) or treated with IFN-α for 14 h. After treatment, total cell extracts containing increased amounts of proteins (∼100 µg/lane) were analyzed for the constitutive and induced levels of IFI16 and actin proteins. A long exposure was taken to detect the signal in all lanes. ( B ) Control THP-1 cells (lanes 1 and 2) or cell population from well # 9 (lanes 3 and 4) as shown in the panel (a) were either left untreated (lanes 1 and 3) or treated with IFN-α for 14 h (lanes 2 and 4). After the treatment, total RNA was analyzed for the steady-state levels of mRNA for the indicated genes. ( C ) Control THP-1 cells (lanes 1–4) or cells infected with virus expressing the shIFI16 mRNA (lanes 5–8; population # 9) were either left untreated (lanes 1 and 5) or treated with IFN-β (1,000 u/ml; lanes 2 and 6), LPS (100 ng/ml; lanes 3 and 7), or dsRNA (10 µg/ml; lanes 4 and 8) for 14 h. After the treatment, total cell lysates containing equal amounts of protein (∼100 µg/lane) were analyzed by immunoblotting using specific antibodies to the indicated proteins. FC, indicates the fold change in the levels of the activated caspase-1 (the p20 band) with respect to the control (lane 1). ( D ) Control THP-1 cells or cells infected with virus expressing the shIFI16 mRNA (population # 9) were either left untreated (white columns) or treated with IFN-β (1,000 u/ml; columns 2–5 and 7–10) for 14 h. After the treatment, total RNA levels were analyzed for the indicated genes by the quantitative TaqMan real-time PCR. The ratio of the test gene to actin mRNA was calculated in units (one unit being the ratio of the test gene to actin mRNA). Results are mean values of triplicate experiments and error bars represent standard deviation.

Article Snippet: THP-1 cells were either infected with control lentiviral particles (sc-108080; from Santa Cruz Biotech, Santa Cruz,, CA) or the lentiviral particles expressing shIFI16 RNA (sc-35633-V, Santa Cruz Biotech) in a six well plate as suggested by the supplier.

Techniques: Infection, Control, Virus, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation