Journal: Journal of Personalized Medicine

Article Title: IGFBP2 Drives Regulatory T Cell Differentiation through STAT3/IDO Signaling Pathway in Pancreatic Cancer

doi: 10.3390/jpm12122005

Figure Lengend Snippet: IGFBP2 overexpression is associated with Treg infiltration in the tumor microenvironment and disease progression in human PDAC. ( A ) IGFBP2 and FOXP3 amounts in PDAC and noncancerous pancreatic tissue specimens. Magnification: 400×. White arrows point to IGFBP2 + PDAC cells; red arrows point to FOXP3 + cells as Tregs. ( B ) Distributions of T stage in the high- and low-IGFBP2 expression groups ( n = 50; *** p < 0.001 by χ 2 test). ( C ) Kaplan–Meier curves comparing overall survival rates in PDAC cases with high and low IGFBP2 levels ( n = 50; p < 0.01 by log-rank test). ( D ) Counts of FOXP3 + cells as Tregs in the PDAC microenvironment with reduced and elevated IGFBP2 amounts ( n = 50; *** p < 0.001 by the Student’s t -test). ( E ) CD4 + CD25 + FOXP3 + cells (Tregs) assessed flow-cytometrically in PDAC tissue samples from freshly collected surgical specimens with reduced and elevated IGFBP2 amounts ( n = 10; ** p < 0.01 by the Student’s t -test). ( F ) CD8 + CD45 + T cells assessed flow-cytometrically in the latter PDAC tissue specimens ( n = 10; * p < 0.05 by the Student’s t -test). Data are mean ± SD from 3 or more assays performed independently.

Article Snippet: Stable IGFBP2 and IDO overexpression cell lines (MDA-PATC148BP2 and MDA-PATC148IDO) were generated by infecting MDA-PATC148 cells with human IGFBP2 and IDO lentiviral particles (GeneCopoeia), respectively, utilizing polybrene, with subsequent incubation under puromycin pressure for 3 weeks.

Techniques: Over Expression, Biomarker Discovery, Expressing

Journal: Journal of Personalized Medicine

Article Title: IGFBP2 Drives Regulatory T Cell Differentiation through STAT3/IDO Signaling Pathway in Pancreatic Cancer

doi: 10.3390/jpm12122005

Figure Lengend Snippet: IGFBP2 increases Treg infiltration in the tumor microenvironment and promotes disease progression in mouse PDAC. ( A ) Panc02 cells stably overexpressing IGFBP2 were generated. IGFBP2 mRNA amounts were assessed by qRT-PCR, normalized to GAPDH (*** p < 0.001 by Student’s t -test). C57BL/6 mice were used for establishing an orthotropic PDAC model. The animals were divided into two groups and injected with IGFBP2-overexpressing (IGFBP2) or empty vector-transfected control (EV) Panc02 cells. Following 4 weeks, euthanasia was performed, and tumor extraction was carried out. Tumor weights ( B ), tumor areas ( C ), and tumor nodules in the liver ( D ) for both mouse groups are shown ( n = 8/group; * p < 0.05 ** p < 0.01 by the Student’s t -test). ( E ) The same orthotropic pancreatic carcinoma model was repeated, and the overall survival rates were analyzed ( n = 15/group; p < 0.01 by log-rank test). ( F ) Flow cytometry analysis of Ki-67 in tumor cells from both groups of C57BL/6 mice ( n = 8 per group; ** p < 0.01 by the Student’s t -test). CD4 + CD25 + FOXP3 + Tregs ( G ) and CD8 + CD45 + T cells ( H ) in the PDAC microenvironment in both mouse groups, assessed flow-cytometrically, are shown ( n = 8/group; * p < 0.05 ** p < 0.01 by the Student’s t -test). Data are mean ± SD from 3 or more assays performed independently.

Article Snippet: Stable IGFBP2 and IDO overexpression cell lines (MDA-PATC148BP2 and MDA-PATC148IDO) were generated by infecting MDA-PATC148 cells with human IGFBP2 and IDO lentiviral particles (GeneCopoeia), respectively, utilizing polybrene, with subsequent incubation under puromycin pressure for 3 weeks.

Techniques: Biomarker Discovery, Stable Transfection, Generated, Quantitative RT-PCR, Injection, Plasmid Preparation, Transfection, Control, Extraction, Flow Cytometry

Journal: Journal of Personalized Medicine

Article Title: IGFBP2 Drives Regulatory T Cell Differentiation through STAT3/IDO Signaling Pathway in Pancreatic Cancer

doi: 10.3390/jpm12122005

Figure Lengend Snippet: IGFBP2 alters T cell differentiation to promote an immunosuppressive phenotype. ( A ) Relative IGFBP2 mRNA expression levels of different PDAC cell lines were assessed by qRT-PCR, normalized to GAPDH (*** p < 0.001 by ANOVA for PATC53 or the Student’s t -test for PTAC148). ( B ) Flow cytometry analysis of human CD4 + T cells cultured without or with TGF-β, or co-cultured with PDAC cells with differential expression of IGFBP2 for CD4 + FOXP3 + Tregs. ( C ) Activated CD8 + T cells were co-cultured with the Tregs inducted by TGF-β or PDAC cell lines to verify their T cell suppressive function (** p < 0.01 by ANOVA for PATC53 or the Student’s t -test for PTAC148). ( D ) Human CD8 + T cells co-cultured with PDAC cells with differential expression of IGFBP2 assessed flow-cytometrically for apoptotic CD8 + T cells. ( E – H ) Cytotoxicity, proliferation, Ki-67, and cell cycle analyses were performed on the same CD8 + T cells described in ( D ) (** p < 0.01 by ANOVA for PATC53 or the Student’s t -test for PTAC148). Data are mean ± SD from 3 or more assays performed independently.

Article Snippet: Stable IGFBP2 and IDO overexpression cell lines (MDA-PATC148BP2 and MDA-PATC148IDO) were generated by infecting MDA-PATC148 cells with human IGFBP2 and IDO lentiviral particles (GeneCopoeia), respectively, utilizing polybrene, with subsequent incubation under puromycin pressure for 3 weeks.

Techniques: Cell Differentiation, Expressing, Quantitative RT-PCR, Flow Cytometry, Cell Culture, Quantitative Proteomics

Journal: Journal of Personalized Medicine

Article Title: IGFBP2 Drives Regulatory T Cell Differentiation through STAT3/IDO Signaling Pathway in Pancreatic Cancer

doi: 10.3390/jpm12122005

Figure Lengend Snippet: IGFBP2 activates STAT3 and increases IDO expression in human PDAC cells. ( A ) IGFBP2 and IDO amounts in PDAC and noncancerous pancreatic tissue specimens. Magnification: 400×. ( B ) IDO amounts in the high- and low-STAT3 groups of specimens in the ICGC PDAC-AU database ( n = 91; p = 0.014 by the Wilcoxon rank sum test). ( C ) Correlation between IDO and IGFBP2 expression for each PDAC tissue from freshly collected surgical specimens, assessed by linear regression. ( D ) IDO + cells in PDAC tissue specimens with low- and high-IGFBP2 expression, assessed flow-cytometrically. ( E ) Levels of tryptophan and L-kynurenine in PDAC tissue samples evaluated by ELISA ( n = 16; ** p < 0.01 by the Student’s t-test). ( F ) Immunoblot of MDA-PATC53 cells after transfection with negative control (siR-ctrl) and IGFBP2 (GFBP2 and -2) siRNAs IGFBP2, respectively (left panel). Immunoblot of MDA-PATC148 cells upon transfection with negative control (EV) and human IGFBP2 (BP2) lentiviruses for IGFBP2 overexpression (right panel). ( G ) IDO mRNA amounts were assessed by qRT-PCR, normalized to GAPDH (** p < 0.01 by ANOVA for PATC53 or the Student’s t-test for PTAC148). Data are mean ± SD from 3 or more assays performed independently.

Article Snippet: Stable IGFBP2 and IDO overexpression cell lines (MDA-PATC148BP2 and MDA-PATC148IDO) were generated by infecting MDA-PATC148 cells with human IGFBP2 and IDO lentiviral particles (GeneCopoeia), respectively, utilizing polybrene, with subsequent incubation under puromycin pressure for 3 weeks.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Negative Control, Over Expression, Quantitative RT-PCR

Journal: Journal of Personalized Medicine

Article Title: IGFBP2 Drives Regulatory T Cell Differentiation through STAT3/IDO Signaling Pathway in Pancreatic Cancer

doi: 10.3390/jpm12122005

Figure Lengend Snippet: IGFBP2 alters T cell differentiation by inducing IDO expression in human PDAC. ( A ) Relative IDO mRNA expression levels of different PDAC cell lines were assessed by qRT-PCR, normalized to GAPDH (*** p < 0.001 by ANOVA for PATC53 or the Student’s t -test for PTAC148). ( B ) Flow cytometry analysis of human CD4 + T cells cultured without or with TGF-β or co-cultured with PDAC cell lines differentially expressing IDO for CD4 + FOXP3 + Tregs. ( C ) Activated CD8 + T cells were co-cultured with the Tregs inducted by TGF-β or PDAC cell lines to verify their T cell suppressive function (** p < 0.01 by ANOVA for PATC53 or the Student’s t -test for PTAC148). ( D ) Human CD8 + T cells co-cultured with PDAC cell lines differentially expressing IDO were assessed flow-cytometrically for apoptotic CD8 + T cells. ( E – H ) Cytotoxicity, proliferation, Ki-67, and cell cycle analysis was performed on the same CD8 + T cells as in ( D ) (** p < 0.01 by ANOVA for PATC53 or the Student’s t -test for PTAC148). Data are mean ± SD from 3 or more assays performed independently.

Article Snippet: Stable IGFBP2 and IDO overexpression cell lines (MDA-PATC148BP2 and MDA-PATC148IDO) were generated by infecting MDA-PATC148 cells with human IGFBP2 and IDO lentiviral particles (GeneCopoeia), respectively, utilizing polybrene, with subsequent incubation under puromycin pressure for 3 weeks.

Techniques: Cell Differentiation, Expressing, Quantitative RT-PCR, Flow Cytometry, Cell Culture, Cell Cycle Assay

Journal: Journal of Personalized Medicine

Article Title: IGFBP2 Drives Regulatory T Cell Differentiation through STAT3/IDO Signaling Pathway in Pancreatic Cancer

doi: 10.3390/jpm12122005

Figure Lengend Snippet: IGFBP2 induces IDO production in human PDAC cells via STAT3 signaling. ( A ) Western blot of high endogenous IGFBP2 MDA-PATC53 cells or IGFBP2-overexpressing PATC148 BP2 after transfection with negative control (siR-ctrl) and STAT3 (siR-STAT3-1 and -2) siRNAs, respectively. STAT3, pSTAT3, and IDO protein amounts were assessed by Western blot, with beta tubulin as a reference protein. IDO mRNA amounts ( B , C ) in the above cells were assessed by qRT-PCR, with GAPDH as a reference gene (** p < 0.01 by ANOVA). ( D ) Low endogenous IGFBP2 MDA-PATC148 cells underwent transfection with wild type and Y705-mutant STAT3 plasmids, respectively. The protein levels of STAT3, pSTAT3 and IDO were assessed by Western blot, with beta tubulin as a reference protein. IDO mRNA amounts ( E ) in the latter cells were determined by qRT-PCR, with GAPDH utilized for normalization (** p < 0.01 by ANOVA). Data are mean ± SD from 3 or more assays performed independently. ( F ) IGFBP2 promotes tumor progression via alternative polarization of macrophages in PDAC in a STAT3 pathway-dependent manner.

Article Snippet: Stable IGFBP2 and IDO overexpression cell lines (MDA-PATC148BP2 and MDA-PATC148IDO) were generated by infecting MDA-PATC148 cells with human IGFBP2 and IDO lentiviral particles (GeneCopoeia), respectively, utilizing polybrene, with subsequent incubation under puromycin pressure for 3 weeks.

Techniques: Western Blot, Transfection, Negative Control, Quantitative RT-PCR, Mutagenesis