Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 1: PDL1 expression in clinical brain tumor samples, glioma cell lines and CSF samples from the immunocompetent glioma mice model. A) Graph (left) represents PDL1 mRNA expression in brain tumors and melanomas (cBioPortal (http://www.cbioportal.org)). Note that the average PDL1 mRNA level in GBM is similar to the average PDL1 mRNA level in melanomas. The PDL1 values in GBM and low-grade gliomas which exceed the average PDL1 mRNA level in melanomas are highlighted in the red box. Kaplan-Meier plot (right) illustrates survival rates of glioma patients with high and low PDL1 expression levels; 9% versus 24% of 2 year survival for high and low PDL1 expression; the difference is significant, P=0.02 (data has been obtained from the Human Pathology Atlas). B) Immunohistochemical detection of PDL1 in the tissue microarray of normal and brain tumor samples. The images were taken at 40x magnification. C) Western blot illustrates PDL1 and Actin protein levels in control and brain tumor clinical samples. Graph represents PDL1 to Actin ratios for corresponding protein samples; 0.87 ± 0.32 (n=7) versus 0.19 ± 0.1 (n=7) for tumor and normal samples, respectively, the difference is significant, P=0.003. Two samples (marked by light grey color) of patients with hemorrhage have been excluded from the average. D) Western blots illustrate PDL1 protein levels in established and PDGx glioma cell lines (left) and in the extracellular media collected from these cell lines (right). The protein content of the collected media was concentrated six folds before an analysis (see method). The graph (right) provides PDL1 concentrations in CSF samples from control and tumor-bearing mice (see method). The immunocompetent glioma mice model with GL261 cells was utilized for this experiment. E) Images illustrate interactions of tumor neuro spheres from XD456 (a) and from two parental U251-IDH1-R132H cell lines with different PDL1 expression levels (b) with primary T-cells. Note that tumor neuro spheres with high PDL1 levels keep their integrity after 48 hours of interaction with primary T-cells (illustrated in the insert). The graph represents the average percent of neuro spheres after 48 hours of interaction with T-cells, 89 ± 7% (n=4) and 14 ± 5% (n=4) for cell lines with high and low PDL1 levels, respectively. The difference is significant, P=0.0002. Primary T-cells were loaded with calcein, AM (green) before experiments for visualization.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Microarray, Western Blot, Control

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 2: HIF1A accumulation evokes PDL1 up-regulation in glioma cell lines. A) HIF1A accumulations induced by CoCl2 treatment of U251 and U87 cell lines evoke PDL1 up-regulation in protein and mRNA levels. Western blot illustrates HIF1A and PDL1 levels in nuclear and cytoplasmic fractions, respectively, in control and after treatment with CoCl2 (85uM). Lamin A/C and alpha Tubulin were utilized to verify nuclear and cytoplasmic fractions, respectively. Graphs illustrate normalized PDL1/ GAPDH mRNA ratios after cell treatment with CoCl2 (85uM) at different time points. In each experiment, data has been normalized to the corresponding ratios in untreated cells, results are presented as mean ± S.D. B) Transcription initiation complexes encoded by pAC154-dual-dCas9VP160 plasmids, guided by sgRNAs to the HIF1A binding domains in the first intron of the PDL1 gene, evoke PDL1 overexpression. Western blot illustrates PDL1 and Actin protein levels in U251 cells after transfection with plasmids encoding control (scrambled sequence) sgRNA or HIF1A sgRNAs. Note that transcription initiation complexes guided by sgRNAs to the HIF1A binding domains (A), (B), and (A) with (B) simultaneously increased PDL1 expression by 1.5, 3.9 and 1.8 folds, respectively, compared to the control (PDL1 expression in the presence of transcription initiation complexes guided by scrambled sgRNA). In each experiment, PDL1 expression was normalized to the Actin expression.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Western Blot, Control, Binding Assay, Over Expression, Transfection, Sequencing, Expressing

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 3: MLN4924 treatment induces up-regulation of HIF1A and PDL1 in glioma cell lines. A) The inhibitory dose- response curves for MLN4924 in established, PDGx and PDGX-stem glioma cell lines. The IC50s are 0.3 ± 0.2 uM (n=4), 2.7 ± 1 uM (n=6), 3 ± 2 uM (n=3), 3 ± 1 uM (n=4), 2.9 ± 0.5 uM (n=4), 0.8 ± 0.2 uM (n =4), 0.2 ± 0.1uM (n=4) for LN221, U251, U87, XD456, JX10, XD456-stem, X14P-stem cell lines, respectively, after treatment with MLN4924 for 5 days. B) Western blots illustrate HIF1A and PDL1 protein levels in nuclear and cytoplasmic fractions in the control and after treatment with MLN4924 (1 uM, 5 days). LaminA/C and alpha Tubulin were utilized to verify nuclear and cytoplasmic fractions, respectively. C) The graph illustrates normalized PDL1/18S mRNA ratios after treatment with MLN4924 (1uM, 5 days) for different cell lines. Note the significant enhancement of the PDL1/18 mRNA ratio for all cell lines after MLN4924 treatment: 8 ± 3, 25 ± 5, 5 ± 1, 8 ± 3, 4.5 ± fold increase compared to the corresponding control values for U251, Ln229, U87, XD456, JX6 cell lines, respectively, P<0.05, n=3.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Western Blot, Control

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 4: Glioma cells treated with MLN4924 decrease T-cell proliferation via utilization of PD1/PDL1 signaling pathway. A) The graph illustrates an inhibition of T-cell proliferation after T-cell encounter with U251 cells (1); with U251 cells in the presence of an inhibitor of PD1/PDL1 interaction (2); with U251 cells treated with MLN4924 (1uM, for 4 days) (3); with U251 cells treated with MLN4924 (1uM, for 4 days) and in the presence of an inhibitor of PD1/PDL1 interaction (4). After MLN4924 treatment, glioma cells were washed and placed in the media with/and without an inhibitor of PD1/PDL1 interaction (4 uM). Note, that glioma cells treated with MLN4924 induce a stronger decrease of T-cell proliferation compared to untreated cells (55 ± 8% (n=4) versus 30 ± 8% (n=4), respectively, the difference is significant with P=0.0005). The reduction of T-cell proliferation induced by glioma cells treated with MLN4924 is inhibited in the presence of an inhibitor of PD1/PDL1 interaction (P=0.0009, n=4). B) The graph illustrates an inhibition of T-cell proliferation after T-cell encounter with XD456 cells (1); with UXD456 cells in the presence of an inhibitor of PD1/PDL1 interaction (2); with XD456 cells treated with MLN4924 (1uM, for 4 days) (3); with XD456 cells treated with MLN4924 (1uM, for 4 days) and in the presence of an inhibitor of PD1/PDL1 interaction (4). After MLN4924 treatment, glioma cells were washed and placed in media with/and without an inhibitor of PD1/PDL1 interaction. Note, that glioma cells treated with MLN4924 induce a stronger decrease of T-cell proliferation compared to untreated cells (74 ± 8% (n=4) versus 41 ± 7% (n=4), respectively, the difference is significant with P=0.0003). The reduction of T-cell proliferation induced by glioma cells treated with MLN4924 is inhibited in the presence of an inhibitor of PD1/PDL1 interaction (P=0.007, n=4).

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Inhibition

Journal: Cell reports

Article Title: Redox regulation of age-associated defects in generation and maintenance of T cell self-tolerance and immunity to foreign antigens

doi: 10.1016/j.celrep.2022.110363

Figure Lengend Snippet:

Article Snippet: CellsDirect one-Step RT-PCR kit (Invitrogen, Catalog number 11753–100) was used for reverse transcription and specific target amplification (RT-PCR) for each single-cell and universal mouse total RNA of 200 pg.

Techniques: Recombinant, Expressing, Microarray, Software

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Ascites in peritoneal carcinomatosis (PC) is biologically active (A) Kaplan-Meier curve showing survival of PC patients who underwent cytoreductive surgery (n = 121), stratified by the presence or absence of ascites. (B and C) Proliferation curves of (B) Colo-205 and (C) SNU-C1 cells treated with varying concentrations of ascites, measured by CellTitre-Glo assay. Data are represented as cell viability at day 5 relative to day 0. Dotted line represents cell viability of Colo-205 treated with 10% fetal bovine serum (FBS) at day 5 relative to day 0. Graph shows mean ± SD. (D and E) Migration of (D) Colo-205 and (E) SNU-C1 cells pre-treated with serum-free media (SFM), 10% FBS medium or 5% cell-free ascites (CFA) medium, assessed by transwell migration assay. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with SFM and 10% FBS or 5% CFA was denoted by ∗. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with 10% FBS and SFM or 5% CFA was denoted by # . # p < 0.05, ## p < 0.01, ### p < 0.001. Graph shows mean ± SD. (F) Enriched signaling pathways in colorectal PC cell lines upon treatment with CFA, identified by comparing gene expressions of cells treated with 5% versus 0.1% CFA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G and H) Treatment of (G) Colo-205 and (H) SNU-C1 cells with 5% CFA activated STAT3 signaling pathway through phosphorylation at Tyr705.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Glo Assay, Migration, Transwell Migration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Identification and validation of prognostic putative activators of STAT3 signaling in ascites (A) Workflow to identify clinically significant putative secreted STAT3 activators. (B) Kaplan-Meier survival curve illustrating poorer survival in patients expressing three biomarkers as compared to patients expressing 0–2 biomarkers. (C) Characterization of cytokine profiles in colorectal PC ascites and benign ascites. Top 25% most highly abundant cytokines in colorectal PC ascites (by mean abundance) are shown. Heatmap displays Z score of normalized mean pixel density of duplicate cytokine spots on the array. (D) Downregulated signaling pathways upon PAI-1 inhibition in Colo-205 cells exposed to CFA with high PAI-1 levels (PC085), CFA with low PAI-1 levels (PC249) and no PAI-1 (FBS control), identified using RNA microarray. Only signaling pathways with differential downregulation are presented. IL6-JAK-STAT3 signaling pathway was significantly downregulated in high PAI-1 CFA-treated cells upon PAI-1 inhibition. Normalized enrichment scores <0 indicate pathway suppression and scores >0 indicate pathway activation. (D) is representative of two independent biological experiments. ∗p < 0.05.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Expressing, Inhibition, Microarray, Activation Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Correlating PAI-1 level in ascites and intracellular STAT3 activation in cancer cells revealed distinct subgroups associated with differing susceptibility to PAI-1 inhibition (A) PAI-1 prevalence in colorectal PC ascites (n = 54). (B) Correlation between colorectal PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.4691, p = 0.0003). (C) PAI-1 prevalence in ascites of various histological PC subtypes (n = 156). † indicates benign ascites. (D) Correlation between various histological PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.6476, p < 0.0001). (A–D) PAI-1 concentrations are plotted on a log 2 scale to transform skewed data to normal distribution. p-STAT3(Y705) level was shown as optical density reading at 450 nm (OD450). (E) Untransformed values of PAI-1 and p-STAT3(Y705) levels from (D) were used for stratification strategy to identify patient subpopulations who might benefit from PAI-1 inhibition. Using 20 ng/mL PAI-1 level and 0.2 OD450 p-STAT3(Y705) level as cut-off values, three distinct subgroups of samples were observed: (i) high PAI-1 and high p-STAT3 levels, termed PAI-1 paracrine addicted (PPA) group (yellow region), (ii) low PAI-1 and high p-STAT3 levels, termed co-activators predominant (CAP) group (pink region) and (iii) low PAI-1 and low p-STAT3 levels, termed alternative pathways activation (APA) group (blue region). Each dot represents one patient ascites. Colors in each panel represent the various histological PC subtypes. All histological subtypes with less than five samples are grouped into other histological subtypes.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Activation Assay, Inhibition

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Cells dependent on PAI-1 to activate STAT3 are most susceptible to PAI-1 inhibition (A) Effect of TM5441 (PAI-1 inhibitor) on CFA-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow), CAP group (pink), APA group (blue) and FBS (control, black) demonstrated a left shift, indicating responsiveness to PAI-1 inhibition. (B) Differential sensitivity to TM5441 corresponding to the three subgroups, with PPA (n = 18) being the most sensitive to PAI-1 inhibition, followed by CAP (n = 59) and APA (n = 17). Graph shows mean ± SD. (C–E) Effect of (C) Napabucasin (STAT3 inhibitor), (D) BEZ235 (dual PI3K/mTOR inhibitor) and (E) Mitomycin C (conventional chemotherapeutic agent – DNA crosslinker) on the three subgroups of ascites-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow; n = 3), CAP group (pink; n = 3), APA group (blue; n = 1) and FBS (control, black). Targeting PAI-1, a dominant paracrine factor in ascites, was more effective than targeting downstream signaling pathway activated by ascites, proliferation pathway or DNA synthesis. (F) Evaluation of STAT3 suppression in Colo-205 cells treated with PPA CFA (PC085 and PC383), CAP CFA (PC249), APA CFA (PC010) and various concentrations of TM5441 by ELISA. STAT3 activation was shown as p-STAT3(Y705) and total STAT3 ratio at the indicated concentration relative to DMSO vehicle. Cells exposed to PPA CFA relied on PAI-1 to activate STAT3 as they required lower concentrations of TM5441 to suppress STAT3 activation. Graph shows mean ± SEM. Data in (A-E) are representative of at least three independent biological experiments and (F) is representative of two independent biological experiments. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Inhibition, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Activation Assay, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet:

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Sequencing, Microarray, Cell Culture, Software