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

Journal: Science Advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: ( A and B ) Representative images (A) and quantification (B) of MM231 breast cancer cell invasion in inverted collagen/fibronectin matrices in the presence of conditioned medium from HUVECs transfected with siRNAs against IGFBP2 (siIGFBP2_1 and siIGFBP2_2) or siNTC. Scale bars, 50 μm. n = 3 biological replicates performed in triplicate, with three stacksper transwell; one-way ANOVA with Tukey correction; ** P < 0.01 and *** P < 0.001; ns, not significant. ( C and D ) Representative Western blot (C) and densitometry analysis (D) of TIFs stably overexpressing mTurquoise2 (mT2) or IGFBP2 ( n = 4; one-sample t test; * P < 0.05). ( E ) Enzyme-linked immunosorbent assay (ELISA) for human IGFBP2 in concentrated conditioned media from TIFs overexpressing mT2 or IGFBP2, measured in duplicate ( n = 3 biological replicates; two-tailed Student’s t test; ** P < 0.01). ( F ) Representative images and quantification of fibroblast-contracted 3D collagen invasion assays of MM231 cancer cell invasion in mT2 or IGFBP2 TIF-contracted matrices after 14 days and stained for Pan-CK (brown). Scale bars, 100 μm. n = 3 biological replicates, triplicate matrices, eight regions per condition per replicate; one-way ANOVA with Tukey correction; *** P < 0.001. ( G and H ) Representative hematoxylin and eosin (H&E) images (G) and quantification (H) of MM231 cell invasion into the mouse dermis from subcutaneous xenografts of MM231 co-injected with TIFs overexpressing either mT2 (control) or IGFBP2. Scale bars, 500 μm; insets, 50 μm. n = 10 mice (mT2) or 12 mice (IGFBP2).

Article Snippet: Primary antibodies in AdvanBlock-Fluor were incubated overnight at 4°C; IGFBP2 (R&D Systems, AF674), IGF-II (Millipore, 05-166-MI), GAPDH (Hytest, 5G4MAB6C5), and GFP (Thermo Fisher Scientific, A11122).

Techniques: Transfection, Western Blot, Stable Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Injection, Control

Journal: Science Advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: ( A ) Three representative images [(i) to (iii)] of human breast tissue stained for IGFBP2 (magenta) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (cyan). Autofluorescence (AF) signal is green. Scale bars, 100 μm; insets, 10 μm. n = 2 adjacent healthy breast tissue sections from ductal carcinoma in situ (DCIS) patient samples. ( B ) Representative images of primary human breast pre- and mature adipocytes with immunofluorescence staining for IGFBP2/PPARγ/actin or Nile Red/vimentin. Scale bars, 50 μm; insets, 10 μm. n = 4 normal reduction mammoplasty patient samples from which preadipocytes were isolated, cultured, and differentiated into mature adipocytes. ( C ) Fold change in gene expression between pre- and mature adipocytes, normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), and detected by quantitative reverse transcription polymerase chan reaction [adipocytes from n = 4 patient samples, differentiated as in (B), in triplicate; one-sample t test; ** P < 0.01 and *** P < 0.001]. PPARG , peroxisome proliferator activated receptor gamma; LIPE , lipase E; FABP4 , fatty acid binding protein 4; CFD , complement factor D. ( D ) ELISA for human IGFBP2 from pre- and mature adipocyte conditioned media, compared to the adipocyte culture media [AM-1; adipocytes from n = 4 patient samples processed as in (B) and media collected; two-tailed Student’s t test with Welch’s correction; *** P < 0.001]. ( E and F ) Representative images (E) and quantification (F) of MM231 cell invasion in inverted collagen/fibronectin matrices in the presence of concentrated conditioned media from adipocytes or adipocyte growth medium, AM-1, or full culture medium with an equivalent volume of PBS added. Scale bars, 50 μm. adipocytes from n = 4 patient samples, processed as in (B) and media collected, performed in triplicate with three stacks per transwell; one-way ANOVA with Tukey correction; ** P < 0.01. ( G and H ) Representative images (G) and quantification (H) of a fibroblast-contracted 3D collagen matrix cancer cell invasion assay monitoring MM231 cancer cell invasion ± mature adipocyte coculture and stained for either Pan-CK or the proliferation marker Ki67. Scale bars, 100 μm. n = 3 biological replicates, triplicate matrices, eight regions per condition per replicate; one-way ANOVA with Tukey correction; *** P < 0.001).

Article Snippet: Primary antibodies in AdvanBlock-Fluor were incubated overnight at 4°C; IGFBP2 (R&D Systems, AF674), IGF-II (Millipore, 05-166-MI), GAPDH (Hytest, 5G4MAB6C5), and GFP (Thermo Fisher Scientific, A11122).

Techniques: Staining, In Situ, Immunofluorescence, Isolation, Cell Culture, Gene Expression, Reverse Transcription, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Invasion Assay, Marker

Journal: Science Advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: ( A ) Schematic of the proteomics experimental setup. LC-MS/MS, liquid chromatography–tandem mass spectrometry. IP, immunoprecipitation. ( B and C ) Representative Western blot (B) and densitometry analysis (C) after silencing of IGF2 (IGF-II gene) using siRNAs in MM231 cells ( n = 4 biological replicates; one-sample t test; * P < 0.05 and *** P < 0.001). ( D and E ) Representative images (D) and quantification (E) of MM231 cell invasion in inverted collagen/fibronectin matrices after IGF2 silencing. Scale bars, 50 μm. n = 3 biological replicates, performed in duplicate with three stacks per transwell; one-way ANOVA with Tukey correction; *** P < 0.001. ( F and G ) Representative images (F) and quantification (G) of MM231 cell invasion in inverted collagen/fibronectin matrices treated with PBS, IGF-II (10 ng/ml), IgG1κ (10 μg/ml), or anti–IGF-II (10 μg/ml). Scale bars, 50 μm. n = 3 biological replicates, performed in duplicate with three stacks per transwell; one-way ANOVA with Tukey correction; *** P < 0.001. ( H and I ) Representative IGF-II Western blot (H) and quantification (I) of TIF, MM231, MM468, and HCC1937 cells ( n = 6 biological replicates; one-sample t test; *** P < 0.001). ( J ) ELISA for human IGF-II in conditioned media from TIF, MM231, MM468, and HCC1937 cells ( n = 4 biological replicates; one-way ANOVA with Dunnett’s correction; * P < 0.05 and *** P < 0.001). ( K and L ) Matrigel invasion assays for MM231 (K) and MM468 (L) cells treated with IgG1κ or anti–IGF-II ( n = 3, eight fields of view (FOVs) per chamber, two to three invasion chambers per condition per replicate; two-tailed Student’s t test with Welch’s correction; ** P < 0.01). Scale bars, 100 μm. ( M ) Schematic of the proposed mechanism for IGFBP2 inhibition of invasion through disruption of breast cancer IGF-II autocrine signaling.

Article Snippet: Primary antibodies in AdvanBlock-Fluor were incubated overnight at 4°C; IGFBP2 (R&D Systems, AF674), IGF-II (Millipore, 05-166-MI), GAPDH (Hytest, 5G4MAB6C5), and GFP (Thermo Fisher Scientific, A11122).

Techniques: Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Inhibition, Disruption

Journal: Science Advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: ( A ) Representative H&E-stained breast tissue samples from healthy patients, patients with DCIS, or patients with IDC. Scale bars, 200 μm. ( B ) Quantification of adipocytes per section from healthy ( n = 8 patients, three to eight sections per patient), DCIS ( n = 6 patients, one to four sections per patient), and IDC ( n = 3 patients, one to two sections per patient). Kruskal-Wallis test using Dunn’s test to correct for multiple comparisons (** P < 0.01 and *** P < 0.001). ( C ) Representative image from a patient sample stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and keratin-8/keratin-14 (KRT8/14; red). Scale bar, 100 μm. n = 8 normal reduction mammoplasty patient samples. Autofluorescence is given in green. ( D ) Representative image of human DCIS breast cancer tissue stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and KRT8/14 (red). Scale bar, 100 μm ( n = 4 DCIS patient samples). Autofluorescence is given in green. ( E ) Representative image of human IDC breast cancer tissue stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and KRT8/14 (red; n = 3 IDC patient samples). Scale bar, 100 μm. Autofluorescence is given in green. The brightness of IGFBP2 staining was increased for display purposes only. ( F ) Quantification of IGFBP2 per adipocyte from healthy ( n = 8 patients, 143 to 280 adipocytes per patient), DCIS ( n = 6 patients, 39 to 410 adipocytes per patient), and IDC ( n = 3 patients, 75 to 283 adipocytes per patient). Kruskal-Wallis test using Dunn’s test to correct for multiple comparisons (*** P < 0.001).

Article Snippet: Primary antibodies in AdvanBlock-Fluor were incubated overnight at 4°C; IGFBP2 (R&D Systems, AF674), IGF-II (Millipore, 05-166-MI), GAPDH (Hytest, 5G4MAB6C5), and GFP (Thermo Fisher Scientific, A11122).

Techniques: Staining